Mechanism Elucidation and Inhibitor Discovery against ...

University of South FloridaScholar Commons

Graduate Theses and Dissertations Graduate School

November 2017

Mechanism Elucidation and Inhibitor Discoveryagainst Serine and Metallo-Beta-LactamasesInvolved in Bacterial Antibiotic ResistanceOrville A PembertonUniversity of South Florida oap2007gmailcom

Follow this and additional works at httpsscholarcommonsusfeduetd

Part of the Biochemistry Commons Microbiology Commons and the Molecular BiologyCommons

This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons It has been accepted for inclusion inGraduate Theses and Dissertations by an authorized administrator of Scholar Commons For more information please contactscholarcommonsusfedu

Scholar Commons CitationPemberton Orville A Mechanism Elucidation and Inhibitor Discovery against Serine and Metallo-Beta-Lactamases Involved inBacterial Antibiotic Resistance (2017) Graduate Theses and Dissertationshttpsscholarcommonsusfeduetd7435

Mechanism Elucidation and Inhibitor Discovery against Serine and Metallo--Lactamases

Involved in Bacterial Antibiotic Resistance

by

Orville A Pemberton

A dissertation submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

with a concentration in Allergy Immunology and Infectious Disease

Department of Molecular Medicine

Morsani College of Medicine

University of South Florida

Major Professor Yu Chen PhD Burt Anderson PhD

Gloria Ferreira PhD

James Leahy PhD

Date of Approval October 11 2017

Keywords Carbapenemase -Lactam Antibiotics X-ray Crystallography Molecular Docking

Drug Design

Copyright copy 2017 Orville A Pemberton

Acknowledgments

I would first like to thank my God and Savior Jesus Christ for getting me through

graduate school I could not have done it without Him I would also like to thank my mother

father and sister for their sacrifices and unwavering support throughout my life I also extend my

deepest gratitude to Dr Yu Chen for all the knowledge wisdom and guidance that he has left

with me these past five years I am grateful for everything that I have learned and experienced as

a member of his lab I would especially like to thank all my lab members both past and present

that have helped me tremendously throughout the years Dr Derek Nichols Dr Emmanuel

Smith Dr Xiujun Zhang Dr Eric Lewandowski Kyle Kroeck Michael Kemp Afroza Akhtar

Cody Johnson Michael Sacco Connor Fries Nicholas Torelli Sabbir Mirza and Jocelyn

Idrovo

I would also like to thank the Department of Molecular Medicine for helping me

complete my doctoral studies And lastly I would like to thank Dr Burt Anderson Dr Gloria

Ferreira and Dr James Leahy for having served on my committee and for extending to me their

time support and advice

i

Table of Contents

List of Tablesiv

List of Figuresv

List of Abbreviationsvii

Abstract ix

Chapter 1 Introduction1

11 Bacterial Antibiotic Resistance1

12 Resistance to -Lactam Antibiotics3

13-Lactamase Classification5

14 Carbapenemases9

15 Clinically Approved BLIs11

16 Fragment-Based Drug Discovery13

17 Molecular Docking13

18 X-ray Crystallography14

19 Structure-Based Drug Design Targeting -Lactamases15

110 Conclusions 15

111 Note to Reader 116

112 References31

Chapter 2 Molecular Basis of Substrate Recognition and Product Release by the Klebsiella

pneumoniae Carbapenemase (KPC-2)39

21 Overview39

22 Introduction39

23 Results and Discussion40

231 Product complex with cefotaxime41

232 Product complex with faropenem45

233 Unique KPC-2 structural features for inhibitor discovery46

24 Conclusions48

25 Materials and Methods48

251 Cloning expression and purification of KPC-248

252 Crystallization and soaking experiments49

253 Data collection and structure determinations50


27 References60

ii

Chapter 3 Studying Proton Transfer in the Mechanism and Inhibition of Serine -Lactamase

Acylation63

31 Overview63

32 Introduction64


331 Crystal structure of avibactam bound to CTX-M-1467

332 Ultrahigh resolution reveals the protonation states of Lys73

and Glu16667

34 Conclusions68


351 Expression and purification of CTX-M-1469

352 Crystallization and structure determination70


37 References76

Chapter 4 The Influence of Binding Kinetics on Inhibition of KPC-2 by Vaborbactam in

Countering Bacterial Resistance78

41 Overview78

42 Introduction79


431 Vaborbactam binding kinetics81

432 Vaborbactam potentiation of aztreonam in KPC-2 and

CTX-M-15 producing E coli83

433 Impact of S130G mutation on vaborbactam inhibition83

434 Structure determination of vaborbactam with KPC-285

44 Conclusions86


451 Generation of KPC-2 mutants91

452 Determination of MIC values and vaborbactam

potentiation experiments91

453 Evaluation of KPC-2 mutant proteins expression

level in PAM1154 strain92

454 Purification of KPC-2 WT and mutant proteins

for biochemical studies92

455 Determination of Km and kcat values for

nitrocefin cleavage by KPC-2 and mutant proteins93

456 Determination of Km and kcat values for meropenem

cleavage by KPC-2 and mutant proteins94

457 Determination of vaborbactam Ki values for

KPC-2 and mutant proteins using NCF as substrate94

458 Determination of vaborbactam Ki values for KPC-2

and mutant proteins using meropenem as substrate94

459 Stoichiometry of KPC-2 and mutant proteins inhibition94

4510 Determination of vaborbactam k2K inactivation

constant for WT KPC-2 and mutant proteins95

4511 Determination of Koff rates of enzyme activity

iii

recovery after KPC-2 and mutantsrsquo inhibition by vaborbactam96

4512 Crystallization experiments96

4513 Data collection and structure determination97



48 References106

Chapter 5 Leveraging Protein Promiscuity in Ligand Binding to Develop Broad-Spectrum

Carbapenemase Inhibitors112

51 Overview112

52 Introduction112


531 Structure-based inhibitor discovery against the

serine carbapenemase KPC-2 115

532 Inhibition of the MBLs NDM-1 and VIM-2118

533 Correlation of KPC-2 NDM-1 and VIM-2 inhibition120

534 Susceptibility of clinical isolates to compounds 9 11 and 13121

54 Conclusions122


551 KPC-2 expression and purification125

552 NDM-1 expression and purification126

553 VIM-2 expression and purification127

554 Molecular docking128

555 Steady-state kinetic analysis128




57 References139

Chapter 6 Summary143

Appendix 1 Copyright Permissions145

Appendix 2 Chapter 4 Supplementary Data154


iv

List of Tables Table 21 X-ray data collection and refinement statistics for KPC-2 apo

and hydrolyzed products58

Table 22 Hydrolyzed products hydrogen bond distances within KPC-259

Table 41 Stoichiometry of KPC-2 inhibition by various BLIs102

Table 42 Kinetic parameters of inhibition of KPC-2 and CTX-M-15

by vaborbactam and avibactam103

Table 43 Concentration-response of aztreonam potentiation by vaborbactam

against engineered E coli strains expressing KPC-2 and

CTX-M-15104

Table 44 Concentration-response of carbenicillin and piperacillin potentiation

by vaborbactam and avibactam against KPC-2 and KPC-2 S130G

producing strain of P aeruginosa105

Table 51 Nitrocefin inhibition assay of phosphonate compounds tested against

KPC-2 NDM-1 and VIM-2137

Table 52 In vitro activity of phosphonate compounds when combined with imipenem138

v

List of Figures

Figure 11 Antibiotic targets18

Figure 12 Mechanisms of antibiotic resistance19

Figure 13 Classes of -lactam antibiotics20

Figure 14 Strategies for -lactam resistance21

Figure 15 Ambler classification scheme of -lactamases22

Figure 16 Catalytic mechanism of class A -lactamases23

Figure 17 Zinc coordinating residues of MBL subclasses24

Figure 18 Catalytic mechanism of subclass B1 and B3 MBLs25

Figure 19 Comparison of carbapenems to penicillins and cephalosporins26

Figure 110 Commonly used BLIs27

Figure 111 General scheme for FBDD28

Figure 112 Growing number of protein structures deposited

in the PDB29

Figure 113 Structure-based design of a CTX-M BLI30

Figure 21 Chemical structures of intact and hydrolyzed cephalosporin

and penem -lactam antibiotics51

Figure 22 Superimposition of apo KPC-2 structures52

Figure 23 KPC-2 cefotaxime product complex53

Figure 24 Two molecules of hydrolyzed cefotaxime in the KPC-2 active site54

Figure 25 KPC-2 faropenem product complex55

vi

Figure 26 Comparison of KPC-2 active site with the narrow-spectrum

-lactamase TEM-1 and the ESBL CTX-M-1456

Figure 27 Molecular interactions of hydrolyzed cefotaxime and

faropenem in the active site of KPC-2 from LigPlot+57

Figure 31 Chemical structure and numbering of atoms of avibactam71

Figure 32 General mechanism of avibactam inhibition against serine -lactamases72

Figure 33 Crystal structure of avibactam bound to CTX-M-1473

Figure 34 Schematic diagram of CTX-M-14avibactam interactions74

Figure 35 Protonation states of active site residues in

CTX-M-14avibactam complex75

Figure 41 Chemical structures of clinically available and

promising new BLIs98

Figure 42 Vaborbactam bound to KPC-2 active site99

Figure 43 Superimposition of KPC-2vaborbactam and

CTX-M-15vaborbactam structures100

Figure 44 Vaborbactam induces a backbone conformational change

near the oxyanion hole101

Figure 51 Clinical and pre-clinical serine carbapenemase and MBL inhibitors132

Figure 52 X-ray crystal structures of KPC-2 bound to phosphonate inhibitors133

Figure 53 X-ray crystal structures of NDM-1 and VIM-2 bound to

phosphonate inhibitors134

Figure 54 Correlation of -lactamase inhibition135

Figure 55 Superimposition of phosphonate inhibitor and -lactam product complexes136

vii

List of Abbreviations

ALSAdvanced Light Source

AMAAspergillomarasmine A

APSAdvanced Photon Source

BATSI Boronic acid transition state inhibitor

BLI-Lactamase inhibitor

BSABovine serum albumin

CDCCenters for Disease Control and Prevention

CHDLCarbapenem-hydrolyzing class D -lactamase

CRECarbapenem-resistant Enterobacteriaceae

CTXCefotaximase

DBODiazabicyclooctane

DMSO Dimethyl sulfoxide

DTTDithiothreitol

EDTAEthylenediaminetetraacetic acid

EIEnzyme-inhibitor

ESBLExtended-spectrum -lactamase

FBDDFragment-based drug discovery

FDAFood and Drug Administration

HBHydrogen bond

viii

HTSHigh-throughput screening

IPTGIsopropyl -D-1-thiogalactopyranoside

KPCKlebsiella pneumoniae carbapenemase

LBLysogeny broth

MBLMetallo--lactamase

MDMolecular dynamics

MICMinimum inhibitory concentration

NAGN-acetylglucosamine

NAMN-acetylmuramic acid

NCFNitrocefin

NDMNew Delhi MBL

NMRNuclear magnetic resonance

OXAOxacillinase

PBPPenicillin-binding protein

PDBProtein Data Bank

PEGPolyethylene glycol

RMSDRoot-mean-square deviation

SARStructurendashactivity relationship

SBDDStructure-based drug design

SDS-PAGESodium dodecyl sulfate polyacrylamide gel electrophoresis

TCEPTris(2-carboxyethyl)phosphine

VIMVerona integron-encoded MBL

WTWild-type

ix

Abstract

The emergence and proliferation of Gram-negative bacteria expressing -lactamases is a

significant threat to human health -Lactamases are enzymes that degrade the -lactam

antibiotics (eg penicillins cephalosporins monobactams and carbapenems) that we use to treat

a diverse range of bacterial infections Specifically -lactamases catalyze a hydrolysis reaction

where the -lactam ring common to all -lactam antibiotics and responsible for their

antibacterial activity is opened leaving an inactive drug There are two groups of -lactamases

serine enzymes that use an active site serine residue for -lactam hydrolysis and metalloenzymes

that use either one or two zinc ions for catalysis Serine enzymes are divided into three classes

(A C and D) while there is only one class of metalloenzymes class B Clavulanic acid

sulbactam and tazobactam are -lactam-based BLIs that demonstrate activity against class A

and C -lactamases however they have no activity against the class A KPC and MBLs NDM

and VIM Avibactam and vaborbactam are novel BLIs approved in the last two years that have

activity against serine carbapenemases (eg KPC) but do not inhibit MBLs The overall goals

of this project were to use X-ray crystallography to study the catalytic mechanism of serine -

lactamases with -lactam antibiotics and to understand the mechanisms behind the broad-

spectrum inhibition of class A -lactamases by avibactam and vaborbactam This project also set

out to find novel inhibitors using molecular docking and FBDD that would simultaneously

inactivate serine -lactamases and MBLs commonly expressed in Gram-negative pathogenic

bacteria

x

The first project involved examining the structural basis for the class A KPC-2 -

lactamase broad-spectrum of activity that includes cephalosporins and carbapenems Three

crystal structures were solved of KPC-2 (1) an apo-structure at 115 Aring (2) a complex structure

with the hydrolyzed cephalosporin cefotaxime at 145 Aring and (3) a complex structure with the

hydrolyzed penem faropenem at 140 Aring These complex structures show how alternative

conformations of Ser70 and Lys73 play a role in the product release step The large and shallow

active site of KPC-2 can accommodate a wide variety of -lactams including the bulky

oxyimino side chain of cefotaxime and also permits the rotation of faropenemrsquos 6-1R-

hydroxyethyl group to promote carbapenem hydrolysis Lastly the complex structures highlight

that the catalytic versatility of KPC-2 may expose a potential opportunity for drug discovery

The second project focused on understanding the stability of the BLI avibactam against

hydrolysis by serine -lactamases A 083 Aring crystal structure of CTX-M-14 bound by avibactam

revealed that binding of the inhibitor impedes a critical proton transfer between Glu166 and

Lys73 This results in a neutral Glu166 and neutral Lys73 A neutral Glu166 is unable to serve as

a general base to activate the catalytic water for the hydrolysis reaction Overall this structure

suggests that avibactam can influence the protonation state of catalytic residues

The third project centered on vaborbactam a cyclic boronic acid inhibitor of class A and

C -lactamases including the serine class A carbapenemase KPC-2 To characterize

vaborbactam inhibition binding kinetic experiments MIC assays and mutagenesis studies were

performed A crystal structure of the inhibitor bound to KPC-2 was solved to 125 Aring These data

revealed that vaborbactam achieves nanomolar potency against KPC-2 due to its covalent and

extensive non-covalent interactions with conserved active site residues Also a slow off-rate and

xi

long drug-target residence time of vaborbactam with KPC-2 strongly correlates with in vitro and

in vivo activity

The final project focused on discovering dual action inhibitors targeting serine

carbapenemases and MBLs Performing molecular docking against KPC-2 led to the

identification of a compound with a phosphonate-based scaffold Testing this compound using a

nitrocefin assay confirmed that it had micromolar potency against KPC-2 SAR studies were

performed on this scaffold which led to a nanomolar inhibitor against KPC-2 Crystal structures

of the inhibitors complexed with KPC-2 revealed interactions with active site residues such as

Trp105 Ser130 Thr235 and Thr237 which are all important in ligand binding and catalysis

Interestingly the phosphonate inhibitors that displayed activity against KPC-2 also displayed

activity against the MBLs NDM-1 and VIM-2 Crystal structures of the inhibitors complexed

with NDM-1 and VIM-2 showed that the phosphonate group displaces a catalytic hydroxide ion

located between the two zinc ions in the active site Additionally the compounds form extensive

hydrophobic interactions that contribute to their activity against NDM-1 and VIM-2 MIC assays

were performed on select inhibitors against clinical isolates of Gram-negative bacteria

expressing KPC-2 NDM-1 and VIM-2 One phosphonate inhibitor was able to reduce the MIC

of the carbapenem imipenem 64-fold against a K pneumoniae strain producing KPC-2 The

same phosphonate inhibitor also reduced the MIC of imipenem 4-fold against an E coli strain

producing NDM-1 Unfortunately no cell-based activity was observed for any of the

phosphonate inhibitors when tested against a P aeruginosa strain producing VIM-2 Ultimately

this project demonstrated the feasibility of developing cross-class BLIs using molecular docking

FBDD and SAR studies

1

Chapter 1

Introduction

11 Bacterial Antibiotic Resistance

Bacteria are single-celled organisms that are ubiquitous throughout the earth living in

diverse environments ranging from hot springs and glaciers to the human body The majority of

bacteria are non-pathogenic however there are certain bacteria that have the ability to cause

disease in humans [1 2] These pathogenic bacteria cause a variety of illnesses such as meningitis

pneumonia gastroenteritis and sepsis Usually these illnesses are life-threatening but the

discovery of drugs known as antibiotics has dramatically decreased the rates of death Antibiotics

are drugs that selectively target bacteria and either inhibit or kill them by stopping a vital cellular

process (Fig 11) [3] The modern antibiotic era began almost ninety years ago with the discovery

of penicillin by Alexander Fleming In 1928 Fleming serendipitously observed mold growing in

dishes of Staphylococcus bacteria Around this mold no bacterial growth occurred This led

Fleming to hypothesize that the mold was secreting some substance that inhibited bacterial growth

Ultimately the mold was identified as Penicillium rubens and the name ldquopenicillinrdquo was used to

describe the antibacterial substance [4 5] Several years later large-scale production of penicillin

was carried out which provided the medical community a treatment option for a wide variety of

bacterial infections [5] Subsequently many other classes of antibiotics such as aminoglycosides

sulfonamides tetracyclines macrolides and quinolones were discovered and developed that added

to our antibiotic arsenal [6 7]

2

Despite the successes surrounding the introduction of penicillin there were some concerns

about its use In 1940 researchers reported that an Escherichia coli strain could inactivate

penicillin by producing an enzyme known as penicillinase [8] This observation was also

documented in 1942 when hospitalized patients infected with strains of Staphylococcus aureus did

not respond to penicillin treatment [9 10] Over the last fifty years resistance to penicillin has

skyrocketed with an estimated 90-95 of S aureus clinical isolates throughout the world

demonstrating resistance to penicillin [11] This resistance trend was also mirrored against other

antibiotics For example aminoglycosides were discovered in 1946 and in the same year

resistance was already observed tetracyclines were discovered in 1944 and by 1950 resistance

was seen in the clinic [12]

According to the Centers for Disease Control and Prevention (CDC) almost all bacterial

infections are becoming increasingly resistant to the antibiotic therapy of choice [13] In 2013 the

CDC stated that the human race has entered the ldquopost-antibiotic erardquo in which infections typically

cured by antibiotics are now becoming lethal [14] The CDC estimates that each year in the United

States at least 2 million people contract a bacterial infection that is resistant to one or more

antibiotics designed to treat that infection Approximately 23000 individuals die each year as a

direct result of those antibiotic-resistant infections though this number may be an underestimate

because deaths attributed to HIV cancer or surgery may in fact be due to an antibiotic-resistant

bacterial infection [13] Antibiotic resistance poses not only a health threat but also an economic

impact In many cases individuals diagnosed with an antibiotic-resistant infection will often

require longer hospital stays prolonged treatment and more doctor visits than individuals with an

infection that responds to antibiotic treatment As a result studies have estimated the economic

impact of antibiotic resistance to be in the range of $6-60 billion annually [15 16] Therefore

3

antibiotic resistance is quickly becoming an emerging global health epidemic and economic

burden

12 Resistance to -Lactam Antibiotics

Bacteria can utilize numerous mechanisms to become resistant to antibiotic treatment The

four main mechanisms of antibiotic resistance include (1) enzymatic inactivation or modification

of the drug (2) target modification (3) decreased drug penetration and (4) bypass of pathways

(Fig 12) [17] The first antibiotic resistance mechanism described was enzymatic inactivation of

penicillin by penicillinases [18] Penicillin belongs to a class of antibiotics known as -lactams

which also includes cephalosporins monobactams and carbapenems (Fig 13) [19] All -lactam

antibiotics possess a four-membered -lactam ring at the core of their structure that is crucial to

their antibacterial activity [20 21] -Lactam antibiotics are broad-spectrum antibiotics and work

by interfering with the synthesis of the bacterial cell wall The cell wall is vital to the survival of a

bacterium for several reasons Overall the major functions of the bacterial cell wall are to help

maintain the cell shape and protect the cell from osmotic changes that it may encounter in the

environment [22-24] The bacterial cell wall is composed of peptidoglycan an organic polymer

consisting of an interlocking network of the polysaccharides N-acetylglucosamine (NAG) and N-

acetylmuramic acid (NAM) cross-linked by short peptides [24] The extensive cross-linking of

peptidoglycan greatly contributes to the strength of the bacterial cell wall This cross-linking

process is catalyzed by a group of enzymes known as penicillin-binding protein (PBPs) PBPs are

also known as DD-transpeptidases since they are responsible for forming the peptide cross bridge

between chains of NAG and NAM [25 26]

4

Some bacteria can be classified according to the chemical and physical properties of their

cell wall This method is based on the Gram stain developed by Danish physician Hans Christian

Gram in 1884 The Gram stain differentiates between Gram-positive and Gram-negative bacteria

by staining these cells violet or pink Gram-positive bacteria have a thick layer of peptidoglycan

in their cell wall and thus can retain the crystal violet dye when stained whereas Gram-negative

bacteria have a thinner peptidoglycan layer which cannot retain the crystal violet dye when

decolorized and are subsequently counterstained pink [27] The Gram stain is a very useful

technique in identifying bacteria however not all bacteria can be Gram stained For example

Mycobacterium species cannot be Gram stained due to the presence of mycolic acids in their cell

walls which prevent uptake of the dyes used [28]

The bacterial cell wall presents a promising target to antibiotics for a variety of reasons

Firstly human cells lack cell walls meaning that cell wall synthesis inhibitors will selectively

target only bacterial cells [29] Secondly the cell wall is essential to the survival of a bacterium

and inhibiting its synthesis will ultimately result in cell death [22-24] -Lactam antibiotics

interfere with the cross-linking reaction that gives the cell wall its rigidity -Lactam antibiotics

resemble the natural substrate of PBPs the D-Ala-D-Ala group found at the end of the pentapeptide

precursors of peptidoglycan This structurally similarity enables -lactam antibiotics to bind to

PBPs and irreversibly acylate an active site serine residue [25]

Although -lactam antibiotics have been a mainstay of treatment for a diverse range of

bacterial infections they are becoming less effective due to the emergence of antibiotic resistance

In general there are three strategies that bacteria use to become resistant to -lactam antibiotics

(1) the synthesis of -lactam degrading enzymes known as -lactamases (2) alteration of PBP

targets and (3) the active transport of -lactam molecules out of the cell via membrane-associated

5

proteins known as efflux pumps (Fig 14) [25] The most common mechanism of -lactam

resistance among Gram-negative bacteria is the production of -lactamases that degrade the

antibiotic before it can reach its PBP target [19 30] -Lactamases provide antibiotic resistance to

bacteria by catalyzing the hydrolysis of the four-membered -lactam ring that is shared by all -

lactam antibiotics Once the -lactam ring is hydrolyzed the antibiotic is no longer effective [19]

What makes -lactamases even more worrisome is the fact that the genes encoding them are

commonly located on mobile genetic elements such as plasmids and transposons which facilitates

their spread among bacteria further contributing to the problem of antibiotic resistance [31]

13 -Lactamase Classification

-Lactamases can be classified using two different schemes The first scheme classifies -

lactamases according to their substrate profiles and inhibition properties This groups -lactamases

into penicillinases cephalosporinases extended-spectrum -lactamases (ESBLs) and

carbapenemases Also taken into consideration is whether the -lactamases are inhibited by the -

lactamase inhibitors (BLIs) clavulanic acid sulbactam and tazobactam Additionally inhibition

by the chelating agent ethylenediaminetetraacetic acid (EDTA) indicates the -lactamases are

metalloenzymes [32] The second and more commonly used classification scheme for -

lactamases is known as the Ambler classification which groups -lactamases into four classes (A

B C and D) based upon their primary structure (Fig 15) [32 33] Classes A C and D include

enzymes that use an active site serine residue to carry out hydrolysis of -lactam substrates

whereas class B -lactamases (divided into subclasses B1 B2 and B3) are metalloenzymes that

use one or two zinc ions to perform -lactam hydrolysis [32-34]

6

The most abundant -lactamases belong to class A comprising more than 550 enzymes

Among these enzymes the most frequently encountered -lactamases include TEM SHV CTX-

M and KPC [32 35 36] Like PBPs class A -lactamases form an acylndashenzyme complex with -

lactam antibiotics however unlike PBPs class A -lactamases can carry out a deacylation

reaction liberating the enzyme and resulting in the release of a hydrolyzed -lactam product (Fig

16) [36-38] There are key residues that are implicated in the acylation and deacylation reaction

of class A -lactamase catalysis It has been proposed that Glu166 serves as the general base in

the acylation part of catalysis by deprotonating the catalytic Ser70 via a water molecule before

Ser70 attacks the -lactam ring [39] Glu166 is also involved in the deacylation reaction where it

serves as a base to activate a hydrolytic water molecule [40] Mutagenesis of Glu166 impairs

deacylation however Glu166 mutant enzymes are still able to form acylndashenzyme complexes [39]

This suggests that another active site residue Lys73 which is located near other catalytic residues

(Ser70 Ser130 and Glu166) can act as the general base during the acylation step Ultrahigh

resolution and neutron crystal structures of the Toho-1 E166A acylndashintermediate confirmed that

Lys73 was neutral and can serve as the general base for the acylation reaction [41]

Class C -lactamases are another major cause of -lactam resistance among bacterial

pathogens Members of the class C family include the clinically relevant -lactamases ADC

AmpC CMY and FOX [32 42] Overall class C -lactamases behave very similarly to class A

-lactamases in terms of catalysis Class C -lactamases use an active site serine residue (Ser64)

to attack the -lactam ring Studies have suggested that two other residues play an important role

in the activation of Ser64 for nucleophilic attack Lys67 and Tyr150 Other studies have also

proposed that the substrate may play a role in activation of Ser64 [43 44] For the deacylation

7

reaction computational studies have suggested a Tyr150Lys67 general base mechanism Tyr150

is largely protonated during the acylndashenzyme complex in which prior to -lactam hydrolysis

transfers a proton to Lys67 This proton transfer allows Tyr150 to align towards and activate the

catalytic water while maintaining a hydrogen bond (HB) with Lys67 The catalytic water then

carries out a nucleophilic attack on the carbonyl carbon of the acylndashenzyme complex resulting in

-lactam hydrolysis [45]

Class D -lactamases like classes A and C are serine enzymes but interestingly the

catalytic mechanism of class D -lactamases differs markedly [46 47] Class D -lactamases are

commonly referred to as OXA enzymes due to their ability to hydrolyze oxacillin a penicillin

derivative Class D -lactamases include over 400 members with clinically important enzymes

such as OXA-2440 OXA-48 and OXA-58 found in a variety of Gram-positive and Gram-

negative pathogens [48-51] Class D -lactamases use an active site Ser70 to carry out nucleophilic

attack on the -lactam ring however an unusual modification of Lys73 is observed in various

crystal structures Lys73 undergoes carbamylation which has been demonstrated to be essential

for both the enzyme acylation and deacylation steps of catalysis [46 47 51 52] This post-

translational modification of Lys73 is unique to class D -lactamases and has not been observed

in any other class of -lactamase [52] Another unique property of class D -lactamases is their

susceptibility to inhibition by sodium chloride Research has suggested that the presence of a Tyr

residue at position 144 is correlated with inhibition by sodium chloride as mutating Tyr144 to Phe

alleviates sodium chloride inhibition [53 54]

Although class B -lactamases catalyze the same reaction as class A C and D -

lactamases the mechanism by which they accomplish this does not involve an active site serine

8

residue or formation of a covalent intermediate [55] Additionally class B -lactamases have no

sequence or structural similarity to class A C and D -lactamases [56] Rather than using an active

site serine residue class B -lactamases are metalloenzymes that use zinc for activity Class B -

lactamases or metallo--lactamases (MBLs) have as little as 25 sequence identity between some

members however multiple crystal structures show that all MBLs have a common fold known

as an sandwich fold with the active site located between domains [57] The active site of

MBLs supports six residues that can coordinate either one or two zinc ions used in catalysis (Fig

17) MBLs are divided into three subclasses (B1 B2 and B3) which differ from one another

based on amino acid sequence B1 and B3 enzymes are maximally active when two zincs are

bound in the active site In B1 and B3 enzymes the first zinc ion (called Zn1) is coordinated by

three His residues However the residues coordinating the second zinc ion (called Zn2) differ

between B1 and B3 enzymes In B1 enzymes Zn2 is coordinated by Asp Cys and His whereas

in B3 enzymes Zn2 is coordinated by Asp His and His [57] B2 enzymes unlike B1 and B3

enzymes are most active with only one zinc in the active site and binding of a second zinc ion is

inhibitory For B2 enzymes the inhibitory Zn1 binding site is formed by Asn His and His while

the activating Zn2 binding site is formed by Asp Cys and His [58] B1 and B3 enzymes have a

broad-substrate profile that includes all -lactam antibiotics except aztreonam while B2 enzymes

selectively hydrolyze carbapenems [59]

B1 enzymes are the most clinically relevant and researched MBLs and include members

such as NDM-1 IMP-1 and VIM-2 [55] Catalysis of B1 enzymes begins with binding of the -

lactam substrate to the zinc metal center with the carbonyl oxygen of the four-membered -lactam

ring interacting with Zn1 and the carboxyl group on the five- or six-membered fused ring

interacting with Zn2 (Fig 18) A nucleophilic hydroxide ion stabilized and located between Zn1

9

and Zn2 is positioned for its attack on the carbonyl carbon of the -lactam ring Attack of the -

lactam ring leads to the formation of a tetrahedral intermediate [57] Ultimately this tetrahedral

intermediate is broken down however the mechanism by which this occurs is still debated One

proposed mechanism involves direct coordination of the substrate departing amine nitrogen to the

zinc ion Another hypothesis is that a metal-bound water molecule donates a proton to the amine

nitrogen leaving group leading to cleavage of the C-N bond [56]

14 Carbapenemases

Among all the -lactam antibiotics carbapenems have the broadest spectrum of activity

against Gram-positive and Gram-negative bacterial pathogens and are often considered the last

line of therapy against multi-drug resistant infections [60] Carbapenems (eg imipenem

meropenem doripenem and ertapenem) are inherently stable against many different serine -

lactamases due to a unique chemical property (Fig 19) -Lactams such as penicillins and

cephalosporins have an acylamino substituent attached to the -lactam ring however

carbapenems depart from this trend by having a hydroxyethyl side chain which is key to their

potency [60] The hydroxyethyl group of carbapenems is attached in the trans-configuration which

differs from the cis-configuration found in the side chains of penicillins and cephalosporins (Fig

19) [61] Studies have shown that the trans-hydroxyethyl group prevents the attack of the catalytic

water on the acyl linkage due to electrostatic and steric hindrance thereby trapping the -lactamase

at the acylndashintermediate stage [60]

Unfortunately some -lactamases have evolved to include carbapenems in their substrate

profile These -lactamases commonly belong to classes A (eg KPC GES NMC-A IMI and

SME) B (NDM VIM IMP and SPM) and D (OXA-23 -2440 -48 -51 -58 and -143)

10

[556263] Class C -lactamases are not typically classified as carbapenemases due to their weak

hydrolytic activity against carbapenems however there are a few cases where carbapenemase

activity has been observed in class C enzymes most notably with CMY-10 [60 64]

Of all the class A carbapenemases the Klebsiella pneumoniae carbapenemase (KPC) is the

most frequently encountered in clinic and causes the most health concern due to its location on

mobile genetic elements such as plasmids and transposons Although KPC is commonly found in

K pneumoniae it is also found in other Gram-negative pathogens especially those of the

Enterobacteriaceae family [62 65] In fact carbapenem-resistant Enterobacteriaceae (CRE) have

been classified by the CDC as ldquoan important emerging threat to public healthrdquo [66] There are 23

variants of KPC (KPC-2 to KPC-24) with KPC-2 being the most prevalent variant [67 68]

Compared with noncarbapenemases (eg CTX-M SHV TEM) the active site of KPC-2 is larger

and shallower allowing for the accommodation of a wide variety of -lactams even those with

ldquobulkyrdquo side chains This property enables KPC-2 to hydrolyze penicillins cephalosporins

monobactams carbapenems and even the -lactam derived BLIs [69]

Carbapenem-hydrolyzing class D -lactamases (CHDLs) are a clinical problem due to their

ability to compromise the use of carbapenem antibiotics Most CHDLs are found in isolates of

Acinetobacter baumannii however some CHDLs are found in K pneumoniae and to a lesser

extent other members of the Enterobacteriaceae family [63] OXA-48 is one of the main CHDLs

isolated around the world and is commonly found in Mediterranean countries Western Europe

and Africa [70] OXA-48 has the highest catalytic efficiency for imipenem out of all the known

class D -lactamases This observation is reflected in OXA-48-producing K pneumoniae strains

exhibiting multi-drug resistance patterns especially towards carbapenems [71] Further

11

complicating matters OXA-48 is poorly inhibited by clavulanic acid sulbactam and tazobactam

[70]

All MBLs can hydrolyze carbapenems and are not inhibited by the classical serine BLIs

The B1 enzymes NDM and VIM are commonly produced by CRE and Pseudomonas aeruginosa

two of the biggest threats to clinical therapies [55] To date 16 variants of NDM have been

identified with the major worldwide variant being NDM-1 and 46 variants of VIM have been

discovered with the most frequent variant being VIM-2 [55 67 72] Interestingly all MBLs are

unable to hydrolyze the monobactam aztreonam which would be an effective therapy against

bacteria solely producing MBLs however MBL-producing bacteria commonly produce serine -

lactamases as well which can hydrolyze aztreonam Therefore aztreonam alone is usually not a

reliable treatment option for MBL-producing bacteria [59 73]

15 Clinically Approved BLIs

A tremendous amount of research has been carried out to discover BLIs that would

hopefully restore the efficacy of the -lactam antibiotics The first breakthrough occurred during

the mid-1970s via a natural product screen that identified a compound produced by Streptomyces

clavuligerus [74] This compound was named clavulanic acid and possessed a structure similar to

penicillin including the -lactam ring Clavulanic acid is a potent inhibitor of many clinically

important class A -lactamases and acts synergistically with -lactams to kill -lactamase

producing bacteria Further research led to the identification of two more BLIs sulbactam and

tazobactam that increased the spectrum of activity to include class C -lactamases (Fig 110) [75]

However there are a few drawbacks to clavulanic acid sulbactam and tazobactam which include

(1) these inhibitors on their own possess no antibacterial activity [76] (2) the presence of a -

12

lactam ring makes these inhibitors susceptible to -lactamase mediated-resistance and (3) they

have no activity against the serine carbapenemases (eg KPC-2 and OXA-48) and MBLs (eg

NDM-1 and VIM-2) [77]

To help address the shortcomings of clavulanic acid sulbactam and tazobactam novel

BLIs are necessary The first of these novel inhibitors was avibactam a synthetic

diazabicyclooctane (DBO) non--lactam inhibitor (Fig 110) Unlike the previous inhibitors

avibactam can inhibit class A (including KPC-2) class C and some class D -lactamases through

a covalent reversible mechanism [75 78] In 2015 the Food and Drug Administration (FDA)

approved the combination of the third-generation cephalosporin ceftazidime with avibactam

(marketed as Avycaz) to treat certain multi-drug resistant bacterial infections [77-79]

Unfortunately avibactam has no activity against MBLs and some mutations in KPC appear to

confer resistance to ceftazidimeavibactam [80 81] Another novel BLI called vaborbactam

(formerly RPX7009) is a cyclic boronic acid compound with potent activity against KPC-2 and

other class A and C enzymes (Fig 110) [75 77 82] Boronic acid compounds have long been

established as potent inhibitors of serine -lactamases due to their ability to form covalent adducts

with the active site serine residue [83] Similarly vaborbactam forms a reversible covalent bond

with the active site serine residue [77] In 2017 the FDA approved the combination of meropenem

(a carbapenem) with vaborbactam marketed as Vabomere for adults with complicated urinary

tract infections including pyelonephritis caused by CRE [84] However like avibactam

vaborbactam has no activity against MBLs [77] Therefore additional research is required to find

an inhibitor with dual serine -lactamase and MBL activity

13

16 Fragment-Based Drug Discovery

Discovering novel inhibitors against protein targets is a challenging and time-consuming

endeavor The drug discovery process can be generally summarized into four stages (1) target

identification and validation (2) lead discovery (3) lead optimization and (4) clinical trials [85]

Stumbles can be had at any of these stages however the hardest part is usually the second stage

of lead discovery [86] The starting point for lead candidates includes natural products high-

throughput screening (HTS) of large compound libraries and fragment-based drug discovery

(FBDD) [87] HTS and FBDD are the most common methods used for lead identification both

having certain advantages and limitations The HTS method began in the late 1980s due to a shift

from phenotypic to target-based drug discovery [88] In HTS a large number of compounds (~

105 ndash 106) are screened to assess for biological activity against a target This has led to the

identification of drug-like and lead-like compounds with desirable pharmacological and biological

activity Although HTS uses a large compound library it only samples a small fraction of the

chemical space of small molecules FBDD is a powerful tool that has recently emerged and has

been crucial in the small-molecule drug design process Fragments are low-molecular-weight (lt

300 Da) compounds that usually have low binding affinity for the target [89-91] These fragments

are then modified grown and linked together to create a lead compound with high affinity and

selectivity (Fig 111) Compared to HTS FBDD has the advantage of sampling a much wider

chemical space thus producing a significantly higher hit rate [91]

17 Molecular Docking

Molecular docking has become an increasingly important computational tool that is vital

to drug discovery The goal of molecular docking is to predict the ligand binding mode to a protein

14

structure Many variables play a role in the prediction of ligand-protein interactions which include

the flexibility of the ligand and target electrostatic interactions van der Waals interactions

formation of HBs and presence of water molecules [92 93] The sum of these interactions is

determined by a scoring function which estimates binding affinities between the ligand and target

(Fig 111) [94] Numerous molecular docking programs have been developed for both academic

and commercial use that utilize different algorithms for ligand docking Some of these programs

include AutoDock [95] DOCK [96] Glide [97] and SwissDock [98]

18 X-ray Crystallography

X-ray crystallography is a powerful technique that allows researchers to analyze the three-

dimensional structure of macromolecules such as proteins X-ray crystallography is a multifaceted

technique involving large scale expression of the protein of interest purifying the protein and

setting up multiple crystallization trials in order to find suitable crystals for X-ray diffraction

Ultimately solved protein structures are deposited into the Protein Data Bank (PDB) which

currently contains over 100000 protein structures solved using X-ray crystallography (Fig 112)

[99] X-ray crystallography is an indispensable technique in FBDD and lead optimization

Analyzing the three-dimensional structure of a protein target bound by a ligand provides valuable

insights into the interactions that drive binding affinity as well as highlighting the potential areas

for modification of the ligand in an effort to increase activity This approach is known as structure-

based drug design (SBDD) since it combines the power of many techniques such as X-ray

crystallography nuclear magnetic resonance (NMR) medicinal chemistry and FBDD to guide

and assist drug development [100]

15

19 Structure-Based Drug Design Targeting -Lactamases

FBDD molecular docking and SBDD have all been used to discover novel inhibitors

against serine -lactamases and MBLs [101] One notable example of this success is against the

class A -lactamase CTX-M-9 [102] For CTX-M-9 FBDD led to the discovery of a tetrazole-

based fragment inhibitor with a Ki of 21 M A complex crystal structure of this compound in the

active site of CTX-M-9 revealed two binding hot spots that could be exploited to increase affinity

The fragment was grown by adding substituents and ultimately the affinity was improved more

than 200-fold against CTX-M-9 (Fig 113) [102]

110 Conclusions

Bacterial antibiotic resistance is quickly becoming a global health threat A common

mechanism of bacterial antibiotic resistance is the production of -lactamase enzymes that can

deactivate the -lactam antibiotics (eg penicillins cephalosporins monobactams and

carbapenems) There are four classes of -lactamases that differ from one another based on their

amino acid sequence and mechanism of action Class A C and D -lactamases are enzymes that

use an active site serine residue to perform -lactam hydrolysis whereas class B -lactamases are

metalloenzymes that use one or two zinc ions to catalyze the same reaction One of the most

alarming resistance trends is the emergence of carbapenemases that can deactivate virtually all -

lactam antibiotics Carbapenemases such as KPC-2 (class A) NDM-1VIM-2 (class B) and OXA-

48 (class D) have been reported in numerous Gram-negative pathogens To address the issue of

carbapenemases inhibitors such as avibactam and vaborbactam were developed however these

inhibitors fall short since they are active only against serine carbapenemases and not MBLs This

16

dissertation will expand on the understanding of serine -lactamase catalysis and inhibition

(Chapters 2 3 and 4) Also presented will be the effort of finding a dual action serine

carbapenemase and MBL inhibitor using molecular docking FBDD and structure-activity

relationship (SAR) studies (Chapter 5)


Figure 11 in this chapter was previously published by [12] Lewis K (2013) Platforms

for antibiotic discovery Nature Reviews Drug Discovery 12(5) 371-387 and has been reproduced

with permission (See Appendix 1)




Figure 13 in this chapter was previously published by [57] Palzkill T (2012) Metallo--

lactamase structure and function Annals of the New York Academy of Sciences 1277(1) 91-104

and has been reproduced with permission (See Appendix 1)

Figure 14 in this chapter was previously published by [25] Wilke M S Lovering A L

amp Strynadka N C (2005) -Lactam antibiotic resistance A current structural perspective

Current Opinion in Microbiology 8(5) 525-533 and has been reproduced with permission (See

Appendix 1)

Figure 16 in this chapter was previously published by [39] Vandavasi V G Weiss K

L Cooper J B Erskine P T Tomanicek S J Ostermann A Coates L (2016) Exploring

the mechanism of -lactam ring protonation in the class A -lactamase acylation mechanism using

17

neutron and X-ray crystallography Journal of Medicinal Chemistry 59(1) 474-479 and has been

reproduced with permission (See Appendix 1)







Figure 19 in this chapter was previously published by [60] Papp-Wallace K M

Endimiani A Taracila M A amp Bonomo R A (2011) Carbapenems past present and future

Antimicrobial Agents and Chemotherapy 55(11) 4943-4960 and has been reproduced with

permission (See Appendix 1)

Figure 112 in this chapter was previously published by [99] Shi Y (2014) A glimpse of

structural biology through X-ray crystallography Cell 159(5) 995-1014 and has been reproduced


Figure 113 in this chapter was previously published by [102] Nichols D A Jaishankar

P Larson W Smith E Liu G Beyrouthy R Chen Y (2012) Structure-based design of

potent and ligand-efficient inhibitors of CTX-M class A -lactamase Journal of Medicinal

Chemistry 55(5) 2163-2172 and has been reproduced with permission (See Appendix 1)

18

Figure 11 Antibiotic targets Targets for antibiotics in bacteria include DNA

synthesis protein synthesis cell wall synthesis and folic acid metabolism Adapted

with permission from [12] See Appendix 1

19

Figure 12 Mechanisms of antibiotic resistance The main mechanisms of antibiotic

resistance include enzyme inactivation or modification target modification decreased

drug penetration increased drug efflux and bypass of pathways Adapted with

permission from [12] See Appendix 1

20

Figure 13 Classes of ‐lactam antibiotics (A) Core structure of penicillins (B) Core structure of cephalosporins (C) Core structure of monobactams (D) Core structure of carbapenems Different R‐groups distinguish various penicillin cephalosporin monobactam and carbapenem derivatives Adapted with permission from [57] See Appendix 1

21

Figure 14 Strategies for -lactam resistance -Lactam resistance can be

mediated by -lactamases alterations in PBPs and efflux pumps Adapted with


22

Figure 15 Ambler classification scheme of -lactamases Schematic diagram of Ambler

classification system

-Lactamases

Serine Enzymes Metalloenzymes

C A D B

B1 B2 B3

23

Figure 16 Catalytic mechanism of class A -lactamases Class A -lactamase catalysis proceeds

through a two-step acylationdeacylation reaction that leads to -lactam hydrolysis The acylation reaction

initiates with the formation of a pre-covalent substrate complex (1) General base-catalyzed nucleophilic

attack on the -lactam carbonyl by Ser70rsquos hydroxyl group proceeds through a tetrahedral intermediate

(2) to a transiently stable acylndashenzyme adduct (3) In the deacylation phase the acylndashenzyme adduct (3)

undergoes general base-catalyzed attack by a hydrolytic water molecule to form a second tetrahedral

intermediate (4) which collapses to a post-covalent product complex (5) from which the hydrolyzed -

lactam product is released Adapted with permission from [39] See Appendix 1

24

Figure 17 Zinc coordinating residues of MBL subclasses (A) Subclass B1 (B) subclass B2 and (C) subclass B3 Adapted with permission from [57] See Appendix 1

25

Figure 18 Catalytic mechanism of subclass B1 and B3 MBLs The zinc ions are labeled and interactions are shown with dashed lines (A) Cephalosporin substrate binds to the active site and interacts with both Zn1 and Zn2 via the carbonyl oxygen and carboxyl groups respectively The bound hydroxide is positioned to attack the carbonyl carbon of the substrate (B) Anionic intermediate bound in the active site via stabilizing interactions provided by Zn2 Adapted with permission from [57] See Appendix 1

26

Figure 19 Comparison of carbapenems to penicillins and cephalosporins (A) A 1--methyl (red) prevents breakdown from the renal enzyme dehydropeptidase I (DHP-I) (B) The pyrrolidine ring (red) increases stability and spectrum against multiple bacteria (C) Penicillin cephalosporin and carbapenem chemical structures Carbapenem has a five-membered ring as does penicillin but a carbon is located at position 1 instead of a sulfur (D) All carbapenems have a hydroxyethyl group at C-6 (E) The R configuration of the hydroxyethyl increases the -lactams potency (F) The trans-configuration of carbapenems at the C-5mdashC-6 bond increases their potency and stability against -lactamases as compared to penicillins and cephalosporins which have a cis-configuration Adapted with permission from [60] See Appendix 1

27

Figure 110 Commonly used BLIs -Lactam (clavulanic acid sulbactam and tazobactam) and

non--lactam-based (avibactam and vaborbactam) BLIs

Clavulanic acid Sulbactam

Avibactam Vaborbactam

Tazobactam

28

Figure 111 General scheme for FBDD In FBDD a library of small molecules is placed into the binding site of a target using molecular docking A scoring function ranks the molecules and compounds are purchased or synthesized for biochemical testing Low affinity inhibitors are selected and false positives are removed The low affinity inhibitors are modified and linked together to create an inhibitor with high affinity for the target

29

Figure 112 Growing number of protein structures deposited in the PDB (A) The number of protein structures in the PDB are rapidly growing every year As of 2017 there are over 100000 protein structures found in the PDB (B) Membrane proteins account for less than 5 of all deposited protein structures Adapted with permission from [99] See Appendix 1

30

Figure 113 Structure-based design of a CTX-M-9 BLI Structure-based design was used in the development of a tetrazole-containing inhibitor of CTX-M-9 displaying nanomolar potency and cell-based activity Adapted with permission from [102] See Appendix 1

31

112 References

1 Talaro K P amp Chess B (2015) Foundations in microbiology (9th ed) New York NY

McGraw-Hill Education

2 Alberts B Wilson J H amp Hunt T (2007) Molecular biology of the cell (5th ed) New

York NY Garland Science

3 Dougherty T J amp Pucci M J (2012) Antibiotic discovery and development (1st ed)

Boston MA Springer

4 Tan S amp Tatsumura Y (2015) Alexander Fleming (1881ndash1955) Discoverer of

penicillin Singapore Medical Journal 56(07) 366-367 doi1011622smedj2015105

5 American Chemical Society Discovery and development of penicillin (2017) Retrieved

from

httpwwwacsorgcontentacseneducationwhatischemistrylandmarksflemingpenicilli

nhtml

6 Kohanski M A Dwyer D J amp Collins J J (2010) How antibiotics kill bacteria From

targets to networks Nature Reviews Microbiology 8(6) 423-435

doi101038nrmicro2333

7 Skoumlld O (2000) Sulfonamide resistance Mechanisms and trends Drug Resistance

Updates 3(3) 155-160 doi101054drup20000146

8 Lobanovska M amp Pilla G (2017) Penicillinrsquos discovery and antibiotic resistance

Lessons for the future The Yale Journal of Biology and Medicine 90(1) 135ndash145

9 Lowy F D (2003) Antimicrobial resistance The example of Staphylococcus aureus

Journal of Clinical Investigation 111(9) 1265-1273 doi101172jci18535

10 Munch-Petersen E amp Boundy C (1962) Yearly incidence of penicillin-resistant

staphylococci in man since 1942 Bulletin of the World Health Organization 26(2) 241ndash

252

11 Sakoulas G amp Moellering J R (2008) Increasing antibiotic resistance among

methicillin‐resistant Staphylococcus aureus strains Clinical Infectious Diseases 46(S5)

doi101086533592

12 Lewis K (2013) Platforms for antibiotic discovery Nature Reviews Drug Discovery

12(5) 371-387 doi101038nrd3975

13 Centers for Disease Control and Prevention (2017) About antimicrobial resistance

Retrieved from httpswwwcdcgovdrugresistanceabouthtml

14 Michael C A Dominey-Howes D amp Labbate M (2014) The antimicrobial resistance

crisis Causes consequences and management Frontiers in Public Health 2

doi103389fpubh201400145

15 Ventola C L (2015) The antibiotic resistance crisis Part 1 Causes and threats Pharmacy

and Therapeutics 40(4) 277ndash283

16 Michaelidis C I Fine M J Lin C J Linder J A Nowalk M P Shields R K

Smith K J (2016) The hidden societal cost of antibiotic resistance per antibiotic

prescribed in the United States An exploratory analysis BMC Infectious Diseases 16(1)

doi101186s12879-016-1990-4

32

17 Hawkey P M (1998) The origins and molecular basis of antibiotic resistance BMJ

317(7159) 657-660 doi101136bmj3177159657

18 Abraham E P amp Chain E (1940) An enzyme from bacteria able to destroy penicillin

Nature 146(3713) 837-837 doi101038146837a0

19 Kong K Schneper L amp Mathee K (2010) Beta-lactam antibiotics From antibiosis to

resistance and bacteriology APMIS 118(1) 1-36 doi101111j1600-0463200902563x

20 Tahlan K amp Jensen S E (2013) Origins of the -lactam rings in natural products The

Journal of Antibiotics 66(7) 401-410 doi101038ja201324

21 Oates J A Wood A J Donowitz G R amp Mandell G L (1988) Beta-lactam

antibiotics New England Journal of Medicine 318(7) 419-426

doi101056nejm198802183180706

22 Silhavy T J Kahne D amp Walker S (2010) The bacterial cell envelope Cold Spring

Harbor Perspectives in Biology 2(5) doi101101cshperspecta000414

23 Huang K C Mukhopadhyay R Wen B Gitai Z amp Wingreen N S (2008) Cell shape

and cell-wall organization in Gram-negative bacteria Proceedings of the National

Academy of Sciences 105(49) 19282-19287 doi101073pnas0805309105

24 Cho H Uehara T amp Bernhardt T (2014) Beta-lactam antibiotics induce a lethal

malfunctioning of the bacterial cell wall synthesis machinery Cell 159(6) 1300-1311

doi101016jcell201411017

25 Wilke M S Lovering A L amp Strynadka N C (2005) -Lactam antibiotic resistance

A current structural perspective Current Opinion in Microbiology 8(5) 525-533

doi101016jmib200508016

26 Lee W McDonough M A Kotra L P Li Z Silvaggi N R Takeda Y Mobashery

S (2001) A 12- Å snapshot of the final step of bacterial cell wall biosynthesis Proceedings

of the National Academy of Sciences 98(4) 1427-1431 doi101073pnas9841427

27 Coico R (2005) Gram staining Current Protocols in Microbiology

doi1010029780471729259mca03cs00

28 Kieser K J amp Rubin E J (2014) How sisters grow apart Mycobacterial growth and

division Nature Reviews Microbiology 12(8) 550-562 doi101038nrmicro3299

29 Bartlett J D amp Jaanus S D (2007) Clinical ocular pharmacology (5th ed) St Louis

MO Butterworth Heinemann

30 Sandanayaka V amp Prashad A (2002) Resistance to -lactam antibiotics Structure and

mechanism based design of -lactamase inhibitors Current Medicinal Chemistry 9(12)

1145-1165 doi1021740929867023370031

31 Fisher J F Meroueh S O amp Mobashery S (2005) Bacterial resistance to -lactam

antibiotics Compelling opportunism compelling opportunity ChemInform 36(24)

doi101002chin200524267

32 Bush K amp Jacoby G A (2009) Updated functional classification of -lactamases

Antimicrobial Agents and Chemotherapy 54(3) 969-976 doi101128aac01009-09

33 Hall B G (2005) Revised Ambler classification of -lactamases Journal of

Antimicrobial Chemotherapy 55(6) 1050-1051 doi101093jacdki130

33

34 Crowder M W Spencer J amp Vila A J (2006) Metallo--lactamases Novel weaponry

for antibiotic resistance in bacteria Accounts of Chemical Research 39(10) 721-728

doi101021ar0400241

35 Philippon A Slama P Deacuteny P amp Labia R (2015) A structure-based classification of

class A -lactamases a broadly diverse family of enzymes Clinical Microbiology

Reviews 29(1) 29-57 doi101128cmr00019-15

36 Bush K amp Fisher J F (2011) Epidemiological expansion structural studies and clinical

challenges of new -lactamases from Gram-negative bacteria Annual Review of

Microbiology 65(1) 455-478 doi101146annurev-micro-090110-102911

37 Fisher J amp Mobashery S (2009) Three decades of the class A -lactamase acyl-enzyme

Current Protein amp Peptide Science 10(5) 401-407 doi102174138920309789351967

38 Hata M Tanaka Y Fujii Y Neya S amp Hoshino T (2005) A theoretical study on the

substrate deacylation mechanism of class C -lactamase The Journal of Physical

Chemistry B 109(33) 16153-16160 doi101021jp045403q

39 Vandavasi V G Weiss K L Cooper J B Erskine P T Tomanicek S J Ostermann

A Coates L (2016) Exploring the mechanism of -lactam ring protonation in the class

A -lactamase acylation mechanism using neutron and X-ray crystallography Journal of

Medicinal Chemistry 59(1) 474-479 doi101021acsjmedchem5b01215

40 Minasov G Wang X amp Shoichet B K (2002) An ultrahigh resolution structure of

TEM-1 -lactamase suggests a role for Glu166 as the general base in acylation Journal of

the American Chemical Society 124(19) 5333-5340 doi101021ja0259640

41 Vandavasi V G Langan P S Weiss K L Parks J M Cooper J B Ginell S L amp

Coates L (2016) Active-site protonation states in an acyl-enzyme intermediate of a class

A -lactamase with a monobactam substrate Antimicrobial Agents and Chemotherapy

61(1) e01636-16 doi101128aac01636-16

42 Bhattacharya M Toth M Antunes N T Smith C A amp Vakulenko S B (2014)

Structure of the extended-spectrum class C -lactamase ADC-1 from Acinetobacter

baumannii Acta Crystallographica Section D Biological Crystallography 70(3) 760-771

doi101107s1399004713033014

43 Chen Y McReynolds A amp Shoichet B K (2009) Re-examining the role of Lys67 in

class C -lactamase catalysis Protein Science 18(3)662-9 doi101002pro60

44 Sharma S amp Bandyopadhyay P (2011) Investigation of the acylation mechanism of

class C beta-lactamase pKa calculation molecular dynamics simulation and quantum

mechanical calculation Journal of Molecular Modeling 18(2) 481-492

doi101007s00894-011-1087-3

45 Chen Y Minasov G Roth T A Prati F amp Shoichet B K (2006) The deacylation

mechanism of AmpC -lactamase at ultrahigh resolution Journal of the American

Chemical Society 128(9) 2970-2976 doi101021ja056806m

46 Strynadka N C Paetzel M Danel F Castro L D Mosimann S C amp Page M G

(2000) Crystal structure of the class D -lactamase OXA-10 Nature Structural Biology

7(10) 918-925 doi10103879688

34

47 Smith C Antunes N Stewart N Toth M Kumarasiri M Chang M Vakulenko S

(2013) Structural basis for carbapenemase activity of the OXA-23 -lactamase from

Acinetobacter baumannii Chemistry amp Biology 20(9) 1107-1115

doi101016jchembiol201307015

48 Mathers A J Peirano G amp Pitout J D (2015) Escherichia coli ST131 The

quintessential example of an international multiresistant high-risk clone Advances in

Applied Microbiology 90 109-154 doi101016bsaambs201409002

49 Szarecka A Lesnock K R Ramirez-Mondragon C A Nicholas H B amp Wymore T

(2011) The class D -lactamase family Residues governing the maintenance and diversity

of function Protein Engineering Design and Selection 24(10) 801-809

doi101093proteingzr041

50 Evans B A amp Amyes S G (2014) OXA -lactamases Clinical Microbiology Reviews

27(2) 241-263 doi101128cmr00117-13

51 Toth M Antunes N T Stewart N K Frase H Bhattacharya M Smith C A amp

Vakulenko S B (2015) Class D -lactamases do exist in Gram-positive bacteria Nature

Chemical Biology 12(1) 9-14 doi101038nchembio1950

52 Lohans C T Wang D Y Jorgensen C Cahill S T Clifton I J McDonough M A

Schofield C J (2017) 13C-Carbamylation as a mechanistic probe for the inhibition of

class D -lactamases by avibactam and halide ions Org Biomol Chem 15(28) 6024-

6032 doi101039c7ob01514c

53 Poirel L Naas T amp Nordmann P (2009) Diversity epidemiology and genetics of class

D -lactamases Antimicrobial Agents and Chemotherapy 54(1) 24-38

doi101128aac01512-08

54 Antunes N amp Fisher J (2014) Acquired class D β-lactamases Antibiotics 3(3) 398-

434 doi103390antibiotics3030398

55 Mojica M F Bonomo R A amp Fast W (2016) B1-Metallo--lactamases Where do we

stand Current Drug Targets 17(9) 1029-1050

doi1021741389450116666151001105622

56 Page M I amp Badarau A (2008) The mechanisms of catalysis by metallo--lactamases

Bioinorganic Chemistry and Applications 2008 1-14 doi1011552008576297

57 Palzkill T (2012) Metallo--lactamase structure and function Annals of the New York

Academy of Sciences 1277(1) 91-104 doi101111j1749-6632201206796x

58 Garau G Bebrone C Anne C Galleni M Fregravere J amp Dideberg O (2005) A metallo-

-lactamase enzyme in action Crystal structures of the monozinc carbapenemase CphA

and its complex with biapenem Journal of Molecular Biology 345(4) 785-795

doi101016jjmb200410070

59 Bebrone C Delbruck H Kupper M B Schlomer P Willmann C Frere J

Hoffmann K M (2009) The structure of the dizinc subclass B2 metallo--lactamase

CphA reveals that the second inhibitory zinc ion binds in the histidine site Antimicrobial

Agents and Chemotherapy 53(10) 4464-4471 doi101128aac00288-09

35

60 Papp-Wallace K M Endimiani A Taracila M A amp Bonomo R A (2011)

Carbapenems Past present and future Antimicrobial Agents and Chemotherapy 55(11)

4943-4960 doi101128aac00296-11

61 De Luca F Benvenuti M Carboni F Pozzi C Rossolini G M Mangani S amp

Docquier J (2011) Evolution to carbapenem-hydrolyzing activity in noncarbapenemase

class D -lactamase OXA-10 by rational protein design Proceedings of the National


62 Queenan A M amp Bush K (2007) Carbapenemases The versatile -lactamases Clinical

Microbiology Reviews 20(3) 440-458 doi101128cmr00001-07

63 Antunes N T Lamoureaux T L Toth M Stewart N K Frase H amp Vakulenko S

B (2014) Class D -lactamases Are they all carbapenemases Antimicrobial Agents and

Chemotherapy 58(4) 2119-2125 doi101128aac02522-13

64 Lee J H amp Lee S H (2006) Carbapenem resistance in Gram-negative pathogens

Emerging non-metallo-carbapenemases Research Journal of Microbiology 1(1) 1-22

doi103923jm2006122

65 Arnold R S Thom K A Sharma S Phillips M Kristie Johnson J amp Morgan D J

(2011) Emergence of Klebsiella pneumoniae carbapenemase-producing bacteria

Southern Medical Journal 104(1) 40-45 doi101097smj0b013e3181fd7d5a

66 Centers for Disease Control and Prevention (2017) Carbapenem-resistant

Enterobacteriaceae (CRE) infection patient FAQs Retrieved from

httpswwwcdcgovhaiorganismscrecre-patientfaqhtml

67 Lahey Clinic (2016) -Lactamase classification and amino acid sequences for TEM SHV

and OXA extended-spectrum and inhibitor resistant enzymes Retrieved from

httpwwwlaheyorgstudiesotherasptable1

68 Mehta S C Rice K amp Palzkill T (2015) Natural variants of the KPC-2 carbapenemase

have evolved increased catalytic efficiency for ceftazidime hydrolysis at the cost of enzyme

stability PLOS Pathogens 11(6) e1004949 doi101371journalppat1004949

69 Papp-Wallace K M Bethel C R Distler A M Kasuboski C Taracila M amp

Bonomo R A (2010) Inhibitor resistance in the KPC-2 -lactamase a preeminent

property of this class A -lactamase Antimicrobial Agents and Chemotherapy 54(2) 890-

897 doi101128aac00693-09

70 Nordmann P Naas T amp Poirel L (2011) Global spread of carbapenemase-producing

Enterobacteriaceae Emerging Infectious Diseases 17(10) 1791-1798

doi103201eid1710110655

71 Poirel L Potron A amp Nordmann P (2012) OXA-48-like carbapenemases The

phantom menace Journal of Antimicrobial Chemotherapy 67(7) 1597-1606

doi101093jacdks121

72 Weide T Saldanha S A Minond D Spicer T P Fotsing J R Spaargaren M Fokin

V V (2010) NH-123-triazole inhibitors of the VIM-2 metallo--lactamase ACS

Medicinal Chemistry Letters 1(4) 150-154 doi101021ml900022q

73 Wenzler E Deraedt M F Harrington A T amp Danizger L H (2017) Synergistic

activity of ceftazidime-avibactam and aztreonam against serine and metallo--lactamase-

36

producing Gram-negative pathogens Diagnostic Microbiology and Infectious Disease

88(4) 352-354 doi101016jdiagmicrobio201705009

74 Reading C amp Cole M (1977) Clavulanic acid A beta-lactamase-inhibiting beta-lactam

from Streptomyces clavuligerus Antimicrobial Agents and Chemotherapy 11(5) 852-857

doi101128aac115852

75 Bush K amp Bradford P A (2016) -lactams and -lactamase inhibitors An overview

Cold Spring Harbor Perspectives in Medicine 6(8) a025247

doi101101cshperspecta025247

76 Drawz S M amp Bonomo R A (2010) Three decades of -lactamase inhibitors Clinical


77 Wong D amp Van Duin D (2017) Novel beta-lactamase inhibitors Unlocking their

potential in therapy Drugs 77(6) 615-628 doi101007s40265-017-0725-1

78 Ehmann D E Jahic H Ross P L Gu R Hu J Kern G Fisher S L (2012)

Avibactam is a covalent reversible non--lactam -lactamase inhibitor Proceedings of

the National Academy of Sciences 109(29) 11663-11668 doi101073pnas1205073109

79 Zasowski E J Rybak J M amp Rybak M J (2015) The -lactams strike back

Ceftazidime-avibactam Pharmacotherapy The Journal of Human Pharmacology and Drug

Therapy 35(8) 755-770 doi101002phar1622

80 Papp-Wallace K M Winkler M L Taracila M A amp Bonomo R A (2015) Variants

of -lactamase KPC-2 that are resistant to inhibition by avibactam Antimicrobial Agents

and Chemotherapy 59(7) 3710-3717 doi101128aac04406-14

81 Haidar G Clancy C J Shields R K Hao B Cheng S amp Nguyen M H (2017)

Mutations in blaKPC-3 that confer ceftazidime-avibactam resistance encode novel KPC-3

variants that function as extended-spectrum -lactamases Antimicrobial Agents and

Chemotherapy 61(5) e02534-16 doi101128aac02534-16

82 Hecker S J Reddy K R Totrov M Hirst G C Lomovskaya O Griffith D C

Dudley M N (2015) Discovery of a cyclic boronic acid -lactamase inhibitor (RPX7009)

with utility vs class A serine carbapenemases Journal of Medicinal Chemistry 58(9)

3682-3692 doi101021acsjmedchem5b00127

83 Crompton I E Cuthbert B K Lowe G amp Waley S G (1988) -Lactamase inhibitors

The inhibition of serine -lactamases by specific boronic acids Biochemical Journal

251(2) 453-459 doi101042bj2510453

84 The Medicines Company (2017 August 30) The Medicines Company announces FDA

approval of VABOMEREtrade (meropenem and vaborbactam) Retrieved from

httpwwwthemedicinescompanycominvestorsnewsmedicines-company-announces-

fda-approval-vabomereE284A2-meropenem-and-vaborbactam

85 Phatak S S Stephan C C amp Cavasotto C N (2009) High-throughput and in silico

screenings in drug discovery Expert Opinion on Drug Discovery 4(9) 947-959

doi10151717460440903190961

86 Aubeacute J (2012) Drug repurposing and the medicinal chemist ACS Medicinal Chemistry

Letters 3(6) 442-444 doi101021ml300114c

37

87 Chilingaryan Z Yin Z amp Oakley A J (2012) Fragment-based screening by protein

crystallography Successes and pitfalls International Journal of Molecular Sciences

13(12) 12857-12879 doi103390ijms131012857

88 Janzen W (2014) Screening technologies for small molecule discovery The state of the

art Chemistry amp Biology 21(9) 1162-1170 doi101016jchembiol201407015

89 Mashalidis E H Śledź P Lang S amp Abell C (2013) A three-stage biophysical

screening cascade for fragment-based drug discovery Nature Protocols 8(11) 2309-2324

doi101038nprot2013130

90 Scott D E Coyne A G Hudson S A amp Abell C (2012) Fragment-based approaches

in drug discovery and chemical biology Biochemistry 51(25) 4990-5003

doi101021bi3005126

91 Bienstock R J (2011) Library Design Search Methods and Applications of Fragment-

Based Drug Design ACS Symposium Series doi101021bk-2011-1076

92 Meng X Zhang H Mezei M amp Cui M (2011) Molecular docking A powerful

approach for structure-based drug discovery Current Computer Aided-Drug Design 7(2)

146-157 doi102174157340911795677602

93 Roberts B C amp Mancera R L (2008) Ligandminusprotein docking with water molecules

Journal of Chemical Information and Modeling 48(2) 397-408 doi101021ci700285e

94 Liu J He X amp Zhang J Z (2013) Improving the scoring of proteinndashligand binding

affinity by including the effects of structural water and electronic polarization Journal of

Chemical Information and Modeling 53(6) 1306-1314 doi101021ci400067c

95 Trott O amp Olson A J (2009) AutoDock Vina Improving the speed and accuracy of

docking with a new scoring function efficient optimization and multithreading Journal of

Computational Chemistry 31(2) 455-461 doi101002jcc21334

96 Moustakas D T Lang P T Pegg S Pettersen E Kuntz I D Brooijmans N amp

Rizzo R C (2006) Development and validation of a modular extensible docking

program DOCK 5 Journal of Computer-Aided Molecular Design 20(10-11) 601-619

doi101007s10822-006-9060-4

97 Friesner R A Banks J L Murphy R B Halgren T A Klicic J J Mainz D T

Shenkin P S (2004) Glide A new approach for rapid accurate docking and scoring 1

Method and assessment of docking accuracy Journal of Medicinal Chemistry 47(7) 1739-

1749 doi101021jm0306430

98 Grosdidier A Zoete V amp Michielin O (2011) SwissDock a protein-small molecule

docking web service based on EADock DSS Nucleic Acids Research 39(suppl) W270-

W277 doi101093nargkr366

99 Shi Y (2014) A glimpse of structural biology through X-ray crystallography Cell

159(5) 995-1014 doi101016jcell201410051

100 Navia M A amp Peattie D A (1993) Structure-based drug design Applications in

immunopharmacology and immunosuppression Immunology Today 14(6) 296-302

doi1010160167-5699(93)90049-q

101 Nichols D A Renslo A R amp Chen Y (2014) Fragment-based inhibitor discovery

against -lactamase Future Medicinal Chemistry 6(4) 413-427 doi104155fmc1410

38

102 Nichols D A Jaishankar P Larson W Smith E Liu G Beyrouthy R Chen Y

(2012) Structure-based design of potent and ligand-efficient inhibitors of CTX-M class A

-lactamase Journal of Medicinal Chemistry 55(5) 2163-2172 doi101021jm2014138

39

Chapter 2

Molecular Basis of Substrate Recognition and Product Release by the Klebsiella

pneumoniae Carbapenemase (KPC-2)

21 Overview

CRE are resistant to many β-lactam antibiotics thanks in part to the production of the KPC-

2 class A -lactamase Here I present the first product complex crystal structures of KPC-2 with

β-lactam antibiotics containing hydrolyzed cefotaxime (cephalosporin) and hydrolyzed faropenem

(penem) These complex crystal structures provide experimental insights into the substrate

recognition by KPC-2 and its unique broad-spectrum -lactamase activity These structures also

represent the first product complexes for a wild-type (WT) serine β-lactamase which helped

elucidate the product release mechanism of these enzymes

22 Introduction

The KPC-2 class A β-lactamase poses a serious threat to nearly all β-lactam antibiotics [1]

KPC was first identified in North Carolina in 1996 and has since spread to many other countries

[2] Although initially identified in K pneumoniae KPC has also been discovered in other Gram-

negative bacterial pathogens mainly belonging to the Enterobacteriaceae family [3] The blaKPC

gene encodes a 293 residue protein with 23 variants (KPC-2 through KPC-24) that differ from one

another by only one or two amino acid changes [4] KPC-2 is the most prevalent carbapenemase

in the United States and it has been termed the ldquoversatile β-lactamaserdquo due to its large and shallow

40

active site permitting the efficient hydrolysis of virtually all β-lactam antibiotics [5] However

the question still remains as to what specific interactions between β-lactams and the KPC-2 active

site allow for such a broad substrate profile [6 7] The reaction catalyzed by class A β-lactamases

proceeds through two steps acylation and deacylation [3] The nucleophilic attack of Ser70 on the

substrate β-lactam ring results in a covalent acylndashenzyme complex Subsequently the catalytic

water is activated by Glu166 and cleaves the acylndashenzyme linkage leading to the formation of the

hydrolyzed product One common way to capture a β-lactam complex along the reaction pathway

of class A β-lactamases is to mutate a key catalytic residue such as Ser70 or Glu166 to

accommodate the newly generated carboxylate group [8] The complex structures presented herein

represent the first product complexes using a WT serine β-lactamase with all native active site

residues intact This provides an accurate picture of the protein microenvironment that is

responsible for catalysis particularly during product release

23 Results and Discussion

I attempted to obtain complex crystal structures with a variety of β-lactams including the

cephalosporins ceftazidime cefotaxime cefoxitin and nitrocefin the penicillins ampicillin

cloxacillin and benzylpenicillin the carbapenems biapenem imipenem and meropenem the

penem faropenem and the monobactam aztreonam Ultimately I was able to obtain two crystal

structures of cefotaxime and faropenem as hydrolyzed products in the active site of KPC-2 (Fig

21) As discussed herein the unique features of KPC-2 and the two substrates may have

contributed to the capture of these two complexes

41

231 Product complex with cefotaxime

KPC-2 was crystallized in the space group P22121 with one copy of KPC-2 in the

asymmetric unit (Table 21) KPC-2 crystals routinely diffracted to 115ndash15 Aring resolution The

apo-structure is similar to that of previously determined KPC-2 models crystallized in different

space groups (Fig 22) [9 10] The product complex structure with cefotaxime was determined to

145 Aring resolution with final Rwork and Rfree values of 168 and 212 respectively The electron

density for the product is well-defined in the active site as seen in the unbiased FondashFc map (Fig

23A) The occupancy value of the hydrolyzed product was refined to 086 There are some

differences in the active site when comparing the cefotaxime complex structure to the apoenzyme

mainly with residues Ser70 Trp105 and Leu167 (Fig 23B) because of interactions between the

hydrolyzed product and the enzyme The complex structure illustrates how the bulky oxyimino

group of third-generation cephalosporins and the newly generated carboxylate group of the product

are accommodated by the KPC-2 active site It represents the first experimental structure of KPC-

2 in complex with a clinically used β-lactam antibiotic and sheds new light on the extensive

interactions between cefotaxime and the active site residues The C4 carboxylate group resides in

a subpocket formed by Thr235 Gly236 Thr237 and Ser130 This region is highly conserved in

serine β-lactamases and PBPs as shown by previous crystallographic studies of these enzymes

and their complexes [11 12] The six-membered dihydrothiazine group of the hydrolyzed product

forms a pindashpi stacking interaction with Trp105 which has been demonstrated to be important in

-lactam substrate recognition [13] The interactions with the substrate also result in a single

conformation of Trp105 which is observed to have two conformations in the apoenzyme (Fig

23B) Meanwhile cefotaximersquos acylamide side chain is nestled in a pocket formed by Thr237

Cys238 Gly239 Leu167 and Asn170 (Fig 23A) The aminothiazole moiety forms extensive

42

nonpolar interactions with Leu167 in addition to van der Waals contacts with Asn170 Cys238

and Gly239 In particular compared with the apo-structure the alkyl side chain of Leu167 moves

closer to interact with the substrate (Fig 23B) In comparison the oxyimino group is largely

solvent exposed and establishes relatively few interactions with the protein mainly with

Thr237C2 and Gly239C (Fig 23A)

As the first product complex with a WT serine β-lactamase this KPC-2 structure also

captures structural features not seen in previous β-lactamase complexes particularly concerning

the conformations of Ser70 Lys73 Ser130 and the substrate carbonyl group In class A β-

lactamases Ser70 carries out nucleophilic attack on the β-lactam ring of the substrate [14] In the

apoenzyme Ser70 forms a HB with Lys73 which has been suggested as a potential base in the

acylation step [15 16] In my structure the presence of the newly generated C8 carboxylate group

a result of the catalytic waterrsquos attack on the carbon atom of the acylndashenzyme carbonyl group

causes Ser70 to adopt a conformation that places its side chain hydroxyl group in the oxyanion

hole formed by the backbone amide groups of Ser70 and Thr237 and previously occupied by the

carbonyl oxygen of the acylndashenzyme intermediate (Fig 23B D) This conformational change

abolishes the HB between Ser70 and Lys73 and establishes a new HB between Ser70 and the

substrate ring nitrogen an interaction not observed at any other stage of the reaction (Fig 23B)

Meanwhile the side chain of Lys73 moves to form a HB with Ser130

Unlike most previous β-lactamase structures Ser130 adopts two conformations (Fig 23A)

[17] Conformation 1 maintains a weak HB with Lys73 is in close proximity to the ring nitrogen

and is the conformation usually observed in class A β-lactamases [18] conformation 2 swings

closer to Lys234 and the substrate C4 carboxylate group establishing more favorable HBs with

the latter two functional groups Additionally the amide nitrogen of the cefotaxime product forms

43

a weak HB with the backbone carbonyl group of Thr237 (Fig 23A) In a previous CTX-M-14

E166A complex structure with a ruthenocene-conjugated penicillin (PDB ID code 4XXR) [19]

Ser130 adopts conformation 1 and serves as a HB acceptor and donor in its interactions with the

protonated ring nitrogen and a neutral Lys73 respectively (Fig 23C) In my current structure

Lys73 appears to be protonated and is within HB distance (26ndash29 Aring) to three HB acceptors

including Ser130O Asn132O1 and the substrate C8 carboxylate group In comparison the

distance between Lys73N and Ser130O of conformation 1 is 31 Aring suggesting a weak HB

contact Ser130O of conformation 1 is also too far away from the ring nitrogen (35 Aring) for a HB

The weakened interactions between Ser130 and Lys73 or the substrate ring nitrogen likely

contribute to Ser130rsquos adoption of conformation 2 which highlights Ser130rsquos role in binding the

substrate carboxylate group Taken together the alternative conformations of Ser70 Lys73 and

Ser130 underscore the important noncatalytic roles these residues play in the product release

process

Previous studies have suggested that steric clash and electrostatic repulsion caused by the

newly generated C8 carboxylate group of cefotaxime is responsible for the expulsion of the

hydrolyzed product from the active site [8 20] My structure supports this hypothesis and provides

important new structural details on potential unfavorable interactions that lead to product

expulsion In my structure possible clashes between the C8 carboxylate group and Ser70 are

alleviated by Ser70rsquos adoption of an alternative and likely high energy conformation with a side

chain χ1 angle of 10deg in comparison to the more favorable 60deg in Ser70rsquos usual conformation In

the complex structure of the AmpC class C β-lactamase S64G mutant with hydrolyzed cephalothin

(PDB ID code 1KVL) the electrostatic repulsion between the C8 and C4 carboxylate groups

caused the C4 carboxylate group to move out of the active site resulting from the rotation of the

44

dihydrothiazine ring and leading to an increase in distance between these two negatively charged

groups [21] In my structure the C4 carboxylate group remains in the active site albeit in a likely

less stable state due to the electrostatic repulsion Although the C8 carboxylate group can establish

new interactions with Lys73 these contacts are accompanied by the loss of HBs between the

substrate and the oxyanion hole between Ser70 and Lys73 and for class A β-lactamases by

possible additional repulsion between the C8 carboxylate group and Glu166 (Fig 23B) Another

unique feature of the product complex is the lack of a HB between the substrate amide group with

Asn132 even though the substrate amide group forms a HB with the backbone O atom of Thr237

(Fig 23A) The HB with Asn132 is present in nearly all complex structures of serine β-lactamases

with β-lactams containing the amide group including the aforementioned CTX-M-14 E166A

product complex (PDB ID code 4XXR) and a Toho-1 E166Acefotaxime complex (PDB ID code

1IYO) structure (Fig 23D) [8 19 22] Instead in this structure the carbonyl oxygen is pushed

out of the active site and exposed to solvent (Fig 23C D) The loss of the HB between the ligand

amide group and Asn132 further suggests that the interactions between the product and the enzyme

are less favorable compared with the Michaelis substrate complex which may facilitate substrate

turnover during catalysis

I observed an additional molecule of hydrolyzed cefotaxime near the active site with a

refined occupancy value of 072 (Fig 24) This molecule did not make as many interactions in the

active site as seen in the other hydrolyzed molecule There is a HB between Lys270 and the C4

carboxylate group of the second hydrolyzed product as well as a HB between the aminothiazole

group of the first hydrolyzed product and the C4 carboxylate of the second hydrolyzed product I

believe that the presence of this second hydrolyzed product is a crystal-packing artifact and does

45

not play a role in catalysis Though interestingly it may have partially stabilized the first

hydrolyzed product inside the active site in the crystal

232 Product complex with faropenem

Faropenem belongs to the penem class of β-lactam antibiotics (Fig 21) Penems share the

structural characteristics of both penicillins and cephalosporins [24] Like carbapenems penems

have a hydroxyethyl group attached to the β-lactam ring in the trans-configuration that provides

stability against many serine β-lactamases [5] I captured a single hydrolyzed molecule of

faropenem in the active site of KPC-2 (Fig 25A) The complex structure with faropenem was

solved to 140 Aring resolution with final Rwork and Rfree values of 150 and 188 respectively

(Table 21) The occupancy value of the hydrolyzed product was refined to 084 Compared to the

product complex with cefotaxime the hydrolyzed faropenem induces similar conformations of

Ser70 and Trp105 and makes many similar interactions in the active site (Fig 25B) For instance

the C7 carboxylate group which is analogous to the cefotaxime C8 carboxylate group forms HBs

with Ser130 Thr235 and Thr237 conformation 2 of Ser130 forms a HB with the C3 carboxylate

group which is analogous to the cefotaxime C4 carboxylate group (Fig 25A) Unlike the

cefotaxime complex the ring nitrogen forms a HB with conformation 1 of Ser130 rather than

Ser70 likely as a result of the smaller five-membered dihydrothiazole ring in faropenem in

comparison to the six-membered dihydrothiazine ring in cefotaxime

One important observation in this faropenem structure is the conformation of the

hydroxyethyl side chain The faropenem structure shows that the hydroxyethyl side chain has

undergone rotation abolishing its interaction with Asn132 Previous studies have shown that in

noncarbapenemases such as SED-1 (PDB ID code 3BFF) the hydroxyethyl group of carbapenems

46

forms a HB with Asn132 and the catalytic water consequently deactivating the catalytic water

molecule and leaving these enzymes unable to hydrolyze the acylndashenzyme linkage [23] However

in carbapenemases like KPC-2 the active site is enlarged permitting rotation of the hydroxyethyl

group thus abolishing its interaction with Asn132 and allowing hydrolysis of carbapenem

antibiotics (Fig 25C) [23 25-27] Importantly in the current structure Leu167 is in favorable

van der Waal contact with the methyl group of faropenemrsquos hydroxyethyl moiety suggesting that

Leu167 may play a catalytic role in inducing the rotation of this side chain group to facilitate

carbapenem hydrolysis Although position 167 is not well-conserved among class A β-lactamases

it appears that a leucine is conserved at this position in class A carbapenemases [11] When

comparing the cefotaxime and faropenem product complexes movements are observed in Val103

the Pro104-Trp105 loop and Leu167 (Fig 25D) These movements are likely due to the different

interactions between the faropenem tetrahydrofuran group and Trp105 and between the

cefotaxime aminothiazole group and Leu167 In addition because of the different chiralities of the

C6C7 atoms linked to the C7C8 carboxylate group the newly generated carboxylate group is

positioned slightly differently between the two product complexes Taken together these findings

highlight the flexibility of the KPC-2 active site for accommodating a variety of β-lactam side

chains

233 Unique KPC-2 structural features for inhibitor discovery

Despite extensive structural studies of numerous serine β-lactamases my structures

represent the only product complexes with a WT enzyme All previous studies relied on mutations

such as S70G to stabilize the interactions between the product and the enzyme active site [8] The

many unfavorable features of the enzymendashproduct contacts have made it difficult to capture such

47

complexes with WT enzymes even for KPC-2 as demonstrated by the failures to obtain similar

complexes using many other β-lactam antibiotics The successes with my current two complexes

may have been due to the particular side chains of these two substrates and some unique properties

of carbapenemases such as KPC-2 In comparison to other β-lactam compounds the oxyimino side

chain of cefotaxime and the tetrahydrofuran side group of faropenem enhance the interactions

between the product and KPC-2 and avoids excessive bulkiness that may cause steric clashes with

protein residues in a crystal-packing environment More importantly compared with narrow-

spectrum β-lactamases (eg TEM-1) and ESBLs (eg CTX-M-14) KPC-2 and other class A

carbapenemases contain several key features and residues that result in an enlarged active site

including a Cys69ndashCys238 disulfide bond Leu167 Pro104 and Trp105 Movements of Ser70 and

Asn170 are also observed in the KPC-2 structure There is a larger distance between Ser70 and

Asn170 in KPC-2 as compared to that in noncarbapenemases like TEM-1 and CTX-M-14 which

may facilitate rotation of the hydroxyethyl group of carbapenems that endows them with stability

against hydrolysis by many serine -lactamases [9] Importantly the KPC-2 active site also

appears to be more hydrophobic with larger nonpolar surfaces provided by Pro104 Trp105

Leu167 and Val240 in comparison to their counterparts Glu104 Tyr105 Pro167 and Asp240 in

TEM-1 (Fig 26A) and Asn104 Tyr105 Pro167 and Asp240 in CTX-M-14 (Fig 26B) These

features may allow KPC-2 to bind to a wide range of β-lactam substrates with a relatively open

active site They are mirrored by similar observations in MBLs that also harbor expanded active

sites with a large number of hydrophobic residues [28] Such features may enable carbapenemases

to hydrolyze nearly all β-lactam antibiotics while also exposing a weakness that can facilitate the

engineering of high affinity inhibitors against these enzymes

48

24 Conclusions

In this work I present the first crystal structures of the KPC-2 class A β-lactamase in

complex with two clinically used β-lactam antibiotics cefotaxime and faropenem The structures

underscore the role of the enlarged and shallow active site in accommodating the bulky cefotaxime

oxyimino side chain the rotation of faropenemrsquos 6α-1R-hydroxyethyl group and particularly

Leu167rsquos critical contribution in catalysis These structures demonstrate how alternative

conformations of Ser70 and Lys73 facilitate product release and capture unique substrate

conformations at this important milestone of the reaction Additionally these structures highlight

the increased druggability of the KPC-2 active site which provides an evolutionary advantage for

the enzymersquos catalytic versatility but also an opportunity for drug discovery

25 Materials and Methods


The KPC-2 gene sequence (residues 25-293) was cloned into the pET-GST vector between

NdeI and BstBI restriction sites as an N-terminal His-tagged fusion protein The construct was

verified by DNA sequencing and transformed into BL21 (DE3) competent cells for protein

expression For His-tagged KPC-2 bacteria were grown overnight at 30 degC with shaking in 50 mL

LB broth supplemented with 50 gmL kanamycin Two liters of LB broth supplemented with 50

gmL kanamycin 200 mM sorbitol and 5 mM betaine were each inoculated with 10 mL of

overnight bacterial culture and grown at 37 degC until reaching an OD600=06-08 Protein expression

was then initiated by the addition of 05 mM IPTG followed by growth for 16 hrs at 20 degC Cells

49

were pelleted by centrifugation and stored at -80 degC until further use The His-tagged KPC-2 was

purified by nickel affinity chromatography and gel filtration Briefly the cell pellets were thawed

and re-suspended in 40 mL of buffer A (20 mM Tris-HCl pH 80 300 mM NaCl 20 mM

imidazole) with one complete protease inhibitor cocktail tablet (Roche) and disrupted by

sonication followed by centrifugation at 45000 RPM to clarify the lysate After centrifugation

the supernatant was filtered before loading onto a 5 mL HisTrap HP affinity column (GE

Healthcare Life Sciences USA) pre-equilibrated with buffer A His-tagged KPC-2 was eluted by

a linear imidazole gradient (20 mM to 500 mM) Fractions were analyzed by SDS-PAGE

Fractions containing His-tagged KPC-2 were pooled and loaded onto a Superdex75 1660 gel

filtration column (GE Healthcare Life Sciences) pre-equilibrated with 20 mM Tris-HCl pH 80

300 mM NaCl Protein concentration was determined by absorbance at 280 nm using an extinction

coefficient of 39545 SDSPAGE analysis indicated that the eluted protein was more than 95

pure The protein containing fractions were pooled and concentrated to 10-20 mgmL The protein

was aliquoted and then flash frozen in liquid nitrogen and stored at -80 degC

252 Crystallization and soaking experiments

Crystals of His-tagged KPC-2 were grown at 20 degC using hanging-drop vapor diffusion

Protein solutions (10-20 mgml) in 20 mM Tris-HCl pH 80 300 mM NaCl were mixed 12 (vv)

with a reservoir solution containing 2 M ammonium sulfate and 5 (vv) ethanol Droplets (15

L) were microseeded with 05 L of diluted seed stock Crystals typically began to form within

two weeks To obtain the ligand bound structures KPC-2 crystals were soaked in a solution

containing 144 M sodium citrate and 30 mM cefotaximefaropenem for 30 minutes The soaked

50

crystals were cryo-protected in a solution containing 115 M sodium citrate 20 (vv) glycerol

and flash-frozen in liquid nitrogen

253 Data collection and structure determinations

Data for the KPC-2 complex structures were collected using beamlines 22-ID-D and 23-

ID-D at the Advanced Photon Source (APS) Argonne Illinois Diffraction data were indexed and

integrated with iMosflm [29] and scaled with SCALA from the CCP4 suite [30] Phasing was

performed using molecular replacement with the program Phaser [31] with the truncated KPC-2

structure (PDB ID code 3C5A) Structure refinement was performed using phenixrefine [32] and

model building in WinCoot [33] The unbiased Fo-Fc electron density maps were generated prior

to refinement with compound The program eLBOW [34] in Phenix was used to obtain geometry

restraint information The final model qualities were assessed using MolProbity [35] Figures were

generated in PyMOL 13 (Schroumldinger) [36] in which structural alignments were generated using

pairwise scores LigPlot+ v145 was used to generate ligand-protein interactions [37]

26 Note to Reader 1

Portions of this chapter have been reproduced from Pemberton O A Zhang X amp Chen

Y (2017) Molecular basis of substrate recognition and product release by the Klebsiella

pneumoniae carbapenemase (KPC-2) Journal of Medicinal Chemistry 60(8) 3525-3530 with

permission of the 2017 American Chemical Society (see Appendix 1)

51

Figure 21 Chemical structures of intact and hydrolyzed cephalosporin and penem -lactam

antibiotics Structures of cephalosporins (cephalothin cefotaxime) carbapenem (meropenem) and

penem (faropenem) β-lactam antibiotics

52

Figure 22 Superimposition of apo KPC-2 structures Green (PDB ID code 3C5A)

cyan (PDB ID code 2OV5) purple (PDB ID code 3DW0) current apo-structure (yellow)

53

Figure 23 KPC-2 cefotaxime product complex The protein and ligand of

the complex are shown in white and yellow respectively HBs are shown as

black dashed lines (A) Unbiased FondashFc electron density map (gray) of

hydrolyzed cefotaxime contoured to 2σ (B) Superimposition of KPC-2 product

complex onto apoprotein (green) with details showing the interactions

involving Ser70 Lys73 the cefotaxime ring nitrogen and the newly formed

C8 carboxylate group PyMOL alignment RMSD = 0153 Aring over 272 residues

(C) Superimposition of KPC-2 product complex and CTX-M-14 E166A

penilloate product complex with a ruthenocene-conjugated penicillin (green

with residues unique to CTX-M-14 E166A labeled in green) in stereo view

The ruthenium atom is shown as a gray sphere PyMOL alignment RMSD =

0617 Aring over 272 residues (D) Superimposition of KPC-2 product complex

with Toho-1 E166A acylndashenzyme complex with cefotaxime (green with

residues unique to Toho-1 E166A labeled in green) PyMOL alignment RMSD

= 0602 Aring over 264 residues

54

Figure 24 Two molecules of hydrolyzed cefotaxime in the KPC-2 active site Hydrolyzed

cefotaxime 1 and 2 (yellow) are shown in the active site of KPC-2 The unbiased Fo-Fc electron density

map (gray) is contoured at 2 σ HBs are shown as black dashed lines The refined occupancy values

of cefotaxime 1 and 2 are 086 and 072 respectively

T235 T237 C238

S130

K73

K270

1 2

55

Figure 25 KPC-2 faropenem product complex The protein and ligand of

the faropenem product complex are shown in white and yellow respectively

HBs are shown as black dashed lines (A) The unbiased FondashFc electron density

map (gray) of hydrolyzed faropenem contoured at 2σ (B) Superimposition of

KPC-2 faropenem complex onto apoprotein (green) with details showing the

interactions involving Ser70 Lys73 and the newly formed faropenem C7

carboxylate group PyMOL alignment RMSD = 0141 Aring over 270 residues (C)

Superimposition of KPC-2 product complex with SED-1 faropenem acylndash

enzyme (green) in stereo view The deacylation water (red sphere) from SED-1

is shown making interactions with Glu166 and the hydroxyethyl group of

faropenem PyMOL alignment RMSD = 0555 Aring over 262 residues (D)

Superimposition of faropenemcefotaxime product complexes showing the

movement of the Pro104-Trp105 loop and alternative conformations for

Val103 and Leu167 (KPC-2faropenem whiteyellow KPC-2cefotaxime

green) PyMOL alignment RMSD = 0101 Aring over 270 residues

56

Figure 26 Comparison of KPC-2 active site with the narrow-spectrum β-

lactamase TEM-1 and the ESBL CTX-M-14 (A) KPC-2 complexed with

cefotaxime superimposed onto TEM-1 (KPC-2 white TEM-1 green

cefotaxime yellow) (B) KPC-2 complexed with faropenem superimposed

onto CTX-M-14 (KPC-2 white CTX-M-14 green faropenem yellow)

57

Figure 27 Molecular interactions of hydrolyzed cefotaxime and faropenem in the active site of KPC-2 from

LigPlot+ (A) Hydrolyzed cefotaxime and (B) hydrolyzed faropenem

A B

58

Statistics for the highest-resolution shell are shown in parentheses

Table 21 X-ray data collection and refinement statistics for KPC-2 apo and hydrolyzed products

Structure Apo KPC-2 KPC-2 with Hydrolyzed

Cefotaxime

KPC-2 with Hydrolyzed

Faropenem

Data Collection

Space group P22121 P22121 P22121

Cell dimensions

a b c (Aring) 5580 6020 7862 5693 5878 7684 5714 5913 7738

(deg) 90 90 90 90 90 90 90 90 90

Resolution (Aring) 4550-115 (121-115) 4574-145 (153-145) 4698-140 (148-140)

No of unique reflections 93824 (12888) 45653 (6335) 52337 (7543)

Rmerge () 3073 (362) 127 (684) 95 (888)

Iσ(I) 110 (20) 91 (23) 99 (22)

Completeness () 991 (944) 985 (956) 999 (100)

Multiplicity 65 (33) 65 (53) 70 (66)

Refinement

Resolution (Aring) 3214-115 4089-145 3630-140

Rwork () 122 (261) 168 (319) 150 (257)

Rfree () 139 (269) 212 (345) 188 (275)

No atoms

Protein 2136 2090 2109

LigandIon 26 60 26

Water 350 255 223

B-factors (Aring2)

protein 1408 1558 2242

ligandion 2832 2202 3711

water 3196 3091 3678

RMS deviations

Bond lengths (Aring) 0009 0005 0005

Bond angles (deg) 108 085 081

Ramachandran plot

favored () 9888 9888 9887

allowed () 112 112 113

outliers () 0 0 0

PDB code 5UL8 5UJ3 5UJ4

59

Interactions of KPC-2 with hydrolyzed cefotaxime

Distance (Aring)

Ser70 OG ndash Cefotaxime N23 295

Lys73 NZ - Cefotaxime O21 258

Ser130 OG ndash Cefotaxime O21 300


Thr235 OG1 - Cefotaxime O27 270

Thr237 OG1 ndash Cefotaxime O26 255

Thr237 O ndash Cefotaxime N07 321

Interactions of KPC-2 with hydrolyzed faropenem

Distance (Aring)

Ser70 OG ndash Faropenem O19 263

Lys73 NZ ndash Faropenem O19 316


Ser130 OG ndash Faropenem N06 310


Asn 132 ND2 ndash Faropenem O20 295

Thr235 OG1 ndash Faropenem O09 264


Table 22 Hydrolyzed products hydrogen bond distances within

KPC-2 HB distances between KPC-2 active site residues and

hydrolyzed cefotaxime and faropenem products calculated from LigPlot+

60

27 References


Enterobacteriaceae Emerging Infectious Diseases 17(10) 1791ndash1798

doi103201eid1710110655

2 Brown N G Chow D amp Palzkill T (2013) BLIP-II Is a highly potent inhibitor of

Klebsiella pneumoniae carbapenemase (KPC-2) Antimicrobial Agents and


3 Walther-Rasmussen J amp Hoiby N (2007) Class A carbapenemases Journal of

Antimicrobial Chemotherapy 60(3) 470-482 doi101093jacdkm226







6 Gupta N Limbago B M Patel J B amp Kallen A J (2011) Carbapenem-resistant

Enterobacteriaceae Epidemiology and prevention Clinical Infectious Diseases 53(1) 60-

67 doi101093cidcir202




897 doi101128aac00693-09

8 Delmas J Leyssene D Dubois D Birck C Vazeille E Robin F amp Bonnet R

(2010) Structural insights into substrate recognition and product expulsion in CTX-M

enzymes Journal of Molecular Biology 400(1) 108-120 doi101016jjmb201004062

9 Ke W Bethel C R Thomson J M Bonomo R A amp Van den Akker F (2007)

Crystal structure of KPC-2 Insights into carbapenemase activity in class A -lactamases

Biochemistry 46(19) 5732-5740 doi101021bi700300u

10 Petrella S Ziental-Gelus N Mayer C Renard M Jarlier V amp Sougakoff W (2008)

Genetic and structural insights into the dissemination potential of the extremely broad-

spectrum class A -lactamase KPC-2 identified in an Escherichia coli strain and an

Enterobacter cloacae strain isolated from the same patient in France Antimicrobial Agents


11 Majiduddin F K amp Palzkill T (2005) Amino acid residues that contribute to substrate

specificity of class A -lactamase SME-1 Antimicrobial Agents and Chemotherapy 49(8)

3421-3427 doi101128aac4983421-34272005

12 Massova I amp Mobashery S (1998) Kinship and diversification of bacterial penicillin-

binding proteins and -lactamases Antimicrobial Agents and Chemotherapy 42(1) 1ndash17

13 Papp-Wallace K M Taracila M Wallace C J Hujer K M Bethel C R Hornick J

M amp Bonomo R A (2010) Elucidating the role of Trp105 in the KPC-2 -lactamase

Protein Science 19(9) 1714-1727 doi101002pro454

61




15 Meroueh S O Fisher J F Schlegel H B amp Mobashery S (2005) Ab initio QMMM

study of class A -lactamase acylation Dual participation of Glu166 and Lys73 in a

concerted base promotion of Ser70 Journal of the American Chemical Society 127(44)

15397-15407 doi101021ja051592u

16 Nichols D A Hargis J C Sanishvili R Jaishankar P Defrees K Smith E W Chen

Y (2015) Ligand-induced proton transfer and low-barrier hydrogen bond revealed by X-

ray crystallography Journal of the American Chemical Society 137(25) 8086-8095

doi101021jacs5b00749

17 Sun T Bethel C R Bonomo R A amp Knox J R (2004) Inhibitor-resistant class A -

lactamases Consequences of the Ser130-to-Gly mutation seen in apo and tazobactam

structures of the SHV-1 variant Biochemistry 43(44) 14111-14117

doi101021bi0487903

18 Helfand M S Bethel C R Hujer A M Hujer K M Anderson V E amp Bonomo R

A (2003) Understanding resistance to -lactams and -lactamase inhibitors in the SHV

-lactamase Lessons from the mutagenesis of SER-130 Journal of Biological Chemistry

278(52) 52724-52729 doi101074jbcm306059200

19 Lewandowski E M Skiba J Torelli N J Rajnisz A Solecka J Kowalski K amp

Chen Y (2015) Antibacterial properties and atomic resolution X-ray complex crystal

structure of a ruthenocene conjugated -lactam antibiotic Chemical Communications

(Cambridge England) 51(28) 6186ndash6189 doi101039c5cc00904a

20 Leyssene D Delmas J Robin F Cougnoux A Gibold L amp Bonnet R (2011)

Noncovalent complexes of an inactive mutant of CTX-M-9 with the substrate piperacillin

and the corresponding product Antimicrobial Agents and Chemotherapy 55(12) 5660ndash

5665 doi101128aac00245-11

21 Beadle B M Trehan I Focia P J amp Shoichet B K (2002) Structural milestones in

the reaction pathway of an amide hydrolase Structure 10(3) 413-424 doi101016s0969-

2126(02)00725-6

22 Shimamura T Ibuka A Fushinobu S Wakagi T Ishiguro M Ishii Y amp Matsuzawa

H (2002) Acyl-intermediate structures of the extended-spectrum class A -lactamase

Toho-1 in complex with cefotaxime cephalothin and benzylpenicillin Journal of

Biological Chemistry 277(48) 46601-46608 doi101074jbcm207884200



4943ndash4960 doi101128AAC00296-11

24 Milazzo I (2003) Faropenem a new oral penem Antibacterial activity against selected

anaerobic and fastidious periodontal isolates Journal of Antimicrobial Chemotherapy

51(3) 721-725 doi101093jacdkg120

25 Fonseca F Chudyk E I Van der Kamp M W Correia A Mulholland A J amp

Spencer J (2012) The basis for carbapenem hydrolysis by class A -lactamases A

62

combined investigation using crystallography and simulations Journal of the American

Chemical Society 134(44) 18275-18285 doi101021ja304460j

26 Stewart N K Smith C A Frase H Black D J amp Vakulenko S B (2015) Kinetic

and structural requirements for carbapenemase activity in GES-type -lactamases

Biochemistry 54(2) 588-597 doi101021bi501052t

27 Nukaga M Bethel C R Thomson J M Hujer A M Distler A Anderson V E

Bonomo R A (2008) Inhibition of class A -lactamases by carbapenems

Crystallographic observation of two conformations of meropenem in SHV-1 Journal of


28 Chiou J Leung T Y amp Chen S (2014) Molecular mechanisms of substrate recognition

and specificity of New Delhi metallo--lactamase Antimicrobial Agents and


29 Battye T G Kontogiannis L Johnson O Powell H R amp Leslie A G (2011)

iMOSFLM A new graphical interface for diffraction-image processing with MOSFLM

Acta Crystallographica Section D Biological Crystallography 67(4) 271-281

doi101107s0907444910048675

30 Collaborative Computational Project Number 4 (1994) The CCP4 suite programs for

protein crystallography Acta Crystallographica Section D Biological Crystallography

50(5) 760-763 doi101107s0907444994003112

31 McCoy A J Grosse-Kunstleve R W Adams P D Winn M D Storoni L C amp

Read R J (2007) Phaser crystallographic software Journal of Applied Crystallography

40(4) 658ndash674 doi101107S0021889807021206

32 Afonine P V Grosse-Kunstleve R W Echols N Headd J J Moriarty N W

Mustyakimov M Adams P D (2012) Towards automated crystallographic structure

refinement with phenixrefine Acta Crystallographica Section D Biological

Crystallography 68(4) 352ndash367 doi101107S0907444912001308

33 Emsley P amp Cowtan K (2004) Coot Model-building tools for molecular graphics Acta

Crystallographica Section D Biological Crystallography 60(12) 2126-2132

doi101107s0907444904019158

34 Moriarty N W Grosse-Kunstleve R W amp Adams P D (2009) electronic Ligand

Builder and Optimization Workbench (eLBOW) A tool for ligand coordinate and restraint

generation Acta Crystallographica Section D Biological Crystallography 65(10) 1074-

1080 doi101107s0907444909029436

35 Davis I W Leaver-Fay A Chen V B Block J N Kapral G J Wang X

Richardson D C (2007) MolProbity All-atom contacts and structure validation for

proteins and nucleic acids Nucleic Acids Research 35(Web Server) W375-W383

doi101093nargkm216

36 DeLano WL (2002) The PyMOL userrsquos manual DeLano Scientific San Carlos CA pp

452

37 Wallace A C Laskowski R A amp Thornton J M (1995) LIGPLOT A program to

generate schematic diagrams of protein-ligand interactions Protein Engineering Design

and Selection 8(2) 127-134 doi101093protein82127

63

Chapter 3

Studying Proton Transfer in the Mechanism and Inhibition of Serine -

Lactamase Acylation

31 Overview

-Lactam antibiotics have long been considered a cornerstone of therapy for many life-

threatening bacterial infections Unfortunately the production of -lactam degrading enzymes

known as -lactamases threatens their clinical use A recent breakthrough in antibacterial drug

discovery was the broad-spectrum BLI known as avibactam Avibactam is a unique BLI because

even though it can rapidly acylate a wide range of -lactamases the covalent bond that it forms

with the catalytic serine residue is particularly stable to hydrolysis Previous crystallographic

studies with class A -lactamases reveal that the catalytic water and Glu166 the general base for

deacylation appear to be intact in the complex and capable of attacking the covalent linkage with

the inhibitor Here I present a 083 Aring resolution crystal structure of the class A -lactamase

CTX-M-14 bound to avibactam showing a unique active site protonation state trapped by the

inhibitor including a neutral Glu166 and a neutral Lys73 This structure suggests that the unique

side chain of avibactam (ie hydroxylamine-O-sulfonate) stabilizes a neutral Lys73 preventing

it from extracting a proton from Glu166 Subsequently Glu166 is not able to serve as the general

base to activate the catalytic water for the hydrolysis reaction My structure illustrates how

inhibitor binding can change the protonation states of catalytic residues

64

32 Introduction

-Lactamase production is the most common mechanism that Gram-negative bacteria use

to destroy -lactam antibiotics before they reach their targets the PBPs which are responsible

for cell wall synthesis [1] -Lactamases are divided into two groups according to the mechanism

that they use to catalyze -lactam hydrolysis Serine enzymes use an active site serine residue in

the covalent catalysis of the -lactam hydrolysis whereas metalloenzymes use zinc ions for

catalysis There are three classes of serine enzymes (A C and D) while there is only one class

of metalloenzymes class B [2]

Class A -lactamases are the most prevalent class of -lactamase in hospital acquired

infections and are usually found in a variety of Gram-negative bacteria such as members of the

Enterobacteriaceae family [3 4] Class A -lactamases most commonly provide resistance to

penicillins and firstsecond-generation cephalosporins Fortunately bacteria producing these

enzymes generally remain susceptible to third-generation cephalosporins and carbapenems

however this situation is quickly changing with the emergence of class A -lactamases

displaying hydrolytic activity against third-generation cephalosporins and carbapenems [5 6]

ESBLs can catalyze the hydrolysis of penicillins and firstsecondthird-generation

cephalosporins Currently the class A CTX-M -lactamases are the most commonly identified

ESBLs worldwide with CTX-M-15 being the most frequent variant [2 7] Carbapenemases have

an even broader spectrum of activity than ESBLs and can hydrolyze virtually all -lactam

antibiotics The KPC class A -lactamase is the most common mechanism of carbapenem

resistance in the United States Of the many KPC variants KPC-2 is the most frequently

identified and characterized variant [2 8 9]

65

The emergence of -lactamases prompted the search for novel inhibitors that could

reverse bacterial resistance against -lactam antibiotics The first clinically available BLIs

inhibitors were clavulanic acid sulbactam and tazobactam which are potent inhibitors of class

A (including CTX-M) and C -lactamases [10] These inhibitors all contain a -lactam core

structure enabling them to bind to the active site of class A and C -lactamases and form a

highly stable intermediate that permanently inactivates the enzyme Specifically clavulanic acid

sulbactam and tazobactam are suicide inhibitors of serine -lactamases because they covalently

bond to the catalytic serine residue and abolish enzymatic activity However these inhibitors can

be hydrolyzed by the more versatile carbapenemases (eg KPC-2 and MBLs) and consequently

have no activity against these enzymes [10 11]

Avibactam is a rationally designed BLI targeting serine -lactamases that includes

ESBLs (CTX-M-15) and serine carbapenemases (KPC-2) in its spectrum of activity Avibactam

has some unique properties that differentiates it from clavulanic acid sulbactam and

tazobactam (1) avibactam does not contain a -lactam ring but instead possesses a DBO

structure that incorporates features of -lactams into its scaffold (Fig 31) and (2) avibactam is a

covalent reversible inhibitor of serine -lactamases which is a radical departure from the

irreversible inhibition seen in the -lactam-based BLIs (Fig 32) [10-12]

To understand the mechanism by which avibactam possesses broad-spectrum of activity

against class A serine -lactamases a high-resolution X-ray crystal structure of avibactam bound

to the CTX-M-15 class A -lactamase was solved [12] This structure revealed that the catalytic

serine (Ser70) of this enzyme carries out nucleophilic attack on the carbonyl of the five-

membered cyclic urea resulting in opening of the avibactam ring and acylation of the enzyme

66

(Fig 32) [12] Acylation of CTX-M-15 can proceed through one of two pathways The first

pathway involves Glu166 activating Ser70 for nucleophilic attack via a water molecule The

second pathway proposes that Lys73 serves as the general base responsible for Ser70 activation

Mutagenesis of Glu166 to a Gln demonstrated a comparable avibactam acylation rate to WT

whereas mutating Lys73 to an Ala almost completely abolished avibactam acylation [13] These

data suggest that Lys73 is the general base during acylation and plays a direct role in activating

Ser70 during avibactam acylation however there is still not a clear consensus as to which

pathway predominates [13]

Ser130 is a residue that plays a critical role in avibactam acylation and subsequently

deacylation During acylation Ser130 behaves as a general acid and donates a proton to the N6

nitrogen of avibactam following ring opening [14] The reversible nature of avibactam inhibition

derives from its ability for recyclization (ie ring closure) after acylation (Fig 32) [13 15] In

order for avibactam recyclization to occur the N6 nitrogen must be deprotonated During

deacylation Ser130 now behaves as a general base extracting a proton from the N6 nitrogen

which facilitates ring closure [13] The intact avibactam molecule can then go on an acylate other

-lactamases or even the same -lactamase [15]

The complex that avibactam forms with serine -lactamases is very stable to hydrolysis

In fact recyclization of avibactam is favored over hydrolysis [16 17] Key questions remain as

to why the covalent bond avibactam forms with serine -lactamases is so stable against

hydrolysis despite the presences of a water molecule perfectly positioned to attack the bond [12

13] Performing ultrahigh resolution X-ray crystallography on the CTX-M-14 class A -

lactamase I attempt to answer this question by directly observing the protonation states of

Lys73 Ser130 and Glu166 residues implicated in a proton transfer process

67



The crystal structure of avibactam bound to CTX-M-14 was solved to 083 Aring resolution

In the active site electron density corresponding to avibactam was identified (Fig 33) The

electron density shows that there is a covalent linkage between avibactam and the catalytic

Ser70 Besides the acylndashenzyme covalent bond there are four other key interactions observed

between the inhibitor and enzyme which are the highly polar sulfate moiety carboxamide group

C7 carbonyl group and piperidine ring (Fig 34) The sulfate moiety binds in a polar region of

CTX-M-14 that commonly recognizes the carboxylate group on -lactam substrates [18-20]

The sulfate group has two conformations with the dominant conformation establishing HBs with

Thr235 and Ser237 and interacts via attractive charges with Lys234 and Arg276 The

carboxamide group forms HBs with the sidechains of Asn104 and Asn132 The C7 carbonyl

group of avibactam is positioned within the oxyanion hole formed by the backbone amides of

Ser70 and Ser237 The piperidine ring forms a modest Van der Waals interaction with Tyr105

In addition to these interactions Ser130 is within HB distance (290 Aring) of the N6 nitrogen

which is hypothesized to play a role in the acylation and recyclization of avibactam as a general

acidbase [14]

332 Ultrahigh resolution reveals the protonation states of Lys73 and Glu166

The 083 Aring resolution crystal structure of CTX-M-14avibactam enabled direct

observations of the protonation state of two key catalytic residues Lys73 and Glu166 (Fig 35)

The unbiased Fo-Fc map shows that side chain amine nitrogen (N) of Lys73 has two protons

indicating that it is neutral The conformation of Lys73 is positioned to form a strong HB (28 Aring)

68

with Ser130 HG where the proton on the Ser130 side chain is clearly visible in the Fo-Fc

electron density map This observation is consistent with molecular dynamic (MD) simulations

studying avibactam recyclization during which dynamic stabilities of the HB between Ser130

and Lys73 were investigated [16] These simulations showed that for 99 of the time a HB is

present between Lys73 N and Ser130 HG with some snapshots showing an N middotmiddotmiddot H distance as

short as 15 Aring suggesting that a proton transfer from Ser130 to Lys73 is possible [16] This

proton transfer would coincide with Ser130 behaving as a general base to extract a proton from

the N6 nitrogen thereby promoting recyclization of avibactam

From the crystal structure it appears that the protonation state of Glu166 is closely tied

with that of Lys73 Analyzing the Fo-Fc map showed a protonated and neutral Glu166 In order

for the catalytic water to become activated for nucleophilic attack on the covalent linkage

Glu166 must be able to transfer its proton to Lys73 to then act as a general base However this

process is energetically unfavorable based on MD simulations [21] Instead the Lys73-Ser130

dyad can serve as a better general base to activate the N6 nitrogen of avibactam for

intramolecular nucleophilic attack on the carbamyl linkage to reform the N6-C7 bond and

generate the intact avibactam molecule (Fig 32) [13 16]

34 Conclusions

Together with previous high-resolution crystal structures of avibactam with class A -

lactamases this new data allows us to track the transfer of a proton throughout the entire

acylation process and demonstrate the essential role of Lys73 in transferring the proton from

Ser70 to Ser130 and ultimately to the N6 nitrogen of avibactam Also revealed was that a

protonated Glu166 is unable to transfer a proton to Lys73 and subsequently cannot act as the

69

general base to catalyze hydrolysis of the EI complex In conjunction with computational

analysis this structure also sheds new light on the stability and reversibility of the avibactam

acylndashenzyme complex highlighting the effect of substrate functional groups in influencing the

protonation states of catalytic residues and subsequently the progression of the reaction



The CTX-M-14 gene was cloned into a modified plasmid vector pET-9a Ecoli BL21

(DE3) transformed with the plasmid pET-blaCTX-M-14 was cultured in LB broth containing

kanamycin at 100 μgmL at 37 degC for 5 h Overexpression of the CTX-M-encoding gene was

induced with 05 mM IPTG overnight at 20 degC Cells were harvested by centrifugation (10000 g

for 10 min at 4 degC) and then disrupted by ultrasonic treatment (four times for 30 sec each time at

20 W) The extract was clarified by centrifugation at 48000 g for 60 min at 4 degC After addition

of 2 μg of DNase I (Roche) the supernatant was dialyzed overnight against 20 mM MESndashNaOH

(pH 60) The purification was carried out by ion-exchange chromatography on a fast-flow CM

column (Amersham Pharmacia Biotech) in 20 mM MES buffer (pH 60) and eluted with a linear

0ndash015 M NaCl gradient The enzyme was more than 95 homogeneous as judged by

Coomassie blue staining after SDS-PAGE The purified protein was dialyzed against 5 mM Trisndash

HCl buffer (pH 70) and concentrated to 20 mgmL for crystallization

70

352 Crystallization and structure determination

CTX-M-14 was crystallized in 10 M potassium phosphate buffer (pH 83) from hanging

drops at 20 degC The final concentration of the protein in the drop ranged from 65 to 10 mgmL

Avibactam complex crystals were obtained through soaking methods with a compound

concentration of 50 mM and soaking times varying from 3 h to 24 h Crystals were cryo-

protected in 10 M potassium phosphate buffer (pH 83) and 30 sucrose (wv) and flash frozen

in liquid nitrogen Diffraction was measured at 23-ID-B of GMCAAPS at Advanced Photon

Source (APS) Argonne Illinois Diffraction data were indexed integrated and scaled with

HKL2000 [22] The complex structure was refined with phenixrefine [23] from the PHENIX

suite Model rebuilding was performed using WinCoot [24] Figures were prepared using

PyMOL 13 (Schroumldinger) [25] and Discovery Studio Visualizer [26]

36 Note to Reader 1

Figure 32 in this chapter was previously published by [12] Lahiri S D Mangani S

Durand-Reville T Benvenuti M De Luca F Sanyal G amp Docquier J (2013) Structural

insight into potent broad-spectrum inhibition with reversible recyclization mechanism

Avibactam in complex with CTX-M-15 and Pseudomonas aeruginosa AmpC -lactamases

Antimicrobial Agents and Chemotherapy 57(6) 2496-2505 doi101128aac02247-12 and has

been reproduced with permission (See Appendix 1)

71

4

3

2

1

6 7

8

5

Figure 31 Chemical structure and numbering of atoms of avibactam

72

Figure 32 General mechanism of avibactam inhibition against serine -lactamases

Adapted with permissions from [12] See Appendix 1

73

K234

S130 K73

Y105

T235

S237

N170

E166

N132

S70

Figure 33 Crystal structure of avibactam bound to CTX-M-14 The Fo-Fc omit electron

density map around avibactam is represented in blue and contoured at 2

74

Figure 34 Schematic diagram of CTX-M-14avibactam interactions Various interactions

that avibactam forms with CTX-M-14 and avibactam are displayed in a schematic diagram

generated by Discovery Studio Visualizer

75

S70 S130

K73

E166

N170

Wat1

Figure 35 Protonation states of active site residues in CTX-M-14avibactam complex The

2Fo-Fc electron density map is represented in blue and contoured at 3 The Fo-Fc electron

density map is represented in red and contoured at 2 Red sphere represents a water molecule

Hydrogen atoms can be observed on K73 S130 and E166 indicating a neutral K73 and E166

76

37 References



1145-1165 doi1021740929867023370031



3 Avci F G Altinisik F E Vardar Ulu D Ozkirimli Olmez E amp Sariyar Akbulut B

(2016) An evolutionarily conserved allosteric site modulates beta-lactamase activity

Journal of Enzyme Inhibition and Medicinal Chemistry 31(sup3) 33-40

doi1010801475636620161201813



doi101002chin200524267

5 Rawat D amp Nair D (2010) Extended-spectrum -lactamases in Gram negative

bacteria Journal of Global Infectious Diseases 2(3) 263 doi1041030974-777x68531

6 Queenan A M amp Bush K (2007) Carbapenemases The versatile -lactamases

Clinical Microbiology Reviews 20(3) 440-458 doi101128cmr00001-07

7 Poirel L Nordmann P Ducroz S Boulouis H Arne P amp Millemann Y (2013)

Extended-spectrum -lactamase CTX-M-15-producing Klebsiella pneumoniae of

sequence type ST274 in companion animals Antimicrobial Agents and Chemotherapy

57(5) 2372-2375 doi101128aac02622-12

8 Mehta S C Rice K amp Palzkill T (2015) Natural variants of the KPC-2

carbapenemase have evolved increased catalytic efficiency for ceftazidime hydrolysis at

the cost of enzyme stability PLOS Pathogens 11(6) e1004949

doi101371journalppat1004949

9 Papp-Wallace K M Winkler M L Taracila M A amp Bonomo R A (2015)

Variants of -lactamase KPC-2 that are resistant to inhibition by avibactam


10 Bush K amp Bradford P A (2016) -Lactams and -lactamase inhibitors An overview



11 Drawz S M amp Bonomo R A (2010) Three decades of -lactamase inhibitors


12 Lahiri S D Mangani S Durand-Reville T Benvenuti M De Luca F Sanyal G amp

Docquier J (2013) Structural insight into potent broad-spectrum inhibition with

reversible recyclization mechanism Avibactam in complex with CTX-M-15 and

Pseudomonas aeruginosa AmpC -lactamases Antimicrobial Agents and Chemotherapy

57(6) 2496-2505 doi101128aac02247-12

13 King D T King A M Lal S M Wright G D amp Strynadka N C (2015)

Molecular mechanism of avibactam-mediated -lactamase inhibition ACS Infectious

Diseases 1(4) 175-184 doi101021acsinfecdis5b00007

77

14 Winkler M L Papp-Wallace K M Taracila M A amp Bonomo R A (2015)

Avibactam and inhibitor-resistant SHV -lactamases Antimicrobial Agents and


15 Krishnan N P Nguyen N Q Papp-Wallace K M Bonomo R A amp Van den Akker

F (2015) Inhibition of Klebsiella -lactamases (SHV-1 and KPC-2) by avibactam A

structural study PLOS ONE 10(9) e0136813 doi101371journalpone0136813

16 Choi H Paton R S Park H amp Schofield C J (2016) Investigations on recyclisation

and hydrolysis in avibactam mediated serine -lactamase inhibition Org Biomol Chem

14(17) 4116-4128 doi101039c6ob00353b

17 Lahiri S D Johnstone M R Ross P L McLaughlin R E Olivier N B amp Alm R

A (2014) Avibactam and class C -lactamases Mechanism of inhibition conservation

of the binding pocket and implications for resistance Antimicrobial Agents and




and the corresponding product Antimicrobial Agents and Chemotherapy 55(12) 5660-

5665 doi101128aac00245-11

19 Pemberton O A Zhang X amp Chen Y (2017) Molecular basis of substrate

recognition and product release by the Klebsiella pneumoniae carbapenemase (KPC-2)

Journal of Medicinal Chemistry 60(8) 3525-3530 doi101021acsjmedchem7b00158

20 Tondi D Venturelli A Bonnet R Pozzi C Shoichet B K amp Costi M P (2014)

Targeting class A and C serine -lactamases with a broad-spectrum boronic acid

derivative Journal of Medicinal Chemistry 57(12) 5449-5458 doi101021jm5006572

21 Sgrignani J Grazioso G De Amici M amp Colombo G (2014) Inactivation of TEM-1

by avibactam (NXL-104) Insights from quantum mechanicsmolecular mechanics

metadynamics simulations Biochemistry 53(31) 5174-5185 doi101021bi500589x

22 Otwinowski Z amp Minor W (1997) Processing of X-ray diffraction data collected in

oscillation mode Methods in Enzymology 307-326 doi101016s0076-6879(97)76066-

x





24 Emsley P amp Cowtan K (2004) Coot Model-building tools for molecular graphics


doi101107s0907444904019158


452

26 Dassault Systegravemes BIOVIA Discovery Studio Modeling Environment Release 2017

San Diego Dassault Systegravemes 2016

78

Chapter 4

The Influence of Binding Kinetics on Inhibition of KPC-2 by Vaborbactam in

Countering Bacterial Resistance

41 Overview

KPC-2 hydrolyzes nearly all -lactam antibiotics including carbapenems and is

responsible for antibiotic resistance in many pathogenic bacteria particularly CRE The recently

approved avibactam has been used as a KPC-2 inhibitor in the clinic yet resistance has already

emerged Here I demonstrate that vaborbactam a novel boronic-acid based BLI behaves as a

slow tight-binding inhibitor and can effectively reverse bacterial resistance caused by KPC-2

including Ser130 mutants resistant to avibactam In particular vaborbactam displayed

significantly higher cell-based activity against KPC-2 than CTX-M-15 an ESBL even though it

has similar binding affinities for both enzymes Analysis of binding kinetics revealed a correlation

between vaborbactams cell-based activity against KPC-2 and a slower koff value in comparison

to CTX-M-15 The molecular basis of this difference as well as the roles of Ser130 in the

inhibition mechanisms of vaborbactam versus avibactam is illustrated by the first crystal complex

structure of KPC-2 with vaborbactam determined at 125 Aring My studies provide valuable insights

into the use of vaborbactam in combating antibiotic resistance and the importance of binding

kinetics in novel antibiotic development

79

42 Introduction

CRE are an emerging health threat due to their production of carbapenemases which are a

unique group of -lactamases that possess the ability to hydrolyze nearly all -lactam antibiotics

[1] CRE infections are associated with high rates of morbidity and mortality worldwide due to

limited treatment options [2 3] One of the most common mechanisms of carbapenem resistance

among CRE is the production of the KPC-2 class A -lactamase [4] Class A -lactamases like

KPC-2 use a catalytic serine residue to mediate the opening of the -lactam ring similar to class

C and D -lactamases [5] In contrast the last remaining class B -lactamases rely on zinc ions

for activity [6]

To address the growing problem of antibiotic resistance numerous BLIs have been

developed that when combined with a -lactam are therapeutic in the treatment of multi-drug

resistant bacterial infections [7] However some clinically available BLIs such as clavulanic acid

and tazobactam (Fig 41) have poor activity against KPC-2 [8 9] In 2015 a potent non--lactam

based BLI known as avibactam (Fig 41) was approved in combination with the third-generation

cephalosporin ceftazidime to combat bacteria producing KPC-2 AmpC and ESBLs [10-12]

Unfortunately clinicians have observed cases of avibactam resistance due to porin mutations and

plasmid-borne blaKPC-3 mutations [13-15] These observations suggest that additional BLI

discovery is vital against this rapidly changing resistance determinant

Antibiotic development requires both the discovery of novel lead chemical scaffolds and a

deep understanding of how these small molecules interact with their bacterial targets Boronic

acids have undergone extensive investigation as broad-spectrum serine BLIs due to formation of

a stable covalent bond with the active site serine residue and the ability to readily diffuse through

the outer membrane of Gram-negative bacteria facilitating their uptake [16] A boronic acid

80

transition state inhibitor (BATSI) known as S02030 (Fig 41) was strategically designed to target

a wide variety of serine -lactamases including ADC-7 KPC-2 and SHV-1 with nanomolar

potency [17] Most recently a cyclic boronic acid known as vaborbactam has been shown to

possess potent inhibitory activity against many class A and C -lactamases [18] Particularly

vaborbactam can inhibit CTX-M SHV and CMY enzymes Examining vaborbactamrsquos in vitro

activity revealed that it can potentiate meropenem (a carbapenem) against clinical isolates of E

coli Enterobacter cloacae and K pneumoniae expressing a wide range of serine -lactamases

from classes A and C [19]

I have extended my studies of vaborbactam to the clinically important KPC-2 In particular

I am interested in how binding kinetics may influence the activity of vaborbactam The importance

of binding kinetics in drug efficacy has received increasing attention with numerous studies

demonstrating that the residence time ie the duration that the drug occupies the target binding

site is a superior indicator of a drugrsquos in vivo efficacy versus the inhibitor constant Ki [20 21]

However most of these studies focused on how the residence time can influence the

pharmacokinetics of the drug inside animals or humans [22 23] It is largely unknown how binding

kinetics can impact the activity of a drug on the cellular level especially relative to their

bactericidal effects To characterize vaborbactam inhibition of KPC-2 I have performed binding

kinetic experiments MIC assays mutagenesis studies and solved the first crystal structure of

vaborbactam bound to KPC-2 These results provide new insights into the unique properties of

vaborbactam in combating carbapenem resistance caused by KPC-2 as well as the influence of

binding kinetics on the cell-based activities of antibacterial compounds

81


431 Vaborbactam binding kinetics

Previously published crystal structures of vaborbactam with CTX-M-15 and AmpC -

lactamases revealed the formation of a covalent bond between the catalytic serine residue of the

enzyme and the boron atom of vaborbactam [18] In agreement with these findings a Lineweaver-

Burk plot of KPC-2 inhibition by vaborbactam demonstrated that it behaves as competitive

inhibitor of this enzyme (Fig S41)

The number of BLI molecules that are needed to inactivate one molecule of a -lactamase

ie the stoichiometry or partition ratio was also investigated KPC-2 inhibition experiments using

varying BLI concentrations revealed that one mole of vaborbactam was required to inhibit one

mole of KPC-2 (Table 41) Avibactam demonstrated the same stoichiometry while tazobactam

and clavulanic acid required a much higher ratio for complete enzyme inhibition as KPC-2 can

efficiently hydrolyze these suicide inhibitors [7] Slow cleavage of avibactam by KPC-2 was

reported previously [24] thus prompting me to investigate the stability of vaborbactam to this

enzyme Incubation of vaborbactam with KPC-2 for 18 h with subsequent chromatographic

analysis revealed no change in the amount of vaborbactam (data not shown) indicating no effect

of the enzyme on the vaborbactam chemical structure

When studied using a reporter substrate technique typical ldquofast on ndash fast offrdquo or reversible

boronic BLIs (eg m-tolylboronic acid and 2-formylphenylboronic acid [25]) demonstrate a linear

KPC-2 inactivation profile indicating that equilibrium between the enzyme and inhibitor is

quickly established (Fig S42) My initial studies revealed a time-dependent decrease of Ki values

when the incubation time of vaborbactam and KPC-2 was increased from the standard 10 min up

82

to 18 h (Fig S43) That led me to hypothesize that unlike other boronic BLIs vaborbactam may

exhibit progressive inactivation profiles typical for covalent irreversible inhibitors [26] Indeed

vaborbactam kinetic behavior was very similar to that demonstrated by tazobactam with a slower

onset of inhibition and non-linear inhibition curves indicating progressive KPC-2 inactivation (Fig

S44) This result suggests that vaborbactamrsquos interaction with KPC-2 follows a two-step kinetic

mechanism with initial formation of a non-covalent EI complex characterized by the binding

constant K which subsequently proceeds to a covalent interaction between the catalytic Ser70 of

KPC-2 and boron atom of vaborbactam in the EI complex The last step is characterized by the

first-order rate constant k2 Independent determination of these values was impossible due to a

linear relationship between kobs and vaborbactam concentration values up to the highest inhibitor

concentration tested (data not shown) Similar kinetic behavior was previously reported for BLIs

from various structural classes [27] Therefore the second-order rate constant k2K for the onset

of inhibition was determined Vaborbactam and avibactam demonstrated comparable k2K values

of 73 x 103 and 13 x 104 M-1s-1 respectively (Table 42) The recovery of KPC-2 activity after

complete inhibition by vaborbactam was investigated by the jump dilution method [28] This study

demonstrated that vaborbactam inhibition of KPC-2 can be reversed (Fig S45) However the

KPC-2 recovery rate was extremely slow with a koff of 0000017 s-1 indicating an enzyme

residence time of 992 min (Table 42) For comparison avibactam showed an approximately 10-

fold higher rate of activity recovery (koff = 000022 s-1) Knowing the rate for the onset of inhibition

and the koff rate allowed for calculation of the affinity Kd of the covalent complex (Table 42)

The two inhibitors have similar inactivation efficiency against KPC-2 but at least a 10-fold

difference in off-rates As a consequence vaborbactam appears to have a higher affinity for the

83

covalent complex than avibactam Overall vaborbactam functions as a slow tight-binding

inhibitor of KPC-2

Analyzing the inhibition of vaborbactam among -lactamases revealed some key

differences in binding kinetics (Table 42) KPC-2 has a much slower koff (0000017 s-1) as

compared to the ESBL CTX-M-15 (000088 s-1) Ultimately this leads to a much longer residence

for KPC-2 (992 min) versus CTX-M-15 (19 min)

432 Vaborbactam potentiation of aztreonam in KPC-2 and CTX-M-15 producing

E coli

As previously shown vaborbactam has widely varying binding kinetics against KPC-2 and

CTX-M-15 in terms of k2K koff residence time and Kd (Table 42) To observe how this would

translate to in vitro activity we performed MIC experiments with aztreonam (a monobactam) and

varying concentrations of vaborbactam on KPC-2 and CTX-M-15 producing E coli (Table 43)

Interestingly only 015 gmL of vaborbactam was needed to reduce the MIC of aztreonam 64-

fold whereas 10 gmL of vaborbactam was needed to reduce the MIC of aztreonam 64-fold

433 Impact of S130G mutation on vaborbactam inhibition

Ser130 plays an important role in the catalytic mechanism of class A -lactamases

including KPC-2 [7] and mutations at this position significantly affect interactions with substrates

and inhibitors including the recently approved avibactam [24 29] The effect of the S130G

substitution on vaborbactam potentiation of various antibiotics was studied in microbiological

experiments with a strain of P aeruginosa PAM1154 [30] that lacks efflux pumps and carries

plasmids expressing the WT and the mutant blaKPC-2 Consistent with earlier studies KPC-2

84

S130G lost the ability to hydrolyze the majority of -lactam antibiotics [24 29 31] P aeruginosa

producing the KPC-2 S130G variant demonstrated resistance to the penicillins carbenicillin and

piperacillin but not to other -lactams (Table S41) Subsequently carbenicillin and piperacillin

were chosen to study the concentration response to vaborbactam using a checkerboard

methodology (Table 44) As previously reported cells producing KPC-2 S130G were highly

resistant to avibactam potentiation of penicillins [29 31] carbenicillin and piperacillin MICs

against the KPC-2 WT producing strain were reduced 512- to 1024-fold by avibactam at 32 gmL

and only 4- to 8-fold against the strain that produced KPC-2 S130G Notably the S130G

substitution did not affect antibiotic potentiation by vaborbactam

Studies with purified enzymes confirmed that the Ki value for avibactam inhibition of

KPC-2 S130G was almost 6000-fold higher than that for the WT KPC-2 70 M vs 0012 M At

the same time the Ki of vaborbactam decreased from 0034 M for the WT KPC-2 to 0011 M

for the S130G mutant Detailed kinetic studies confirmed the significant effect of the S130G

substitution on acylation efficiency of avibactam [24 31 32] it was decreased almost 1000-fold

(Table 42) Conversely vaborbactam inactivated the KPC-S130G variant with an almost 10-fold

higher efficiency compared to its inactivation of the WT KPC-2 Another unexpected impact of

S130G on vaborbactam kinetics was an over 200-fold increase in the rate of recovery of enzymatic

activity (Table 42) Of note due to an increase in the rate of onset of inhibition (k2K) the Kd

value remained in the nanomolar range providing a possible explanation as to why the S130G

mutation did not impact the antibiotic potentiation activity of vaborbactam

85


To understand how vaborbactam achieves potent inhibition of KPC-2 requires structural

information to supplement our previously obtained structures of vaborbactam bound to CTX-M-

15 and AmpC [18] Therefore I co-crystallized KPC-2 with vaborbactam and solved the complex

structure to 125 Aring (Fig 42 Table S42) KPC-2vaborbactam co-crystals belong to the space

group P22121 with one molecule in the asymmetric unit The Fo-Fc map showed an unambiguous

density in the KPC-2 active site corresponding to two closely related conformations of

vaborbactam differing from one another by a flip of the thiophene ring (Fig 42A) Vaborbactam

forms several HBs and extensive hydrophobic interactions with the enzyme (Fig 42A) The NH

of the amide group of vaborbactam donates a HB to the Thr237 backbone carbonyl while the

carbonyl of the same amide group of vaborbactam accepts a HB from the Asn132 sidechain amide

NH2 The carboxylate group of vaborbactam is extensively coordinated by the hydroxyl side chains

of Ser130 Thr235 and Thr237 The thiophene moiety of the inhibitor re-orients inward to make

hydrophobic interactions with Trp105 The interactions that vaborbactam makes with KPC-2

largely mimic the interactions that the -lactam antibiotics cefotaxime and faropenem form with

KPC-2 [33]

Like most serine hydrolases the KPC-2 active site contains an ldquooxyanion holerdquo which is

a small subpocket surrounded by several HB donor atoms that coordinate the carbonyl group

oxygen of the substrate next to the scissile bond [34 35] In KPC-2 it is formed by the backbone

amide NH groups of residues Ser70 and Thr237 A distinct feature of the vaborbactam complex is

the mode of interaction with the oxyanion hole Typically substrates and inhibitors engage it with

a single acceptor (oxygen atom or hydroxyl) [36-40] vaborbactam on the other hand inserts two

oxygen atoms (exocyclic and endocyclic) into the oxyanion hole so that the Ser70 NH group is

86

interacting mostly with the exocyclic oxygen and the Thr237 NH group is interacting with

endocyclic oxygen (Fig 42A)

Vaborbactam induces some conformational changes in the KPC-2 active site upon binding

(Fig 42B) In the apoenzyme (PDB ID code 5UL8 [33]) Trp105 alternates between two

conformations that flank opposite sides of the active site whereas in the complex structure Trp105

adopts two conformations that are positioned to make hydrophobic contacts with the six-

membered ring and thiophene moiety of vaborbactam One conformation of Trp105 is similar to a

conformation observed in the apoenzyme whereas the other conformation is unique to the

particular complex allowing the protein to establish more non-polar contacts with vaborbactam

In the apoenzyme Ser130 also has two conformations Conformation 1 forms a weak HB (35 Aring)

with Lys73 and is the conformation normally observed in class A -lactamases whereas

conformation 2 establishes a strong HB (27 Aring) with Lys234 [33] In the complex structure Ser130

adopts a single conformation conformation 2 forming a HB with the vaborbactam carboxylate

group

44 Conclusions

Bacterial antibiotic resistance to -lactam antibiotics is a significant health threat

worldwide Of notable health concern is the KPC-2 class A -lactamase due to its broad-substrate

profile that includes penicillins extended-spectrum cephalosporins and carbapenems My

biochemical and structural analyses have provided important insights into the binding kinetics and

molecular interactions underlying the clinical utility of vaborbactam a unique cyclic boron-based

BLI in countering bacterial resistance caused by KPC-2 and its mutants

87

Like other boronic acid inhibitors vaborbactam acts as a covalent yet reversible ligand

for -lactamases [41-43] The apparent Ki values of these inhibitors are usually determined by

steady-state kinetics using the hydrolysis reaction with reporter substrates such as nitrocefin Due

to the formation of a covalent bond these inhibitors frequently have a slow koff and the binding to

the protein may not reach equilibrium during the biochemical assay This is especially true for

vaborbactam whose residence time for KPC-2 is more than 10X longer than the duration of the

experiment usually in the range of 10-20 min As a result this can lead to an underestimate of the

true KdKi value In my previous study using steady-state kinetics vaborbactam displayed similar

activities against both KPC-2 and CTX-M-15 [18] My latest results provide a more accurate

picture of the inhibition of these two enzymes by vaborbactam The Kd value against KPC-2 is

approximately 5X lower than previously reported In comparison the Kd for CTX-M-15 is similar

to previous studies [18] This is likely due to the shorter residence time of vaborbactam for CTX-

M-15 which allows the binding to reach equilibrium during the length of the assay

The longer residence time of vaborbactam for KPC-2 (992 min) in comparison to CTX-

M-15 (19 min) has translated into a significantly improved cell-based activity against the former

enzyme than the latter At 03 gmL vaborbactam decreased the MIC of aztreonam by gt100-fold

against KPC-2 expressing E coli but only 2-fold for CTX-M-15 Recent studies have highlighted

the importance of residence time in the in vivo efficacy of many drugs targeting both human and

bacterial proteins [20-23] For antibacterial targets it has been suggested that the residence time

should be at least comparable to the bacterial generation time (eg 20 min) [7] The residence

time of vaborbactam for both KPC-2 and CTX-M-15 satisfies this criterion The significant

improvement in the cell-based activity suggests that a long residence time is important for the

potency of antibacterial compounds at least in the case of BLIs even without considering the

88

pharmacokinetics inside the human body At the same time it raises the question as to why a

residence time longer than the bacterial generation time can still be beneficial for antibiotic utility

For BLIs this may be due to potential -lactamase activity even after bacterial cell death as a

result of the diffusion of both -lactamases and antibiotics

The high binding affinity and long residence time of vaborbactam for serine -lactamases

is mostly derived from the covalent bond between the catalytic serine and the boron atom

However the different binding kinetics of KPC-2 and CTX-M-15 illustrates the contribution of

non-covalent interactions to the activity of vaborbactam Vaborbactam was previously solved in

the active site of CTX-M-15 to 150 Aring resolution (PDB ID code 4XUZ [18]) There are many

similarities between vaborbactam in KPC-2 and CTX-M-15 (Fig 43) Both the endocyclic and

exocyclic oxygens of the vaborbactam ring enter and interact with the oxyanion hole formed by

the backbone NH groups of Ser70 and Thr237 (in the case of KPC-2) and Ser70 and Ser237 (in

the case of CTX-M-15) The exocyclic oxygen forms a HB with the NH group of Ser70 whereas

the endocyclic oxygen HBs with the NH group of Thr237 in KPC-2 or Ser237 in CTX-M-15

Unlike CTX-M-15 KPC-2 and related carbapenemases possess a disulfide bond between

the adjacent Cys69 and Cys238 that has been demonstrated to be important for activity [44 45]

The presence of this disulfide bond appears to have a structural impact on the architecture of the

KPC-2 active site as compared to the CTX-M-15 active site The disulfide bond results in an

outward shift of Gly239 and Val240 which contributes to the re-orientation of the thiophene

moiety of vaborbactam due to potential steric clashing Interestingly the disulfide bond may also

pre-adjust the backbone conformation around the oxyanion hole to allow insertion of the

endocyclic and exocyclic oxygen atoms of vaborbactam This difference is particularly evident

when comparing CTX-M-15 structures (apo PDB ID code 4HBT [46] vaborbactam PDB ID

89

code 4XUZ [18] and avibactam PDB ID code 4HBU [46]) Indeed vaborbactam binding to

CTX-M-15 is accompanied by ~ 06 Aring ldquobulgingrdquo of the backbone at Ser237 (Fig 44A) enlarging

the oxyanion hole (as compared to less than 02 Aring movement for avibactam) On the other hand

virtually no backbone movement is seen around the oxyanion hole in our KPC-2vaborbactam

structure when compared with an apo structure of KPC-2 (PDB ID code 5UL8 [33]) however

the structure of avibactam bound to KPC-2 (PDB ID code 4ZBE [24]) does result in backbone

shifts upstream at Cys238 and especially Gly239 (Fig 44B) This observation may in part explain

the potency of vaborbactam against class A carbapenemases

One major difference between the KPC-2 and CTX-M-15 vaborbactam complexes is the

conformation of the thiophene moiety In CTX-M-15 the thiophene moiety assumes a linear

conformation whereas in KPC-2 the thiophene moiety bends inward to form hydrophobic

interactions with Trp105 (Fig 43) The presence of the large non-polar tryptophan residue at

position 105 in KPC-2 versus the smaller and more polar tyrosine residue at position 105 in CTX-

M-15 likely contributes to this observation In comparison to Trp105 in KPC-2 Tyr105 in CTX-

M-15 establishes fewer interactions with vaborbactam This is due not only to the smaller size of

Tyr105 but also due to its relative rigidity which prevents it from moving closer to the ligand

like the second conformation of Trp105 In fact this is a general trend observed in several CTX-

M and KPC-2 structures which underscores the versatility of Trp105 in adapting to different

ligands and contributing to the broad-spectrum activity of KPC-2 [33 40 47-49]

Overall the more extensive non-covalent interactions between vaborbactam and KPC-2

may explain its longer residence time compared with CTX-M-15 however the increased

complementarity may have been achieved with a significant entropic cost on the protein and the

ligand and led to the slower on-rate for KPC-2 (Table 42) On the protein side the need for an

90

optimal Trp105 conformation can affect the kon rate albeit on a smaller scale This is reminiscent

of previous studies showing protein conformational changes usually accompany long residence

times [21 50] On the ligand side vaborbactam adopts a very compact conformation in KPC-2

which affords little conformational freedom In comparison significant movement of the

thiophene arm can be accommodated in the extended conformation in the CTX-M-15 active site

The strict conformational requirement for vaborbactam in the KPC-2 active site may be another

factor contributing to the slower on-rate than CTX-M-15

Another interesting observation is that the S130G mutation seems to significantly increase

the on-rate for KPC-2 I have recently found that Ser130 mutations in CTX-M-14 significantly

increase the flexibility of the active site loop containing Ser130 (unpublished data) I hypothesize

that similar effects in KPC-2 may lead to a more open active site which may reduce the

conformational restraints on vaborbactam

Although avibactam has been successfully used to target carbapenem-resistance caused by

KPC-2 its clinical utility is threatened by emerging resistance mutations such as S130G [24 31

32] The Ser130 sidechain plays a significant role in the catalytic mechanism of class A -

lactamases donating a proton to the nitrogen atom upon cleavage of the amide (lactam) bond [29]

Indeed S130G mutant enzymes generally have much lower catalytic efficiency with the

remaining activity attributed to replacement of the Ser130 sidechain by a water molecule

Accordingly avibactam and other inhibitors that depend on an Ser130-mediated acylation

mechanism are strongly impacted by this mutation resulting in substantial resistance [32] Ser130

mutations may be particularly detrimental to avibactam activity because they also affect the

recyclization of the avibactam ring which reverses the acylation reaction and allows the inhibitor

91

to be recycled [51] In comparison the boronate moiety of vaborbactam is not interacting with

Ser130 and therefore formation of the covalent adduct should not be affected by this residue

In conclusion vaborbactam achieves nanomolar potency against KPC-2 due to its covalent

and extensive non-covalent interactions with conserved active site residues Its remarkable

residence time on KPC-2 correlates with its favorable pharmacological properties Ultimately

these in vitro findings corroborate the idea that a slow off-rate and long residence time translate to

potent cell-based activity Vaborbactam has shown promising results when combined with the

carbapenem meropenem and is marketed as Vabomere which has been recently approved by the

FDA Vabomere provides a promising strategy for the treatment of serious Gram-negative

infections caused by bacteria resistant to carbapenem antibiotics


451 Generation of KPC-2 mutants

Mutations in the KPC-2 gene cloned in either pUCP24 or pET28a plasmids were

introduced using ldquoQuikChange Lightning Site-Directed Mutagenesis Kitrdquo (Agilent Technologies)

The presence of desired mutations and the lack of unwanted ones were confirmed by DNA

sequencing of the entire KPC-2 gene

452 Determination of MIC values and vaborbactam potentiation experiments

For microbiological studies KPC-2 WT and its mutant genes were cloned in pUCP24

shuttle vector Resulting plasmids were transformed into P aeruginosa PAM1154 strain MIC

values for various antibiotics were determined by the standard broth microdilution method using

92

cation adjusted Mueller-Hinton Broth [52] Potentiation of antibiotic activity by vaborbactam in

bacterial strains carrying WT and mutants KPC-2 genes were performed using standard checker

board assay [53] All microbiological studies were performed with 15 gmL of gentamycin

present in the media

453 Evaluation of KPC-2 mutant proteins expression level in PAM1154 strain

Bacterial cells carrying plasmids expressing KPC-2 mutants were grown in liquid media

till OD600=07-09 and diluted to final OD600=05 500 L of cell culture were spun down and

resulting pellet was resuspended in 500 L of gel-loading buffer 20 L of cell lysate was loaded

on 8-16 SDS-PAGE After gel transfer membrane was probed with custom produced rat anti-

KPC-2 antibodies and subsequently treated with secondary goat anti-rat HRP-conjugated

antibodies Anti-RNA polymerase -subunit monoclonal antibodies (Abcam ab12087) were

used as loading control

454 Purification of KPC-2 WT and mutant proteins for biochemical studies

For protein expression the full KPC-2 gene coding sequence with its Shine-Dalgarno box

was cloned into the pET28a vector that produced a construct with periplasmic KPC-2 secretion

and a 6xHis-tag on its C-terminus The recombinant plasmids were transformed into E coli

BL21(DE3) pLysS cells 2 mL of overnight culture was inoculated in 1 L of LB media with 50

gmL of kanamycin and 20 gmL of chloramphenicol and grown at 37 oC with 300 RPM shaking

until reaching an OD600=07-08 IPTG was added to 02 mM concentration and cells continued to

grow for an additional 3 h Cells were harvested by centrifugation and the cell pellet was

resuspended in 40 mL of ice-cold 50 mM Tris-HCl pH 80 500 mM sucrose 1 mM EDTA and 1

93

tablet of complete protease inhibitor tablet (Roche) Suspension was incubated on ice with six

cycles of 15 sec vortexing and 5 min pause between them Suspension was centrifuged for 30 min

at 30000 x g supernatant was collected sonicated for 30 sec to reduce viscosity and MgCl2 and

imidazole were added to 2 mM and 5 mM concentrations respectively Lysate was loaded by

gravity flow onto a 1 mL column with HisPur Cobalt Resin (Thermo Scientific) pre-equilibrated

with 50 mM Na-phosphate pH 74 300 mM NaCl 5 mM imidazole buffer Column was washed

with 40 mL of the same buffer and consequently 6xHis-tag protein was eluted with 50 mM Na-

phosphate pH 74 300 mM NaCl 70 mM imidazole buffer All wash and elution fractions were

analyzed by 8-16 SDS-PAGE Fractions containing target protein were pooled concentrated

and dialyzed against 50 mM Na-phosphate pH 70 Purity of all proteins were at least 95 as

determined by SDS-PAGE Protein preparations were aliquoted and stored at -20 oC until further

use

455 Determination of Km and kcat values for nitrocefin cleavage by KPC-2 and

mutant proteins

Purified protein was mixed with various concentrations of nitrocefin (NCF) in 50 mM Na-

phosphate pH 70 01 mgmL bovine serum albumin (BSA) (reaction buffer) and substrate

cleavage was monitored at 490 nm every 10 sec for 10 min on SpectraMax plate reader at 37 oC

Initial rates of NCF cleavage were calculated and used to obtain Km and kcat values with Prism

software (GraphPad)

94

456 Determination of Km and kcat Values for meropenem cleavage by KPC-2 and

mutant proteins

Purified protein was mixed with various concentrations of meropenem in reaction buffer

and substrate cleavage was monitored at 294 nm every 30 sec for 30 min on SpectraMax plate

reader at 37 oC Initial rates of meropenem cleavage were calculated and used to obtain Km and

kcat values with Prism software (GraphPad)

457 Determination of vaborbactam Ki values for KPC-2 and mutant proteins using

NCF as substrate

Protein was mixed with various concentrations of inhibitors in reaction buffer and

incubated for 10 min at 37 oC 50 M NCF was added and substrate cleavage profiles were

recorded at 490 nm every 10 sec for 10 min Ki values were calculated by method of Waley SG

[54]


meropenem as substrate

Protein was mixed with various concentrations of inhibitor in reaction buffer and incubated

for 10 min at 37 oC 100 M meropenem solution was added and substrate cleavage profiles were

recorded at 294 nm every 30 sec for 30 min Ki values were calculated as described in [54]

459 Stoichiometry of KPC-2 and mutant proteins inhibition

Enzyme at 1 M in reaction buffer was mixed with BLI at molar ratios varying from 64 to

00625 After 30 min incubation at 37 oC reaction mixture was diluted 200-fold and enzyme

95

activity was measured with NCF as described above Stoichiometry of inhibition was determined

as a minimal BLIenzyme ratio reducing enzyme activity to at least 10

4510 Determination of vaborbactam k2K inactivation constant for WT KPC-2 and

mutant proteins

Inactivation kinetic parameters were determined by the reporter substrate method [55] for

slow tight binding inhibitor kinetic scheme

K k2

E + I harr EI harr EI

k-2

Protein was quickly mixed with 100 M nitrocefin and various concentrations of BLI in reaction

buffer and absorbance at 490 nm was measured immediately every two seconds for 180 sec on

SpectraMax plate reader (ldquoMolecular Devicesrdquo) at 37 oC Resulting progression curves of OD490

vs time at various BLI concentrations were imported into Prism software (ldquoGraphPadrdquo) and pseudo

first-order rate constants kobs were calculated using the following equation

P=V0(1-e-kobst)kobs where Vo - uninhibited KPC-2 rate Kobs values calculated at various

vaborbactam concentrations were fitted in the following equation

kobs=k-2+k2K[I](1+[NCF]Km(NCF)) where

k2K - inactivation constant

[I] ndash inhibitor concentration

[NCF] ndash nitrocefin concentration

Km(NCF) - Michaelis constant of NCF for KPC-2

96

4511 Determination of Koff rates of enzyme activity recovery after KPC-2 and

mutantsrsquo inhibition by vaborbactam

Purified enzyme at 1 M concentration in reaction buffer was mixed with BLIs at 8-fold

higher concentration than its stoichiometry ratio (determined in preliminary stoichiometry

experiments) After 30 min incubation at 37 oC reaction mixture was diluted 30000-fold in

reaction buffer and 100 L of diluted enzyme was mixed with 100 L of 400 M NCF in reaction

buffer Absorbance at 490 nm was recorded every minute during 4 h at 37 oC Resulting reaction

profiles were fitted into the following equation using Prism software (GraphPad) to obtain Koff

values P=Vst+(Vo-Vs)(1-e-kofft)koff where

Vs ndash uninhibited enzyme velocity measured in the reaction with enzyme and no inhibitor

Vo ndash completely inhibited enzyme velocity measured in the reaction with no enzyme and NCF

only

4512 Crystallization experiments

KPC-2 was expressed and purified as previously described [33] Vaborbactam was dissolved in

DMSO as a 200 mM stock solution and stored at -20 oC Prior to crystallization setup 5 mM

vaborbactam was gently mixed with 116 mgmL His-tag KPC-2 and incubated at room

temperature for 5 min Crystal trays for His-tag KPC-2 were set with 500 L wells and crystal

drops consisted of 05 L protein and 1 L of crystallization solution (2 M ammonium sulfate and

5 (vv) ethanol) Droplets (15 L) were microseeded with 05 L of diluted seed stock

Crystallization trays were stored at 20 oC and crystals typically appeared within 5 days Crystals

were cryo-protected in mother liquor supplemented with 20 (vv) glycerol and flash frozen with

liquid nitrogen

97

4513 Data collection and structure determination

Data for the KPC-2vaborbactam structure was collected at the Advanced Photon Source (APS)

beamline 19-ID Diffraction data was indexed and integrated with iMosflm [56] and scaled with

SCALA [57] from the CCP4 suite [58] Phasing was performed using molecular replacement with

the program Phaser [59] with the KPC-2 structure (PDB ID code 5UL8 [33]) Structure refinement

was performed using phenixrefine [60] in the Phenix suite [61] and model building in WinCoot

[62] The program eLBOW [63] in the Phenix suite was used to obtain geometry restraint

information The final model quality was assessed using MolProbity [64] Figures were prepared

using PyMOL 13 (Schroumldinger) [65]

46 Note to Reader 1

The binding kinetic experiments MIC assays and mutagenesis studies were contributed

by The Medicines Company which are the developers of vaborbactam and Vabomere

(vaborbactammeropenem)

47 Note to Reader 2

Supplementary figures (Fig S41 S42 S43 S44 and S45) and tables (Table S41 and

S42) are located in Appendix 2

98

Clavulanic acid Avibactam Tazobactam

Figure 41 Chemical structures of clinically available and promising new BLIs

S02030 Vaborbactam

99

Figure 42 Vaborbactam bound to KPC-2 active site (A) The unbiased Fo-F

c map (gray) of vaborbactam in the active site

of KPC-2 contoured at 3 KPC-2 residues and the vaborbactam molecule are green HB are depicted by dashed lines (B)

Superimposition of KPC-2vaborbactam (green) and KPC-2 apo (PDB ID code 5UL8 purple)

T237 T235

C69 C238

L167

P104

W105

S130 K73

N132

K234

E166

S70

A

T235 T237

S70

N132

L167

K73

W105

S130

K234

B

100

T235

T237 S237

C69 C238 G238

N132

P104 N104

V240 G240 G239

S70

S130

W105 Y105

Figure 43 Superimposition of KPC-2vaborbactam and CTX-M-15vaborbactam structures KPC-

2vaborbactam (green) with CTX-M-15vaborbactam (PDB ID code 4XUZ purple) Red arrow indicates a

backbone shift

101

A

C69

S70

T235

S237

G238

G240

C69

C238

G239

T237

T235

Figure 44 Vaborbactam induces a backbone conformational changes near the oxyanion hole (A) CTX-M-15 apo

(PDB ID code 4HBT purple) avibactam (PDB ID code 4HBU blue) and vaborbactam (PDB ID code 4XUZ green)

(B) KPC-2 apo (PDB ID code 5UL8 purple) avibactam (PDB ID code 4ZBE blue) and vaborbactam (green)

B

S70

102

BLI Stoichiometry of KPC-2 inhibition

Vaborbactam 1

Avibactam 1

Tazobactam gt64

Clavulanic acid gt64

Table 41 Stoichiometry of KPC-2 inhibition by various BLIs

103

-Lactamase BLI k2K (M-1 s-1) koff s-1 Residence time min Kd nM

KPC-2 Vaborbactam 73 X 103 0000017 992 23

KPC-2 S130G Vaborbactam 67 X 104 00044 38 66

KPC-2 Avibactam 13 x 104 000022 77 17

KPC-2 S130G Avibactam 90 ND ND ND

CTX-M-15 Vaborbactam 23 x 104 000088 19 38

Table 42 Kinetic parameters of inhibition of KPC-2 and CTX-M-15 by vaborbactam and avibactam

104

Aztreonam MIC (gmL) in the presence of varied concentrations of vaborbactam (gmL)

-Lactamase 0 015 03 06 125 25 5 10 MPC16

KPC-2 16 025 le0125 le0125 le0125 le0125 le0125 le0125 le015

CTX-M-15 8 8 4 2 1 05 025 le0125 25

Table 43 Concentration-response of aztreonam potentiation by vaborbactam against engineered E coli

strains expressing KPC-2 and CTX-M-15

105

Antibiotic MIC (gmL) in the presence of various concentrations of BLIs (gmL)

Plasmid BLI Antibiotic 0 1 2 4 8 16 32

KPC-2 Vaborbactam Carbenicillin 1024 64 16 8 4 2 1

KPC-2 S130G Vaborbactam Carbenicillin 128 4 2 1 le05 le05 le05

KPC Avibactam Carbenicillin 1024 64 64 32 16 8 2

KPC-2 S130G Avibactam Carbenicillin 128 128 128 64 64 64 32

KPC-2 Vaborbactam Piperacillin 128 1 05 025 le013 le013 le013

KPC-2 S130G Vaborbactam Piperacillin 128 2 05 025 025 le013 le013

KPC-2 Avibactam Piperacillin 128 2 1 05 025 le013 le013

KPC-2 S130G Avibactam Piperacillin 128 128 64 64 64 32 16

Table 44 Concentration-response of carbenicillin and piperacillin potentiation by vaborbactam and avibactam

against KPC-2 and KPC-2 S130G producing strain of P aeruginosa

106

48 References



2 Van Duin D Kaye K S Neuner E A amp Bonomo R A (2013) Carbapenem-resistant

Enterobacteriaceae A review of treatment and outcomes Diagnostic Microbiology and

Infectious Disease 75(2) 115-120 doi101016jdiagmicrobio201211009

3 Falagas M E Lourida P Poulikakos P Rafailidis P I amp Tansarli G S (2013)

Antibiotic treatment of infections due to carbapenem-resistant Enterobacteriaceae

Systematic evaluation of the available evidence Antimicrobial Agents and Chemotherapy

58(2) 654-663 doi101128aac01222-13

4 Savard P amp Perl T (2014) Combating the spread of carbapenemases in

Enterobacteriaceae A battle that infection prevention should not lose Clinical

Microbiology and Infection 20(9) 854-861 doi1011111469-069112748



6 Wang Z Fast W Valentine A M amp Benkovic S J (1999) Metallo--lactamase

Structure and mechanism Current Opinion in Chemical Biology 3(5) 614-622

doi101016S1367-5931(99)00017-4



8 Drawz S M Papp-Wallace K M amp Bonomo R A (2014) New β-lactamase Inhibitors

A Therapeutic Renaissance in an MDR World Antimicrobial Agents and Chemotherapy

58(4) 1835ndash1846 doi101128aac00826-13



10 Coleman K (2011) Diazabicyclooctanes (DBOs) A potent new class of non--lactam -

lactamase inhibitors Current Opinion in Microbiology 14(5) 550-555

doi101016jmib201107026

11 Ehmann D E Jahić H Ross P L Gu R-F Hu J Durand-Reacuteville T F Fisher S

L (2013) Kinetics of avibactam inhibition against class A C and D -lactamases The

Journal of Biological Chemistry 288(39) 27960ndash27971 doi101074jbcM113485979


Ceftazidime-avibactam Pharmacotherapy 35(8) 755ndash770 doi101002phar1622

13 Humphries R M Yang S Hemarajata P Ward K W Hindler J A Miller S A amp

Gregson A (2015) First report of ceftazidime-avibactam resistance in a KPC-3-

expressing Klebsiella pneumoniae isolate Antimicrobial Agents and Chemotherapy

59(10) 6605ndash6607 doi101128AAC01165-15

14 Shields R K Chen L Cheng S Chavda K D Press E G Snyder A Clancy C J

(2017) Emergence of ceftazidime-avibactam resistance due to plasmid-borne blaKPC-3

mutations during treatment of carbapenem-resistant Klebsiella pneumoniae infections

Antimicrobial Agents and Chemotherapy 61(3) e02097ndash16 doi101128AAC02097-16

107

15 Humphries R M amp Hemarajata P (2017) Resistance to ceftazidime-avibactam in

Klebsiella pneumoniae due to porin mutations and the increased expression of KPC-3

Antimicrobial Agents and Chemotherapy 61(6) e00537-17 doi101128aac00537-17

16 Crompton I E Cuthbert B K Lowe G amp Waley S G (1988) Beta-lactamase

inhibitors The inhibition of serine beta-lactamases by specific boronic acids Biochemical

Journal 251(2) 453ndash459

17 Nguyen N Q Krishnan N P Rojas L J Prati F Caselli E Romagnoli C van den

Akker F (2016) Crystal structures of KPC-2 and SHV-1 -lactamases in complex with

the boronic acid transition state analog S02030 Antimicrobial Agents and Chemotherapy

60(3) 1760ndash1766 doi101128AAC02643-15





19 Lapuebla A Abdallah M Olafisoye O Cortes C Urban C Quale J amp Landman

D (2015) Activity of meropenem combined with RPX7009 a novel -lactamase inhibitor

against Gram-negative clinical isolates in New York City Antimicrobial Agents and


20 Chang A Schiebel J Yu W Bommineni G R Pan P Baxter M V Tonge P J

(2013) Rational optimization of drug-target residence time Insights from inhibitor binding

to the S aureus FabI enzyme-product complex Biochemistry 52(24) 4217ndash4228

doi101021bi400413c

21 Cramer J Krimmer S G Fridh V Wulsdorf T Karlsson R Heine A amp Klebe G

(2017) Elucidating the origin of long residence time binding for inhibitors of the

metalloprotease thermolysin ACS Chemical Biology 12(1) 225-233

doi101021acschembio6b00979

22 Lu H England K Ende C am Truglio J J Luckner S Reddy B G Tonge P J

(2009) Slow-onset inhibition of the FabI enoyl reductase from Francisella tularensis

Residence time and in vivo activity ACS Chemical Biology 4(3) 221ndash231

doi101021cb800306y

23 Neckles C Eltschkner S Cummings J E Hirschbeck M Daryaee F Bommineni G

R Tonge P J (2017) Rationalizing the binding kinetics for the inhibition of the

Burkholderia pseudomallei FabI1 enoyl-ACP reductase Biochemistry 56(13) 1865-1878

doi101021acsbiochem6b01048

24 Krishnan N P Nguyen N Q Papp-Wallace K M Bonomo R A amp van den Akker


structural study PLoS ONE 10(9) e0136813 doi101371journalpone0136813

25 Beesley T Gascoyne N Knott-Hunziker V Petursson S Waley S G Jaurin B amp

Grundstroumlm T (1983) The inhibition of class C -lactamases by boronic acids

Biochemical Journal 209(1) 229ndash233

108

26 Powers J C Asgian J L Ekici Ouml D amp James K E (2002) Irreversible inhibitors of

serine cysteine and threonine proteases Chemical Reviews 102(12) 4639-4750

doi101021cr010182v

27 Kurz S G Hazra S Bethel C R Romagnoli C Caselli E Prati F Bonomo R A

(2015) Inhibiting the -lactamase of Mycobacterium tuberculosis (Mtb) with novel

boronic acid transition-state inhibitors (BATSIs) ACS Infectious Diseases 1(6) 234-242

doi101021acsinfecdis5b00003

28 Copeland R A Basavapathruni A Moyer M amp Scott M P (2011) Impact of enzyme

concentration and residence time on apparent activity recovery in jump dilution analysis

Analytical Biochemistry 416(2) 206-210 doi101016jab201105029



-lactamase Journal of Biological Chemistry 278(52) 52724-52729

doi101074jbcm306059200

30 Lomovskaya O Lee A Hoshino K Ishida H Mistry A Warren M S Lee V J

(1999) Use of a genetic approach to evaluate the consequences of inhibition of efflux

pumps in Pseudomonas aeruginosa Antimicrobial Agents and Chemotherapy 43(6)

1340-1346







33 Pemberton O A Zhang X amp Chen Y (2017) Molecular basis of substrate recognition

and product release by the Klebsiella pneumoniae carbapenemase (KPC-2) Journal of


34 Botos I amp Wlodawer A (2007) The expanding diversity of serine hydrolases Current

Opinion in Structural Biology 17(6) 683ndash690 doi101016jsbi200708003

35 Curley K amp Pratt R (2000) The oxyanion hole in serine -lactamase catalysis

Interactions of thiono substrates with the active site Bioorganic Chemistry 28(6) 338-

356 doi101006bioo20001184



2126(02)00725-6

37 Teotico D G Babaoglu K Rocklin G J Ferreira R S Giannetti A M amp Shoichet

B K (2009) Docking for fragment inhibitors of AmpC -lactamase Proceedings of the

National Academy of Sciences of the United States of America 106(18) 7455ndash7460

doi101073pnas0813029106

38 Vercheval L Bauvois C Di Paolo A Borel F Ferrer J Sauvage E Kerff F (2010)

Three factors that modulate the activity of class D -lactamases and interfere with the post-

109

translational carboxylation of Lys70 Biochemical Journal 432(3) 495-506

doi101042bj20101122




40 Langan P S Vandavasi V G Weiss K L Cooper J B Ginell S L amp Coates L

(2016) The structure of Toho1 -lactamase in complex with penicillin reveals the role of

Tyr105 in substrate recognition FEBS Open Bio 6(12) 1170-1177 doi1010022211-

546312132

41 Weston G S Blaacutezquez J Baquero F amp Shoichet B K (1998) Structure-based

enhancement of boronic acid-based inhibitors of AmpC -lactamase Journal of Medicinal

Chemistry 41(23) 4577-4586 doi101021jm980343w

42 Ke W Sampson J M Ori C Prati F Drawz S M Bethel C R van den Akker F

(2011) Novel insights into the mode of inhibition of class A SHV-1 -lactamases revealed

by boronic acid transition state inhibitors Antimicrobial Agents and Chemotherapy 55(1)

174ndash183 doi101128AAC00930-10

43 Rojas L J Taracila M A Papp-Wallace K M Bethel C R Caselli E Romagnoli

C Bonomo R A (2016) Boronic acid transition state inhibitors active against KPC and

other class A -Lactamases Structure-activity relationships as a guide to inhibitor design

Antimicrobial Agents and Chemotherapy 60(3) 1751ndash1759 doi101128AAC02641-15

44 Ke W Bethel C R Thomson J M Bonomo R A amp van den Akker F (2007) Crystal

structure of KPC-2 Insights into carbapenemase activity in class A -lactamases

Biochemistry 46(19) 5732ndash5740 doi101021bi700300u

45 Smith C A Nossoni Z Toth M Stewart N K Frase H amp Vakulenko S B (2016)

Role of the conserved disulfide bridge in class A carbapenemases Journal of Biological

Chemistry 291(42) 22196-22206 doi101074jbcm116749648


Docquier J-D (2013) Structural insight into potent broad-spectrum inhibition with



57(6) 2496ndash2505 doi101128AAC02247-12

47 Doucet N De Wals P amp Pelletier J N (2004) Site-saturation mutagenesis of Tyr-105

reveals its importance in substrate stabilization and discrimination in TEM-1 -lactamase

Journal of Biological Chemistry 279(44) 46295-46303 doi101074jbcm407606200



Protein Science 19(9) 1714ndash1727 doi101002pro454

49 Li M Conklin B C Taracila M A Hutton R A amp Skalweit M J (2012)

Substitutions at position 105 in SHV family -lactamases decrease catalytic efficiency and

cause inhibitor resistance Antimicrobial Agents and Chemotherapy 56(11) 5678-5686

doi101128aac00711-12

110

50 Copeland R A (2011) Conformational adaptation in drugndashtarget interactions and

residence time Future Medicinal Chemistry 3(12) 1491-1501 doi104155fmc11112


A (2014) Avibactam and class C -lactamases Mechanism of inhibition conservation of

the binding pocket and implications for resistance Antimicrobial Agents and

Chemotherapy 58(10) 5704ndash5713 doi101128AAC03057-14

52 Koeth L M (2000) Comparison of cation-adjusted Mueller-Hinton broth with Iso-

Sensitest broth for the NCCLS broth microdilution method Journal of Antimicrobial

Chemotherapy 46(3) 369-376 doi101093jac463369

53 Bonapace C R Bosso J A Friedrich L V amp White R L (2002) Comparison of

methods of interpretation of checkerboard synergy testing Diagnostic Microbiology and

Infectious Disease 44(4) 363-366 doi101016s0732-8893(02)00473-x

54 Waley S G (1985) Kinetics of suicide substrates Practical procedures for determining

parameters Biochemical Journal 227(3) 843ndash849

55 De Meester F Joris B Reckinger G Bellefroid-Bourguignon C Fregravere J amp Waley

S G (1987) Automated analysis of enzyme inactivation phenomena Application to -

lactamases and DD-peptidases Biochemical Pharmacology 36(14) 2393-2403

doi1010160006-2952(87)90609-5

56 Battye T G G Kontogiannis L Johnson O Powell H R amp Leslie A G W (2011)


Acta Crystallographica Section D Biological Crystallography 67(4) 271ndash281

doi101107S0907444910048675

57 Evans P (2006) Scaling and assessment of data quality Acta Crystallographica Section

D Biological Crystallography 62(1) 72-82 doi101107S0907444905036693

58 Winn M D Ballard C C Cowtan K D Dodson E J Emsley P Evans P R

Wilson K S (2011) Overview of the CCP4 suite and current developments Acta

Crystallographica Section D Biological Crystallography 67(4) 235ndash242

doi101107S0907444910045749



40(4) 658ndash674 doi101107S0021889807021206





61 Adams P D Afonine P V Bunkoacuteczi G Chen V B Davis I W Echols N Zwart

P H (2010) PHENIX A comprehensive Python-based system for macromolecular

structure solution Acta Crystallographica Section D Biological Crystallography 66(2)

213ndash221 doi101107S0907444909052925

62 Emsley P Lohkamp B Scott W G amp Cowtan K (2010) Features and development

of Coot Acta Crystallographica Section D Biological Crystallography 66(4) 486-501

doi101107S0907444910007493

111




1080 doi101107s0907444909029436




doi101093nargkm216


452

112

Chapter 5

Leveraging Protein Promiscuity in Ligand Binding to Develop Broad-Spectrum

Carbapenemase Inhibitors

51 Overview

Gram-negative bacterial pathogens expressing the serine -lactamase KPC-2 and the

MBLs NDM-1 and VIM-2 threaten the clinical utility of all -lactam antibiotics These enzymes

have a broad-substrate profile most likely due to the hydrophobicity and flexibility of their active

sites particularly for the metalloenzymes Here I demonstrate that this promiscuity in ligand

recognition may expose a potential weakness that can be exploited through rational drug design

Using a fragment-based approach I report the identification of a series of phosphonate compounds

that represent the first cross-class non-covalent inhibitors of KPC-2 NDM-1 and VIM-2

Although my lead optimization specifically targeted KPC-2 the increase in KPC-2 inhibition was

mirrored by improvement in activity against NDM-1 and particularly VIM-2 These findings

provide novel chemical scaffolds for antibiotic development against bacteria co-producing serine

carbapenemases and MBLs and suggest that protein promiscuity can be leveraged in developing

high affinity cross-class inhibitors

52 Introduction

-Lactam antibiotics are among the oldest and most widely used drugs used to treat a

diverse range of bacterial infections [1] The most common mechanism of -lactam antibiotic

113

resistance involves the production of -lactamase enzymes that hydrolyze them [2] There are four

classes of -lactamases (A B C and D) which differ from one another based on their amino acid

sequence and mechanism of action Class A C and D -lactamases are active-site serine enzymes

whereas class B -lactamases require one or two zinc ions for activity [3 4]

Carbapenems (eg imipenem meropenem doripenem and ertapenem) are the most potent

-lactam antibiotics and are often considered the ldquolast-resort antibioticsrdquo used to treat multi-drug

resistant infections [5-7] Unfortunately the recent emergence of CRE and multi-drug resistant P

aeruginosa threatens the use of all -lactam antibiotics CRE infections are associated with high

rates of morbidity and mortality worldwide due to limited treatment options [8] The CDC

estimates that there are over 9000 cases of CRE and 6700 cases of multi-drug resistant P

aeruginosa infections a year in the United States [9] Carbapenem resistance in CRE and P

aeruginosa is usually mediated by the production of carbapenem hydrolyzing enzymes known as

carbapenemases andor by the combination of porin loss and hyperproduction of AmpC or an

ESBL [10-12] Most carbapenemases are broadly promiscuous enzymes that can catalyze the

hydrolysis of multiple -lactam substrates including not only carbapenems but also penicillins

cephalosporins monobactams and -lactam-based BLIs [7 13] These enzymes belong to either

class A and D -lactamases (serine carbapenemases) or molecular class B (MBLs) [14] Of notable

health concern is the serine carbapenemase KPC-2 (class A) and the MBLs NDM-1 and VIM-2

(class B) all of which are commonly isolated from CRE P aeruginosa and other Gram-negative

pathogens [13]

To address the growing problem of -lactam antibiotic resistance numerous inhibitors

have been developed to target -lactamases However most of these inhibitors target serine -

114

lactamases and none in clinical use are active against both serine -lactamases and MBLs [13] In

general the BLIs clavulanic acid sulbactam and tazobactam display potent activity against many

class A -lactamases with only limited activity against class C and D -lactamases and no activity

against KPC-2 and class B -lactamases [13 15] Recently novel BLIs such as avibactam and

vaborbactam (Fig 51) have been demonstrated to inhibit the class A C and D -lactamases

including KPC-2 but possess no activity against MBLs [16-18] Additionally a naturally produced

polyamino acid called Aspergillomarasmine A (AMA) (Fig 51) derived from the mold

Aspergillus versicolor has been observed to potently inhibit MBLs by acting as a chelating agent

[19 20] Unfortunately there are some drawbacks to AMA which include its non-specific

inhibition as demonstrated by its ability to chelate essential metal ions required by human

metalloproteins and its inability to inhibit serine -lactamases [19 21] Therefore there is a

serious unmet medical need for the development of novel compounds that can simultaneously

target serine carbapenemases and MBLs commonly encountered in multi-drug resistant bacterial

infections

Developing a single cross-class inhibitor that can inactivate both serine carbapenemases

and MBLs has long been considered a challenging feat due to the structural and mechanistic

heterogeneity between these enzymes as well as the difficulties associated with antibacterial

development in general [13 22-24] In the last decade fragment-based approaches have become

an effective approach for inhibitor discovery against challenging targets yet key questions remain

as for its utility in developing cross-class carbapenemase inhibitors in both lead identification and

optimization [25-27] Firstly although fragments usually have low specificity there has been no

report of fragment compounds displaying activity against both serine carbapenemases and MBLs

with the majority of drug discovery efforts focusing on only one of the two groups of enzymes

115

Secondly FBDD has mainly been applied to a single target or multiple targets with high structural

similarity It is unknown whether such an approach can be successfully implemented against two

groups of enzymes with as much structural and mechanistic differences as serine carbapenemases

and MBLs This is particularly true for the lead-optimization process when inhibitors also display

increased specificity [27]

In this study I demonstrate that a fragment-based and structure-guided approach can be

successfully implemented in developing cross-class inhibitors against serine carbapenemases and

MBLs The newly identified inhibitors provide novel scaffolds for antibiotic development

targeting carbapenem resistance as well as valuable insights into how the unique broad-spectrum

activity of carbapenemases can be exploited in FBDD against multiple targets


531 Structure-based inhibitor discovery against the serine carbapenemase KPC-2

My investigation into a broad-spectrum serine carbapenemase and MBL inhibitor began

with molecular docking to KPC-2 This was due to the relatively abundant knowledge of serine -

lactamases and the need for novel inhibitor chemotypes against KPC-2 in spite of the latest drug

discovery efforts (eg avibactam relebactam vaborbactam) [27 28] Docking to the ZINC

database of commercially available compounds led to the identification of a phosphonate based

compound 1 (Table 51)

Using a biochemical assay with nitrocefin as a substrate we observed compound 1 to be a

moderate inhibitor of KPC-2 (Ki = 329 M) To improve compound activity and to probe the

contributions of various functional groups to binding five analogs were purchased and tested

116

against KPC-2 All the purchased compounds displayed micromolar potency against KPC-2

ranging from a Ki of 14 M to 1544 M (Table 51) Compounds 2 and 3 had similar Kirsquos of 134

M and 153 M respectively Compounds 4 and 5 are regioisomers with one methyl group

attached to either the C6 atom (compound 5) or the C7 atom (compound 4) Compound 5 had a Ki

of 1544 M against KPC-2 whereas compound 4 had a Ki of 395 M This suggests that KPC-

2 prefers the methyl substituent on the C7 atom as opposed to the C6 atom This trend becomes

more apparent when comparing compounds 1 and 6 which are also regioisomers having two

methyl groups one attached to C6 and C7 (compound 1) or attached to C5 and C7 (compound 6)

Compound 6 had the best activity with a Ki of 14 M As a result SAR studies were focused on

derivatives of compound 6 in subsequent chemical synthesis efforts

The synthesized compounds 7 through 10 like compound 6 had various substituents

attached to C5 and C7 to investigate how they would impact KPC-2 inhibition At both C5 and C7

positions increasing the size of the substitution (from ndashF -CH3 to ndashBr) appears to improve the

inhibition except for compound 7 with a methoxy group at C5 This replacement resulted in a

decrease in activity as compared to compound 6 (Ki = 57 M) The bromine substitution at C5

compound 9 resulted in a potent inhibitor of KPC-2 (Ki = 047 M)

I was interested in seeing if changing the coumarin scaffold to a similar quinolone and

quinoline scaffold would retain activity against KPC-2 due to the unfavorable pharmacokinetic

properties often associated with the coumarin ring Three quinolone derivates (compounds 11 12

13) and two quinoline derivatives (compounds 14 15) were synthesized and tested for inhibition

Out of all the quinolone derivatives compound 11 had the lowest level of inhibition (Ki = 122

M) possibly due to the lack of substituents on the C5 and C7 atoms A similar result was seen

for compound 12 (Ki = 792 M) which also had no substituents on the C5 and C7 atoms but

117

possessed a methyl group attached to the N1 atom Compound 13 was the most potent quinolone

derivative (Ki = 42 M) demonstrating similar activity to compound 6 due to both having methyl

groups located on C5 and C7 The two quinoline derivatives (compounds 14 15) had the lowest

level of activity against KPC-2 possessing Kirsquos of 4471 M and 4145 M respectively

Together these results suggest that the exact chemical composition of a coumarin ring itself is not

essential for compound activity and demonstrates the potential of these compounds for further

development

To better understand the structural basis of inhibition of the phosphonate based compounds

I determined complex crystal structures with compounds 1 2 3 6 and 9 (eg 1 6 9 Fig 52 and

2 3 Fig S51) at resolutions of 150 Aring and better In the binding site the conformation of the

inhibitors was unambiguously identified in the initial Fo-Fc electron density difference map

contoured at 2 (Fig 52) For all the KPC-2 complex structures the phosphonate moiety

establishes HBs with Ser70 Ser130 Thr235 and Thr237 In addition the phosphonate group

forms a water-mediated HB with the backbone carbonyl of Thr216 and the carbonyl oxygen of

the inhibitors form a water-mediated HB with Arg220 and His274 The coumarin ring system

forms a pi-pi stacking interaction with Trp105 When examining the biochemical activities of

compounds 1 6 and 9 it becomes apparent why compounds 6 and 9 have much better activity

against KPC-2 than compound 1 Moving the C6 methyl group (1) to the C5 position (6 and 9)

allows the coumarin ring to swing towards Trp105 a position that would otherwise cause steric

clashes between the C6 methyl group in 1 and Asn132 Concomitantly there is a conformational

change in Trp105 that allows it to flip upward and closer to the ring system forming stronger

hydrophobic interactions In addition the C5 substitution in compounds 6 and 9 also contributes

to non-polar interactions with Trp105

118


Expanding my inhibitor discovery efforts to two clinically relevant MBLs NDM-1 and

VIM-2 I tested a select list of the phosphonate based coumarin quinolone and quinoline

compounds that displayed activity against KPC-2 Compound 6 was the most potent of the

compounds against NDM-1 (Ki = 338 M) and VIM-2 (Ki = 082 M) As observed in KPC-2

NDM-1 and VIM-2 also prefer substituents on C5 and C7 for effective inhibition This is reflected

in compounds with C5 and C7 substituents (6 8 9 and 13) having greater potency than

compounds lacking C5 and C7 substituents (11 12 14 15) (Table 51) Interestingly a fluorine

atom located on either C5 (compound 10) or both C5 and C7 (compound 8) appears to negatively

influence NDM-1 inhibition This is demonstrated by no inhibition observed with compound 8 and

decreased inhibition of compound 10 as compared with compound 9 that has a bromine atom on

C5 instead of a fluorine

NDM-1 complex structures were determined with compounds 1 2 3 7 8 9 11 12 13

and 14 and VIM-2 complex structures with compounds 8 and 14 All structures were solved at

resolutions of 175 Aring and better (Fig 53 Fig S52) In the binding site the conformation of the


contoured at 2 (Fig 53 Fig S52) The phosphonate group of each inhibitor coordinates with

both zinc ions in NDM-1 and VIM-2 displacing a hydroxide ion that is essential to catalysis [29]

For NDM-1 the phosphonate based coumarin quinolone and quinoline compounds form common

interactions within the active site These interactions include the phosphonate moiety forming HBs

with Asn220 and with Asp124 which also forms a similar interaction with the hydroxide ion in

the apoenzyme and the ring systems of the compounds forming pi-sigma interactions with Met67

as well as non-polar interactions with Trp93 and Val73 Another commonly observed interaction

119

is a pi-pi T-shaped interaction with Phe70 that every compound forms with NDM-1 except

compound 13

Aside from the shared binding features one key structural variation among the binding

poses of these compounds is the two different orientations of the ring system one pointing the ring

carbonyl group away from Val73 (pose 1 eg compound 1 Fig 53A) and the other towards it

(pose 2 eg compound 13 Fig 53B) Pose 1 is favored by compounds with substitution at the

C6 position (eg 2 3 Fig S52) or at the ON1 position (eg 11 12 Fig S52) in order to

enhance interactions with Val73 (eg using the C6 methyl group of 1) or avoid unfavorable

interactions with Phe70 respectively The unfavorable interactions with Phe70 include both

potential steric clashes in the case of the N1 methyl group in 12 and the inability to HB with water

in the case of the N1 hydrogen in 11 Pose 2 is favored by compounds with C5 substitutions for

additional interactions with His122 (eg -F in 8 Fig 53C -Br in 9 Fig 53G) In the case of 13

which has both C5 and N1 substitutions pose 2 binding is observed in the active site to increase

interactions with His122 while Phe70 moves slightly away from the compound to avoid

unfavorable interactions with the hydrogen at the N1 position

Similar to NDM-1 compound 8 in VIM-2 adopts pose 2 and its phosphonate group forms

a HB with Asn210 and Asp118 (Fig 53D) The ring system of compound 8 forms a pi-pi stacking

interaction with Tyr67 and Trp87 and a pi-pi T-shaped interaction with His240 like the contacts

between related compounds and Phe70Trp93His250 in NDM-1 Unique to VIM-2 is the presence

of Arg205 near the active site which can establish a HB with the carbonyl oxygen of compound

8 which may result in tighter binding particularly in comparison to NDM-1 Arg205 is also one

of the reasons why two copies of compound 14 were observed in the active of VIM-2 (Fig 53F)

This is unlike any other inhibitors where only one copy binds to the active site of KPC-2 or NDM-

120

1 The first copy of compound 14 has the phosphonate group forming HBs with the Arg205 side

chain and Asn210 backbone The ring system forms a pi-pi T-shaped interaction with Tyr67 in

addition to a pi-pi stacking interaction with the second copy of compound 14 The second copy

binds in a way similar to 8 but is pushed away from Tyr67 by the first copy The binding affinity

of compound 8 (Ki = 33 M) is roughly 6-fold lower than compound 14 (Ki = 223 M) This

may partially be explained by the more extensive interactions between compound 8 and the

protein It is possible that both copies of 14 are relevant to enzyme inhibition as there appears to

be favorable interactions between the compounds as well which can potentially enhance binding

to the protein

533 Correlation of KPC-2 NDM-1 and VIM-2 inhibition

When comparing the activities of the synthesized phosphonate based inhibitors against

KPC-2 and VIM-2 a trend becomes apparent These synthesized compounds represent the core

phosphonate ring scaffold with and without decorations at both C5 and C7 positions As inhibitors

become more potent against KPC-2 they also demonstrate increasing potency against NDM-1 and

VIM-2 (Fig 54) The correlation reflects the similar binding features of both proteins and the

relatively simple configuration of the compound which reduces the likelihood of steric clashes

Nevertheless it is a striking trend considering the design and synthesis of these compounds were

originally intended for the SAR studies for KPC-2 based on the understanding of the commercially

available analogs

The crystal structures of KPC-2 illustrate how the activity of the compounds are enhanced

as additional functional groups are added to the core scaffold Compared with the smallest

compounds 14 and 15 the additional carbonyl group on 11 and 12 establishes new water-mediated

121

interactions with Arg220 and His274 the subsequent substitutions at C5 and C7 positions in 8

and particularly in 9 led to increased contacts with Leu167 Trp105 and neighboring residues

Similar observations were made in NDM-1 and VIM-2 complex structures For NDM-1 the larger

-Br of 9 makes new contacts with His122 while for VIM-2 the carbonyl group of 8 augments

inhibitor binding through a HB with Arg205


To investigate the effects of these inhibitors on bacterial cells compounds 9 11 and 13

were selected and tested (at 128 gmL) in combination with the carbapenem antibiotic imipenem

against Gram-negative clinical isolates known to produce carbapenemases (Table 52) Compound

9 restored susceptibility to imipenem in a K pneumoniae strain producing KPC-2 and reduced the

MIC by 64-fold It also decreased the MIC for imipenem by 4-fold in an NDM-1 producing E coli

strain and 2-fold in an NDM-1 producing K pneumoniae strain Compound 9 did not have any

activity against NDM-1 producing E cloacae Compared to KPC-2 the lower cell-based activity

of 9 against NDM-1 correlates with the different in vitro activities in biochemical testing The

variation among different bacterial strains underscores the influence of other biological factors on

the cell-based activity of these inhibitors This is further demonstrated by the lack of activity of 9

against a VIM-2 producing P aeruginosa strain even though our nitrocefin inhibition assays

demonstrated compound 9 to be a micromolar inhibitor of VIM-2 (Ki = 13 M) Compound 11

was less effective than compound 9 in reducing the MIC of imipenem against the KPC-2 producing

K pneumoniae It displayed similarly low activity or no effect against NDM-1 producing E coli

K pneumoniae and E cloacae Compound 13 was comparable to compound 9 at reducing the

MIC of imipenem by 64-fold in KPC-2 producing K pneumoniae and 4-fold in NDM-1 producing

122

E coli but was not able to reduce the MIC in NDM-1 producing K pneumoniaeE cloacae and

VIM-2 producing P aeruginosa Overall these results suggest that the phosphonate inhibitors were

able to cross the cell envelope and inhibit the carbapenemases produced by pathogenic Gram-

negative bacteria

54 Conclusions

Serine carbapenemases and MBLs produced by Gram-negative pathogens are a serious

threat to human health due to their multi-drug resistant nature The development of an inhibitor

that can target both serine carbapenemases and MBLs is imperative My biochemical X-ray

crystallographic and cell-based studies have demonstrated for the first time that rational design

of a cross-class non-covalent broad-spectrum inhibitor against serine carbapenemases and MBLs

is an achievable task More importantly my results provide valuable chemical and structural

information as well as important insights for future drug discovery efforts against these enzymes

My inhibitor discovery efforts focused on fragment-based approaches which are uniquely

suitable for identifying novel inhibitor chemotypes for difficult bacterial targets Nevertheless as

evidenced by the lack of published results in this area identifying suitable cross-class fragment

inhibitors for carbapenemases is challenging particularly for chemical scaffolds that enable lead

optimization for both groups of enzymes The phosphonate compounds provide a promising novel

fragment chemotype for this purpose and represent the first non-covalent inhibitors with

comparable binding affinities against both serine -lactamases and MBLs In both KPC-2 and

NDM-1VIM-2 the inhibitors reside in an area usually occupied by the -lactam core of the

substrates and is thus essential for enzyme activity (Fig 55) In KPC-2 the phosphonate moiety

is placed in a subpocket formed by Ser130 Thr235 and Thr237 which also happens to interact

123

with the C3rsquo4rsquo carboxylate group of the -lactam substrate [30] In NDM-1VIM-2 the

phosphonate group interacts with both zinc ions and neighboring residues important for binding to

the same substrate C3rsquo4rsquo carboxylate group as well as the -lactam carbonyl group which is

converted to a carboxylate group after the hydrolysis reaction catalyzed by the -lactamases [4

13] The cell-based activity of these inhibitors further supports these compoundsrsquo potential as lead

scaffolds for antibiotic development In addition the observation of an acetate molecule in the

NDM-1 active site informs future fragment-based lead-optimization efforts by highlighting an

underutilized binding hot spot that also contributes to the binding of the C3rsquo4rsquo carboxylate group

in previous NDM-1 complexes [31 32]

Most significantly my results have underscored the versatility of carbapenemases in ligand

binding and demonstrated that this feature can be a double-edged sword for carbapenemases

presenting a unique opportunity for drug discovery The relative open and hydrophobic features of

carbapenemase active sites particularly in the metalloenzymes have led to the hypothesis that

these binding surfaces may lead to increased druggability [30] Our crystal structures have

illustrated the important contributions of a number of hydrophobic residues to ligand binding such

as Trp105Leu167 in KPC-2 Val73Trp93Met67Phe70 in NDM-1 and Trp87Tyr67 in VIM-2

In addition the conformational variation of the protein residues and ligand binding poses observed

in our structures suggest that residue flexibility and the relative openness of the active site affords

the carbapenemases important adaptability in binding to small molecules Examples of protein

structural flexibility include Trp105 in KPC-2 as shown by its varied conformations in binding

to compounds 1 and 6 and particularly in comparison to the more rigid Tyr105 in other class A

serine -lactamases [33-36] Phe70 and the surrounding loop in NDM-1 as indicated by their

movement in binding to compound 13 and Arg205 in VIM-2 adopting different conformations

124

binding to compounds 8 and 14 and making important HBs in both cases The relative

opennessflat features of MBLs is partially demonstrated by how two drastically different ligand

conformations caused by flipping of the ring system (eg 1 vs 13 Fig 53A B) can both be

accommodated in NDM-1 active site allowing the ligand to maximize its contacts with the protein

while having minimal steric clashes

These unique structural features endow carbapenemases with a certain level of ligand

binding promiscuity The openness of the active site reduces the chances of steric clashes with

small molecules whereas increased hydrophobicity compensates the diminished shape

complementarity Albeit to a lesser extent these properties are reminiscent of other proteins

capable of binding to a wide range of ligands such as efflux pumps [37 38] For carbapenemases

such qualities may explain their ability to bind and hydrolyze nearly all -lactam antibiotics but

may also expose a weakness to novel inhibitor binding For drug discovery against these enzymes

this is particularly valuable for the lead optimization process The correlation between the inhibitor

activity particularly with VIM-2 and KPC-2 is very informative considering that the compounds

were designed and synthesized to target KPC-2 The hydrophobicity flexibility and relative

flatness of the VIM-2 active site enables it to accommodate and adapt to the ligand to maximize

the interactions as the size and complexity of the ligand increases This correlation is also

especially striking when compared to previous studies on CTX-M class A and AmpC class C -

lactamases Both are serine-based enzymes and have more active site similarities than those shared

by KPC-2 and VIM-2 [39] In the previous fragment-based inhibitor discovery efforts the starting

fragment inhibitors have low specificity with mM range affinities against both enzymes But as

the compound size rises and the binding affinity against CTX-M-9 improves to mid-low M the

125

specificity significantly increases with the binding affinity remaining at mM or undetectable for

AmpC

There have been previous efforts to develop cross-class inhibitors of serine -lactamases

and MBLs including the most recent success of a series of cyclic boronate compounds [22 40

41] However these inhibitors usually use two different mechanisms to inhibit serine -lactamases

and MBLs separately and provide relatively little information on rational design of novel cross

class inhibitors particularly concerning the challenges in the lead optimization process In the case

of cyclic boronates they rely on the reactivity of boron groups to form covalent bonds with the

catalytic serine of serine -lactamases and consequently achieve high binding affinity In

comparison the boronates behave as non-covalent inhibitors for MBLs As a result the lead

optimization process would only need to focus on increasing the binding affinity against the

metalloenzymes with some consideration of avoiding steric clashes in serine -lactamase active

sites

In conclusion my studies have uncovered novel cross-class inhibitors against two groups

of clinically important carbapenemases Together with the new insights into ligand binding by

these enzymes and FBDD these compounds provide a promising strategy to develop novel

antibiotics against -lactam resistance in bacteria


551 KPC-2 expression and purification

KPC-2 was expressed and purified as previously described [30]

126

552 NDM-1 expression and purification

The gene encoding NDM-1 (residues 29-270) was cloned into the pET-SUMO vector with

an N-terminal SUMO-tag The plasmid containing SUMO-NDM-1 was transformed into E coli

BL21 (DE3) cells For SUMO-tag NDM-1 bacteria were grown overnight at 30 oC with shaking

in 50 mL LB broth supplemented with 100 gmL ampicillin Two liters of LB broth supplemented

with 100 gmL ampicillin were each inoculated with 10 mL of overnight bacterial culture

Cultures were then grown at 37 oC until an OD600 of 06-07 Protein expression was then initiated

by the addition of IPTG (final concentration 05 mM) followed by growth for 16 h at 20 oC Cells

were pelleted by centrifugation and stored at -80 oC until further use

The SUMO-tag NDM-1 -lactamase was purified by nickel affinity chromatography and

gel filtration Briefly the cell pellets were thawed and re-suspended in 40 mL of buffer A (20 mM

HEPES pH 74 05 M NaCl 20 mM imidazole) with one complete protease inhibitor cocktail

tablet (Roche) and disrupted by sonication followed by ultracentrifugation to clarify the lysate

After ultracentrifugation the supernatant was passed through a 022 m filter before loading onto

a 5 mL HisTrap HP affinity column (GE Healthcare Life Sciences USA) pre-equilibrated with

buffer A SUMO-tag NDM-1 was eluted by a linear imidazole gradient (20 mM to 500 mM)

Fractions were analyzed by SDS-PAGE Fractions containing SUMO-tag NDM-1 were buffer

exchanged into 20 mM HEPES pH 70 100 mM NaCl Cleavage of the SUMO-tag was then

carried out with ULP-1 protease overnight at room temperature and then concentrated using a 10k

NMWL Amicon Ultra-15 Centrifugal Filter Unit The sample was then loaded back onto a nickel

affinity column and the flow through was collected containing the untagged NDM-1 NDM-1 was

concentrated and loaded onto a gel filtration column (GE Healthcare Life Sciences) pre-

equilibrated with 50 mM HEPES 150 mM NaCl and 50 M ZnSO4 Protein concentration was

127

determined by absorbance at 280 nm using an extinction coefficient of 27960 SDS-PAGE

analysis indicated that the eluted protein was more than 95 pure

553 VIM-2 expression and purification

The gene encoding VIM-2 (residues 27-266) was custom synthesized and inserted into a

vector with an N-terminal His-tag The plasmid containing His-tag VIM-2 was transformed into

Rosetta2 (DE3) E coli cells For His-tag VIM-2 bacteria were grown overnight at 30 oC with

shaking in 50 mL LB broth supplemented with 50 gmL kanamycin Two liters of terrific broth

supplemented with 50 gmL kanamycin were each inoculated with 10 mL of overnight bacterial

culture Cultures were then grown at 37 oC until an OD600 of 06-07 Protein expression was then

initiated by the addition of IPTG (final concentration 05 mM) followed by growth for 16 h at 20

oC Cells were pelleted by centrifugation and stored at -80 oC until further use

VIM-2 was purified by nickel affinity chromatography anion exchange and gel filtration

chromatography Briefly the cell pellets were thawed and re-suspended in 40 mL of buffer A (50

mM HEPES pH 75 150 mM NaCl 20 mM imidazole 50 M ZnSO4) with one complete protease

inhibitor cocktail tablet (Roche) and disrupted by sonication followed by ultracentrifugation to

clarify the lysate After ultracentrifugation the supernatant was passed through a 022 m filter

before loading onto a 5 mL HisTrap HP affinity column (GE Healthcare Life Sciences USA) pre-

equilibrated with buffer A VIM-2 was eluted by a linear imidazole gradient (20 mM to 500 mM)

Fractions were analyzed by SDS-PAGE Fractions containing VIM-2 were buffer exchanged into

50 mM Tris-HCl pH 80 150 mM NaCl 1 mM DTT Cleavage of the His-tag was then carried out

with TEV protease overnight at room temperature and then concentrated using a 10k NMWL

Amicon Ultra-15 Centrifugal Filter Unit The sample was then loaded back onto a nickel affinity

128

column and the flow through was collected containing the untagged VIM-2 VIM-2 was then

subjected to anion exchange chromatography using a linear gradient from buffer A (20 mM Tris-

HCl pH 75) to buffer B (20 mM Tris-HCl pH 75 1 M NaCl) VIM-2 was concentrated and loaded

onto a gel filtration column (GE Healthcare Life Sciences) pre-equilibrated with 50 mM HEPES

pH 70 150 mM NaCl 50 M ZnSO4 Protein concentration was determined by absorbance at 280

nm using an extinction coefficient of 25669 SDS-PAGE analysis indicated that the eluted protein

was more than 95 pure

554 Molecular docking

The program DOCK 354 [42] was used to screen the fragment subset of the ZINC

database of small molecules [43] using an in-house apo KPC-2 structure as a receptor template

The conserved catalytic water was left in the active site while all other waters were removed

Docking was performed as previously described [39]

555 Steady-state kinetic analysis

Steady-state kinetic parameters were determined by using a Biotek Cytation Multi-Mode

Reader For KPC-2 each assay was performed in 100 mM Tris-HCl pH 70 001 Triton X-100

at 37 oC Vmax and Km were determined from initial steady-state velocities from nitrocefin read at

a wavelength of 486 nm The kinetic parameters were obtained using the non-linear portion of the

data to the Henri-Michaelis (equation 1) using SigmaPlot 125

V= Vmax [S](Km + [S]) (1)

129

IC50 defined as the inhibitor concentration that results in a 50 reduction of nitrocefin (20 m)

hydrolysis was determined by measurements of initial velocities after mixing 1 nM of KPC-2 with

increasing concentrations of inhibitors The inhibition constant (Ki) was calculated according to

equation 2

Ki= IC50([S]Km + 1) (2)

For NDM-1 and VIM-2 the procedures were the same as above except the assay was performed

in 50 mM HEPES pH 72 50 M ZnSO4 001 Triton X-100 1 gmL bovine serum albumin

(BSA)


KPC-2 was crystallized as described previously [30] KPC-2 crystals were soaked in 144

M sodium citrate containing 10 mM inhibitor for approximately 2 h KPC-2 crystals were then

cryoprotected in a solution containing 115 M sodium citrate 20 (vv) glycerol and flash-frozen

in liquid nitrogen Crystals of NDM-1 were grown at 20 degC using hanging-drop vapor diffusion

Protein solutions (10-20 mgmL) in 50 mM HEPES 150 mM NaCl and 50 M ZnSO4 were mixed

11 (vv) with reservoir solution containing 50 mM potassium phosphate dibasic 10 mM CaCl2

and 25 (wv) PEG8000 Crystals typically formed in two days To obtain the ligand bound

structures NDM-1 crystals were soaked in a solution of 50 mM sodium acetate pH 385 25

(wv) PEG8000 and 10 mM inhibitor for 1 h The soaked crystals were cryo-protected in a solution

containing 50 mM sodium acetate pH 385 25 (wv) PEG8000 and 20 (vv) glycerol and

flash-frozen in liquid nitrogen Crystals of VIM-2 were grown at 20 degC using hanging-drop vapor

130

diffusion Protein solutions (10-20 mgmL) in 50 mM HEPES 150 mM NaCl and 50 M ZnSO4

were mixed 11 (vv) with reservoir solution containing 200 mM calcium acetate 20 (wv)

PEG3350 and 1 mM TCEP Crystals typically formed in two days To obtain the ligand bound

structures VIM-2 crystals were soaked in a solution of 50 mM sodium acetate pH 385 25 (wv)

PEG8000 and 10 mM inhibitor for 1 h The soaked crystals were cryo-protected in a solution


flash-frozen in liquid nitrogen


Data for the KPC-2 NDM-1 and VIM-2 complex structures were collected using

beamlines 22-ID-D and 23-ID-D at the Advanced Photon Source (APS) Argonne Illinois and

beamline 831 at the Advanced Light Source (ALS) Berkeley California Diffraction data were

indexed and integrated with iMosflm [44] and scaled with SCALA [45] from the CCP4 suite [46]

Phasing was performed using molecular replacement with the program Phaser [47] of the Phenix

suite [48] with the KPC-2 structure (PDB ID code 5UL8) NDM-1 (PDB ID code 4TYF) and

VIM-2 (PDB ID code 4BZ3) Structure refinement was performed using phenixrefine [49] of the

Phenix suite and model building in WinCoot [50] The unbiased Fo-Fc electron density maps were

generated prior to refinement with compound The program eLBOW [51] in Phenix was used to

obtain geometry restraint information The final model qualities were assessed using MolProbity

[52] Figures were generated in PyMOL 13 (Schroumldinger) in which structural alignments were

generated using pairwise scores [53]

131

56 Note to Reader 1

Supplementary figures (Fig S51 and S52) are located in Appendix 3

132

Figure 51 Clinical and pre-clinical serine carbapenemase and MBL inhibitors

Avibactam Vaborbactam Aspergillomarasmine A

133

R220 H274

T216

P104

L167 W105

K234

E166

N132

S130 K73

T235

S70

T237

C69 C238

(A)

R220 H274

C238 T237 T235

T216

K234

S130

W105

P104

L167 E166

N132

K73

S70

C69

(B)

C69

C238

H274 R220

T216

K234

S130

W105 N132 E166

P104

L167

K73

T235 T237

S70

(C)

Figure 52 X-ray crystal structures of KPC-2 bound to phosphonate inhibitors (A) compound 1 (B) compound 6 and (C)

compound 9 KPC-2 residues are colored gray compounds are colored in green and the unbiased Fo-Fc map is colored in gray and

contoured at 2

134

R205 Y67

W87

H116 H114

H179 D118

C198

H240 Zn2

Zn1

(D)

N210

C208

W93

L65

M67 F70

N220 H122

H189 H120

H250 D124

Zn2

ACT Zn1

(B)

V73

W93

V73

L65

M67 F70

C208

H250

D124

Zn2

H120 H189

N220 Zn1

H122 ACT

(C)

H250

V73

W93

L65 M67

F70

N220

H189 H120

C208

D124

ACT H122

Zn1

Zn2

(A)

H189

N220

F70 M67

L65

V73 W93

C208

H250 D124

H120

H122 Zn2

Zn1 ACT

(E)

W87

Y67

R205

H179

H114 H116

C198

H240

D118 Zn2 Zn1

(F)

N210

H189 H120

H122

D124

W93

L65

M67

F70

N220 C208

Zn2

Zn1

H250

V73

ACT

(G)

Figure 53 X-ray crystal structures of NDM-1 and VIM-2 bound to phosphonate inhibitors (A) NDM-1 with 1 (B) NDM-1 with

13 (C) NDM-1 with 8 (D) VIM-2 with 8 (E) NDM-1 with 14 (F) VIM-2 with 14 (G) NDM-1 with 9 Fo-Fc map is colored in gray

and contoured at 2 Acetate ion is labeled as ACT and colored in purple

135

(A)

(B)

Figure 54 Correlation of -lactamase inhibition (A) KPC-2 vs NDM-1 inhibition and (B) KPC-2 vs

VIM-2 inhibition with select phosphonate compounds

136

Figure 55 Superimposition of phosphonate inhibitor and -lactam product complexes

Superimposition of (A) KPC-2compound 9 and KPC-2cefotaxime product KPC-2compound 9

complex are colored in gray and green respectively KPC-2cefotaxime product are both colored in

blue Superimposition of (B) NDM-1compound 9 and NDM-1ampicillin product NDM-1compound

9 complex are colored in gray and green respectively NDM-1ampicillin product are both colored in

blue

(A) (B)

T235

T237

S130

H250

D124

H122

N220 H189

H120

C208

ACT

Zn2

Zn1

137

Compound Structure MW Ki (KPC-2) M Ki (NDM-1) M Ki (VIM-2) M

1

2682

329

443

21

2

2702 134

1237

21

3

2842

153

NA

NA

4

2542

395

NA

NA

5

2542

1544

NA

NA

6

2682

14

338

082

7

2842

57

NA

NA

8

2761

89

No inhibition

33

9

3331

047

654

13

10

2722

33

2302

33

11

2392

122

1902

64

12

2532

792

810

75

13

2672

42

562

34

14

2232

4471

7413

223

15

2232

4145

No inhibition

303

Table 51 Nitrocefin inhibition assay of phosphonate compounds tested against KPC-2 NDM-1 and VIM-2

138

Test Organism Phenotype Imip

enem

Imip

enem

Com

pou

nd

9

Imip

enem

Com

pou

nd

11

Imip

enem

Com

pou

nd

13

E coli NDM-1 8 2128 2 128 2128

K pneumoniae KPC-2 64 1128 4 128 1128

K pneumoniae NDM-1 gt64 32 128 32 128 64 128

E cloacae NDM-1 2 2128 2 128 2128

P aeruginosa VIM-2 gt64 gt64 128 gt64128 gt64 128

Table 52 In vitro activity of phosphonate compounds when combined with imipenem Compounds 9 11 and 13 were

tested at 128 gmL fixed concentration with imipenem against KPC-2 NDM-1 and VIM-2 producing bacteria

139

57 References



doi101016jcell201411017








5 Nordmann P Dortet L amp Poirel L (2012) Carbapenem resistance in

Enterobacteriaceae Here is the storm Trends in Molecular Medicine 18(5) 263-272

doi101016jmolmed201203003

6 Trecarichi E M amp Tumbarello M (2017) Therapeutic options for carbapenem-resistant

Enterobacteriaceae infections Virulence 8(4) 470-484

doi1010802150559420171292196



4943ndash4960 doi101128AAC00296-11


Enterobacteriaceae a review of treatment and outcomes Diagnostic Microbiology and


9 Morrill H J Pogue J M Kaye K S amp LaPlante K L (2015) Treatment options for

carbapenem-resistant Enterobacteriaceae infections Open Forum Infectious Diseases

2(2) ofv050-ofv050 doi101093ofidofv050

10 Lutgring J D amp Limbago B M (2016) The problem of carbapenemase-producing-

carbapenem-resistant-Enterobacteriaceae detection Journal of Clinical Microbiology

54(3) 529-534 doi101128jcm02771-15

11 Meletis G (2015) Carbapenem resistance Overview of the problem and future

perspectives Therapeutic Advances in Infectious Disease 3(1) 15-21

doi1011772049936115621709

12 Ho P L Cheung Y Y Wang Y Lo W U Lai E L Chow K H amp Cheng V C

(2016) Characterization of carbapenem-resistant Escherichia coli and Klebsiella

pneumoniae from a healthcare region in Hong Kong European Journal of Clinical

Microbiology amp Infectious Diseases 35(3) 379-385 doi101007s10096-015-2550-3





140

15 Watkins R R Papp-Wallace K M Drawz S M amp Bonomo R A (2013) Novel -

lactamase inhibitors A therapeutic hope against the scourge of multidrug resistance

Frontiers in Microbiology 4 doi103389fmicb201300392

16 Hecker S J Reddy K R Totrov M Hirst G C Lomovskaya O Griffith D C hellip




17 Abboud M I Damblon C Brem J Smargiasso N Mercuri P Gilbert B Fregravere J

(2016) Interaction of avibactam with class B metallo--lactamases Antimicrobial Agents


18 Castanheira M Rhomberg P R Flamm R K amp Jones R N (2016) Effect of the -

lactamase inhibitor vaborbactam combined with meropenem against serine

carbapenemase-producing Enterobacteriaceae Antimicrobial Agents and Chemotherapy

60(9) 5454-5458 doi101128aac00711-16

19 King A M Reid-Yu S A Wang W King D T De Pascale G Strynadka N C

Wright G D (2014) Aspergillomarasmine A overcomes metallo--lactamase antibiotic

resistance Nature 510(7506) 503-506 doi101038nature13445

20 Koteva K King A M Capretta A amp Wright G D (2015) Total synthesis and activity

of the metallo--lactamase inhibitor Aspergillomarasmine A Angewandte Chemie

128(6) 2250-2252 doi101002ange201510057

21 Drawz S M Papp-Wallace K M amp Bonomo R A (2013) New -lactamase inhibitors

A therapeutic renaissance in an MDR world Antimicrobial Agents and Chemotherapy

58(4) 1835-1846 doi101128aac00826-13

22 Brem J Cain R Cahill S McDonough M A Clifton I J Jimeacutenez-Castellanos J

Schofield C J (2016) Structural basis of metallo--lactamase serine--lactamase and

penicillin-binding protein inhibition by cyclic boronates Nature Communications 7

12406 doi101038ncomms12406

23 Silver L L (2011) Challenges of Antibacterial Discovery Clinical Microbiology

Reviews 24(1) 71-109 doi101128cmr00030-10

24 Brown E D amp Wright G D (2016) Antibacterial drug discovery in the resistance era

Nature 529(7586) 336-343 doi101038nature17042

25 Murray C W amp Rees D C (2009) The rise of fragment-based drug discovery Nature

Chemistry 1(3) 187-92 doi101038nchem217

26 Erlanson D A (2011) Introduction to fragment-based drug discovery Topics in Current

Chemistry 1-32 doi101007128_2011_180



doi101021bi3005126





141




31 Zhang H amp Hao Q (2011) Crystal structure of NDM-1 reveals a common -lactam

hydrolysis mechanism The FASEB Journal 25(8) 2574-2582 doi101096fj11-184036

32 King D T Worrall L J Gruninger R amp Strynadka N C (2012) New Delhi metallo-

-lactamase Structural insights into -lactam recognition and inhibition Journal of the

American Chemical Society 134(28) 11362-11365 doi101021ja303579d




34 Bethel C R Taracila M Shyr T Thomson J M Distler A M Hujer K M hellip

Bonomo R A (2011) Exploring the inhibition of CTX-M-9 by -lactamase inhibitors

and carbapenems Antimicrobial Agents and Chemotherapy 55(7) 3465-3475

doi101128aac00089-11

35 Tomanicek S J Standaert R F Weiss K L Ostermann A Schrader T E Ng J D

amp Coates L (2012) Neutron and x-ray crystal structures of a perdeuterated enzyme

inhibitor complex reveal the catalytic proton network of the Toho-1 -lactamase for the

acylation reaction Journal of Biological Chemistry 288(7) 4715-4722

doi101074jbcm112436238





57(6) 2496-2505 doi101128aac02247-12

37 Sun J Deng Z amp Yan A (2014) Bacterial multidrug efflux pumps Mechanisms

physiology and pharmacological exploitations Biochemical and Biophysical Research

Communications 453(2) 254-267 doi101016jbbrc201405090

38 Borges-Walmsley M I McKeegan K S amp Walmsley A R (2003) Structure and

function of efflux pumps that confer resistance to drugs Biochemical Journal 376(2) 313ndash

338 doi101042BJ20020957

39 Chen Y amp Shoichet B K (2009) Molecular docking and ligand specificity in fragment-

based inhibitor discovery Nature Chemical Biology 5(5) 358-364

doi101038nchembio155

40 Johnson J W Gretes M Goodfellow V J Marrone L Heynen M L Strynadka N

C amp Dmitrienko G I (2010) Cyclobutanone analogues of -lactams revisited Insights

into conformational requirements for inhibition of serine- and metallo--lactamases

Journal of the American Chemical Society 132(8) 2558-2560 doi101021ja9086374

41 Cahill S T Cain R Wang D Y Lohans C T Wareham D W Oswin H P Brem

J (2017) Cyclic boronates inhibit all classes of -lactamases Antimicrobial Agents and


142

42 Lorber D M amp Shoichet B K (2005) Hierarchical Docking of Databases of Multiple

Ligand Conformations Current Topics in Medicinal Chemistry 5(8) 739ndash749

43 Irwin J J amp Shoichet B K (2005) ZINC ndash A free database of commercially available

compounds for virtual screening Journal of Chemical Information and Modeling 45(1)

177ndash182 doi101021ci049714




doi101107s0907444910048675






doi101107S0907444910045749



40(4) 658ndash674 doi101107S0021889807021206




213-221 doi101107s0907444909052925






of Coot Acta Crystallographica Section D Biological Crystallography 66(4) 486ndash501

doi101107S0907444910007493



generation Acta Crystallographica Section D Biological Crystallography 65(Pt 10)

1074ndash1080 doi101107S0907444909029436




doi101093nargkm216


452

143

Chapter 6

Summary

Production of -lactamase enzymes by Gram-negative pathogens is a major cause of

bacterial resistance against the commonly used -lactam antibiotics -Lactam resistance mediated

by -lactamases can be overcome through the use of BLIs Clavulanic acid sulbactam and

tazobactam are examples of clinically used BLIs that demonstrate activity against class A and D

-lactamases Unfortunately because these inhibitors possess a -lactam ring they are susceptible

to degradation by -lactamases such as KPC-2 (class A serine enzyme) and NDM-1VIM-2 (class

B metalloenzymes) As a result a better understanding of the catalytic mechanism of -lactamases

is crucial in discovering novel inhibitors active against both serine and metalloenzymes

The first project addressed the cephalosporinase and carbapenemase activity of the serine

carbapenemase KPC-2 Three crystal structures apo hydrolyzed cefotaxime and hydrolyzed

faropenem provided experimental insights into the substrate recognition of KPC-2 and how

alternative conformations of Ser70 and Lys73 promote expulsion of the hydrolyzed product from

the active site The ability of KPC-2 to bind such a wide variety of -lactam substrates can be

exploited through rational drug design to engineer high affinity inhibitors

The second project focused on understanding the proton transfer process involved in the

mechanism of avibactam inhibition against serine -lactamases Ultrahigh resolution X-ray

crystallography of CTX-M-14 bound with avibactam allowed for elucidation of protonation states

144

of the key catalytic residues Lys73 Ser130 and Glu166 Avibactam impedes a critical proton

transfer between Glu166 and Lys73 keeping these residues as neutral species and thereby leaving

a stable EI complex resistant to hydrolysis

The third project investigated the influence of binding kinetics on vaborbactam inhibition

of KPC-2 The binding kinetic experiments revealed that vaborbactam has an extremely slow off-

rate with KPC-2 leading to a residence time of close to 1000 min which is much greater than the

bacterial generation time (~ 20 min) The long residence time of vaborbactam translated to

favorable in vitro and in vivo efficacy A high-resolution crystal structure of vaborbactam bound

to KPC-2 demonstrated the covalent bond with the catalytic serine as well as the extensive non-

covalent interactions that contribute to binding affinity

The final project focused on discovering inhibitors that could simultaneously target serine

-lactamases and MBLs Using molecular docking and FBDD a series of phosphonate-based

compounds were identified that displayed micromolar potency against KPC-2 NDM-1 and VIM-

2 Crystal structures of these inhibitors with KPC-2 NDM-1 and VIM-2 demonstrated their

interactions with conserved active site features The phosphonate-based inhibitors reduced the

MIC of imipenem against a K pneumoniae strain expressing KPC-2 and an E coli strain

expressing NDM-1 Ultimately these phosphonate-based compounds will serve as scaffolds for

the development of potent inhibitors targeting serine -lactamases and MBLs

145

Appendix 1

Copyright Permissions

Copyright permission for

Lewis K (2013) Platforms for antibiotic discovery Nature Reviews Drug Discovery 12(5)

371-387 doi101038nrd3975

146


Palzkill T (2012) Metallo--lactamase structure and function Annals of the New York


147


Wilke M S Lovering A L amp Strynadka N C (2005) -Lactam antibiotic resistance A

current structural perspective Current Opinion in Microbiology 8(5) 525-533

doi101016jmib200508016

148


Vandavasi V G Weiss K L Cooper J B Erskine P T Tomanicek S J Ostermann A

Coates L (2016) Exploring the mechanism of -lactam ring protonation in the class A -

lactamase acylation mechanism using neutron and X-ray crystallography Journal of Medicinal

Chemistry 59(1) 474-479 doi101021acsjmedchem5b01215

149


Papp-Wallace K M Endimiani A Taracila M A amp Bonomo R A (2011) Carbapenems

Past present and future Antimicrobial Agents and Chemotherapy 55(11) 4943-4960

doi101128aac00296-11

150


Shi Y (2014) A glimpse of structural biology through X-ray crystallography Cell 159(5) 995-

1014 doi101016jcell201410051

151


Nichols D A Jaishankar P Larson W Smith E Liu G Beyrouthy R Chen Y (2012)

Structure-based design of potent and ligand-efficient inhibitors of CTX-M class A -lactamase

Journal of Medicinal Chemistry 55(5) 2163-2172 doi101021jm2014138

152


Pemberton O A Zhang X amp Chen Y (2017) Molecular basis of substrate recognition and

product release by the Klebsiella pneumoniae carbapenemase (KPC-2) Journal of Medicinal


153


Lahiri S D Mangani S Durand-Reville T Benvenuti M De Luca F Sanyal G amp

Docquier J (2013) Structural insight into potent broad-spectrum inhibition with reversible

recyclization mechanism Avibactam in complex with CTX-M-15 and Pseudomonas aeruginosa

AmpC -lactamases Antimicrobial Agents and Chemotherapy 57(6) 2496-2505

doi101128aac02247-12

154

Appendix 2

Chapter 4 Supplementary Data

Figure S41 Lineweaver-Burk plot of KPC-2 inhibition by vaborbactam

155

Figure S42 KPC-2 inactivation profile using boronic acid BLIs

156

Figure S43 Time-dependence of vaborbactam inhibition of KPC-2

157

Figure S44 Vaborbactam and tazobactam kinetic behavior with KPC-2

158

Figure S45 Jump dilution can reverse vaborbactam inhibition of KPC-2

159

Table S41 MIC of various -lactams against P aeruginosa expressing KPC-2 and KPC-2 S130G

Plasmid Ceftazidime Cefepime Carbenicillin Piperacillin Meropenem Aztreonam

vector 2 0125 05 025 0125 1

KPC-2 32 128 gt512 128 128 16

KPC-2 S130G 1 0125 128 64 025 025

160

Table S42 X-ray data collection and refinement statistics

Structure KPC-2 with vaborbactam

Data Collection

Space group P22121

Cell dimensions

a b c (Aring) 5577 5997 7772

(deg) 90 90 90

Resolution (Aring) 4748 -125 (1295-125)

No of unique reflections 72556 (7159)

Rmerge () 90 (912)

Iσ(I) 89 (18)

Completeness () 997 (997)

Multiplicity 58 (58)

Refinement

Resolution (Aring) 4748 -125

Rwork () 147 (269)

Rfree () 170 (286)

No atoms

Protein 2160

LigandIon 66

Water 305

B-factors (Aring2)

protein 1498

ligandion 2865

water 3529

RMS deviations

Bond lengths (Aring) 0007

Bond angles (deg) 107

Ramachandran plot

favored () 9888

allowed () 112

outliers () 0

PDB code 5WLA

161

Appendix 3


W105

H274 R220

T216

T235 T237 C238

C69

E166

L167

P104

N132

S130

K234 K73

S70

(A)

P104

N132 L167 W105

S130

T216

T235

R220 H274

T237

C69

C238

K234

K73

S70

E166

(B)

Figure S51 KPC-2 bound to (A) compound 2 and (B) compound 3 Compounds are colored in green and the

unbiased Fo-Fc map is colored in gray and contoured at 2

162

H120

H189

H122

F70 M67

V73

W93

L65

H250

C208

D124

N220

ACT

Zn2

Zn1

(A)

N220

F70 M67

L65

V73 W93

H250

C208

H120

H189

H122

D124

Zn2

ACT Zn1

(B)

L65

M67

F70 V73

W93

H250

C208

D124

N220

H122

H189 H120

ACT

Zn2

Zn1

(C)

H250

C208

H120 H189 H122

N220

D124

W93

V73

L65

M67 F70

Zn2

Zn1

ACT

(D)

Figure S52 NDM-1 bound to (A) compound 2 (B) compound 3 (C) compound 7 (D) compound 11 and (E)

compound 12 Compounds are colored in green and the unbiased Fo-Fc map is colored in gray and contoured at 2

163

L65

F70 M67

V73 W93

D124

H250

C208

H120

H189

H122 N220 ACT

Zn2 Zn1

(E)

Figure S52 Continued


Scholar Commons

November 2017

Mechanism Elucidation and Inhibitor Discovery against Serine and Metallo-Beta-Lactamases Involved in Bacterial Antibiotic Resistance

Orville A Pemberton

Scholar Commons Citation

tmp1546629497pdfD2PfG

Mechanism Elucidation and Inhibitor Discovery against Serine and Metallo--Lactamases

Involved in Bacterial Antibiotic Resistance

by

Orville A Pemberton

A dissertation submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

with a concentration in Allergy Immunology and Infectious Disease

Department of Molecular Medicine

Morsani College of Medicine


Major Professor Yu Chen PhD Burt Anderson PhD

Gloria Ferreira PhD

James Leahy PhD

Date of Approval October 11 2017

Keywords Carbapenemase -Lactam Antibiotics X-ray Crystallography Molecular Docking

Drug Design

Copyright copy 2017 Orville A Pemberton

Acknowledgments










Idrovo





i

Table of Contents

List of Tablesiv

List of Figuresv


Abstract ix





14 Carbapenemases9






110 Conclusions 15


112 References31



21 Overview39

22 Introduction39





24 Conclusions48






27 References60

ii


Acylation63

31 Overview63

32 Introduction64




and Glu16667

34 Conclusions68





37 References76



41 Overview78

42 Introduction79







44 Conclusions86





















iii






48 References106



51 Overview112

52 Introduction112







54 Conclusions122










57 References139





iv









CTX-M-15104







v

List of Figures













in the PDB29








vi
























vii











CTXCefotaximase



DTTDithiothreitol


EIEnzyme-inhibitor




HBHydrogen bond

viii




LBLysogeny broth






NCFNitrocefin

NDMNew Delhi MBL


OXAOxacillinase










WTWild-type

ix

Abstract



















bacteria

x























xi


in vivo activity





















1

Chapter 1

Introduction



















2
























3


burden




















4
























5






















6























7























8
























9







14 Carbapenemases
















10























11


[70]





















12






















13























14






















15









110 Conclusions













16


















Appendix 1)




17




















18




19





20


21




22



-Lactamases


C A D B

B1 B2 B3

23









24


25


26


27





Tazobactam

28


29


30


31

112 References






Boston MA Springer




from


nhtml





Updates 3(3) 155-160 doi101054drup20000146







252



doi101086533592


12(5) 371-387 doi101038nrd3975





doi103389fpubh201400145






doi101186s12879-016-1990-4

32


317(7159) 657-660 doi101136bmj3177159657


Nature 146(3713) 837-837 doi101038146837a0







doi101056nejm198802183180706








doi101016jcell201411017



doi101016jmib200508016





doi1010029780471729259mca03cs00







1145-1165 doi1021740929867023370031



doi101002chin200524267





33



doi101021ar0400241



Reviews 29(1) 29-57 doi101128cmr00019-15



















61(1) e01636-16 doi101128aac01636-16




doi101107s1399004713033014






doi101007s00894-011-1087-3






7(10) 918-925 doi10103879688

34













27(2) 241-263 doi101128cmr00117-13







6032 doi101039c7ob01514c



doi101128aac01512-08





doi1021741389450116666151001105622








doi101016jjmb200410070





35



4943-4960 doi101128aac00296-11












doi103923jm2006122
















897 doi101128aac00693-09



doi103201eid1710110655



doi101093jacdks121






36





doi101128aac115852













Therapy 35(8) 755-770 doi101002phar1622














251(2) 453-459 doi101042bj2510453







doi10151717460440903190961


Letters 3(6) 442-444 doi101021ml300114c

37



13(12) 12857-12879 doi103390ijms131012857





doi101038nprot2013130



doi101021bi3005126





146-157 doi102174157340911795677602












doi101007s10822-006-9060-4




1749 doi101021jm0306430





159(5) 995-1014 doi101016jcell201410051



doi1010160167-5699(93)90049-q



38




39

Chapter 2



21 Overview








22 Introduction








40





















41
























42
























43













process











44























45























46

















chains






47























48

24 Conclusions




















49






















50














26 Note to Reader 1





51




52



53
















54





T235 T237 C238

S130

K73

K270

1 2

55
















56






57



A B

58




Cefotaxime


Faropenem

Data Collection


Cell dimensions

a b c (Aring) 5580 6020 7862 5693 5878 7684 5714 5913 7738

(deg) 90 90 90 90 90 90 90 90 90



Rmerge () 3073 (362) 127 (684) 95 (888)

Iσ(I) 110 (20) 91 (23) 99 (22)

Completeness () 991 (944) 985 (956) 999 (100)

Multiplicity 65 (33) 65 (53) 70 (66)

Refinement


Rwork () 122 (261) 168 (319) 150 (257)

Rfree () 139 (269) 212 (345) 188 (275)

No atoms

Protein 2136 2090 2109

LigandIon 26 60 26

Water 350 255 223

B-factors (Aring2)

protein 1408 1558 2242

ligandion 2832 2202 3711

water 3196 3091 3678

RMS deviations



Ramachandran plot

favored () 9888 9888 9887

allowed () 112 112 113

outliers () 0 0 0


59


Distance (Aring)









Distance (Aring)












60

27 References



doi103201eid1710110655


















897 doi101128aac00693-09














3421-3427 doi101128aac4983421-34272005






61







15397-15407 doi101021ja051592u








doi101021bi0487903




278(52) 52724-52729 doi101074jbcm306059200








5665 doi101128aac00245-11



2126(02)00725-6







4943ndash4960 doi101128AAC00296-11



51(3) 721-725 doi101093jacdkg120



62
















doi101107s0907444910048675



50(5) 760-763 doi101107s0907444994003112



40(4) 658ndash674 doi101107S0021889807021206







doi101107s0907444904019158




1080 doi101107s0907444909029436




doi101093nargkm216


452




63

Chapter 3


Lactamase Acylation

31 Overview
















64

32 Introduction






















65























66
























67

















acidbase [14]






68

















34 Conclusions






69



















70












36 Note to Reader 1







71

4

3

2

1

6 7

8

5


72



73

K234

S130 K73

Y105

T235

S237

N170

E166

N132

S70



74




75

S70 S130

K73

E166

N170

Wat1





76

37 References



1145-1165 doi1021740929867023370031






doi1010801475636620161201813



doi101002chin200524267








57(5) 2372-2375 doi101128aac02622-12

















57(6) 2496-2505 doi101128aac02247-12




77









14(17) 4116-4128 doi101039c6ob00353b








5665 doi101128aac00245-11












x







doi101107s0907444904019158


452



78

Chapter 4



41 Overview















79

42 Introduction








for activity [6]















80






















81























82























83


inhibitor of KPC-2






E coli














84





















85
















KPC-2 [33]








86














group

44 Conclusions







87
























88
























89
























90























91





















92






















93












use


mutant proteins





software (GraphPad)

94


mutant proteins






NCF as substrate




[54]









95




mutant proteins



K k2


k-2













96












only










liquid nitrogen

97










46 Note to Reader 1




47 Note to Reader 2



98



S02030 Vaborbactam

99





T237 T235

C69 C238

L167

P104

W105

S130 K73

N132

K234

E166

S70

A

T235 T237

S70

N132

L167

K73

W105

S130

K234

B

100

T235

T237 S237

C69 C238 G238

N132

P104 N104

V240 G240 G239

S70

S130

W105 Y105



backbone shift

101

A

C69

S70

T235

S237

G238

G240

C69

C238

G239

T237

T235




B

S70

102


Vaborbactam 1

Avibactam 1

Tazobactam gt64



103




KPC-2 Avibactam 13 x 104 000022 77 17




104


-Lactamase 0 015 03 06 125 25 5 10 MPC16


CTX-M-15 8 8 4 2 1 05 025 le0125 25



105













106

48 References









58(2) 654-663 doi101128aac01222-13








doi101016S1367-5931(99)00017-4





58(4) 1835ndash1846 doi101128aac00826-13





doi101016jmib201107026









59(10) 6605ndash6607 doi101128AAC01165-15





107










60(3) 1760ndash1766 doi101128AAC02643-15












doi101021bi400413c








doi101021cb800306y











108



doi101021cr010182v











doi101074jbcm306059200




1340-1346














356 doi101006bioo20001184



2126(02)00725-6




doi101073pnas0813029106



109


doi101042bj20101122







546312132







174ndash183 doi101128AAC00930-10















57(6) 2496ndash2505 doi101128AAC02247-12










doi101128aac00711-12

110


















doi1010160006-2952(87)90609-5




doi101107S0907444910048675






doi101107S0907444910045749



40(4) 658ndash674 doi101107S0021889807021206








213ndash221 doi101107S0907444909052925



doi101107S0907444910007493

111




1080 doi101107s0907444909029436




doi101093nargkm216


452

112

Chapter 5



51 Overview













52 Introduction



113




















pathogens [13]



114














infections










115






















116
























117







development

















118
























119


compound 13






















120









to the protein










available analogs




121























122




negative bacteria

54 Conclusions


















123
























124























125


AmpC











sites








126
























127























128



















V= Vmax [S](Km + [S]) (1)

129




equation 2

Ki= IC50([S]Km + 1) (2)



(BSA)













130





















131

56 Note to Reader 1


132



133

R220 H274

T216

P104

L167 W105

K234

E166

N132

S130 K73

T235

S70

T237

C69 C238

(A)

R220 H274

C238 T237 T235

T216

K234

S130

W105

P104

L167 E166

N132

K73

S70

C69

(B)

C69

C238

H274 R220

T216

K234

S130

W105 N132 E166

P104

L167

K73

T235 T237

S70

(C)



contoured at 2

134

R205 Y67

W87

H116 H114

H179 D118

C198

H240 Zn2

Zn1

(D)

N210

C208

W93

L65

M67 F70

N220 H122

H189 H120

H250 D124

Zn2

ACT Zn1

(B)

V73

W93

V73

L65

M67 F70

C208

H250

D124

Zn2

H120 H189

N220 Zn1

H122 ACT

(C)

H250

V73

W93

L65 M67

F70

N220

H189 H120

C208

D124

ACT H122

Zn1

Zn2

(A)

H189

N220

F70 M67

L65

V73 W93

C208

H250 D124

H120

H122 Zn2

Zn1 ACT

(E)

W87

Y67

R205

H179

H114 H116

C198

H240

D118 Zn2 Zn1

(F)

N210

H189 H120

H122

D124

W93

L65

M67

F70

N220 C208

Zn2

Zn1

H250

V73

ACT

(G)




135

(A)

(B)



136






blue

(A) (B)

T235

T237

S130

H250

D124

H122

N220 H189

H120

C208

ACT

Zn2

Zn1

137


1

2682

329

443

21

2

2702 134

1237

21

3

2842

153

NA

NA

4

2542

395

NA

NA

5

2542

1544

NA

NA

6

2682

14

338

082

7

2842

57

NA

NA

8

2761

89

No inhibition

33

9

3331

047

654

13

10

2722

33

2302

33

11

2392

122

1902

64

12

2532

792

810

75

13

2672

42

562

34

14

2232

4471

7413

223

15

2232

4145

No inhibition

303


138


enem

Imip

enem

Com

pou

nd

9

Imip

enem

Com

pou

nd

11

Imip

enem

Com

pou

nd

13

E coli NDM-1 8 2128 2 128 2128

K pneumoniae KPC-2 64 1128 4 128 1128


E cloacae NDM-1 2 2128 2 128 2128




139

57 References



doi101016jcell201411017













doi1010802150559420171292196



4943ndash4960 doi101128AAC00296-11









54(3) 529-534 doi101128jcm02771-15



doi1011772049936115621709









140














60(9) 5454-5458 doi101128aac00711-16






128(6) 2250-2252 doi101002ange201510057



58(4) 1835-1846 doi101128aac00826-13




12406 doi101038ncomms12406


Reviews 24(1) 71-109 doi101128cmr00030-10






Chemistry 1-32 doi101007128_2011_180



doi101021bi3005126





141















doi101128aac00089-11





doi101074jbcm112436238





57(6) 2496-2505 doi101128aac02247-12






338 doi101042BJ20020957











142





177ndash182 doi101021ci049714




doi101107s0907444910048675






doi101107S0907444910045749



40(4) 658ndash674 doi101107S0021889807021206




213-221 doi101107s0907444909052925







doi101107S0907444910007493




1074ndash1080 doi101107S0907444909029436




doi101093nargkm216


452

143

Chapter 6

Summary


















144



















145

Appendix 1




371-387 doi101038nrd3975

146




147




doi101016jmib200508016

148






149




doi101128aac00296-11

150



1014 doi101016jcell201410051

151





152





153






doi101128aac02247-12

154

Appendix 2



155


156


157


158


159



vector 2 0125 05 025 0125 1

KPC-2 32 128 gt512 128 128 16

KPC-2 S130G 1 0125 128 64 025 025

160



Data Collection

Space group P22121

Cell dimensions

a b c (Aring) 5577 5997 7772

(deg) 90 90 90



Rmerge () 90 (912)

Iσ(I) 89 (18)



Refinement


Rwork () 147 (269)

Rfree () 170 (286)

No atoms

Protein 2160

LigandIon 66

Water 305

B-factors (Aring2)

protein 1498

ligandion 2865

water 3529

RMS deviations



Ramachandran plot

favored () 9888

allowed () 112

outliers () 0

PDB code 5WLA

161

Appendix 3


W105

H274 R220

T216

T235 T237 C238

C69

E166

L167

P104

N132

S130

K234 K73

S70

(A)

P104

N132 L167 W105

S130

T216

T235

R220 H274

T237

C69

C238

K234

K73

S70

E166

(B)



162

H120

H189

H122

F70 M67

V73

W93

L65

H250

C208

D124

N220

ACT

Zn2

Zn1

(A)

N220

F70 M67

L65

V73 W93

H250

C208

H120

H189

H122

D124

Zn2

ACT Zn1

(B)

L65

M67

F70 V73

W93

H250

C208

D124

N220

H122

H189 H120

ACT

Zn2

Zn1

(C)

H250

C208

H120 H189 H122

N220

D124

W93

V73

L65

M67 F70

Zn2

Zn1

ACT

(D)



163

L65

F70 M67

V73 W93

D124

H250

C208

H120

H189

H122 N220 ACT

Zn2 Zn1

(E)



Scholar Commons

November 2017


Orville A Pemberton



Acknowledgments










Idrovo





i

Table of Contents

List of Tablesiv

List of Figuresv


Abstract ix





14 Carbapenemases9






110 Conclusions 15


112 References31



21 Overview39

22 Introduction39





24 Conclusions48






27 References60

ii


Acylation63

31 Overview63

32 Introduction64




and Glu16667

34 Conclusions68





37 References76



41 Overview78

42 Introduction79







44 Conclusions86





















iii






48 References106



51 Overview112

52 Introduction112







54 Conclusions122










57 References139





iv









CTX-M-15104







v

List of Figures













in the PDB29








vi
























vii











CTXCefotaximase



DTTDithiothreitol


EIEnzyme-inhibitor




HBHydrogen bond

viii




LBLysogeny broth






NCFNitrocefin

NDMNew Delhi MBL


OXAOxacillinase










WTWild-type

ix

Abstract



















bacteria

x























xi


in vivo activity





















1

Chapter 1

Introduction



















2
























3


burden




















4
























5






















6























7























8
























9







14 Carbapenemases
















10























11


[70]





















12






















13























14






















15









110 Conclusions













16


















Appendix 1)




17




















18




19





20


21




22



-Lactamases


C A D B

B1 B2 B3

23









24


25


26


27





Tazobactam

28


29


30


31

112 References






Boston MA Springer




from


nhtml





Updates 3(3) 155-160 doi101054drup20000146







252



doi101086533592


12(5) 371-387 doi101038nrd3975





doi103389fpubh201400145






doi101186s12879-016-1990-4

32


317(7159) 657-660 doi101136bmj3177159657


Nature 146(3713) 837-837 doi101038146837a0







doi101056nejm198802183180706








doi101016jcell201411017



doi101016jmib200508016





doi1010029780471729259mca03cs00







1145-1165 doi1021740929867023370031



doi101002chin200524267





33



doi101021ar0400241



Reviews 29(1) 29-57 doi101128cmr00019-15



















61(1) e01636-16 doi101128aac01636-16




doi101107s1399004713033014






doi101007s00894-011-1087-3






7(10) 918-925 doi10103879688

34













27(2) 241-263 doi101128cmr00117-13







6032 doi101039c7ob01514c



doi101128aac01512-08





doi1021741389450116666151001105622








doi101016jjmb200410070





35



4943-4960 doi101128aac00296-11












doi103923jm2006122
















897 doi101128aac00693-09



doi103201eid1710110655



doi101093jacdks121






36





doi101128aac115852













Therapy 35(8) 755-770 doi101002phar1622














251(2) 453-459 doi101042bj2510453







doi10151717460440903190961


Letters 3(6) 442-444 doi101021ml300114c

37



13(12) 12857-12879 doi103390ijms131012857





doi101038nprot2013130



doi101021bi3005126





146-157 doi102174157340911795677602












doi101007s10822-006-9060-4




1749 doi101021jm0306430





159(5) 995-1014 doi101016jcell201410051



doi1010160167-5699(93)90049-q



38




39

Chapter 2



21 Overview








22 Introduction








40





















41
























42
























43













process











44























45























46

















chains






47























48

24 Conclusions




















49






















50














26 Note to Reader 1





51




52



53
















54





T235 T237 C238

S130

K73

K270

1 2

55
















56






57



A B

58




Cefotaxime


Faropenem

Data Collection


Cell dimensions

a b c (Aring) 5580 6020 7862 5693 5878 7684 5714 5913 7738

(deg) 90 90 90 90 90 90 90 90 90



Rmerge () 3073 (362) 127 (684) 95 (888)

Iσ(I) 110 (20) 91 (23) 99 (22)

Completeness () 991 (944) 985 (956) 999 (100)

Multiplicity 65 (33) 65 (53) 70 (66)

Refinement


Rwork () 122 (261) 168 (319) 150 (257)

Rfree () 139 (269) 212 (345) 188 (275)

No atoms

Protein 2136 2090 2109

LigandIon 26 60 26

Water 350 255 223

B-factors (Aring2)

protein 1408 1558 2242

ligandion 2832 2202 3711

water 3196 3091 3678

RMS deviations



Ramachandran plot

favored () 9888 9888 9887

allowed () 112 112 113

outliers () 0 0 0


59


Distance (Aring)









Distance (Aring)












60

27 References



doi103201eid1710110655


















897 doi101128aac00693-09














3421-3427 doi101128aac4983421-34272005






61







15397-15407 doi101021ja051592u








doi101021bi0487903




278(52) 52724-52729 doi101074jbcm306059200








5665 doi101128aac00245-11



2126(02)00725-6







4943ndash4960 doi101128AAC00296-11



51(3) 721-725 doi101093jacdkg120



62
















doi101107s0907444910048675



50(5) 760-763 doi101107s0907444994003112



40(4) 658ndash674 doi101107S0021889807021206







doi101107s0907444904019158




1080 doi101107s0907444909029436




doi101093nargkm216


452




63

Chapter 3


Lactamase Acylation

31 Overview
















64

32 Introduction






















65























66
























67

















acidbase [14]






68

















34 Conclusions






69



















70












36 Note to Reader 1







71

4

3

2

1

6 7

8

5


72



73

K234

S130 K73

Y105

T235

S237

N170

E166

N132

S70



74




75

S70 S130

K73

E166

N170

Wat1





76

37 References



1145-1165 doi1021740929867023370031






doi1010801475636620161201813



doi101002chin200524267








57(5) 2372-2375 doi101128aac02622-12

















57(6) 2496-2505 doi101128aac02247-12




77









14(17) 4116-4128 doi101039c6ob00353b








5665 doi101128aac00245-11












x







doi101107s0907444904019158


452



78

Chapter 4



41 Overview















79

42 Introduction








for activity [6]















80






















81























82























83


inhibitor of KPC-2






E coli














84





















85
















KPC-2 [33]








86














group

44 Conclusions







87
























88
























89
























90























91





















92






















93












use


mutant proteins





software (GraphPad)

94


mutant proteins






NCF as substrate




[54]









95




mutant proteins



K k2


k-2













96












only










liquid nitrogen

97










46 Note to Reader 1




47 Note to Reader 2



98



S02030 Vaborbactam

99





T237 T235

C69 C238

L167

P104

W105

S130 K73

N132

K234

E166

S70

A

T235 T237

S70

N132

L167

K73

W105

S130

K234

B

100

T235

T237 S237

C69 C238 G238

N132

P104 N104

V240 G240 G239

S70

S130

W105 Y105



backbone shift

101

A

C69

S70

T235

S237

G238

G240

C69

C238

G239

T237

T235




B

S70

102


Vaborbactam 1

Avibactam 1

Tazobactam gt64



103




KPC-2 Avibactam 13 x 104 000022 77 17




104


-Lactamase 0 015 03 06 125 25 5 10 MPC16


CTX-M-15 8 8 4 2 1 05 025 le0125 25



105













106

48 References









58(2) 654-663 doi101128aac01222-13








doi101016S1367-5931(99)00017-4





58(4) 1835ndash1846 doi101128aac00826-13





doi101016jmib201107026









59(10) 6605ndash6607 doi101128AAC01165-15





107










60(3) 1760ndash1766 doi101128AAC02643-15












doi101021bi400413c








doi101021cb800306y











108



doi101021cr010182v











doi101074jbcm306059200




1340-1346














356 doi101006bioo20001184



2126(02)00725-6




doi101073pnas0813029106



109


doi101042bj20101122







546312132







174ndash183 doi101128AAC00930-10















57(6) 2496ndash2505 doi101128AAC02247-12










doi101128aac00711-12

110


















doi1010160006-2952(87)90609-5




doi101107S0907444910048675






doi101107S0907444910045749



40(4) 658ndash674 doi101107S0021889807021206








213ndash221 doi101107S0907444909052925



doi101107S0907444910007493

111




1080 doi101107s0907444909029436




doi101093nargkm216


452

112

Chapter 5



51 Overview













52 Introduction



113




















pathogens [13]



114














infections










115






















116
























117







development

















118
























119


compound 13






















120









to the protein










available analogs




121























122




negative bacteria

54 Conclusions


















123
























124























125


AmpC











sites








126
























127























128



















V= Vmax [S](Km + [S]) (1)

129




equation 2

Ki= IC50([S]Km + 1) (2)



(BSA)













130





















131

56 Note to Reader 1


132



133

R220 H274

T216

P104

L167 W105

K234

E166

N132

S130 K73

T235

S70

T237

C69 C238

(A)

R220 H274

C238 T237 T235

T216

K234

S130

W105

P104

L167 E166

N132

K73

S70

C69

(B)

C69

C238

H274 R220

T216

K234

S130

W105 N132 E166

P104

L167

K73

T235 T237

S70

(C)



contoured at 2

134

R205 Y67

W87

H116 H114

H179 D118

C198

H240 Zn2

Zn1

(D)

N210

C208

W93

L65

M67 F70

N220 H122

H189 H120

H250 D124

Zn2

ACT Zn1

(B)

V73

W93

V73

L65

M67 F70

C208

H250

D124

Zn2

H120 H189

N220 Zn1

H122 ACT

(C)

H250

V73

W93

L65 M67

F70

N220

H189 H120

C208

D124

ACT H122

Zn1

Zn2

(A)

H189

N220

F70 M67

L65

V73 W93

C208

H250 D124

H120

H122 Zn2

Zn1 ACT

(E)

W87

Y67

R205

H179

H114 H116

C198

H240

D118 Zn2 Zn1

(F)

N210

H189 H120

H122

D124

W93

L65

M67

F70

N220 C208

Zn2

Zn1

H250

V73

ACT

(G)




135

(A)

(B)



136






blue

(A) (B)

T235

T237

S130

H250

D124

H122

N220 H189

H120

C208

ACT

Zn2

Zn1

137


1

2682

329

443

21

2

2702 134

1237

21

3

2842

153

NA

NA

4

2542

395

NA

NA

5

2542

1544

NA

NA

6

2682

14

338

082

7

2842

57

NA

NA

8

2761

89

No inhibition

33

9

3331

047

654

13

10

2722

33

2302

33

11

2392

122

1902

64

12

2532

792

810

75

13

2672

42

562

34

14

2232

4471

7413

223

15

2232

4145

No inhibition

303


138


enem

Imip

enem

Com

pou

nd

9

Imip

enem

Com

pou

nd

11

Imip

enem

Com

pou

nd

13

E coli NDM-1 8 2128 2 128 2128

K pneumoniae KPC-2 64 1128 4 128 1128


E cloacae NDM-1 2 2128 2 128 2128




139

57 References



doi101016jcell201411017













doi1010802150559420171292196



4943ndash4960 doi101128AAC00296-11









54(3) 529-534 doi101128jcm02771-15



doi1011772049936115621709









140














60(9) 5454-5458 doi101128aac00711-16






128(6) 2250-2252 doi101002ange201510057



58(4) 1835-1846 doi101128aac00826-13




12406 doi101038ncomms12406


Reviews 24(1) 71-109 doi101128cmr00030-10






Chemistry 1-32 doi101007128_2011_180



doi101021bi3005126





141















doi101128aac00089-11





doi101074jbcm112436238





57(6) 2496-2505 doi101128aac02247-12






338 doi101042BJ20020957











142





177ndash182 doi101021ci049714




doi101107s0907444910048675






doi101107S0907444910045749



40(4) 658ndash674 doi101107S0021889807021206




213-221 doi101107s0907444909052925







doi101107S0907444910007493




1074ndash1080 doi101107S0907444909029436




doi101093nargkm216


452

143

Chapter 6

Summary


















144



















145

Appendix 1




371-387 doi101038nrd3975

146




147




doi101016jmib200508016

148






149




doi101128aac00296-11

150



1014 doi101016jcell201410051

151





152





153






doi101128aac02247-12

154

Appendix 2



155


156


157


158


159



vector 2 0125 05 025 0125 1

KPC-2 32 128 gt512 128 128 16

KPC-2 S130G 1 0125 128 64 025 025

160



Data Collection

Space group P22121

Cell dimensions

a b c (Aring) 5577 5997 7772

(deg) 90 90 90



Rmerge () 90 (912)

Iσ(I) 89 (18)



Refinement


Rwork () 147 (269)

Rfree () 170 (286)

No atoms

Protein 2160

LigandIon 66

Water 305

B-factors (Aring2)

protein 1498

ligandion 2865

water 3529

RMS deviations



Ramachandran plot

favored () 9888

allowed () 112

outliers () 0

PDB code 5WLA

161

Appendix 3


W105

H274 R220

T216

T235 T237 C238

C69

E166

L167

P104

N132

S130

K234 K73

S70

(A)

P104

N132 L167 W105

S130

T216

T235

R220 H274

T237

C69

C238

K234

K73

S70

E166

(B)



162

H120

H189

H122

F70 M67

V73

W93

L65

H250

C208

D124

N220

ACT

Zn2

Zn1

(A)

N220

F70 M67

L65

V73 W93

H250

C208

H120

H189

H122

D124

Zn2

ACT Zn1

(B)

L65

M67

F70 V73

W93

H250

C208

D124

N220

H122

H189 H120

ACT

Zn2

Zn1

(C)

H250

C208

H120 H189 H122

N220

D124

W93

V73

L65

M67 F70

Zn2

Zn1

ACT

(D)



163

L65

F70 M67

V73 W93

D124

H250

C208

H120

H189

H122 N220 ACT

Zn2 Zn1

(E)



Scholar Commons

November 2017


Orville A Pemberton



i

Table of Contents

List of Tablesiv

List of Figuresv


Abstract ix





14 Carbapenemases9






110 Conclusions 15


112 References31



21 Overview39

22 Introduction39





24 Conclusions48






27 References60

ii


Acylation63

31 Overview63

32 Introduction64




and Glu16667

34 Conclusions68





37 References76



41 Overview78

42 Introduction79







44 Conclusions86





















iii






48 References106



51 Overview112

52 Introduction112







54 Conclusions122










57 References139





iv









CTX-M-15104







v

List of Figures













in the PDB29








vi
























vii











CTXCefotaximase



DTTDithiothreitol


EIEnzyme-inhibitor




HBHydrogen bond

viii




LBLysogeny broth






NCFNitrocefin

NDMNew Delhi MBL


OXAOxacillinase










WTWild-type

ix

Abstract



















bacteria

x























xi


in vivo activity





















1

Chapter 1

Introduction



















2
























3


burden




















4
























5






















6























7























8
























9







14 Carbapenemases
















10























11


[70]





















12






















13























14






















15









110 Conclusions













16


















Appendix 1)




17




















18




19





20


21




22



-Lactamases


C A D B

B1 B2 B3

23









24


25


26


27





Tazobactam

28


29


30


31

112 References






Boston MA Springer




from


nhtml





Updates 3(3) 155-160 doi101054drup20000146







252



doi101086533592


12(5) 371-387 doi101038nrd3975





doi103389fpubh201400145






doi101186s12879-016-1990-4

32


317(7159) 657-660 doi101136bmj3177159657


Nature 146(3713) 837-837 doi101038146837a0







doi101056nejm198802183180706








doi101016jcell201411017



doi101016jmib200508016





doi1010029780471729259mca03cs00







1145-1165 doi1021740929867023370031



doi101002chin200524267





33



doi101021ar0400241



Reviews 29(1) 29-57 doi101128cmr00019-15



















61(1) e01636-16 doi101128aac01636-16




doi101107s1399004713033014






doi101007s00894-011-1087-3






7(10) 918-925 doi10103879688

34













27(2) 241-263 doi101128cmr00117-13







6032 doi101039c7ob01514c



doi101128aac01512-08





doi1021741389450116666151001105622








doi101016jjmb200410070





35



4943-4960 doi101128aac00296-11












doi103923jm2006122
















897 doi101128aac00693-09



doi103201eid1710110655



doi101093jacdks121






36





doi101128aac115852













Therapy 35(8) 755-770 doi101002phar1622














251(2) 453-459 doi101042bj2510453







doi10151717460440903190961


Letters 3(6) 442-444 doi101021ml300114c

37



13(12) 12857-12879 doi103390ijms131012857





doi101038nprot2013130



doi101021bi3005126





146-157 doi102174157340911795677602












doi101007s10822-006-9060-4




1749 doi101021jm0306430





159(5) 995-1014 doi101016jcell201410051



doi1010160167-5699(93)90049-q



38




39

Chapter 2



21 Overview








22 Introduction








40





















41
























42
























43













process











44























45























46

















chains






47























48

24 Conclusions




















49






















50














26 Note to Reader 1





51




52



53
















54





T235 T237 C238

S130

K73

K270

1 2

55
















56






57



A B

58




Cefotaxime


Faropenem

Data Collection


Cell dimensions

a b c (Aring) 5580 6020 7862 5693 5878 7684 5714 5913 7738

(deg) 90 90 90 90 90 90 90 90 90



Rmerge () 3073 (362) 127 (684) 95 (888)

Iσ(I) 110 (20) 91 (23) 99 (22)

Completeness () 991 (944) 985 (956) 999 (100)

Multiplicity 65 (33) 65 (53) 70 (66)

Refinement


Rwork () 122 (261) 168 (319) 150 (257)

Rfree () 139 (269) 212 (345) 188 (275)

No atoms

Protein 2136 2090 2109

LigandIon 26 60 26

Water 350 255 223

B-factors (Aring2)

protein 1408 1558 2242

ligandion 2832 2202 3711

water 3196 3091 3678

RMS deviations



Ramachandran plot

favored () 9888 9888 9887

allowed () 112 112 113

outliers () 0 0 0


59


Distance (Aring)









Distance (Aring)












60

27 References



doi103201eid1710110655


















897 doi101128aac00693-09














3421-3427 doi101128aac4983421-34272005






61







15397-15407 doi101021ja051592u








doi101021bi0487903




278(52) 52724-52729 doi101074jbcm306059200








5665 doi101128aac00245-11



2126(02)00725-6







4943ndash4960 doi101128AAC00296-11



51(3) 721-725 doi101093jacdkg120



62
















doi101107s0907444910048675



50(5) 760-763 doi101107s0907444994003112



40(4) 658ndash674 doi101107S0021889807021206







doi101107s0907444904019158




1080 doi101107s0907444909029436




doi101093nargkm216


452




63

Chapter 3


Lactamase Acylation

31 Overview
















64

32 Introduction






















65























66
























67

















acidbase [14]






68

















34 Conclusions






69



















70












36 Note to Reader 1







71

4

3

2

1

6 7

8

5


72



73

K234

S130 K73

Y105

T235

S237

N170

E166

N132

S70



74




75

S70 S130

K73

E166

N170

Wat1





76

37 References



1145-1165 doi1021740929867023370031






doi1010801475636620161201813



doi101002chin200524267








57(5) 2372-2375 doi101128aac02622-12

















57(6) 2496-2505 doi101128aac02247-12




77









14(17) 4116-4128 doi101039c6ob00353b








5665 doi101128aac00245-11












x







doi101107s0907444904019158


452



78

Chapter 4



41 Overview















79

42 Introduction








for activity [6]















80






















81























82























83


inhibitor of KPC-2






E coli














84





















85
















KPC-2 [33]








86














group

44 Conclusions







87
























88
























89
























90























91





















92






















93












use


mutant proteins





software (GraphPad)

94


mutant proteins






NCF as substrate




[54]









95




mutant proteins



K k2


k-2













96












only










liquid nitrogen

97










46 Note to Reader 1




47 Note to Reader 2



98



S02030 Vaborbactam

99





T237 T235

C69 C238

L167

P104

W105

S130 K73

N132

K234

E166

S70

A

T235 T237

S70

N132

L167

K73

W105

S130

K234

B

100

T235

T237 S237

C69 C238 G238

N132

P104 N104

V240 G240 G239

S70

S130

W105 Y105



backbone shift

101

A

C69

S70

T235

S237

G238

G240

C69

C238

G239

T237

T235




B

S70

102


Vaborbactam 1

Avibactam 1

Tazobactam gt64



103




KPC-2 Avibactam 13 x 104 000022 77 17




104


-Lactamase 0 015 03 06 125 25 5 10 MPC16


CTX-M-15 8 8 4 2 1 05 025 le0125 25



105













106

48 References









58(2) 654-663 doi101128aac01222-13








doi101016S1367-5931(99)00017-4





58(4) 1835ndash1846 doi101128aac00826-13





doi101016jmib201107026









59(10) 6605ndash6607 doi101128AAC01165-15





107










60(3) 1760ndash1766 doi101128AAC02643-15












doi101021bi400413c








doi101021cb800306y











108



doi101021cr010182v











doi101074jbcm306059200




1340-1346














356 doi101006bioo20001184



2126(02)00725-6




doi101073pnas0813029106



109


doi101042bj20101122







546312132







174ndash183 doi101128AAC00930-10















57(6) 2496ndash2505 doi101128AAC02247-12










doi101128aac00711-12

110


















doi1010160006-2952(87)90609-5




doi101107S0907444910048675






doi101107S0907444910045749



40(4) 658ndash674 doi101107S0021889807021206








213ndash221 doi101107S0907444909052925



doi101107S0907444910007493

111




1080 doi101107s0907444909029436




doi101093nargkm216


452

112

Chapter 5



51 Overview













52 Introduction



113




















pathogens [13]



114














infections










115






















116
























117







development

















118
























119


compound 13






















120









to the protein










available analogs




121























122




negative bacteria

54 Conclusions


















123
























124























125


AmpC











sites








126
























127























128



















V= Vmax [S](Km + [S]) (1)

129




equation 2

Ki= IC50([S]Km + 1) (2)



(BSA)













130





















131

56 Note to Reader 1


132



133

R220 H274

T216

P104

L167 W105

K234

E166

N132

S130 K73

T235

S70

T237

C69 C238

(A)

R220 H274

C238 T237 T235

T216

K234

S130

W105

P104

L167 E166

N132

K73

S70

C69

(B)

C69

C238

H274 R220

T216

K234

S130

W105 N132 E166

P104

L167

K73

T235 T237

S70

(C)



contoured at 2

134

R205 Y67

W87

H116 H114

H179 D118

C198

H240 Zn2

Zn1

(D)

N210

C208

W93

L65

M67 F70

N220 H122

H189 H120

H250 D124

Zn2

ACT Zn1

(B)

V73

W93

V73

L65

M67 F70

C208

H250

D124

Zn2

H120 H189

N220 Zn1

H122 ACT

(C)

H250

V73

W93

L65 M67

F70

N220

H189 H120

C208

D124

ACT H122

Zn1

Zn2

(A)

H189

N220

F70 M67

L65

V73 W93

C208

H250 D124

H120

H122 Zn2

Zn1 ACT

(E)

W87

Y67

R205

H179

H114 H116

C198

H240

D118 Zn2 Zn1

(F)

N210

H189 H120

H122

D124

W93

L65

M67

F70

N220 C208

Zn2

Zn1

H250

V73

ACT

(G)




135

(A)

(B)



136






blue

(A) (B)

T235

T237

S130

H250

D124

H122

N220 H189

H120

C208

ACT

Zn2

Zn1

137


1

2682

329

443

21

2

2702 134

1237

21

3

2842

153

NA

NA

4

2542

395

NA

NA

5

2542

1544

NA

NA

6

2682

14

338

082

7

2842

57

NA

NA

8

2761

89

No inhibition

33

9

3331

047

654

13

10

2722

33

2302

33

11

2392

122

1902

64

12

2532

792

810

75

13

2672

42

562

34

14

2232

4471

7413

223

15

2232

4145

No inhibition

303


138


enem

Imip

enem

Com

pou

nd

9

Imip

enem

Com

pou

nd

11

Imip

enem

Com

pou

nd

13

E coli NDM-1 8 2128 2 128 2128

K pneumoniae KPC-2 64 1128 4 128 1128


E cloacae NDM-1 2 2128 2 128 2128




139

57 References



doi101016jcell201411017













doi1010802150559420171292196



4943ndash4960 doi101128AAC00296-11









54(3) 529-534 doi101128jcm02771-15



doi1011772049936115621709









140














60(9) 5454-5458 doi101128aac00711-16






128(6) 2250-2252 doi101002ange201510057



58(4) 1835-1846 doi101128aac00826-13




12406 doi101038ncomms12406


Reviews 24(1) 71-109 doi101128cmr00030-10






Chemistry 1-32 doi101007128_2011_180



doi101021bi3005126





141















doi101128aac00089-11





doi101074jbcm112436238





57(6) 2496-2505 doi101128aac02247-12






338 doi101042BJ20020957











142





177ndash182 doi101021ci049714




doi101107s0907444910048675






doi101107S0907444910045749



40(4) 658ndash674 doi101107S0021889807021206




213-221 doi101107s0907444909052925







doi101107S0907444910007493




1074ndash1080 doi101107S0907444909029436




doi101093nargkm216


452

143

Chapter 6

Summary


















144



















145

Appendix 1




371-387 doi101038nrd3975

146




147




doi101016jmib200508016

148






149




doi101128aac00296-11

150



1014 doi101016jcell201410051

151





152





153






doi101128aac02247-12

154

Appendix 2



155


156


157


158


159



vector 2 0125 05 025 0125 1

KPC-2 32 128 gt512 128 128 16

KPC-2 S130G 1 0125 128 64 025 025

160



Data Collection

Space group P22121

Cell dimensions

a b c (Aring) 5577 5997 7772

(deg) 90 90 90



Rmerge () 90 (912)

Iσ(I) 89 (18)



Refinement


Rwork () 147 (269)

Rfree () 170 (286)

No atoms

Protein 2160

LigandIon 66

Water 305

B-factors (Aring2)

protein 1498

ligandion 2865

water 3529

RMS deviations



Ramachandran plot

favored () 9888

allowed () 112

outliers () 0

PDB code 5WLA

161

Appendix 3


W105

H274 R220

T216

T235 T237 C238

C69

E166

L167

P104

N132

S130

K234 K73

S70

(A)

P104

N132 L167 W105

S130

T216

T235

R220 H274

T237

C69

C238

K234

K73

S70

E166

(B)



162

H120

H189

H122

F70 M67

V73

W93

L65

H250

C208

D124

N220

ACT

Zn2

Zn1

(A)

N220

F70 M67

L65

V73 W93

H250

C208

H120

H189

H122

D124

Zn2

ACT Zn1

(B)

L65

M67

F70 V73

W93

H250

C208

D124

N220

H122

H189 H120

ACT

Zn2

Zn1

(C)

H250

C208

H120 H189 H122

N220

D124

W93

V73

L65

M67 F70

Zn2

Zn1

ACT

(D)



163

L65

F70 M67

V73 W93

D124

H250

C208

H120

H189

H122 N220 ACT

Zn2 Zn1

(E)



Scholar Commons

November 2017


Orville A Pemberton



ii


Acylation63

31 Overview63

32 Introduction64




and Glu16667

34 Conclusions68





37 References76



41 Overview78

42 Introduction79







44 Conclusions86





















iii






48 References106



51 Overview112

52 Introduction112







54 Conclusions122










57 References139





iv









CTX-M-15104







v

List of Figures













in the PDB29








vi
























vii











CTXCefotaximase



DTTDithiothreitol


EIEnzyme-inhibitor




HBHydrogen bond

viii




LBLysogeny broth






NCFNitrocefin

NDMNew Delhi MBL


OXAOxacillinase










WTWild-type

ix

Abstract



















bacteria

x























xi


in vivo activity





















1

Chapter 1

Introduction



















2
























3


burden




















4
























5






















6























7























8
























9







14 Carbapenemases
















10























11


[70]





















12






















13























14






















15









110 Conclusions













16


















Appendix 1)




17




















18




19





20


21




22



-Lactamases


C A D B

B1 B2 B3

23









24


25


26


27





Tazobactam

28


29


30


31

112 References






Boston MA Springer




from


nhtml





Updates 3(3) 155-160 doi101054drup20000146







252



doi101086533592


12(5) 371-387 doi101038nrd3975





doi103389fpubh201400145






doi101186s12879-016-1990-4

32


317(7159) 657-660 doi101136bmj3177159657


Nature 146(3713) 837-837 doi101038146837a0







doi101056nejm198802183180706








doi101016jcell201411017



doi101016jmib200508016





doi1010029780471729259mca03cs00







1145-1165 doi1021740929867023370031



doi101002chin200524267





33



doi101021ar0400241



Reviews 29(1) 29-57 doi101128cmr00019-15



















61(1) e01636-16 doi101128aac01636-16




doi101107s1399004713033014






doi101007s00894-011-1087-3






7(10) 918-925 doi10103879688

34













27(2) 241-263 doi101128cmr00117-13







6032 doi101039c7ob01514c



doi101128aac01512-08





doi1021741389450116666151001105622








doi101016jjmb200410070





35



4943-4960 doi101128aac00296-11












doi103923jm2006122
















897 doi101128aac00693-09



doi103201eid1710110655



doi101093jacdks121






36





doi101128aac115852













Therapy 35(8) 755-770 doi101002phar1622














251(2) 453-459 doi101042bj2510453







doi10151717460440903190961


Letters 3(6) 442-444 doi101021ml300114c

37



13(12) 12857-12879 doi103390ijms131012857





doi101038nprot2013130



doi101021bi3005126





146-157 doi102174157340911795677602












doi101007s10822-006-9060-4




1749 doi101021jm0306430





159(5) 995-1014 doi101016jcell201410051



doi1010160167-5699(93)90049-q



38




39

Chapter 2



21 Overview








22 Introduction








40





















41
























42
























43













process











44























45























46

















chains






47























48

24 Conclusions




















49






















50














26 Note to Reader 1





51




52



53
















54





T235 T237 C238

S130

K73

K270

1 2

55
















56






57



A B

58




Cefotaxime


Faropenem

Data Collection


Cell dimensions

a b c (Aring) 5580 6020 7862 5693 5878 7684 5714 5913 7738

(deg) 90 90 90 90 90 90 90 90 90



Rmerge () 3073 (362) 127 (684) 95 (888)

Iσ(I) 110 (20) 91 (23) 99 (22)

Completeness () 991 (944) 985 (956) 999 (100)

Multiplicity 65 (33) 65 (53) 70 (66)

Refinement


Rwork () 122 (261) 168 (319) 150 (257)

Rfree () 139 (269) 212 (345) 188 (275)

No atoms

Protein 2136 2090 2109

LigandIon 26 60 26

Water 350 255 223

B-factors (Aring2)

protein 1408 1558 2242

ligandion 2832 2202 3711

water 3196 3091 3678

RMS deviations



Ramachandran plot

favored () 9888 9888 9887

allowed () 112 112 113

outliers () 0 0 0


59


Distance (Aring)









Distance (Aring)












60

27 References



doi103201eid1710110655


















897 doi101128aac00693-09














3421-3427 doi101128aac4983421-34272005






61







15397-15407 doi101021ja051592u








doi101021bi0487903




278(52) 52724-52729 doi101074jbcm306059200








5665 doi101128aac00245-11



2126(02)00725-6







4943ndash4960 doi101128AAC00296-11



51(3) 721-725 doi101093jacdkg120



62
















doi101107s0907444910048675



50(5) 760-763 doi101107s0907444994003112



40(4) 658ndash674 doi101107S0021889807021206







doi101107s0907444904019158




1080 doi101107s0907444909029436




doi101093nargkm216


452




63

Chapter 3


Lactamase Acylation

31 Overview
















64

32 Introduction






















65























66
























67

















acidbase [14]






68

















34 Conclusions






69



















70












36 Note to Reader 1







71

4

3

2

1

6 7

8

5


72



73

K234

S130 K73

Y105

T235

S237

N170

E166

N132

S70



74




75

S70 S130

K73

E166

N170

Wat1





76

37 References



1145-1165 doi1021740929867023370031






doi1010801475636620161201813



doi101002chin200524267








57(5) 2372-2375 doi101128aac02622-12

















57(6) 2496-2505 doi101128aac02247-12




77









14(17) 4116-4128 doi101039c6ob00353b








5665 doi101128aac00245-11












x







doi101107s0907444904019158


452



78

Chapter 4



41 Overview















79

42 Introduction








for activity [6]















80






















81























82























83


inhibitor of KPC-2






E coli














84





















85
















KPC-2 [33]








86














group

44 Conclusions







87
























88
























89
























90























91





















92






















93












use


mutant proteins





software (GraphPad)

94


mutant proteins






NCF as substrate




[54]









95




mutant proteins



K k2


k-2













96












only










liquid nitrogen

97










46 Note to Reader 1




47 Note to Reader 2



98



S02030 Vaborbactam

99





T237 T235

C69 C238

L167

P104

W105

S130 K73

N132

K234

E166

S70

A

T235 T237

S70

N132

L167

K73

W105

S130

K234

B

100

T235

T237 S237

C69 C238 G238

N132

P104 N104

V240 G240 G239

S70

S130

W105 Y105



backbone shift

101

A

C69

S70

T235

S237

G238

G240

C69

C238

G239

T237

T235




B

S70

102


Vaborbactam 1

Avibactam 1

Tazobactam gt64



103




KPC-2 Avibactam 13 x 104 000022 77 17




104


-Lactamase 0 015 03 06 125 25 5 10 MPC16


CTX-M-15 8 8 4 2 1 05 025 le0125 25



105













106

48 References









58(2) 654-663 doi101128aac01222-13








doi101016S1367-5931(99)00017-4





58(4) 1835ndash1846 doi101128aac00826-13





doi101016jmib201107026









59(10) 6605ndash6607 doi101128AAC01165-15





107










60(3) 1760ndash1766 doi101128AAC02643-15












doi101021bi400413c








doi101021cb800306y











108



doi101021cr010182v











doi101074jbcm306059200




1340-1346














356 doi101006bioo20001184



2126(02)00725-6




doi101073pnas0813029106



109


doi101042bj20101122







546312132







174ndash183 doi101128AAC00930-10















57(6) 2496ndash2505 doi101128AAC02247-12










doi101128aac00711-12

110


















doi1010160006-2952(87)90609-5




doi101107S0907444910048675






doi101107S0907444910045749



40(4) 658ndash674 doi101107S0021889807021206








213ndash221 doi101107S0907444909052925



doi101107S0907444910007493

111




1080 doi101107s0907444909029436




doi101093nargkm216


452

112

Chapter 5



51 Overview













52 Introduction



113




















pathogens [13]



114














infections










115






















116
























117







development

















118
























119


compound 13






















120









to the protein










available analogs




121























122




negative bacteria

54 Conclusions


















123
























124























125


AmpC











sites








126
























127























128



















V= Vmax [S](Km + [S]) (1)

129




equation 2

Ki= IC50([S]Km + 1) (2)



(BSA)













130





















131

56 Note to Reader 1


132



133

R220 H274

T216

P104

L167 W105

K234

E166

N132

S130 K73

T235

S70

T237

C69 C238

(A)

R220 H274

C238 T237 T235

T216

K234

S130

W105

P104

L167 E166

N132

K73

S70

C69

(B)

C69

C238

H274 R220

T216

K234

S130

W105 N132 E166

P104

L167

K73

T235 T237

S70

(C)



contoured at 2

134

R205 Y67

W87

H116 H114

H179 D118

C198

H240 Zn2

Zn1

(D)

N210

C208

W93

L65

M67 F70

N220 H122

H189 H120

H250 D124

Zn2

ACT Zn1

(B)

V73

W93

V73

L65

M67 F70

C208

H250

D124

Zn2

H120 H189

N220 Zn1

H122 ACT

(C)

H250

V73

W93

L65 M67

F70

N220

H189 H120

C208

D124

ACT H122

Zn1

Zn2

(A)

H189

N220

F70 M67

L65

V73 W93

C208

H250 D124

H120

H122 Zn2

Zn1 ACT

(E)

W87

Y67

R205

H179

H114 H116

C198

H240

D118 Zn2 Zn1

(F)

N210

H189 H120

H122

D124

W93

L65

M67

F70

N220 C208

Zn2

Zn1

H250

V73

ACT

(G)




135

(A)

(B)



136






blue

(A) (B)

T235

T237

S130

H250

D124

H122

N220 H189

H120

C208

ACT

Zn2

Zn1

137


1

2682

329

443

21

2

2702 134

1237

21

3

2842

153

NA

NA

4

2542

395

NA

NA

5

2542

1544

NA

NA

6

2682

14

338

082

7

2842

57

NA

NA

8

2761

89

No inhibition

33

9

3331

047

654

13

10

2722

33

2302

33

11

2392

122

1902

64

12

2532

792

810

75

13

2672

42

562

34

14

2232

4471

7413

223

15

2232

4145

No inhibition

303


138


enem

Imip

enem

Com

pou

nd

9

Imip

enem

Com

pou

nd

11

Imip

enem

Com

pou

nd

13

E coli NDM-1 8 2128 2 128 2128

K pneumoniae KPC-2 64 1128 4 128 1128


E cloacae NDM-1 2 2128 2 128 2128




139

57 References



doi101016jcell201411017













doi1010802150559420171292196



4943ndash4960 doi101128AAC00296-11









54(3) 529-534 doi101128jcm02771-15



doi1011772049936115621709









140














60(9) 5454-5458 doi101128aac00711-16






128(6) 2250-2252 doi101002ange201510057



58(4) 1835-1846 doi101128aac00826-13




12406 doi101038ncomms12406


Reviews 24(1) 71-109 doi101128cmr00030-10






Chemistry 1-32 doi101007128_2011_180



doi101021bi3005126





141















doi101128aac00089-11





doi101074jbcm112436238





57(6) 2496-2505 doi101128aac02247-12






338 doi101042BJ20020957











142





177ndash182 doi101021ci049714




doi101107s0907444910048675






doi101107S0907444910045749



40(4) 658ndash674 doi101107S0021889807021206




213-221 doi101107s0907444909052925







doi101107S0907444910007493




1074ndash1080 doi101107S0907444909029436




doi101093nargkm216


452

143

Chapter 6

Summary


















144



















145

Appendix 1




371-387 doi101038nrd3975

146




147




doi101016jmib200508016

148






149




doi101128aac00296-11

150



1014 doi101016jcell201410051

151





152





153






doi101128aac02247-12

154

Appendix 2



155


156


157


158


159



vector 2 0125 05 025 0125 1

KPC-2 32 128 gt512 128 128 16

KPC-2 S130G 1 0125 128 64 025 025

160



Data Collection

Space group P22121

Cell dimensions

a b c (Aring) 5577 5997 7772

(deg) 90 90 90



Rmerge () 90 (912)

Iσ(I) 89 (18)



Refinement


Rwork () 147 (269)

Rfree () 170 (286)

No atoms

Protein 2160

LigandIon 66

Water 305

B-factors (Aring2)

protein 1498

ligandion 2865

water 3529

RMS deviations



Ramachandran plot

favored () 9888

allowed () 112

outliers () 0

PDB code 5WLA

161

Appendix 3


W105

H274 R220

T216

T235 T237 C238

C69

E166

L167

P104

N132

S130

K234 K73

S70

(A)

P104

N132 L167 W105

S130

T216

T235

R220 H274

T237

C69

C238

K234

K73

S70

E166

(B)



162

H120

H189

H122

F70 M67

V73

W93

L65

H250

C208

D124

N220

ACT

Zn2

Zn1

(A)

N220

F70 M67

L65

V73 W93

H250

C208

H120

H189

H122

D124

Zn2

ACT Zn1

(B)

L65

M67

F70 V73

W93

H250

C208

D124

N220

H122

H189 H120

ACT

Zn2

Zn1

(C)

H250

C208

H120 H189 H122

N220

D124

W93

V73

L65

M67 F70

Zn2

Zn1

ACT

(D)



163

L65

F70 M67

V73 W93

D124

H250

C208

H120

H189

H122 N220 ACT

Zn2 Zn1

(E)



Scholar Commons

November 2017


Orville A Pemberton



iii






48 References106



51 Overview112

52 Introduction112







54 Conclusions122










57 References139





iv









CTX-M-15104







v

List of Figures













in the PDB29








vi
























vii











CTXCefotaximase



DTTDithiothreitol


EIEnzyme-inhibitor




HBHydrogen bond

viii




LBLysogeny broth






NCFNitrocefin

NDMNew Delhi MBL


OXAOxacillinase










WTWild-type

ix

Abstract



















bacteria

x























xi


in vivo activity





















1

Chapter 1

Introduction



















2
























3


burden




















4
























5






















6























7























8
























9







14 Carbapenemases
















10























11


[70]





















12






















13























14






















15









110 Conclusions













16


















Appendix 1)




17




















18




19





20


21




22



-Lactamases


C A D B

B1 B2 B3

23









24


25


26


27





Tazobactam

28


29


30


31

112 References






Boston MA Springer




from


nhtml





Updates 3(3) 155-160 doi101054drup20000146







252



doi101086533592


12(5) 371-387 doi101038nrd3975





doi103389fpubh201400145






doi101186s12879-016-1990-4

32


317(7159) 657-660 doi101136bmj3177159657


Nature 146(3713) 837-837 doi101038146837a0







doi101056nejm198802183180706








doi101016jcell201411017



doi101016jmib200508016





doi1010029780471729259mca03cs00







1145-1165 doi1021740929867023370031



doi101002chin200524267





33



doi101021ar0400241



Reviews 29(1) 29-57 doi101128cmr00019-15



















61(1) e01636-16 doi101128aac01636-16




doi101107s1399004713033014






doi101007s00894-011-1087-3






7(10) 918-925 doi10103879688

34













27(2) 241-263 doi101128cmr00117-13







6032 doi101039c7ob01514c



doi101128aac01512-08





doi1021741389450116666151001105622








doi101016jjmb200410070





35



4943-4960 doi101128aac00296-11












doi103923jm2006122
















897 doi101128aac00693-09



doi103201eid1710110655



doi101093jacdks121






36





doi101128aac115852













Therapy 35(8) 755-770 doi101002phar1622














251(2) 453-459 doi101042bj2510453







doi10151717460440903190961


Letters 3(6) 442-444 doi101021ml300114c

37



13(12) 12857-12879 doi103390ijms131012857





doi101038nprot2013130



doi101021bi3005126





146-157 doi102174157340911795677602












doi101007s10822-006-9060-4




1749 doi101021jm0306430





159(5) 995-1014 doi101016jcell201410051



doi1010160167-5699(93)90049-q



38




39

Chapter 2



21 Overview








22 Introduction








40





















41
























42
























43













process











44























45























46

















chains






47























48

24 Conclusions




















49






















50














26 Note to Reader 1





51




52



53
















54





T235 T237 C238

S130

K73

K270

1 2

55
















56






57



A B

58




Cefotaxime


Faropenem

Data Collection


Cell dimensions

a b c (Aring) 5580 6020 7862 5693 5878 7684 5714 5913 7738

(deg) 90 90 90 90 90 90 90 90 90



Rmerge () 3073 (362) 127 (684) 95 (888)

Iσ(I) 110 (20) 91 (23) 99 (22)

Completeness () 991 (944) 985 (956) 999 (100)

Multiplicity 65 (33) 65 (53) 70 (66)

Refinement


Rwork () 122 (261) 168 (319) 150 (257)

Rfree () 139 (269) 212 (345) 188 (275)

No atoms

Protein 2136 2090 2109

LigandIon 26 60 26

Water 350 255 223

B-factors (Aring2)

protein 1408 1558 2242

ligandion 2832 2202 3711

water 3196 3091 3678

RMS deviations



Ramachandran plot

favored () 9888 9888 9887

allowed () 112 112 113

outliers () 0 0 0


59


Distance (Aring)









Distance (Aring)












60

27 References



doi103201eid1710110655


















897 doi101128aac00693-09














3421-3427 doi101128aac4983421-34272005






61







15397-15407 doi101021ja051592u








doi101021bi0487903




278(52) 52724-52729 doi101074jbcm306059200








5665 doi101128aac00245-11



2126(02)00725-6







4943ndash4960 doi101128AAC00296-11



51(3) 721-725 doi101093jacdkg120



62
















doi101107s0907444910048675



50(5) 760-763 doi101107s0907444994003112



40(4) 658ndash674 doi101107S0021889807021206







doi101107s0907444904019158




1080 doi101107s0907444909029436




doi101093nargkm216


452




63

Chapter 3


Lactamase Acylation

31 Overview
















64

32 Introduction






















65























66
























67

















acidbase [14]






68

















34 Conclusions






69



















70












36 Note to Reader 1







71

4

3

2

1

6 7

8

5


72



73

K234

S130 K73

Y105

T235

S237

N170

E166

N132

S70



74




75

S70 S130

K73

E166

N170

Wat1





76

37 References



1145-1165 doi1021740929867023370031






doi1010801475636620161201813



doi101002chin200524267








57(5) 2372-2375 doi101128aac02622-12

















57(6) 2496-2505 doi101128aac02247-12




77









14(17) 4116-4128 doi101039c6ob00353b








5665 doi101128aac00245-11












x







doi101107s0907444904019158


452



78

Chapter 4



41 Overview















79

42 Introduction








for activity [6]















80






















81























82























83


inhibitor of KPC-2






E coli














84





















85
















KPC-2 [33]








86














group

44 Conclusions







87
























88
























89
























90























91





















92






















93












use


mutant proteins





software (GraphPad)

94


mutant proteins






NCF as substrate




[54]









95




mutant proteins



K k2


k-2













96












only










liquid nitrogen

97










46 Note to Reader 1




47 Note to Reader 2



98



S02030 Vaborbactam

99





T237 T235

C69 C238

L167

P104

W105

S130 K73

N132

K234

E166

S70

A

T235 T237

S70

N132

L167

K73

W105

S130

K234

B

100

T235

T237 S237

C69 C238 G238

N132

P104 N104

V240 G240 G239

S70

S130

W105 Y105



backbone shift

101

A

C69

S70

T235

S237

G238

G240

C69

C238

G239

T237

T235




B

S70

102


Vaborbactam 1

Avibactam 1

Tazobactam gt64



103




KPC-2 Avibactam 13 x 104 000022 77 17




104


-Lactamase 0 015 03 06 125 25 5 10 MPC16


CTX-M-15 8 8 4 2 1 05 025 le0125 25



105













106

48 References









58(2) 654-663 doi101128aac01222-13








doi101016S1367-5931(99)00017-4





58(4) 1835ndash1846 doi101128aac00826-13





doi101016jmib201107026









59(10) 6605ndash6607 doi101128AAC01165-15





107










60(3) 1760ndash1766 doi101128AAC02643-15












doi101021bi400413c








doi101021cb800306y











108



doi101021cr010182v











doi101074jbcm306059200




1340-1346














356 doi101006bioo20001184



2126(02)00725-6




doi101073pnas0813029106



109


doi101042bj20101122







546312132







174ndash183 doi101128AAC00930-10















57(6) 2496ndash2505 doi101128AAC02247-12










doi101128aac00711-12

110


















doi1010160006-2952(87)90609-5




doi101107S0907444910048675






doi101107S0907444910045749



40(4) 658ndash674 doi101107S0021889807021206








213ndash221 doi101107S0907444909052925



doi101107S0907444910007493

111




1080 doi101107s0907444909029436




doi101093nargkm216


452

112

Chapter 5



51 Overview













52 Introduction



113




















pathogens [13]



114














infections










115






















116
























117







development

















118
























119


compound 13






















120









to the protein










available analogs




121























122




negative bacteria

54 Conclusions


















123
























124























125


AmpC











sites








126
























127























128



















V= Vmax [S](Km + [S]) (1)

129




equation 2

Ki= IC50([S]Km + 1) (2)



(BSA)













130





















131

56 Note to Reader 1


132



133

R220 H274

T216

P104

L167 W105

K234

E166

N132

S130 K73

T235

S70

T237

C69 C238

(A)

R220 H274

C238 T237 T235

T216

K234

S130

W105

P104

L167 E166

N132

K73

S70

C69

(B)

C69

C238

H274 R220

T216

K234

S130

W105 N132 E166

P104

L167

K73

T235 T237

S70

(C)



contoured at 2

134

R205 Y67

W87

H116 H114

H179 D118

C198

H240 Zn2

Zn1

(D)

N210

C208

W93

L65

M67 F70

N220 H122

H189 H120

H250 D124

Zn2

ACT Zn1

(B)

V73

W93

V73

L65

M67 F70

C208

H250

D124

Zn2

H120 H189

N220 Zn1

H122 ACT

(C)

H250

V73

W93

L65 M67

F70

N220

H189 H120

C208

D124

ACT H122

Zn1

Zn2

(A)

H189

N220

F70 M67

L65

V73 W93

C208

H250 D124

H120

H122 Zn2

Zn1 ACT

(E)

W87

Y67

R205

H179

H114 H116

C198

H240

D118 Zn2 Zn1

(F)

N210

H189 H120

H122

D124

W93

L65

M67

F70

N220 C208

Zn2

Zn1

H250

V73

ACT

(G)




135

(A)

(B)



136






blue

(A) (B)

T235

T237

S130

H250

D124

H122

N220 H189

H120

C208

ACT

Zn2

Zn1

137


1

2682

329

443

21

2

2702 134

1237

21

3

2842

153

NA

NA

4

2542

395

NA

NA

5

2542

1544

NA

NA

6

2682

14

338

082

7

2842

57

NA

NA

8

2761

89

No inhibition

33

9

3331

047

654

13

10

2722

33

2302

33

11

2392

122

1902

64

12

2532

792

810

75

13

2672

42

562

34

14

2232

4471

7413

223

15

2232

4145

No inhibition

303


138


enem

Imip

enem

Com

pou

nd

9

Imip

enem

Com

pou

nd

11

Imip

enem

Com

pou

nd

13

E coli NDM-1 8 2128 2 128 2128

K pneumoniae KPC-2 64 1128 4 128 1128


E cloacae NDM-1 2 2128 2 128 2128




139

57 References



doi101016jcell201411017













doi1010802150559420171292196



4943ndash4960 doi101128AAC00296-11









54(3) 529-534 doi101128jcm02771-15



doi1011772049936115621709









140














60(9) 5454-5458 doi101128aac00711-16






128(6) 2250-2252 doi101002ange201510057



58(4) 1835-1846 doi101128aac00826-13




12406 doi101038ncomms12406


Reviews 24(1) 71-109 doi101128cmr00030-10






Chemistry 1-32 doi101007128_2011_180



doi101021bi3005126





141















doi101128aac00089-11





doi101074jbcm112436238





57(6) 2496-2505 doi101128aac02247-12






338 doi101042BJ20020957











142





177ndash182 doi101021ci049714




doi101107s0907444910048675






doi101107S0907444910045749



40(4) 658ndash674 doi101107S0021889807021206




213-221 doi101107s0907444909052925







doi101107S0907444910007493




1074ndash1080 doi101107S0907444909029436




doi101093nargkm216


452

143

Chapter 6

Summary


















144



















145

Appendix 1




371-387 doi101038nrd3975

146




147




doi101016jmib200508016

148






149




doi101128aac00296-11

150



1014 doi101016jcell201410051

151





152





153






doi101128aac02247-12

154

Appendix 2



155


156


157


158


159



vector 2 0125 05 025 0125 1

KPC-2 32 128 gt512 128 128 16

KPC-2 S130G 1 0125 128 64 025 025

160



Data Collection

Space group P22121

Cell dimensions

a b c (Aring) 5577 5997 7772

(deg) 90 90 90



Rmerge () 90 (912)

Iσ(I) 89 (18)



Refinement


Rwork () 147 (269)

Rfree () 170 (286)

No atoms

Protein 2160

LigandIon 66

Water 305

B-factors (Aring2)

protein 1498

ligandion 2865

water 3529

RMS deviations



Ramachandran plot

favored () 9888

allowed () 112

outliers () 0

PDB code 5WLA

161

Appendix 3


W105

H274 R220

T216

T235 T237 C238

C69

E166

L167

P104

N132

S130

K234 K73

S70

(A)

P104

N132 L167 W105

S130

T216

T235

R220 H274

T237

C69

C238

K234

K73

S70

E166

(B)



162

H120

H189

H122

F70 M67

V73

W93

L65

H250

C208

D124

N220

ACT

Zn2

Zn1

(A)

N220

F70 M67

L65

V73 W93

H250

C208

H120

H189

H122

D124

Zn2

ACT Zn1

(B)

L65

M67

F70 V73

W93

H250

C208

D124

N220

H122

H189 H120

ACT

Zn2

Zn1

(C)

H250

C208

H120 H189 H122

N220

D124

W93

V73

L65

M67 F70

Zn2

Zn1

ACT

(D)



163

L65

F70 M67

V73 W93

D124

H250

C208

H120

H189

H122 N220 ACT

Zn2 Zn1

(E)



Scholar Commons

November 2017


Orville A Pemberton



iv









CTX-M-15104







v

List of Figures













in the PDB29








vi
























vii











CTXCefotaximase



DTTDithiothreitol


EIEnzyme-inhibitor




HBHydrogen bond

viii




LBLysogeny broth






NCFNitrocefin

NDMNew Delhi MBL


OXAOxacillinase










WTWild-type

ix

Abstract



















bacteria

x























xi


in vivo activity





















1

Chapter 1

Introduction



















2
























3


burden




















4
























5






















6























7























8
























9







14 Carbapenemases
















10























11


[70]





















12






















13























14






















15









110 Conclusions













16


















Appendix 1)




17




















18




19





20


21




22



-Lactamases


C A D B

B1 B2 B3

23









24


25


26


27





Tazobactam

28


29


30


31

112 References






Boston MA Springer




from


nhtml





Updates 3(3) 155-160 doi101054drup20000146







252



doi101086533592


12(5) 371-387 doi101038nrd3975





doi103389fpubh201400145






doi101186s12879-016-1990-4

32


317(7159) 657-660 doi101136bmj3177159657


Nature 146(3713) 837-837 doi101038146837a0







doi101056nejm198802183180706








doi101016jcell201411017



doi101016jmib200508016





doi1010029780471729259mca03cs00







1145-1165 doi1021740929867023370031



doi101002chin200524267





33



doi101021ar0400241



Reviews 29(1) 29-57 doi101128cmr00019-15



















61(1) e01636-16 doi101128aac01636-16




doi101107s1399004713033014






doi101007s00894-011-1087-3






7(10) 918-925 doi10103879688

34













27(2) 241-263 doi101128cmr00117-13







6032 doi101039c7ob01514c



doi101128aac01512-08





doi1021741389450116666151001105622








doi101016jjmb200410070





35



4943-4960 doi101128aac00296-11












doi103923jm2006122
















897 doi101128aac00693-09



doi103201eid1710110655



doi101093jacdks121






36





doi101128aac115852













Therapy 35(8) 755-770 doi101002phar1622














251(2) 453-459 doi101042bj2510453







doi10151717460440903190961


Letters 3(6) 442-444 doi101021ml300114c

37



13(12) 12857-12879 doi103390ijms131012857





doi101038nprot2013130



doi101021bi3005126





146-157 doi102174157340911795677602












doi101007s10822-006-9060-4




1749 doi101021jm0306430





159(5) 995-1014 doi101016jcell201410051



doi1010160167-5699(93)90049-q



38




39

Chapter 2



21 Overview








22 Introduction








40





















41
























42
























43













process











44























45























46

















chains






47























48

24 Conclusions




















49






















50














26 Note to Reader 1





51




52



53
















54





T235 T237 C238

S130

K73

K270

1 2

55
















56






57



A B

58




Cefotaxime


Faropenem

Data Collection


Cell dimensions

a b c (Aring) 5580 6020 7862 5693 5878 7684 5714 5913 7738

(deg) 90 90 90 90 90 90 90 90 90



Rmerge () 3073 (362) 127 (684) 95 (888)

Iσ(I) 110 (20) 91 (23) 99 (22)

Completeness () 991 (944) 985 (956) 999 (100)

Multiplicity 65 (33) 65 (53) 70 (66)

Refinement


Rwork () 122 (261) 168 (319) 150 (257)

Rfree () 139 (269) 212 (345) 188 (275)

No atoms

Protein 2136 2090 2109

LigandIon 26 60 26

Water 350 255 223

B-factors (Aring2)

protein 1408 1558 2242

ligandion 2832 2202 3711

water 3196 3091 3678

RMS deviations



Ramachandran plot

favored () 9888 9888 9887

allowed () 112 112 113

outliers () 0 0 0


59


Distance (Aring)









Distance (Aring)












60

27 References



doi103201eid1710110655


















897 doi101128aac00693-09














3421-3427 doi101128aac4983421-34272005






61







15397-15407 doi101021ja051592u








doi101021bi0487903




278(52) 52724-52729 doi101074jbcm306059200








5665 doi101128aac00245-11



2126(02)00725-6







4943ndash4960 doi101128AAC00296-11



51(3) 721-725 doi101093jacdkg120



62
















doi101107s0907444910048675



50(5) 760-763 doi101107s0907444994003112



40(4) 658ndash674 doi101107S0021889807021206







doi101107s0907444904019158




1080 doi101107s0907444909029436




doi101093nargkm216


452




63

Chapter 3


Lactamase Acylation

31 Overview
















64

32 Introduction






















65























66
























67

















acidbase [14]






68

















34 Conclusions






69



















70












36 Note to Reader 1







71

4

3

2

1

6 7

8

5


72



73

K234

S130 K73

Y105

T235

S237

N170

E166

N132

S70



74




75

S70 S130

K73

E166

N170

Wat1





76

37 References



1145-1165 doi1021740929867023370031






doi1010801475636620161201813



doi101002chin200524267








57(5) 2372-2375 doi101128aac02622-12

















57(6) 2496-2505 doi101128aac02247-12




77









14(17) 4116-4128 doi101039c6ob00353b








5665 doi101128aac00245-11












x







doi101107s0907444904019158


452



78

Chapter 4



41 Overview















79

42 Introduction








for activity [6]















80






















81























82























83


inhibitor of KPC-2






E coli














84





















85
















KPC-2 [33]








86














group

44 Conclusions







87
























88
























89
























90























91





















92






















93












use


mutant proteins





software (GraphPad)

94


mutant proteins






NCF as substrate




[54]









95




mutant proteins



K k2


k-2













96












only










liquid nitrogen

97










46 Note to Reader 1




47 Note to Reader 2



98



S02030 Vaborbactam

99





T237 T235

C69 C238

L167

P104

W105

S130 K73

N132

K234

E166

S70

A

T235 T237

S70

N132

L167

K73

W105

S130

K234

B

100

T235

T237 S237

C69 C238 G238

N132

P104 N104

V240 G240 G239

S70

S130

W105 Y105



backbone shift

101

A

C69

S70

T235

S237

G238

G240

C69

C238

G239

T237

T235




B

S70

102


Vaborbactam 1

Avibactam 1

Tazobactam gt64



103




KPC-2 Avibactam 13 x 104 000022 77 17




104


-Lactamase 0 015 03 06 125 25 5 10 MPC16


CTX-M-15 8 8 4 2 1 05 025 le0125 25



105













106

48 References









58(2) 654-663 doi101128aac01222-13








doi101016S1367-5931(99)00017-4





58(4) 1835ndash1846 doi101128aac00826-13





doi101016jmib201107026









59(10) 6605ndash6607 doi101128AAC01165-15





107










60(3) 1760ndash1766 doi101128AAC02643-15












doi101021bi400413c








doi101021cb800306y











108



doi101021cr010182v











doi101074jbcm306059200




1340-1346














356 doi101006bioo20001184



2126(02)00725-6




doi101073pnas0813029106



109


doi101042bj20101122







546312132







174ndash183 doi101128AAC00930-10















57(6) 2496ndash2505 doi101128AAC02247-12










doi101128aac00711-12

110


















doi1010160006-2952(87)90609-5




doi101107S0907444910048675






doi101107S0907444910045749



40(4) 658ndash674 doi101107S0021889807021206








213ndash221 doi101107S0907444909052925



doi101107S0907444910007493

111




1080 doi101107s0907444909029436




doi101093nargkm216


452

112

Chapter 5



51 Overview













52 Introduction



113




















pathogens [13]



114














infections










115






















116
























117







development

















118
























119


compound 13






















120









to the protein










available analogs




121























122




negative bacteria

54 Conclusions


















123
























124























125


AmpC











sites








126
























127























128



















V= Vmax [S](Km + [S]) (1)

129




equation 2

Ki= IC50([S]Km + 1) (2)



(BSA)













130





















131

56 Note to Reader 1


132



133

R220 H274

T216

P104

L167 W105

K234

E166

N132

S130 K73

T235

S70

T237

C69 C238

(A)

R220 H274

C238 T237 T235

T216

K234

S130

W105

P104

L167 E166

N132

K73

S70

C69

(B)

C69

C238

H274 R220

T216

K234

S130

W105 N132 E166

P104

L167

K73

T235 T237

S70

(C)



contoured at 2

134

R205 Y67

W87

H116 H114

H179 D118

C198

H240 Zn2

Zn1

(D)

N210

C208

W93

L65

M67 F70

N220 H122

H189 H120

H250 D124

Zn2

ACT Zn1

(B)

V73

W93

V73

L65

M67 F70

C208

H250

D124

Zn2

H120 H189

N220 Zn1

H122 ACT

(C)

H250

V73

W93

L65 M67

F70

N220

H189 H120

C208

D124

ACT H122

Zn1

Zn2

(A)

H189

N220

F70 M67

L65

V73 W93

C208

H250 D124

H120

H122 Zn2

Zn1 ACT

(E)

W87

Y67

R205

H179

H114 H116

C198

H240

D118 Zn2 Zn1

(F)

N210

H189 H120

H122

D124

W93

L65

M67

F70

N220 C208

Zn2

Zn1

H250

V73

ACT

(G)




135

(A)

(B)



136






blue

(A) (B)

T235

T237

S130

H250

D124

H122

N220 H189

H120

C208

ACT

Zn2

Zn1

137


1

2682

329

443

21

2

2702 134

1237

21

3

2842

153

NA

NA

4

2542

395

NA

NA

5

2542

1544

NA

NA

6

2682

14

338

082

7

2842

57

NA

NA

8

2761

89

No inhibition

33

9

3331

047

654

13

10

2722

33

2302

33

11

2392

122

1902

64

12

2532

792

810

75

13

2672

42

562

34

14

2232

4471

7413

223

15

2232

4145

No inhibition

303


138


enem

Imip

enem

Com

pou

nd

9

Imip

enem

Com

pou

nd

11

Imip

enem

Com

pou

nd

13

E coli NDM-1 8 2128 2 128 2128

K pneumoniae KPC-2 64 1128 4 128 1128


E cloacae NDM-1 2 2128 2 128 2128




139

57 References



doi101016jcell201411017













doi1010802150559420171292196



4943ndash4960 doi101128AAC00296-11









54(3) 529-534 doi101128jcm02771-15



doi1011772049936115621709









140














60(9) 5454-5458 doi101128aac00711-16






128(6) 2250-2252 doi101002ange201510057



58(4) 1835-1846 doi101128aac00826-13




12406 doi101038ncomms12406


Reviews 24(1) 71-109 doi101128cmr00030-10






Chemistry 1-32 doi101007128_2011_180



doi101021bi3005126





141















doi101128aac00089-11





doi101074jbcm112436238





57(6) 2496-2505 doi101128aac02247-12






338 doi101042BJ20020957











142





177ndash182 doi101021ci049714




doi101107s0907444910048675






doi101107S0907444910045749



40(4) 658ndash674 doi101107S0021889807021206




213-221 doi101107s0907444909052925







doi101107S0907444910007493




1074ndash1080 doi101107S0907444909029436




doi101093nargkm216


452

143

Chapter 6

Summary


















144



















145

Appendix 1




371-387 doi101038nrd3975

146




147




doi101016jmib200508016

148






149




doi101128aac00296-11

150



1014 doi101016jcell201410051

151





152





153






doi101128aac02247-12

154

Appendix 2



155


156


157


158


159



vector 2 0125 05 025 0125 1

KPC-2 32 128 gt512 128 128 16

KPC-2 S130G 1 0125 128 64 025 025

160



Data Collection

Space group P22121

Cell dimensions

a b c (Aring) 5577 5997 7772

(deg) 90 90 90



Rmerge () 90 (912)

Iσ(I) 89 (18)



Refinement


Rwork () 147 (269)

Rfree () 170 (286)

No atoms

Protein 2160

LigandIon 66

Water 305

B-factors (Aring2)

protein 1498

ligandion 2865

water 3529

RMS deviations



Ramachandran plot

favored () 9888

allowed () 112

outliers () 0

PDB code 5WLA

161

Appendix 3


W105

H274 R220

T216

T235 T237 C238

C69

E166

L167

P104

N132

S130

K234 K73

S70

(A)

P104

N132 L167 W105

S130

T216

T235

R220 H274

T237

C69

C238

K234

K73

S70

E166

(B)



162

H120

H189

H122

F70 M67

V73

W93

L65

H250

C208

D124

N220

ACT

Zn2

Zn1

(A)

N220

F70 M67

L65

V73 W93

H250

C208

H120

H189

H122

D124

Zn2

ACT Zn1

(B)

L65

M67

F70 V73

W93

H250

C208

D124

N220

H122

H189 H120

ACT

Zn2

Zn1

(C)

H250

C208

H120 H189 H122

N220

D124

W93

V73

L65

M67 F70

Zn2

Zn1

ACT

(D)



163

L65

F70 M67

V73 W93

D124

H250

C208

H120

H189

H122 N220 ACT

Zn2 Zn1

(E)



Scholar Commons

November 2017


Orville A Pemberton



v

List of Figures













in the PDB29








vi
























vii











CTXCefotaximase



DTTDithiothreitol


EIEnzyme-inhibitor




HBHydrogen bond

viii




LBLysogeny broth






NCFNitrocefin

NDMNew Delhi MBL


OXAOxacillinase










WTWild-type

ix

Abstract



















bacteria

x























xi


in vivo activity





















1

Chapter 1

Introduction



















2
























3


burden




















4
























5






















6























7























8
























9







14 Carbapenemases
















10























11


[70]





















12






















13























14






















15









110 Conclusions













16


















Appendix 1)




17




















18




19





20


21




22



-Lactamases


C A D B

B1 B2 B3

23









24


25


26


27





Tazobactam

28


29


30


31

112 References






Boston MA Springer




from


nhtml





Updates 3(3) 155-160 doi101054drup20000146







252



doi101086533592


12(5) 371-387 doi101038nrd3975





doi103389fpubh201400145






doi101186s12879-016-1990-4

32


317(7159) 657-660 doi101136bmj3177159657


Nature 146(3713) 837-837 doi101038146837a0







doi101056nejm198802183180706








doi101016jcell201411017



doi101016jmib200508016





doi1010029780471729259mca03cs00







1145-1165 doi1021740929867023370031



doi101002chin200524267





33



doi101021ar0400241



Reviews 29(1) 29-57 doi101128cmr00019-15



















61(1) e01636-16 doi101128aac01636-16




doi101107s1399004713033014






doi101007s00894-011-1087-3






7(10) 918-925 doi10103879688

34













27(2) 241-263 doi101128cmr00117-13







6032 doi101039c7ob01514c



doi101128aac01512-08





doi1021741389450116666151001105622








doi101016jjmb200410070





35



4943-4960 doi101128aac00296-11












doi103923jm2006122
















897 doi101128aac00693-09



doi103201eid1710110655



doi101093jacdks121






36





doi101128aac115852













Therapy 35(8) 755-770 doi101002phar1622














251(2) 453-459 doi101042bj2510453







doi10151717460440903190961


Letters 3(6) 442-444 doi101021ml300114c

37



13(12) 12857-12879 doi103390ijms131012857





doi101038nprot2013130



doi101021bi3005126





146-157 doi102174157340911795677602












doi101007s10822-006-9060-4




1749 doi101021jm0306430





159(5) 995-1014 doi101016jcell201410051



doi1010160167-5699(93)90049-q



38




39

Chapter 2



21 Overview








22 Introduction








40





















41
























42
























43













process











44























45























46

















chains






47























48

24 Conclusions




















49






















50














26 Note to Reader 1





51




52



53
















54





T235 T237 C238

S130

K73

K270

1 2

55
















56






57



A B

58




Cefotaxime


Faropenem

Data Collection


Cell dimensions

a b c (Aring) 5580 6020 7862 5693 5878 7684 5714 5913 7738

(deg) 90 90 90 90 90 90 90 90 90



Rmerge () 3073 (362) 127 (684) 95 (888)

Iσ(I) 110 (20) 91 (23) 99 (22)

Completeness () 991 (944) 985 (956) 999 (100)

Multiplicity 65 (33) 65 (53) 70 (66)

Refinement


Rwork () 122 (261) 168 (319) 150 (257)

Rfree () 139 (269) 212 (345) 188 (275)

No atoms

Protein 2136 2090 2109

LigandIon 26 60 26

Water 350 255 223

B-factors (Aring2)

protein 1408 1558 2242

ligandion 2832 2202 3711

water 3196 3091 3678

RMS deviations



Ramachandran plot

favored () 9888 9888 9887

allowed () 112 112 113

outliers () 0 0 0


59


Distance (Aring)









Distance (Aring)












60

27 References



doi103201eid1710110655


















897 doi101128aac00693-09














3421-3427 doi101128aac4983421-34272005






61







15397-15407 doi101021ja051592u








doi101021bi0487903




278(52) 52724-52729 doi101074jbcm306059200








5665 doi101128aac00245-11



2126(02)00725-6







4943ndash4960 doi101128AAC00296-11



51(3) 721-725 doi101093jacdkg120



62
















doi101107s0907444910048675



50(5) 760-763 doi101107s0907444994003112



40(4) 658ndash674 doi101107S0021889807021206







doi101107s0907444904019158




1080 doi101107s0907444909029436




doi101093nargkm216


452




63

Chapter 3


Lactamase Acylation

31 Overview
















64

32 Introduction






















65























66
























67

















acidbase [14]






68

















34 Conclusions






69



















70












36 Note to Reader 1







71

4

3

2

1

6 7

8

5


72



73

K234

S130 K73

Y105

T235

S237

N170

E166

N132

S70



74




75

S70 S130

K73

E166

N170

Wat1





76

37 References



1145-1165 doi1021740929867023370031






doi1010801475636620161201813



doi101002chin200524267








57(5) 2372-2375 doi101128aac02622-12

















57(6) 2496-2505 doi101128aac02247-12




77









14(17) 4116-4128 doi101039c6ob00353b








5665 doi101128aac00245-11












x







doi101107s0907444904019158


452



78

Chapter 4



41 Overview















79

42 Introduction








for activity [6]















80






















81























82























83


inhibitor of KPC-2






E coli














84





















85
















KPC-2 [33]








86














group

44 Conclusions







87
























88
























89
























90























91





















92






















93












use


mutant proteins





software (GraphPad)

94


mutant proteins






NCF as substrate




[54]









95




mutant proteins



K k2


k-2













96












only










liquid nitrogen

97










46 Note to Reader 1




47 Note to Reader 2



98



S02030 Vaborbactam

99





T237 T235

C69 C238

L167

P104

W105

S130 K73

N132

K234

E166

S70

A

T235 T237

S70

N132

L167

K73

W105

S130

K234

B

100

T235

T237 S237

C69 C238 G238

N132

P104 N104

V240 G240 G239

S70

S130

W105 Y105



backbone shift

101

A

C69

S70

T235

S237

G238

G240

C69

C238

G239

T237

T235




B

S70

102


Vaborbactam 1

Avibactam 1

Tazobactam gt64



103




KPC-2 Avibactam 13 x 104 000022 77 17




104


-Lactamase 0 015 03 06 125 25 5 10 MPC16


CTX-M-15 8 8 4 2 1 05 025 le0125 25



105













106

48 References









58(2) 654-663 doi101128aac01222-13








doi101016S1367-5931(99)00017-4





58(4) 1835ndash1846 doi101128aac00826-13





doi101016jmib201107026









59(10) 6605ndash6607 doi101128AAC01165-15





107










60(3) 1760ndash1766 doi101128AAC02643-15












doi101021bi400413c








doi101021cb800306y











108



doi101021cr010182v











doi101074jbcm306059200




1340-1346














356 doi101006bioo20001184



2126(02)00725-6




doi101073pnas0813029106



109


doi101042bj20101122







546312132







174ndash183 doi101128AAC00930-10















57(6) 2496ndash2505 doi101128AAC02247-12










doi101128aac00711-12

110


















doi1010160006-2952(87)90609-5




doi101107S0907444910048675






doi101107S0907444910045749



40(4) 658ndash674 doi101107S0021889807021206








213ndash221 doi101107S0907444909052925



doi101107S0907444910007493

111




1080 doi101107s0907444909029436




doi101093nargkm216


452

112

Chapter 5



51 Overview













52 Introduction



113




















pathogens [13]



114














infections










115






















116
























117







development

















118
























119


compound 13






















120









to the protein










available analogs




121























122




negative bacteria

54 Conclusions


















123
























124























125


AmpC











sites








126
























127























128



















V= Vmax [S](Km + [S]) (1)

129




equation 2

Ki= IC50([S]Km + 1) (2)



(BSA)













130





















131

56 Note to Reader 1


132



133

R220 H274

T216

P104

L167 W105

K234

E166

N132

S130 K73

T235

S70

T237

C69 C238

(A)

R220 H274

C238 T237 T235

T216

K234

S130

W105

P104

L167 E166

N132

K73

S70

C69

(B)

C69

C238

H274 R220

T216

K234

S130

W105 N132 E166

P104

L167

K73

T235 T237

S70

(C)



contoured at 2

134

R205 Y67

W87

H116 H114

H179 D118

C198

H240 Zn2

Zn1

(D)

N210

C208

W93

L65

M67 F70

N220 H122

H189 H120

H250 D124

Zn2

ACT Zn1

(B)

V73

W93

V73

L65

M67 F70

C208

H250

D124

Zn2

H120 H189

N220 Zn1

H122 ACT

(C)

H250

V73

W93

L65 M67

F70

N220

H189 H120

C208

D124

ACT H122

Zn1

Zn2

(A)

H189

N220

F70 M67

L65

V73 W93

C208

H250 D124

H120

H122 Zn2

Zn1 ACT

(E)

W87

Y67

R205

H179

H114 H116

C198

H240

D118 Zn2 Zn1

(F)

N210

H189 H120

H122

D124

W93

L65

M67

F70

N220 C208

Zn2

Zn1

H250

V73

ACT

(G)




135

(A)

(B)



136






blue

(A) (B)

T235

T237

S130

H250

D124

H122

N220 H189

H120

C208

ACT

Zn2

Zn1

137


1

2682

329

443

21

2

2702 134

1237

21

3

2842

153

NA

NA

4

2542

395

NA

NA

5

2542

1544

NA

NA

6

2682

14

338

082

7

2842

57

NA

NA

8

2761

89

No inhibition

33

9

3331

047

654

13

10

2722

33

2302

33

11

2392

122

1902

64

12

2532

792

810

75

13

2672

42

562

34

14

2232

4471

7413

223

15

2232

4145

No inhibition

303


138


enem

Imip

enem

Com

pou

nd

9

Imip

enem

Com

pou

nd

11

Imip

enem

Com

pou

nd

13

E coli NDM-1 8 2128 2 128 2128

K pneumoniae KPC-2 64 1128 4 128 1128


E cloacae NDM-1 2 2128 2 128 2128




139

57 References



doi101016jcell201411017













doi1010802150559420171292196



4943ndash4960 doi101128AAC00296-11









54(3) 529-534 doi101128jcm02771-15



doi1011772049936115621709









140














60(9) 5454-5458 doi101128aac00711-16






128(6) 2250-2252 doi101002ange201510057



58(4) 1835-1846 doi101128aac00826-13




12406 doi101038ncomms12406


Reviews 24(1) 71-109 doi101128cmr00030-10






Chemistry 1-32 doi101007128_2011_180



doi101021bi3005126





141















doi101128aac00089-11





doi101074jbcm112436238





57(6) 2496-2505 doi101128aac02247-12






338 doi101042BJ20020957











142





177ndash182 doi101021ci049714




doi101107s0907444910048675






doi101107S0907444910045749



40(4) 658ndash674 doi101107S0021889807021206




213-221 doi101107s0907444909052925







doi101107S0907444910007493




1074ndash1080 doi101107S0907444909029436




doi101093nargkm216


452

143

Chapter 6

Summary


















144



















145

Appendix 1




371-387 doi101038nrd3975

146




147




doi101016jmib200508016

148






149




doi101128aac00296-11

150



1014 doi101016jcell201410051

151





152





153






doi101128aac02247-12

154

Appendix 2



155


156


157


158


159



vector 2 0125 05 025 0125 1

KPC-2 32 128 gt512 128 128 16

KPC-2 S130G 1 0125 128 64 025 025

160



Data Collection

Space group P22121

Cell dimensions

a b c (Aring) 5577 5997 7772

(deg) 90 90 90



Rmerge () 90 (912)

Iσ(I) 89 (18)



Refinement


Rwork () 147 (269)

Rfree () 170 (286)

No atoms

Protein 2160

LigandIon 66

Water 305

B-factors (Aring2)

protein 1498

ligandion 2865

water 3529

RMS deviations



Ramachandran plot

favored () 9888

allowed () 112

outliers () 0

PDB code 5WLA

161

Appendix 3


W105

H274 R220

T216

T235 T237 C238

C69

E166

L167

P104

N132

S130

K234 K73

S70

(A)

P104

N132 L167 W105

S130

T216

T235

R220 H274

T237

C69

C238

K234

K73

S70

E166

(B)



162

H120

H189

H122

F70 M67

V73

W93

L65

H250

C208

D124

N220

ACT

Zn2

Zn1

(A)

N220

F70 M67

L65

V73 W93

H250

C208

H120

H189

H122

D124

Zn2

ACT Zn1

(B)

L65

M67

F70 V73

W93

H250

C208

D124

N220

H122

H189 H120

ACT

Zn2

Zn1

(C)

H250

C208

H120 H189 H122

N220

D124

W93

V73

L65

M67 F70

Zn2

Zn1

ACT

(D)



163

L65

F70 M67

V73 W93

D124

H250

C208

H120

H189

H122 N220 ACT

Zn2 Zn1

(E)



Scholar Commons

November 2017


Orville A Pemberton



Mechanism Elucidation and Inhibitor Discovery against ...

Documents

Transcript of Mechanism Elucidation and Inhibitor Discovery against ...