The Career of Christopher T. Walshchemlabs.princeton.edu/.../sites/6/TDB-walsh.pdf · 1972-1987...

1

The Career ofChristopher T. Walsh

Supergroup Meeting May 9, 2007

Teresa D. Beeson

Outline

! Who is Christopher T. Walsh?

! Research

! Vancomycin Resistance

! Biosynthesis of Natural Products

! Siderophores

! Suicide Substrates

2

Biography

Christopher T. Walsh

Hamilton Kuhn Professor of Biological Chemistry and Molecular Pharmacology (Harvard Medical School)

Biography

1965 A.B. in biology (Harvard Univ.)

C. Walsh, J.H. Law, and E.O. Wilson. "Purification of the Fire Ant TrailSubstance." Nature 1965, 207, 320-321.

1970 Ph.D. in life sciences (Rockefeller Univ.)

Advisor Leonard B. Spector

6 publications, all first author

"The Mechanism of Action of the Citrate Cleavage Enzyme." Ph.D.Dissertation, The Rockefeller University.

3

Biography 1970-1972 Postdoctoral Fellow (Brandeis Univ.)

Advisor Robert H. Abeles

8 publications

1972-1987 Professor of Chemistry and Biology (MIT)

1987-1995 Chair, Dept. of Biological Chemistry and Molecular Pharmacology (Harvard Medical School)

1991-present Hamilton Kuhn Professor of Biological Chemistry and Molecular Pharmacology (Harvard Medical School)

1992-1995 President of Dana Farber Cancer Institute

Biography

Authored over 650 publications since 1965

Average of over 15 publicatons per year for 42 years

Former students and post docs include:

Michael Marletta (Berkeley) Robert Pascal (Princeton) Peter Schultz (Scripps) Greg Verdine (Harvard) Yian Shi (Colorado State) Thomas Wandless (Stanford)

4

Affiliations National Academy of Sciences

Institute of Medicine

American Academy of Arts and Sciences.

American Academy of Microbiology

Pharmaceutical consultant (Merck, Roche, and Abbot)

Scientific advisor (Microbia, Genzyme, Immunogen, KosanBiosciences, and Millennium)

Board of Directors (Critical Therapeutics, Kosan,Immunogen, Leukosite. Microbia, Transform, and Vicuron)

Awards

Eli Lilly Award in Biochemistry

Arthur C. Cope Scholar Award in Organic Chemistry

Repligen Award in Biological Chemistry

Alfred Bader Award in Bioorganic and Bioinorganic Chemistry

Promega Biotechnology Research Award from the AmericanSociety for Microbiology (ASM).

5

Suicide Substrates

Suicide Substrates

! What is a suicide substrate?

Walsh, C. T. Tetrahedron, 1982, 38, 871.

! A competitive inhibitor that is converted to an irreversible inhibitor

at the active site of the enzyme

! The enzyme causes it's own destruction by unmasking a latent

functional group

Alkylated Enzyme

N

OH

HOMe

O

HN

OH

Me

O

N

O

Me

O

HN

OH

Me

O

AcetaminophenTylenol

P450

NADPH, O2 – H2O

Imino quinone

P450

6

Suicide Substrates

! Suicide substrates in medicine

Walsh, C. T. Tetrahedron, 1982, 38, 871.

! Chemotherapy

! Can be used study the role of proteins in vitro or in vivo

HN

NH

5-Fluorouracil

! Suicide substrates for research

O

O

FHO NH2

O

F F

NH2

DFMO

O

O

OH

Me

Me

H H

H

Formestane

! Antibiotics

N

S

O

OHO

OO

Sulbactam

NH

OO

NH2

D-cycloserine

Suicide Substrates

! Enzyme Target: E. coli B Alanine Racemase

Wang, E.; Walsh, C. T. Biochemistry, 1978, 17, 1313.

! Alanine racemase epimerizes L-alanine

! Natural Antibiotics for Alanine Racemase

! D-Ala-D-Ala binding site for Vancomycin

NH2

MeOH

O

NH2

MeOH

O

NH2

MeOH

O

L-alanine D-alanine

alanine racemase

! D-Alanine incorporated into bacterial cell walls

! Racemases not found in mammalian cells

NH

OO

NH2

D-cycloserine O-carbamoyl-D-serine

H2N O OH

O

NH2

O

7

Suicide Substrates

! Haloalanines also shown to be bacteriocidal


! Time-dependant inhibition of alanine racemase

! What is the mode of enzyme deactivation?

NH2

OH

O

NH2

OH

O

D-!-chloroalanine D-!-fluoroalanine

! Deuterated analogues more potent antibiotics

Cl F

NH2

OH

O

F

NH2

OH

O

Cl

DD D D

Suicide Substrates

! Observations from the Walsh Lab


! Time-dependant inactivation of alanine racemase by fluoroalanine

! Enzyme activity was never regained after filtration or competitive

binding

! 14C-Labeled fluoroalanine caused 14C-labeling of enzyme

! Haloalanines deemed to be irreversible inhibitors

8

Suicide Substrates

! Observations from the Walsh Lab


! Alanine racemase did not epimerize haloalanines

! D-haloalanines much faster than L-haloalanines but...

! Turnovers before inactivation ~800 for all haloalanines

NH2

MeOH

O

NH2

MeOH

O

NH2

MeOH

O

L-alanine D-alanine

alanine racemase

NH2

OH

O

MeOH

O

Pyruvate

alanine racemase

O

NH3 Cl–Cl

! Is a common inactivator or "suicide substrate" formed?

Suicide Substrates

! Explaining the observations

NH3

Cl


O–

O

Alkylated Enzyme

O–

O

NH3

x

! Simple nucleophilic attack by enzyme we would expect less

turnovers before inactivation for Cl than F

! Haloalanines must be suicide substrates

9

Aldimine complex

N

H NH

H

Cl

H

Me

OP

O–O

HO

Pyridoxal-P

N

H O

H

Me

OP

O–O

HO

OHOH

Suicide Substrates


NH3

Cl

–H+

N

H NH

Cl

H

Me

OP

O–O

HO

OH

Enamino acid complex Electrophilic species

N

H NH

H

Me

OP

O–O

HO

OH– Cl–


O–

O

O–

O

O–

O

O–

O

Suicide Substrates


Electrophilic species

N

H NH

H

Me

OP

O–O

HO

OH

NH3

H2O H2O

O

Me

Pyruvate


O–

O

O– O–

OO

10

Suicide Substrates


Electrophilic species

N

H NH

H

Me

OP

O–O

HO

OH

NH3

H2O H2O

O

Me

Pyruvate

N

H NH

H

Me

OP

O–O

HO

OH

Enzyme nucleophilic attack

H2O

O

Alkylated Enzyme


O–

O

O–

O

O–

O

O–

O

O–

O

Suicide Substrates

! Natural D-serine antibiotics determined to be suicide substrates


! Turnovers per inactivation also ~800

NH

OO

NH2

D-cycloserine O-carbamoyl-D-serine

H2N O OH

O

NH2

O

O-acetyl-D-serine

Me O OH

O

NH2

O

! D-serine does not inactivate alanine racemase

D-serine

HO OH

O

NH2

11

Suicide Substrates

! Propargyl suicide substrates operate in a similar manner

Johnston, M.; Jankowski, D.; Marcotte, P.; Tanaka, H.; Esaki, N.; Soda, K.; Walsh, C. T. J. Am. Chem. Soc., 1979, 21, 4690.

N

H NH

H

OP

O–O

HO

OH

O–

O

Me N

H NH

H

OP

O–O

HO

OH

O–

O

•

Me N

NH

H

OP

O–O

HO

OH

O–

O

Me

N

N

H

OP

O–O

HO

OH

O–

O

Me

Me

H

N

N

H

OP

O–O

HO

OH

O–

O

Me

Me

H

H+

NH3

O–

O

Me

SR

Suicide Substrates

! Allyl sulfoxide suicide substrates undergo 2,3-rearrangement

Johnston, M.; Raines, R.; Walsh, C.; Firestone, R. A. J. Am. Chem. Soc., 1980, 4241.

N

NH

H

–HO3POOH

O–

O

Me

S

R

O

Cl

N

NH

H

–HO3POOH

O–

O

Me

S

R

O

!-Elim

N

NH

H

–HO3POOH

O–

O

Me

OS

R

O

O–

OOH

sulfenate ester

12

Vancomycin Resistance


! Background: Vancomycin's mode of Action

Walsh, C. T.; Fisher, S. L.; Park, I-S.; Prahalad, M.; Wu, Z. Chemistry & Biology, 1996, 3, 21.

Disaccharide pentapeptide units are cross-linked to build rigid bacterial cell walls

Vancomycin-PG-D-Ala-D-Ala complex sterically prevents further cell wall assemply

13


! The Key Missing H-Bond


! Key H-bond lost due to replacement of D-Alanine for D-Lactate

! 1000-fold reduction in Kd


! 5 Genes Necessary for Vancomycin Resistance


Protein

vanR vanS vanH vanA vanXPvanR PvanH

! All 5 encoded proteins isolated and characterized by Walsh Lab

Activity Function

VanR

VanS

VanH

VanA

VanX

Transmembrane histidine kinase

Response regulator

D-specific !-keto acid reductase

Depsipeptide ligase for D-Ala-D-lactate

D-Ala-D-Ala dipeptidase

Initiates signal transduction

Activates vanH,A,X transcription

Generates D-lactate

Generates D-Ala-D-lactate

Removes D-Ala-D-Ala

14


! VanR and VanS: Enteroccocal Bacteria's "Sensor"


Phosphate transferred to VanR, which causes conformational change

VanR-PO3 is DNA-binder to promoter region of vanH,A,X, induces transcription

In the absence of Vancomycin, VanH,A,X genes are not transcribed

VanS located in the bacterial cell wall, phosphorylated upon external signal


! Key Change from Alanine to Lactate


MeO

O

O

MeO

O

OH

VanH

Pyruvate D-Lactate

H2NO

O

Me

VanA

D-Ala-D-Lactate

MeO

O

OH

D-Lactate

O

Me

O

H2N

HN

O

Me

D-Ala-D-Ala

O

Me

O

H2N

HN

O

Me

D-Ala-D-Ala

O

Me

O

VanX H2N

2 D-Ala

O

Me

O

2

15


! The Key Missing H-Bond


! Key H-bond lost due to replacement of D-Alanine for D-Lactate

! 1000-fold reduction in Kd

Siderophores

16

Siderophores

! “Bacterial-Host Iron Wars”

Fischbach, M. A; Lin, H.; Liu, D.; Walsh, C. T. Nature Chem.Bio., 2006, 2, 132.

! Iron required for bacterial growth

! Concentration of Fe(III) in water 10–18 M

! Concentration of Fe(III) in human serum ~10–24 M

! Bacterial growth requires cytoplasmic Fe(III) concentration of ~10–6 M

! Bacteria secrete "siderophores" to scavange iron

! "Sidero" means iron; "phore" means to carry in greek

! Low molecular weight molecules with high Fe(III) affinity

Siderophores


17

Siderophores

! Common siderophores

O

O O O

O

O

HN

HN

NH

O

O

O

OH

OH

OH

OH

HO

HO

Enterobactin

E. coli

Salmonella

Me

N

HN NH

HO

O O

OH

N

Me

OH

O

HO

O O

O O

OH HO

N

HNO

NH

O

O

OH

O

N

OH

O

O

N

O

OH

Me

Mycobactin J

M. paratuberculosis

"tuberculosis"

N

S

OH

NH

S OH

N

SO

OHMe

Yersiniabactin

Y. pestis

"plague"

Aerobactin

E. coli

H

Siderophores

! Siderophores studied by the Walsh Lab

! Mycobactin from Mycobacterium tuberculosis

! Pyochelin from Pseudomonas aeruginosa

! Yersiniabactin from Yersinia pestis "plague"

! Vibriobactin from Vibrio cholerae "cholera"

! Enterobactin from E. coli and salmonella

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Siderophores

! Enterobactin-mediated iron aquisition


O

O O O

O

O

HN

HN

NH

O

O

O

OH

OH

OH

OH

HO

HO

Enterobactin

Fe(III) Kd = 10–49 M

Siderophores

! Enterobactin-mediated iron aquisition


O

O O O

O

O

HN

HN

NH

O

O

O

OH

OH

OH

OH

HO

HO

Enterobactin

Fe(III) Kd = 10–49 M

"It can rip iron out of stainless steel" – C. T. Walsh

19

Siderophores


! Production of iron scavangers alone not sufficient

! Iron-free form must be secreted through bacterial cell wall

! Fe(III) scavanged from surrounding environment

! Iron-bound form imported through bacterial cell wall

Siderophores

! E. coli turn on 15 genes when iron deficient


! EntA-F: Synthesis of enterobactin from chorismate and serine

! EntS: Export out of cell wall

! FepA-G: Import of iron-bound enterobactin

! Fes: Release of Fe(III) from enterobactin

20

Siderophores

! Immune system's response to enterobactin


! Siderocalin, from neutrophils, binds enterobactin

! Serum albumin binds enterobactin

! Enterobactin sequestered and rendered ineffective

Siderophores

! "Bacteria fight back"


! IroB and IroE: modification of enterobactin before export

! iroA: 5 gene cluster, encodes 5 proteins

! IroN: Import of iron-bound modified-enterobactin

! IroC: Export of modified enterobactin

! IroD: Release of iron from complex

21

Siderophores



! IroB C-glycosylation of enterobaction

O

O O O

O

O

HN

HN

NH

O

O

O

OH

OH

OH

OH

HO

HO

Enterobactin

O

O O O

O

O

HN

HN

NH

O

O

O

OH

OH

OH

OH

HO

HO

O

O

OH

OHOH

HO

OH

OHHO

HO

Salmochelin S4 (DGE)

IroB

! Biologically very rare, extremely stable

Siderophores



! IroE linearization of Salmochelin followed by IroC-mediated export

O

O O O

O

O

HN

HN

NH

O

O

O

OH

OH

OH

OH

HO

HO

O

O

OH

OHOH

HO

OH

OHHO

HO

Salmochelin S4 (DGE)

IroE

O

O HO O

O

O

HN

HN

NH

O

O

O

OH

OH

OH

OH

HO

HO

O

O

OH

OHOH

HO

OH

OHHO

HO

OH

Salmochelin S2

! Salmochelin S2 30-fold lower lipid partition coefficient than enterobactin

22

Siderophores



! IroD exhaustively hydrolyzes iron-bound Salmochelins

IroD

O HN

O

OH

OH

OH

O

OH

HOHO

OH

OH

HN

O

OH

OH

OH

O

OH

Fe(III)

Salmochelin SX

2

Iron-bound Salmochelin S2

! Steric bulk and hydrophilic nature of glucoses prevent siderocalin binding

O

O

NHO

OHO

HO

O

HN

O

O

O

O

HN

OO O

OO

O

HO

HO OH

HO

23

Natural ProductBiosynthesis

Nature's Assembly Lines

Fischbach, M. A.; Walsh, C. T. Chem. Rev., 2006, 106, 3468.

24

Nature's Assembly Lines


! Many biologically-active natural products fall into 3 classes

! Polyketides (PK)

! Non-ribosomal peptides (NRP)

! Hybrids of polyketides and non-ribosomal peptides

! Comprised mainly of malonyl-CoA, methylmalonyl-CoA

! Building blocks of amino acids and aryl acids

HO S

O O

CoA HO S

O O

CoA

Me

P OH

O

N

N N

N

NH2

O

O–

–O O

P

O

O

O–

P

O

O–

O

HN

HN

S

OH

OO

s-CoA =

Natural Product Assembly Lines

! Yersiniabactin is a NRP-PK hybrid siderophore

Miller, D. A.; Luo, L.; Hillson, N.; Keating, T. A.; Walsh, C. T. Chemistry & Biology, 2002, 9, 333.


OH

S

HN

N

S

HO

S

N

O

HO

Me

H

Yersiniabactin

! Yersiniabactin has been synthesized in 35 chemical steps

! Enzymolic assembly line uses 15 steps

Ino, A.; Murabayashi, A. Tetrahedron, 2001, 57, 1897.

25


! Functional "domains" within proteins form assembly line


TE

E

Ox

AL

CE

Hal

Cy

Cyp

T

KS

AT

C

CLF

ER

DH

MT

Re

AMT

thiolation

ketosynthase

acyltransferase

condensation

chain length factor

adenylation

thioesterase

Enoylreductase

dehydratase

ketoreductase

cyclization

condensation/epimerization

epimerization

methyltransferase

reductase

oxidase

acyl-CoA ligase

aminotransferase

halogenase

cyclopropanase

A

KR


! Acylation of "T" thiolation domain starts NRP and PK assembly


TT

AACy Cy

T

S–

T

SH

A

B

A


OOH

OH OH

O

N

N N

N

NH2

OP

O

O

O–

P

O

O–

OP

O

O–

–O

ATP OH OH

O

N

N N

N

NH2

OP

O

O–

O

OOH

T

ACyT

S

A

O

HO

OH

~ ~

~

26


! Cyclization "Cy" domains catalyze thiazoline/oxazoline formation



T

ACyT

S

A

O

HO

~S

O

HS

N

H

H

B

T

ACyT

A ~S

O

B

HS NH

O

HO

T

ACyT

A ~S

O

B

S NH

OHHO

T

ACyT

A ~S

O

B

S N

OH

B B


! Additional cyclization "Cy" domains & tandem heterocyclizations



T

ACyT

A

S

O

SN

HO

CyT

S

O

H2N

HS

T

ACyT

A

S

O

S N

CyT

N

S

~ ~

HO

27

T

AT

OH

S

O–

O

O

KST

S

O

Me

SH

S

O

Me

SH


! PK ketosynthase "Ks" domains catalyze C–C bond formation


T T

SHS–

AT

SCoA–O

O O

AT

OH O

O–

O

O

KS KST

AcS

T

AcS

T

AT

S

O–

KST T

AT

S

KST

O

O

Me

MT MT MT

MT MT


~ ~ ~

~ ~

S

O

S N

SH

N

SHO


! PK ketosynthase "Ks" domain also catalyzes chain transfer



TAT

S

O–

O

O

KS

MT ~KRT

ACyT

A

S

O

S N

CyT~

NRP Assembly Line PK Assembly Line

TAT

S

O–

O

O

KS

MT ~KR

Hybrid NRP-PK

N

SHO

28


! Methyltransferase "MT" domains transfer Me from SAM-Me



TAT

KS

MT ~KR

OH

S N

N

SHO

S

O

TAT

KS

MT ~KR

O

S N

N

SHO

S

O

2 Me-SAM


! Ketoreductase "KR" domains reduce ketones with NADPH



TAT

KS

MT ~KR

O

S N

N

SHO

S

O

TAT

KS

MT ~KR

OH

S N

N

SHO

S

O

NADPH

N

H HO

NH2

29


! Cyclization then methylation yields quaternary stereocenter



TAT

KS

MTKR

OH

S N

N

SHO

S

O

Cy TMT

S

O

NH2

HS

TAT

KS

MTKR

OH

S N

N

SHO

Cy TMT

TE TECyclization

Methylation

S

N

O

S

Me

HO


! Terminal thioesterase "TE" hydrolyzes ester linkage



TAT

KS

MTKR

OH

S N

N

SHO

Cy TMT

TEYbtU

post-translationalreduction

S

N

O

S

Me

OH

S

HN

N

S

HO

S

N

O

HO

Me

H

Yersiniabactin

30


! How big are the assembly lines?



! Most are >1 MDa or 10,000 amino acids

! Tyrocidine synthetase has 30 domains in 3 proteins, ~1 MDa

! Largest known is PK ECO-02301

! 122 domains

! 9 proteins

! 4.7 MDa, 47,000 amino acid residues

Macrocyclization of Natural Products

! Macrocycles Constrain, Induce Bioactivity

Tseng, C. C.; Bruner, S. D.; Kohli, R. H.; Marahiel, M. A.; Walsh, C. T.; Sieber, S. A. Biochem, 2002, 41, 13350.

! Structures of cyclic natural products from NRPS (surfactin, daptomycin, iturin, ramoplanin, and tyrocidine) and PKS (erythromycin) systems.

31

Macrocyclization of Antibiotics

! Thioesterase Identified as Catalyst for Macrocyclization

Trauger, J. W.; Kohli, R. H.; Mootz, H. D.; Marahiel, M. A.; Walsh, C. T. Nature, 2000, 407, 215.

! HPLC analysis shows disappearance of pentapeptide and appearance of Tyrocidine A in the presence of TyrC Thioesterase


! Tyrocidine Thioesterase Catalyzes Macrocyclization


a) Tyrocidine non-ribosomal peptide synthetaseb) Proposed mechanism and experimental system used as a probe

32


! Thioesterase Proposed to Catalyze Gramicidin Formation


! Proposed thioesterase catalyzed dimerization and macrocyclization


! Thioesterase Catalyzes Macrolactonization to Epothilone C

Boddy, C. N.; Schneider, T. L.; Hotta, K.; Walsh, C. T.; Khosla, C. J. Am. Chem. Soc., 2000, 125, 3428.

33

What Now?

Combinatorial Biosynthesis

! Solid-phase macrocyclization using thioesterase

Kohli, R. M.; Walsh, C. T.; Burkart, M. D. Nature, 2002, 418, 658.

PEGA O O

Leu–Orn–Val–Tyr–Gln–Asn–DPhe–Phe–Pro–DPhe-NH2

Tyrocidine A

Isolated thioesterase domain

TE

34


! D-Phe replaced by natural and unnatural amino acids


PEGA O O

Leu–Orn–Val–Tyr–Gln–Asn–XXX–Phe–Pro–XXX-NH2

! Library of over 300 new antibiotics synthesized


! New antibiotics show different activity spectrum


Eukaryotic (MHC, µM) Human erythrocytes

Gram negative (MIC, µM) E. coli P. aeruginosa P. putida

Gram positive (MIC, µM) B. subtilis MRSA S. epidermidis

4

>290290

>290

122

60

1203030

274

150

2903774

5189

Tyrocidine A !A02 !F10

! Higher selectivity for baterial over human cells

35

Summary

! Chris Walsh's publication record puts us all to shame

! Research encompasses vast number of enzymes

! The 5 genes of vancomycin resistance

! Assembly-Line enzymology of natural products

! "Bacterial-Host iron wars" Siderophores

! "Trojan Horse" suicide substrates

The Career of Christopher T. Walshchemlabs.princeton.edu/.../sites/6/TDB-walsh.pdf · 1972-1987...

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Transcript of The Career of Christopher T. Walshchemlabs.princeton.edu/.../sites/6/TDB-walsh.pdf · 1972-1987...