Sungkyunkwan University School of Medicine

AminoglycosidesAminoglycosides

Kwan Soo Ko

Sungkyunkwan University School of Medicine

StreptomycinThe first aminoglycosideFrom Streptomyces griseu by Waksman SA (1943)

Selman Abraham Waksman (1888-1973)

"for his discovery of streptomycin, the first antibiotic effective against tuberculosis“ (Nobel Prize, 1952)


g ( , )

Aminoglycosides Source Year reported

Streptomycin Streptomyces griseus 1944

Neomycin Streptomyces fradiae 1949

Kanamycin Streptomyces kanamyceticus 1957

Paromomycin Streptomyces fradiae 1959

Gentamicin Micromonospora purpurea & Micromonospora echinospora 1963

Tobramycin Streptomyces tenebrarius 1967Tobramycin Streptomyces tenebrarius 1967

Amikacin Streptomyces kanamyceticus 1972

Netilmicin Micronomospora inyoensis 1975Netilmicin Micronomospora inyoensis 1975

Stectinomycin Streptomyces spectabilis 1961

Sisomicin Micromonospora inyoensis 1970Sisomicin Micromonospora inyoensis 1970

Dibekacin Streptomyces kanamyceticus 1971

I i i Mi 1978


Isepamicin Micromonospora purpurea 1978

AminocyclitolAminocyclitol

E ti l 6 b d i ith i b tit tEssential 6-membered ring with amino group substituents

Glycosidic bonds between the aminocyclitol and two orGlycosidic bonds between the aminocyclitol and two or more amino-containing or non-amino-containing sugars

→ Aminoglycosides g y


Central aminocyclitolCentral aminocyclitol

Streptomycin OthStreptomycinSpectinomycin

Others

streptidin 2-deoxystreptidine


Streptomycin1944 Neomycin

1949Kanamycin

1957

Paromonmycin1965

Gentamicin C1963

Ribostamycin1970

Tobramycin1970

Sisomicin1970

Spectinomycin1971

Burirosin1971

Dibekacin1971

Lividomycin1972

Amikacin1972

Gentamicin B1972

Arbekcain1973

Netilmicin1974


50 Years of ICAAC, 1961-2010 Isepamicin1977

Antimicrobial activity• Bind with high avidity to a region of highly conserved

Antimicrobial activityg y g g y

nucleotides in the mRNA decoding region of the 30S ribosomes

Changes consistent with aminoglycoside binding to anaminoglycoside binding to an asymetric interval loop within the A site

Conformational change in two AConformational change in two A residues (A1492 & A1493), resulting in interference with mRNA


translation and translocation

Other biological activities• The subjects of ongoing study

Other biological activitiesj g g y

- genetic disease from point mutations producing premature stop codons

) C ti fib i (t b d teg) Cystic fibrosis (transmembrane conductance regulator protein), Duchenn’s muscular dystrophy, the Hurler syndrome and nephrogenic diabetes insipidusHurler syndrome, and nephrogenic diabetes insipidus

• Aminoglycosides can suppress premature stop codons and restore physiologically active amounts of functional protein in CF

• Aminoglycosides bind to and modulate the function of other RNA or RNA-related molecules


other RNA or RNA related molecules

Enzymatic inactivation

• ATP-dependent phosphorylation of a hydroxyl group by

Enzymatic inactivation

p p p y y y g p ya phosphotransferase (APH)

ATP d d d l i f h d l b• ATP-dependent adenylation of a hydroxyl group by a nucleotidyltransferase (ANT)

• Acetyl coenzyme A-dependent acetylation of an amino group by an acetyltransferase (AAC)g p y y ( )

Loss of antibacterial activity


Mechanisms of antimicrobial activityMechanisms of antimicrobial activity

Aminoglycosides require aerobic energy to enter the cell and bind to ribosome

Combination of trapping of high concentrations of drug, RNA mistranslation with aberrant protein productionRNA mistranslation with aberrant protein production, and cell membrane dysfunction

→ Bacterial cell death Bacterial cell death


Binding to ribosomes- PrerequisitePrerequisite- Reversible, so bacteriostatic effect- Additional unidentified mechanisms of bactericidal activity

Stimulation of hydroxyl radical formation in bacteria as aStimulation of hydroxyl radical formation in bacteria as a function of metabolism-related depletion of reduced NADH, destabilization of iron-sulfur clusters, and stimulation of thedestabilization of iron sulfur clusters, and stimulation of the Fenton reaction ?


Initial BindingInitial Binding

Initial binding of aminoglycosides to the cell surface→ two energy-dependent uptake phases→ binding to ribosomes→ binding to ribosomes

Rapid & energy-independent p gy p

In gram-negatives, - Cationic aminoglycosides bind to negatively charged

residues in the LPS polar heads of phospholipids andresidues in the LPS, polar heads of phospholipids, and anionic outer membrane protein


Competitively displace cell wall Mg2+ and Ca2+ bridges(normally link adjacent LPS molecules)

↓

Rearrangement of LPS with subsequent bleeding of OMFormation of transient holes in CWFormation of transient holes in CWDisruption of CW’s normal permeability function

After initial binding

Transport of aminoglycosides across the bacterial cytoplasmic membrane by energy-dependent mechanism


Energy-dependent phasesEnergy-dependent phases

Two phases

Initial slow energy dependent phase (EDP I)Initial slow energy-dependent phase (EDP-I)- Transports the drug into the cytosol- Be inhibited by divalent cations, elevated osmolarity, low Onset of bacterial killingy , y,

pH, and anaerobic conditionsOnset of bacterial killing

Subsequent rapid energy-dependent phase (EDP-II)- Binding to ribosomes


Aminoglycoside ResistanceAminoglycoside Resistance

Intrinsic resistance- Nonenzymatic or enzymaticy y

A i d i tAcquired resistance- Reduced entry or efflux- Enzymatic modificaiton- Enzymatic modificaiton


Intrinsic resistanceIntrinsic resistance

Anaerobic bacteria- For entrance of aminoglycosides to a bacterial cell, an

active electron transport chain to generate an electrical Nonenzymaticpotential difference across the membrane is required

Mutations at the 16S rRNAMutations at the 16S rRNA- M. tuberculosis to streptomycin as a result of point

mutations in ribosomal protein S12 and in the 16S rRNAp- M. abscess and M. chelonae to amikacin

M h l i difi i h 16S RNAMethylating enzymes modifiying the 16S rRNA- In a growing number of aminoglycoside-resistant clinical

isolates worldwide


isolates worldwide

Acquired resistanceAcquired resistance

Combination of decreased drug uptake, efflux pump, and enzymatic modification of drug

Aminoglycosides induce biofilm formationdifficult to treat chronic infections- difficult to treat chronic infections


Low-level aminoglycoside resistanceg yattributed to impaired cell wall permeability- result of drug efflux mechanisms

MexXY efflux pumpIn P aeruginosa- In P. aeruginosa,

- Necessary for inhibitory effect of divalent cations- Adaptive resistancep

- transient resistance to aminoglycosides following the rapid, early, concentration-dependent killing of susceptible bacteria - refractory state last beyond the postantibiotic effective period into the time of regrowthperiod into the time of regrowth

- In vitro in animal models & in patients with CF


Exposure of susceptible bacteria to aminoglycosidesExposure of susceptible bacteria to aminoglycosides

Two types of ressistant subpopulations

Activation of MexXY(adaptive resistance)

Small colony variantswith deficient energy dependent(adaptive resistance) with deficient energy-dependent

uptake of aminoglycosides


MexXY Small colony variants


Enzymatic Modification

• Amino groups by N-acetyltransferases (AAC)

Enzymatic Modification

g p y y ( )- acetyl-coenzyme A as donor

H d l b O l id l f (ANT) O• Hydroxyl grous by O-nucleotidyltransferases (ANT) or O-phosphotransferases (APH)- ATP as donor- ATP as donor

Poor binding to ribosomes & high levels of resistanceg g


Aac (6’)-IaAac (6 ) Ia

Acetylating enzmexAcetylating enzmexthat modifies aminoglycosides at the 6’ position;same resistance profile with that of Aac(6’)-Ib,same resistance profile with that of Aac(6 ) Ib,

but unique enzyme


Tetracyclines & ChloramphenicolTetracyclines & Chloramphenicol

Kwan Soo Ko


Tetracyclinesy

Broad-spectrum bacteriostatic antibioticsBroad-spectrum bacteriostatic antibiotics- gram-positive bacteria- gram-negative bacteriag g- intracellular organisms such as chlamydiae, mycoplasmas,

rickettsiae, & protozoa

Low costP it f j id ff tPaucity of major side effects


In 1945 by screening organisms from soilBenjamin M. DuggarStreptomyces aureofaciensAuromycin (chlortetracycline)


Chlortetracycline1945

Tetracycline1952

Minocycline1968

Tigecycline1998

Streptomyces aureofaciens(B. Duggar)

Oxytetracycline1949

Doxycycline1966

Streptomyces rimosus(Finlay et al.) 50 Years of ICAAC, 1961-2010


Chopra & Robert. MMBR (2001)


Hydronaphthacene nucleus with four fused ringswith four fused rings


Glycylcyclines


Inhibition of bacterial protein synthesisInhibition of bacterial protein synthesis

By binding the 30S ribosomal subunit; reversible (bacteriostatic)

Blocks the association of the aminoacyl-tRNA t t it RNA ibtRNA to acceptor site on mRNA-ribosome complex→ prevents the addition of new amino acid→ prevents the addition of new amino acid


Enter the outer membrane of gram-negative bacteria by passive diffusion through porin channels OmpF and p g p pOmpC

→ Dissociation of complex in periplasmic spacet th i b b diff i→ enter the inner membrane by diffusion

(lipophilic tetracyclines)

Lipophilic form enters the cytoplasmic space of gram-y gpositive bacteria driven by a process dependent on the ΔpH


High affinity for bacterial ribosomal subunit 30S

Inhibition of mitochondrial protein synthesis

& weak interaction with 80S ribosomal subunit of

k t

protein synthesisby binding 70S ribosomal subunits

eukaryotes

Absence of accumulation andEfficacy of tetracyclines in eukaryotic parasitesAbsence of accumulation and

toxic effects in eukaryotesy p

Doxycycline & tetracycline against malaria


against malaria

Resistance to tetracyclinesResistance to tetracyclines

By acquisition of genes on mobile elements- 33 different genes of tetracycline resistance

29, tetracycline resistance gene (tet) family3, oxytetracycline resistance gene (otr) family

By point mutations in ribosomal RNAor by activity of innate bacterial efflux proteins (rare)or by activity of innate bacterial efflux proteins (rare)

Decrease in porin content of OM (low-level)


Tetracycline resistance genesTetracycline resistance genes

Two main mechanisms- Efflux pump or ribosomal protection (or sometimes both)

Enzymatic inactivation by tet(X), found only in Bacteroides


Mechanisms of resistance to tetracyclines for ycharacterized tet and otr genes

Mechanism GenesMechanism GenesEfflux tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H), tet(I), tet(J),

tet(Z), tet(30), tet(31)tet(K), tet(L)

otr(B), tcr3

t tP(A)tetP(A)

tet(V)

tet(Y)tet(Y)

Ribosomal protection tet(M), tet(O), tet(S), tet(W)

tet(Q), tet(T)

otr(A), tetP(B), tet

Enzymatic tet(X)


Unknown tet(U), otr(C)

Efflux pumpsEfflux pumps

Membrane-associated proteins exporting tetracyclines from the cell, reducing the intracellular concentrations and making them ineffective

M t i t t t t li b t t t d li dMost, resistance to tetracycline but not to doxycycline and minocycline

Six different classes of efflux proteins- based on amino acid sequence identity


Group 1- Tet(A), Tet(B), Tet(C), Tet (D), Tet(E), Tet(G), Tet(H),

Tet(Z), probably Tet(I), Tet(J), and Tet(30)- 12 predicted transmembrane α-helices with long central

nonconserved cytoplasmic loops (helices 6 7)nonconserved cytoplasmic loops (helices 6-7)- In gram-negative bacteria, except for Tet(C)

Group 2- Tet(K) and Tet(L)Tet(K) and Tet(L)- 14 predicted transmembrane α-helices- Resistance to tetracycline & chlortetracycline- In gram-positive bacteria


Group 3- Otr(B) & TcrC- In Streptomyces spp.- Topology similar to group2

Group 4- Tet A(P) from Clostridium spp.Tet A(P) from Clostridium spp.- 12 predicted transmembrane α-helices

Group 5- Tet(V) from Mycobacterium smegmatics

Group 6- Unnamed determinants from Corynebacterium striatum


Unnamed determinants from Corynebacterium striatum

Ribosomal protection proteins (RPPs)Ribosomal protection proteins (RPPs)

Cytoplasmic proteins protecting ribosome from the action of both first- and second-generation tetracycline

→ wider spectrum of resistance than efflux proteins

R l t t li f t t it b GDP d d tRelease tetracyclines from target site by GDP-dependent mechanism

Conformational modification preventing tetracycline bindingConformational modification preventing tetracycline binding without interfering with protein synthesis

Mainly in gram-positive bacterian and some nonenteric gram-negative bacteria and anaerobes


GlycylcyclinesGlycylcyclines(Tigecycline)


TigecyclineTigecycline

Higher binding affinities for ribosome than tetracycline (5x)→ be unaffected by bacterial ribosomal protection proteins

Evade efflux pumps present in tetracycline-resistant strains

* Overexpression of multidrug efflux pumps→ decreased susceptibility to tigecyclinedec eased suscept b ty to t gecyc e

A. baumanniiK. pneumoniae producing carbapenemasESBL-producing Enterobacteriaceae


RIBOSOMAL PROTECTION

Tetracycline blocked from binding

Tigecycline is still able to bind to ribosomeg to bind to ribosome


EFFLUX PUMP PROTECTION

Efflux Efflux pump pump

Tetracycline pumped out of cell

Tigecycline cannot be pumped out of cell


Tigecycline binding to 30S subunitg y gComparison of Tetracycline and Tigecycline docking

tetracycline Side chain results in increased in binding gstrength

tigecycline


Chloramphenicolp

Was isolated from fermentation of Streptomyces venezuelaeWas isolated from fermentation of Streptomyces venezuelae by scientist from Parke Davis led by Ehrlich in 1947

In clinical use in 1961Association with aplastic anemiaPathogenicity of anerobes & emergence of ampicillinPathogenicity of anerobes & emergence of ampicillin-

resistant H. influenzae → brief resurgence

Toxicity and widespread resistance has severely limited its clinical use in developed countries

- Only as alternative therapy in seriously ill patients & for patients infected with highly resistant pathogens, and as an alternative for anthrax or plague


alternative for anthrax or plague

However,

Broad spectrum of activity (gram-positive and gram-negative bacteria, anaerobes, spirochetes, rickettsiae, chlamydia,

d l )and mycoplasmas)Excellent tissue penetrationInexpensiveInexpensive

First-line therapy for enteric fever and other infections in many developing countriesmany developing countries


Structure (27-2)

The first antibiotic whose chemical synthesis ywas economically and technically practical for large-scale production


g p

Action mechanism of chloramphenicol

Inhibit protein synthesis by reversibly binding to the largerInhibit protein synthesis by reversibly binding to the larger 50S subunit

- Prevent the attachment of amino acid-containing end of the aminoacyl-transfer RNA to its binding region

Association of amino acid substrate with peptidyltransferasePeptide bond formation


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