Challenges of Antibacterial Discovery

39 Pages·2011·2.09 MB·English

CLINICALMICROBIOLOGYREVIEWS,Jan.2011,p.71–109 Vol.24,No.1
0893-8512/11/$12.00 doi:10.1128/CMR.00030-10
Copyright©2011,AmericanSocietyforMicrobiology.AllRightsReserved.
Challenges of Antibacterial Discovery
Lynn L. Silver*
LLSilverConsulting,LLC,955S.SpringfieldAve.,UnitC403,Springfield,NewJersey07081
INTRODUCTION.........................................................................................................................................................72
TheDiscoveryVoid...................................................................................................................................................72
ClassModificationsversusNovelClasses.............................................................................................................72
BACKGROUND............................................................................................................................................................72
EarlyScreening—aBriefandBiasedPhilosophicalHistory.............................................................................72
TheRate-LimitingStepsofAntibacterialDiscovery...........................................................................................74
TheMultitargetHypothesis....................................................................................................................................74 D
ANTIBACTERIALRESISTANCE..............................................................................................................................75 o
EndogenousversusExogenousResistance............................................................................................................75 w
n
AssessingEndogenousResistancePotential.........................................................................................................76 lo
FitnessofResistantMutantsandCompensatoryMutations.............................................................................77 a
TARGETS......................................................................................................................................................................78 d
e
LinkingMICtoTargetInhibition..........................................................................................................................78 d
SupportforEnzymeInhibitionastheAntibacterialMechanism......................................................................78 f
r
SARofenzymeinhibitionandMIC...................................................................................................................78 o
m
Phenotypicprofiling..............................................................................................................................................78

Under-andoverexpressionoftheputativetarget............................................................................................79 h
t
Anarrayofarrays:expression,sensitization,resistance,andsynergy.........................................................79 t
p
Targetalteration...................................................................................................................................................79 :
/
/
RECENTRECORDOFSINGLE-ENZYME-TARGETEDAGENTS....................................................................80 c
m
TheOpacityoftheDiscoveryProcess....................................................................................................................80
r
MurBtoMurFEnzymesasAntibacterialTargets..............................................................................................80 .
a
TargetsofInhibitorsDiscoveredbyEnzymeScreeningorDesign...................................................................83 s
m
UppS.......................................................................................................................................................................83
.
WalK/WalR............................................................................................................................................................83 o
r
LpxC.......................................................................................................................................................................84 g
/
FtsZ.........................................................................................................................................................................85
o
InhibitorsIdentifiedafterPhenotypicandEmpiricalDiscovery.......................................................................86 n

Single-EnzymeTargetsofNovelInhibitorsinClinicalTrials...........................................................................86 M
Peptidyldeformylase............................................................................................................................................86 a
r
Enoyl-reductasesofFASII..................................................................................................................................87 c
h
LeucyltRNAsynthetasesofGram-negativeorganisms...................................................................................88

3
RNApolymeraseinC.difficile.............................................................................................................................88
0
DHFRandiclaprim..............................................................................................................................................89 ,

2
SummaryofChallengesofSingle-TargetDiscovery............................................................................................89
0
MULTITARGETING....................................................................................................................................................89 1
9
SinglePharmacophore,MultipleTargets..............................................................................................................90

b
DualinhibitorsofDNAgyraseandtopoisomeraseIV....................................................................................90
y
DualinhibitorsofGram-positiveDNApolymerases.......................................................................................92 g
(cid:1)-Ketoacyl-ACPsynthasesofFASII.................................................................................................................92 u
e
TargetingSubstratesandCofactors.......................................................................................................................92 s
LipidIIandotherspecificcellwallsubstrates................................................................................................92 t
Avitamincofactorastarget................................................................................................................................93
HybridMolecules:DualPharmacophore,MultipleTargets..............................................................................93
CHEMISTRY.................................................................................................................................................................94
SpectrumIsDuetoPermeabilityasWellasTargetDistribution.....................................................................94
ChemicalLibrariesAreLimiting...........................................................................................................................95
BarrierstoIntracellularAccumulationinGram-NegativeOrganisms............................................................95
Cytoplasmicandoutermembraneshaveorthogonalsievingproperties......................................................95
RNDeffluxpumps.................................................................................................................................................96
FormulationofRulesforIntracellularAccumulation.........................................................................................96
BinningforSARofantibacterials......................................................................................................................96
*Mailing address: LL Silver Consulting, LLC, 955 S. Springfield
Ave.,UnitC403,Springfield,NJ07081.Phone:[phone].E-mail:
[email protected].
71
72 SILVER CLIN.MICROBIOL.REV.
Charge....................................................................................................................................................................97
Methodsforexpandingthedatabase.................................................................................................................98
DealingwithEfflux...................................................................................................................................................98
ABRIEFPRESCRIPTIONFORNATURALPRODUCTSCREENING..............................................................99
CONCLUSIONS...........................................................................................................................................................99
REFERENCES............................................................................................................................................................100
INTRODUCTION (empiricalscreening).Notespeciallyinnovative,butitworked.
Infact,sincethoselastdiscoveriesinthe1980s,therehasbeen
Thechallengestoantibacterialdiscoveryhavekepttheout-
a great deal of creative, rational, technologically cutting-edge
put of novel antibacterial drug classes to extraordinarily low
screeningforandeffortsatdesignofnewantibacterials.Butso
levelsoverthepast25years,eventhoughdiscoveryprograms
far,littlehasreachedseriousdevelopment.Theproblemwith
have been in place at large and small pharmaceutical compa-
the conduct of antibacterial discovery since the early 1980s is
nies as well as academic laboratories over this period. This
notalackofinnovation.
review focuses on the scientific challenges to the discovery of D
ItisdueinlargeparttothisdiscoveryvoidthatBigPharma o
novel small-molecule antibacterials rather than on the com- hasbeenwithdrawingfromresearchinthearea,eventhough w
mercialandregulatoryconsiderations,whicharewellcovered n
inanumberofreviews(186,197,301,303,345).Rate-limiting therehascertainlybeenrecognitionofthecontinuingneedfor lo
newantibacterialstocombattheriseofresistantorganisms. a
stepstothediscoveryprocessarediscussed,andsomeperspec- d
e
tive on avenues to address those limitations is offered. An d
underlying thesis of this review is that the bleak picture of ClassModificationsversusNovelClasses f
r
antibacterial discovery is due to an expenditure of effort and o
Theantibacterialproductpipelinehasnotbeenemptydur- m
resourcesonnon-rate-limitingstepsoftheprocess.Whileitis
ing this time but has been filled with improved versions of h
easy to find compounds that kill bacteria, it is hard to find t
previouslyregisteredclasses.Manyoftheseweretrueimprove- t
novelantibacterialclassesworthyofdevelopment.Ifnewmo- p
lecular entities with desirable properties and specificity had ments, adding bacterial spectrum, safety, simpler dosing regi- ://
mens, and most importantly, activity insusceptible to specific c
been discovered commonly throughout the past 25 years, it m
seems likely that large pharmaceutical companies (Big resistance mechanisms acting on the parent compound. A r.
numberofdrugswithimprovedactivityagainstresistantpatho- a
Pharma) would have viewed the area as productive and con- s
gens, such as oritavancin, iclaprim, and ceftobiprole, have m
tinued with antibacterial discovery. Even if unlimited money
reached the FDA but have met with regulatory problems, .
werepouredintodiscoveryandproblematicregulatoryguide- o
lines were improved and stabilized, then it is probable that largely due to inadequacy of trials in proving noninferiority. rg
Telavancinhasbeenapproved,anddevelopmentofceftaroline /
noveldiscoverywouldstillbestymiedbecausescientificobsta- o
continues. Finding a derivative with an exploitable advantage n
clesremaintobeovercome.
overtheoriginaldrugisnotaneasypathbutisaprocesswhose M
starting material has acceptable pharmacological properties a
r
TheDiscoveryVoid and whose modification may be approached rationally. While c
h
thesamecaveatsofmeetingpharmacologicalandtoxicological
3
Walsh has noted that “no major classes of antibiotics were standards apply to this effort as to novel drug discovery, the 0
introduced”between1962and2000andreferstotheinterim leapismuchgreaterfornoveldiscovery,asitrequiresthatthe , 2
asaninnovationgap(115,378).Thisunderstatestheproblem. leadsmeetatremendousnumberofcriteria. 0
1
The latest registered representatives of novel antibacterial 9
classes,linezolid,daptomycin,andthetopicalagentretapamu- b
lin, were indeed introduced in 2000, 2003, and 2007, respec- BACKGROUND y
g
tively,butthesechemicalclasses(oxazolidinones,acidlipopep- EarlyScreening—aBriefandBiasedPhilosophicalHistory u
tides,andpleuromutilins)werefirstreported(orpatented)in e
s
1978(124),1987(86),and1952(275),respectively.Atimeline The earliest history of antibacterial chemotherapeutic dis- t
(Fig.1)ofdatesofdiscoveryofdistinctclassesofantibacterials covery was via screening dyestuffs and other chemicals for
(asopposedtodatesofintroduction)illustratesthattherehave selectiveantibacterialactivity,yieldingsalvarsanandthesulfa
been no (as yet) successful discoveries of novel agents since drugs. When the folate pathway inhibited by the sulfas was
1987.Thereisadiscoveryvoidofunknownextentratherthan better understood, more directed screening of pyrimidine de-
agap.Whilethereareasmallnumberofnovelcompoundsin rivativesandanalogsforinhibitionofthebacterialfolatepath-
the early clinical phase that might portend the end of this way produced trimethoprim, an inhibitor of dihydrofolate re-
hiatus, in most cases their eventual developmental success is ductase (57, 158, 375). But the so-called “Golden Age” of
unclear. Is the void due to a lack of innovation? While the antibacterialdiscoveryinvolvedscreeningofnaturalproducts,
simpledefinitionofinnovationistheactofintroducingsome- especiallyfermentationbrothsandextractsofmicroorganisms,
thingnew,thewordimpliescreativity,intent,andexperimen- simplyfortheabilitytoinhibitgrowthofbacterialorganismsof
tation. Almost all the discoveries shown in Fig. 1 (with the interest (pathogens or surrogates), without regard to their
exceptionoftrimethoprim,monobactams,fosfomycin,andcar- mechanismofaction.Thishasbeentermedempiricalscreen-
bapenems) were serendipitous, made by screening fermenta- ing (71, 342). Selectivity was generally tested in secondary
tion products or chemicals for inhibition of bacterial growth assays of toxicity in animals. This worked admirably for a
VOL.24,2011 CHALLENGES OF ANTIBACTERIAL DISCOVERY 73
D
o
w
n
lo
a
d
e
d

f
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h
t
t
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/
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m
r
.
a
s
m
.
o
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FIG. 1. Illustrationofthe“discoveryvoid.”Datesindicatedarethoseofreportedinitialdiscoveryorpatent. g
/

o
n

M
number of years, as the most common antibiotics (natural beenfound,Cohenproposedrationalchemotherapyofinfec- a
product-derivedantibacterials)werediscoveredandrediscov- tiousorganismsthroughasearchforinhibitorsofspecificen- rc
h
ered rapidly. The prevalence of production of “common” an- zymesinthetargetorganism(77).This,alongwiththegrowing
3
tibioticsamongstandardActinomyceteshasbeenestimatedby ability to clone genes and manipulate bacterial strains to en- 0
Baltz (22). To efficiently discard such previously described hance whole-cell phenotypic screens for inhibitors of specific ,
2
compounds,methodsofso-called“dereplication”werequickly targets (and eventually allow the production of purified pro- 0
1
developedtoidentifythem(1,104,352). teins which could be used for in vitro screening and assays), 9
In an effort to make dereplication easier, starting by the turned the whole of antibacterial discovery toward more tar- b
y
early1960s(126),screeningmethodsweremodifiedinorderto get-directedscreens.
g
limit the hits to subsets of all possible antibiotic compounds. Muchofearlyindustrialantibacterialscreeningwascarried u
For example, many screens were developed over the years to out by cohesive groups that did natural product fermentation e
s
detect inhibitors of the pathway of peptidoglycan (cell wall) andbothdesignedandranthescreens.Thescientificdirection t
synthesis(126,278,333).Eachtimeahitinsuchascreenwas andprioritizationofresourcesweredonewithinthegroup.But
detected, it could be compared for biological and chemical changesinthepharmaceuticalindustryled,inmanycases,toa
similaritytothepreviouslydiscoveredcellwallsynthesisinhib- modularized system that is still more or less in effect. Drug
itors. Thus, pathway- or rudimentary target-based screening discovery programs for different therapeutic areas (such as
aroseinpartfordereplicationpurposesbutalsobecausecer- infectious diseases, cardiology, oncology, immunology, etc.)
tainpathways(cellwallandproteinsynthesis)appearedtobe are generally organized such that biology and sometimes
common targets for useful antibiotics. Furthermore, it was chemistry are committed to that area, but other functions
early recognized that cell wall inhibition was a very selective (screening, animal testing, pharmacology, structural biology,
antibacterial target. The only clinically useful antibacterial etc.)maybeshared.Sinceresourcesarealwayslimiting,their
classes discovered through directed screening thus far allocationbecamearelativelyhigh-levelmanagementdecision
(monobactams, carbapenems, and fosfomycin) were discov- (often at a remove from bench science), weighing the value
eredinthesecellwallpathwayscreens. to the company of a therapeutic area, the probability of suc-
Importantly, in 1977, at a time when the output of novel cess,theproximitytothe“cuttingedge”ofcurrenttechnology,
antibiotic classes had decreased, the low-hanging fruit having and the ability of the scientists and their managers to push
74 SILVER CLIN.MICROBIOL.REV.
specificprograms.Forexample,antibacterialdiscoverygroups TABLE 1. Systemicmonotherapeuticantibacterials
hadtocompetewithothertherapeuticareasfortheopportu- andtheirtargets
nity(aso-called“slot”)toscreennaturalproducts.Theaward- Class Target Functioninhibited
ingofnaturalproductscreeningslotscametobebasedonthe
(cid:1)-Lactams PBPs Peptidoglycansynthesis
perceived attractiveness of the target and its amenability to Glycopeptides D-Ala-D-AlaoflipidII Peptidoglycansynthesis
downstreambiochemicalandphysicalanalysis.Thoseantibac- Macrolides rRNAof50Sribosomesubunit Proteinsynthesis
Lincosamides rRNAof50Sribosomesubunit Proteinsynthesis
terial screens designed to find primarily novelty (over inhibi- Chloramphenicol rRNAof50Sribosomesubunit Proteinsynthesis
torsofaspecifictarget)wereoftengivenlowpriority.Itcould Oxazolidinones rRNAof50Sribosomesubunit Proteinsynthesis
Tetracyclines rRNAof30Sribosomesubunit Proteinsynthesis
be argued that finding novelty is the goal of natural product Aminoglycosidesa rRNAof30Sribosomesubunit Proteinsynthesis/
screeningforantibacterialsandthatconcentrationonasmall mistranslation
Fluoroquinolones Topoisomerases(DNAgyrase, DNAreplication
numberofpreselected“desirable”targets(forwhichinhibitors
topoisomeraseIV)
might or might not be present) is an inefficient use of the Daptomycin Membranes
natural product resource. Screening strategies for novelty Metronidazole DNA
amongnaturalproductsarenotedinalatersection. aStreptomycin,anaminoglycoside,isanexceptioninthatittargetsaribo- D
Naturalproductscreening(atleastfornovelantibacterials) somalprotein,andsingle-stepresistancecanoccurbymutationinitsgene,rpsL. o
wanedwiththelowoutputofgoodleads,theadventofhigh- w
n
throughput liquid handling-based screening methods, for lo
whichcrudemicrobialfermentationbrothsareapoorfit(since come barriers to bacterial entry and proclivity to be effluxed, a
they require labor- and time-intensive culture isolation, fer- especiallyinGram-negativeorganisms. de
mentation,andextractiontoproducearelativelylimitednum- In regard to target selection, the emphasis here on the im- d

ber of samples), and the rise in the screening of chemical portanceofchoosingtargetsbytheirlowpropensityforrapid fr
o
libraries,especiallycombinatorialchemicals.Antibacterialdis- resistance selection may seem a narrow view of the problem. m
covery largely became limited to screening these chemical li- There are a number of other important considerations in- h
braries.Thiswasnotafruitfulsource,asdiscussedbelow. volved in choosing specific targets for rational antibacterial tt
p
Tosummarize,aftertheGoldenAge,antibacterialdiscovery discoveryprojects.Theseinclude(i)essentialitytotheorgan- :
/
becametargetorientedandlargelyabandonednaturalproduct ismofthefunction,enzyme,orstructuresothatinhibitionof /c
m
sources.BigPharmaevidentlyweighedthecostsofmaintain- enzymeactionorblockageofthefunctionleadstoinhibitionof
r
ingtheresourcesfornaturalproductprogramsagainstthelow bacterialgrowthor,better,death;(ii)conservationofstructure .a
probabilityofusefuloutputandoptedforthesyntheticchem- ofthetargetenzymeacrossbacterialspeciessufficienttopro- sm
icalroute.Targetswerepursuedfirstasameansofdereplica- vide a useful antibacterial spectrum; (iii) a lack of structural .
o
tioninnaturalproductscreeningandlatertoprovidearational homologywiththesameorsimilarfunctionsinthemammalian r
g
basisfordiscoveryandasaroutetoavoidingcross-resistance host in order to avoid mechanism-based toxicity; and (iv) in /
withotherdrugs,asdiscussedbelow. common with other areas of human drug discovery, “drugga- on
bility”ofthechosentarget,inthatthereshouldexistsiteson
M
the target enzyme or structure that small drug-like molecules a
TheRate-LimitingStepsofAntibacterialDiscovery canbindtoand,insodoing,exertabiologicaleffect.Theseare rc
h
importantconsiderationsand,inpractice,generallyleadtothe

3
The direction of novel antibacterial discovery research (as selectionofsingleenzymesastargetstopursue.However,one 0
opposedtothatofimprovinguponestablishedclasses)inthe ofthethesesofthisreviewisthatsingleenzymesmaynotmake ,
2
past20yearshasbeentodeployanarrayofnewtechnologies, good antibacterial targets due to their potential for rapid re- 0
1
basedongenomics,bioinformatics,structuralbiology,andvar- sistance development. This possibility has largely been ne- 9
ious high-throughput methods, in an effort to transform the glected in the course of recent antibacterial discovery, to its b
giant leap of novel discovery into doable quantum steps. In- detriment,andthusitisspotlightedhere. y

g
deed,theallureoftherational,engineering-oriented,stepwise Manychallengestocandidateselectionandsubsequentde- u
application of techniques to make the discovery process a velopment of antibacterials, including pharmacological prop- e
s
turnkey system is understandable. The concept has been to erties, pharmacokinetic/pharmacodynamic (PK/PD) analysis, t
define broad-spectrum (or more species-specific) targets, andtoxicities(bothmechanismandchemistry-based),arecom-
screen for or design inhibitors, and then hope to address the mon to all drug discovery. They have been approached, ad-
subsequentobstaclesofbacterialentry,non-mechanism-based dressed,andovercomebymore-standardizedmedicinalchem-
toxicity,serumbinding,pharmacokinetics,etc.,inapiecemeal istry magic for many generations of successful antibacterials
manner.Butthisapproachhasapparentlynotworked. and other human health drugs and are addressed only mini-
The purpose of this review is to underscore and illustrate mallyinthisreview.
some of those problems unique to the discovery and optimi-
zationofnovelantibacterialagentsthathaveadverselyaffected
TheMultitargetHypothesis
the output of the effort over the past 20 years. These are the
rate-limiting steps of the antibacterial discovery process and Thefactthatsuccessfulsystemicantibacterialshavemultiple
canbedividedintotwomainareas:(i)propertargetselection, molecular targets or targets encoded by multiple genes has
specificallythenecessityofpursuingmoleculartargetsthatare been evident for the past 20 years (50, 73, 204, 337, 339, 340,
notpronetorapidresistancedevelopment;and(ii)limitation 347). This is illustrated in Table 1, where the currently used
ofchemicaldiversity,especiallythatwhichisnecessarytoover- systemic monotherapeutic agents and their targets are listed.
VOL.24,2011 CHALLENGES OF ANTIBACTERIAL DISCOVERY 75
TABLE 2. Mechanismsofantibacterialresistance
Origin Mechanism Examplesofaffecteddrugclasses
Exogenous Class-specificefflux Tetracycline,macrolides
Class-specificdegradation/modification (cid:1)-Lactams,aminoglycosides,chloramphenicol,streptogramin
A,metronidazole(foranaerobes),fosfomycin
Targetprotection/modification Tetracycline,macrolides,lincosamides,oxazolidinones,
streptograminB
Replacementwithreduced-affinitytarget (cid:1)-Lactams,vancomycin,trimethoprim,mupirocin,sulfonamides
Sequestrationoftarget Fluoroquinolones,fusidicacid
Endogenous Singlemutationsreducingtargetaffinity Rifamycin,streptomycin,trimethoprim(forGram-positive
organisms),fusidicacid
Multistepmutationsreducingaffinityor Fluoroquinolones,oxazolidinones,daptomycin,vancomycin,
remodelingoftarget polymyxin,(cid:1)-lactams(fortransformablespecies)
Generaleffluxmechanisms MostclassesforPseudomonas;manyclassesforotherspecies
Reduceduptake(porinorpermeaseloss) Carbapenems,fosfomycin D
Lossofactivation Metronidazole(forH.pylori) o
Upregulationoftarget Fosfomycin w
n
lo
a
d
e
Theseantibacterialsarenotsubjecttohigh-leveltarget-based agents in monotherapy. This is supported by the inverse, that d

resistancebysinglegeneticchangesinthehost.Thehypothesis most single targeted antibacterials in the clinic are indeed fr
o
isthattheseagentsaresuccessfulmonotherapeuticsandnot subject to single-step high-level resistance selection and are m
subject to such resistance because they are multitargeted usedaspartofcombinationtherapies,especiallyintherapyof
h
(339, 340). M. tuberculosis or as topical agents (see Tables 3 and 4 of tt
p
Onlytwoofthecommonlyusedantibacterialclassesactually reference337).Ofcourse,allantibacterialswithevenmoder- :
/
targetmultipledifferentenzymesinagivenspecies.Thebeta- atespectrahavemultiplehomologoustargetsinthattheymust /c
m
lactams target the penicillin binding proteins (PBPs) (34, 93, inhibit enzymes or bind to structures that are present and
r
130,348),andthefluoroquinolones(FQs)targetthecatalytic variedamongthebacterialspeciesofthatspectrum. .a
subunitofDNAgyrase(GyrA)andtopoisomeraseIV(ParC) If success as a monotherapeutic is indeed due to multitar- s
m
(65, 112, 189). The multitarget hypothesis was offered before geting (or targets encoded by multiple genes) and single-tar- .
o
the second target of the FQs, topoisomerase IV, was recog- geted agents, prone as they are to single-step mutation to r
g
nized (112, 189). The FQs had appeared to be an exception target-based resistance (347, 388), are not optimal for mono- /

o
totherule(340),sinceresistancetotheFQswasnotextensive therapy, then there are grave implications for antibacterial
n
in the clinic by the early 1990s. This finding thus served to discovery. The impact of endogenous resistance (that occur- M
supportthehypothesis. ring by antibiotic selection in the pathogen) on antibacterial a
The clinically used agents that target rRNA in their inhibi- drugdiscoveryanddevelopmentiscoveredbelow. rc
h
tionofproteinsynthesisprovideanotheravenueofsupportfor
3
thehypothesis.Theseincludemacrolides,lincosamides,chlor- 0
amphenicol, oxazolidinones, tetracyclines, aminoglycosides, ANTIBACTERIALRESISTANCE ,
2
andpleuromutilins(thelastisnotincludedinTable1because 0
EndogenousversusExogenousResistance
1
itisnotyetusedsystemically).Theyareusefulinmonotherapy 9
againstorganismsthatcontainmultiplecopiesofrRNAgenes. Antibacterialresistancemaybecategorizedasarisingen- b
y
Against the slow-growing mycobacteria, however, which con- dogenously in the pathogen, by mutation and selection, or
g
tain only a single rRNA cistron (36, 188), they are used in exogenously, by transmission to human pathogens from en- u
combinationwithotheragentsbecausesinglebasechangesin vironmental organisms (antibiotic producers, commensals, e
s
therRNAgeneleadtohigh-levelresistance.WithHelicobacter nonhumanpathogens,etc.)byhorizontalgenetransmission t
pylori, which contains 2 rRNA cistrons (55), clarithromycin (HGT) (54, 78, 85, 82, 147, 240, 241, 340).
resistance arises during therapy (2, 376), and heterozygous Thecollectivegenomicrepertoireofpossiblemechanismsof
strainsdisplayaresistantphenotype(376).Ofcourse,anti-H. resistancetoantibacterialagents,viachemicalmodificationor
pyloritherapygenerallyinvolves2ormoreantibacterialagents, breakdown of antibiotics, target protection, efflux, or specific
although not strictly due to resistance development. For en- changes to the target, has been termed the antibiotic “resis-
terococci, it has been shown that MICs of linezolid-resistant tome”(85).Thetypesofexogenousandendogenousresistance
isolates are highly correlated with the percentage of rRNA mechanisms acting on marketed (nonmycobacterial) antibac-
cistronsmutated(237).InawaysimilartotherRNAcase,the terials are summarized in Table 2. Recent reviews have gen-
FQswhichhavedualtargetsinstandardpathogenshaveonly erally emphasized the role of the exogenous resistome and
a single target (DNA gyrase) in both Mycobacterium tubercu- HGTinthespreadofclinicallyimportantantibioticresistance
losis and H. pylori (160) and do yield to single-step resistance (78, 84, 85, 239, 241, 390). Indeed, most of the mechanisms
(178,195). which have played major roles in resistance to the standard
It remains a hypothesis that the low potential for target- monotherapeutic agents have arisen in this way, with the no-
basedresistanceiscausallyrelatedtothesuccessofmultitarget table exception of the FQs. According to the multitarget hy-
76 SILVER CLIN.MICROBIOL.REV.
pothesis,thesedrugsareusefulmonotherapeuticallyprecisely single targeted or subject to high-level resistance via single
because of their low susceptibility to high-level, single-step mutations. It is clear for M. tuberculosis that resistance is not
endogenousresistancedevelopment,althoughchromosomally due to the exogenous resistome, since it lacks plasmids and
encoded resistance via efflux or reduced permeability or does not participate in HGT, but to endogenous resistance
changes to multiple targets (as with FQs) may compromise arisingthroughmutationofindividualclonesofM.tuberculosis
thesedrugsinastepwisefashion(asnotedinTable2).Wood- (131,262,349).Asaconsequenceofsingletargetingofdrugs
fordandEllington(388)discusstheimportanceofmutationin fortheseinfections,successfultherapyforM.tuberculosisand
thedevelopmentofresistanceandmaketheimportantdistinc- HIVhasevolvedtousecombinationsoftheseagents.Indeed,
tion between those antibiotics to which resistance can arise since the standard of care for M. tuberculosis and HIV is
rapidlyinthelaboratory(suchasrifampin,streptomycin,and treatment with combinations, the resistance potential of new
fusidic acid) and compromise their use in monotherapy and single-targetedagentsfortreatmentofthosepathogensisnot
clinicallyusefulmonotherapeuticagents(suchasFQsandlin- asproblematicasitmightbeformore-standardpathogens.In
ezolid)towhichresistancemayariseviastepwisemutation. contrast to the case with standard pathogens, a number of
Studies of resistance genes from antibiotic-producing spe- interestingnewanti-M.tuberculosisagentsareinvariousstages
D
cies that are theorized to be a reservoir for HGT raise the of development (229, 374). Is combination therapy a feasible o
possibility that antibiotics derived from natural products are pathfordevelopmentofnewsingle-targetedagents? w
n
morelikelytobesusceptibletosuchapreexistingsetofresis- Insummary,itseemsthatintheinitialstagesofantibacterial lo
tance mechanisms than are totally synthetic drugs (61). How- discovery,endogenousresistance,thatwhichisselectedforby a
d
ever, it has been shown that among antibiotic-producing gen- theleadcompoundinthepathogenitself,iscritical.Successful
e
era, resistance determinants for the synthetic sulfonamides development of such compounds will depend on whether en- d

(82), oxazolidinones (219, 324), and FQs (85) are not rare. dogenousresistancecompromisesmonotherapy.Whatlevelof fr
o
Thus, the strong implication is that resistance via horizontal resistanceselectioninvitroiscompatiblewithadvancementof m
transfer from environmental organisms will eventually com- aleadtoclinicalcandidatestatus?
h
promise both known and still-undiscovered antibacterial tt
p
agents,whetherderivedfromnaturalproductsorsynthetic. AssessingEndogenousResistancePotential :/
What relevance does this have to the discovery and devel- /c
m
opment of new antibacterial agents? Obviously, novel drugs A number of reviews have described useful methods for
r
intended for development must not be cross-resistant with ascertaining resistance frequency (number of resistant organ- .a
existing therapies. For at least the last 20 years, the general isms in a given population) or rates of resistance (number of s
m
answer to the challenge of avoiding cross-resistance has been mutationaleventsleadingtoresistanceperbacteriumpergen- .
o
tosearchforinhibitorsofmoleculartargetsthathadnotpre- eration) to a given antibacterial in the laboratory (240, 281, r
g
viously been “exploited,” that is, they were not the targets of 314). O’Neill and Chopra (281) give practical information on /

o
previously developed agents (6, 29, 49, 61, 168, 253). This preclinicalevaluationofnovelantibacterials,includingimpor-
n
presupposes that existing resistance mechanisms to the drug tant directions for evaluation of resistance potential in vitro. M
classesinusearetargetdirected—whichsomeare—butmany Martinez et al. (241) emphasize that such measurements a
areclassspecific(Table2).Ofcourse,inhibitorsofthesenew shouldbemadeunderavarietyofgrowthconditions.Several rc
h
targetswouldeventuallyfalltoexogenousresistancefromthe authorsrecommendtheuseofhypermutatorstrainstodeter-
3
resistome.Howlongwouldthattake? mine the range of possible endogenous resistance mutations 0
Fornaturalproducts,therangeoftimesbetweenintroduc- (241, 256, 282). The determination of mutation rates by fluc- ,
2
tion and first report of transmissible resistance in pathogens tuationtests(227,281,314)avoids“jackpots,”whichcanoccur 0
has been very large: resistance arose immediately for (cid:1)-lac- when single saturated cultures are plated and may distort de- 19
tams,as(cid:1)-lactamaseswereseeninStaphylococcusaureusvery terminations of mutation frequency. It also demonstrates, as b
y
soon after the broad introduction of penicillin (191), while originallyintendedbyLuriaandDelbruck(227),thatmutation
g
vancomycin resistance in enterococci took 33 years to be rec- to resistance can occur before selection is applied. Mutation u
ognizedintheclinic(206).Forthesyntheticantibacterials,the frequencies to significant levels of resistance (between 10(cid:2)6 e
s
first transmissible resistance in pathogens was recognized for and 10(cid:2)9) usually indicate a single target. The higher rate t
sulfonamides,fluoroquinolones,andoxazolidinonesin23,11, would generally be due to resistance via loss of a function,
and 6 years, respectively (194, 219, 242). Thus, the prospects whichcanoccurthroughdeletion,insertion,orbasechangesat
forlong-termavoidanceofresistancetoanovelsyntheticagent manysitesinthegeneencodingthatfunction.Thelowerfre-
arenotrosy,butafewyearsmightbeexpectedtoelapsebefore quency(10(cid:2)9)wouldindicatethatresistanceisduetoalimited
exogenous resistance mechanisms come into play. However, numberofallowablebasechangesatasinglesite.
even this short period may be further abbreviated if endoge- The resistance frequency (or rate) depends upon the con-
nousresistanceoccursmorerapidly,perhapsevenduringther- centrationoftheselectinginhibitor.Iftheinhibitorhasasingle
apy,ascouldbeexpectedwithasingleenzymetarget. target,itmayrequireplatingatarelativelyhighmultipleofthe
Thus,itmaybemisleadingtoapplylessonslearnedfromthe MICtodetecttarget-basedresistance,sinceatlowermultiples,
patterns of resistance development via HGT to expectations mutationsthataffectpermeabilityandeffluxfunctionsoccurat
for inhibitors of new targets. It might be more reasonable to relatively high frequencies and may predominate. With some
expectthepatternsofresistancedevelopmenttosingle-enzyme single-enzymeinhibitors,suchasrifampin,singlebasechanges
inhibitors that are seen with drugs used in therapy of M. tu- canraisetheMIC32,000-fold(17).Iftheinhibitorhasmultiple
berculosis or, for that matter, HIV, where the drugs are all targets with various sensitivities to inhibition (but within a
VOL.24,2011 CHALLENGES OF ANTIBACTERIAL DISCOVERY 77
narrowconcentrationrange),aswiththeFQs,thentheincre- described (9, 99, 144, 241) and can be used profitably, but
mentofMICspossiblewithchangestooneofthetargetscan animalmodelsforresistanceselectionandcompetitivefitness
be small and will be related to the difference in intrinsic sen- shouldalsobestandardizedandapplied(39,220,241).Sinceit
sitivitiesofthetargets.Thatis,ifthemostsensitive(primary) is difficult to predict the impact resistance would have in the
target is inhibited sufficiently at an external concentration of clinicwhenaninhibitorisalreadyinhand,itshouldbeappar-
0.01 (cid:3)g/ml to prevent growth (MIC (cid:4) 0.01 (cid:3)g/ml) and the entthatpredictinglowresistancepotentialforagiventargetin
secondary target is inhibited at 0.04 (cid:3)g/ml, then even a 100- the absence of an inhibitor is much more problematic. It is
fold decrease in the sensitivity of the primary target would possibletopredict,however,byusingmicrobialgenetics,that
raisetheMICnohigherthan0.04(cid:3)g/ml.Infact,thisillustrates inhibitionofaparticulartargetmightleadtoabypasseventat
thebenefitofhavingmultipletargets.Eventhoughmutations arelativelyhighfrequency[see“Peptidyldeformylase”below].
inthemostsensitivetarget(GyrAorParC,dependingonthe Although most single-enzyme-targeted agents are used in
speciesanddrugbeingtested)occuratsignificantfrequencies, combination or topically and thus avoid rapid endogenous
high-level FQ resistance requires multiple mutational events resistance development, there are a few exceptions, such as
(98,171,354). fosfomycin (272), which has been used successfully (outside
D
What does a specific frequency portend for the future po- theUnitedStates)againsturinarytractinfections(UTIs).Why o
tential of endogenous resistance development in the clinic? is there a lack of clinically relevant endogenous fosfomycin w
Generally,afrequencyof(cid:5)10(cid:2)10issoughtbecauseorganisms resistance? Fosfomycin targets UDP-N-acetylglucosamine nlo
inaninfectioncanreach109cells/ml(256,281)or1010cellsin enolpyruvyl transferase (MurA), the first committed step of a
d
aninfectedindividual(98).However,thismaynotbestringent peptidoglycan synthesis, forming a covalent adduct with an
e
enough. As noted above, the use of hypermutator strains can activesitecysteine(181).Despiteitscovalentandirreversible d

helptorevealtherangeofendogenousresistance.Whilethese action, its activity appears to be highly selective, and it has a fr
o
can give up to 1,000-fold higher resistance frequencies than verylowtoxicity(50%lethaldose[LD ]inmiceof(cid:6)20g/kg m
50
normal (256), their use may be particularly relevant, since a of body weight when dosed orally [126]). M. tuberculosis is
h
significant percentage of antibiotic-resistant clinical isolates, naturally resistant to fosfomycin due to the existence of an tt
p
especially those from chronic infections, have been shown to aspartate instead of cysteine at that site, and the aspartate- :
/
behypermutablestrains(138,146,241,256,359).Thepressure containing enzyme is highly active in Escherichia coli (190). /c
m
of selection for mutations will itself select for hypermutators Thus, it appears that the cysteine is not required for enzyme
r
(235). activity, and theoretically, a mutation consistent with cell .a
Optimally,itshouldbedetermined(i)whethersinglemuta- growth could occur at that site, leading to fosfomycin resis- s
m
tionaleventsthatraisetheMICaboveclinicallyrelevantdrug tance.However,therehavebeennoreportsofinvitroselection .
o
levels can occur in a target organism and (ii) whether strains of spontaneous murA mutants resistant to fosfomycin (al- r
g
containing these mutations are sufficiently fit and virulent to though one was selected after mutagenesis and counterselec- /

o
survive and be infective in the absence of selective pressure. tionagainstuptakemutants[393]).Rather,selectionwithfos-
n
Thisiseasiersaidthandone. fomycininthelaboratoryresultsinarelativelyhighfrequency M
of resistance due to loss of the (cid:7)-glycerophosphate (glpT ) or a
hexose-P(uhp)activeuptakesystem(272),andthehighrateof rc
FitnessofResistantMutantsandCompensatoryMutations uptakemutants(asgreatas10(cid:2)7pergeneration)mayobscure h
3
Iftheypreexistinapopulation,mutationsconferringresis- selection of murA mutants. These uptake mutants have been 0
tancewillbeselectedforbytreatmentwithadoseofinhibitor reported to be nonvirulent and slow growing (272, 389) and, ,
2
thatkillsofftheparentalstrainbuttowhichthemutantstrain hence, likely to be unstable in an infecting population. Fur- 0
1
is resistant. Under conditions of drug treatment, then, such thermore,fosfomycintreatmentleadstohighurinarylevelsof 9
mutantscompetewellandarefitrelativetotheirdeadsiblings. drug((cid:6)1mg/mlfor12hafteroraldosing[326]),whichwould b
y
In the absence of drug, many forms of resistance can exact a likelykilloffpreexistingmutants(ofthetargetoruptaketype)
g
fitnesscost,suchthatmutantswillbeslowedingrowthrateand resistanttolowerconcentrations;additionally,thetotalorgan- u
will not compete well with the nonmutant, sensitive parental ismal load in uncomplicated UTI is probably (cid:5)108 (based on e
s
strain(9).However,furthercompensatorymutationscanoften calculations for E. coli in reference 272). In the clinic, fosfo- t
occurthatreducethefitnesscostoftheoriginalmutation,and mycin resistance generally is due to covalent formation of a
thesewilltendtostabilizetheresistancemutationinthepop- fosfomycin-glutathione adduct by FosA, FosB, and FosX en-
ulation(8).Itshouldbenotedthathypermutatorsalsowillplay zymeswhichhavespreadbyHGT(52,113,252).
a role in the appearance of these compensatory mutations The fosfomycin case raises the possibility that endogenous
(388). It is the complex balance of these events and the pres- resistance to single-enzyme targets may be avoided if drug
suresofrepeatedselectionwithantibacterialagentsthatcon- levels at the infection site can be kept high without toxicity
troltheoverallrateofevolutionofresistancethatoccursupon and/or the mutants are unfit or of low virulence. Clearly, fos-
clinical introduction of a new agent (241). The occurrence of fomycinhasbeensuccessful(althoughforalimitedindication),
compensatory mutations has been studied for a number of butisthefosfomycin/MurAscenariomorebroadlyapplicable?
drugs, both in vitro and in clinical isolates, by Andersson and Shouldsingle-enzymetargetsbeavoidedaltogether,orhasin
coworkers(forexample,seereferences198,230,231,268,273, vitro analysis of resistance frequencies had an unnecessary
and292).Fornewandnoveldrugcandidates,howshouldthe chillingeffectondiscoveryprogramswithinindustry?Hasthe
problem of fitness be addressed? In vitro methods, including awarenessofthepotentialforresistancetosingle-targetagents
hollow-fiber“pharmacodynamicinfection”models,havebeen ledtotheearlydemiseofprogramsthatwouldotherwisehave
78 SILVER CLIN.MICROBIOL.REV.
proceeded—to optimization or even to the clinic? This is a anddesignprograms,withanemphasisoncompoundsidenti-
chasteningthought. fiedbyinvitrobiochemicalscreensandassaysofenzymeinhi-
bitionorbinding.
TARGETS
LinkingMICtoTargetInhibition
Theeffortspentincataloguinglikelytargetsthroughgenom-
ics, functional genomics, and bioinformatics appears to have While it is not necessary that an antibacterial discovered
beenunsuccessfulinprovidingastartingplaceforthedesired duringtargetedscreeninghitsolelythedesiredtarget,oreven
stepwiseprocesstodiscoveryofanoveldrug. thattargetatall,theraisond’ˆetreoftarget-directedscreening
Asnotedabove,potentialantibacterialtargetswouldtradi- is that specific bacterium-selective and hence nontoxic inhibi-
tionallybedefinedasessential,distinctfromrelatedmamma- tors will be discovered in this manner. While it should be
lian structures/enzymes, present in a useful spectrum of bac- obvious that an inhibitor discovered in a general empirical
teria, such that an inhibitor might be reasonably used for screenforgrowthinhibitionmustbeshowntobeselectiveand
therapy of a clinical indication (such as community-acquired notkillthroughnonspecific(andlikelycytotoxic)activity(such
D
pneumonia[CAP]),andpossessingareasonablepotentialfor asdetergency,alkylation,energypoisoning,etc.),thisisequally o
druggability.Atleastforproteintargets,mostoftheseparam- importantforacompoundidentifiedviainvitroenzymeinhi- w
n
eters (aside from essentiality) can be ascertained by in silico bition.Thislinkagehasnotbeenmadeinanumberofcases(as lo
methods of bioinformatics and structural analysis. Even the shownbelow),andeventualdeterminationthattheantibacte- a
d
potential for multiple targets sharing active site sequence ho- rialactivitywasnotcausallylinkedtoenzymeinhibitionmight
e
mologies or protein motifs may be addressed by ever more havecontributedtoterminationoftheprogram. d

sophisticatedanalyticaltools,asnotedinMultitargeting. fr
o
Thefocusontargetsfordiscoveryledtothedeploymentof m
SupportforEnzymeInhibitionasthe
intensivecampaignsfortargetevaluation,tothedevelopment
AntibacterialMechanism h
of high-throughput screening (HTS) platforms, and to pro- tt
p
gramsofvirtualligandscreeningandrationalstructure-based SAR of enzyme inhibition and MIC. Hits from screens for :
/
drug design (SBDD). While most of the programs discussed enzyme inhibition are generally tested early on for antibacte- /c
m
and tabulated below come from screening efforts, SBDD rial activity. If the hits are chemically tractable, exploratory
r
shouldplaymoreofaroleinthefuture.Virtualligandscreen- medicinal chemistry may be instituted to improve enzyme- .a
ing and a number of its successes in human health drug dis- inhibitorypotencyandsolubilityand,ifnoorpoorwhole-cell s
m
covery are reviewed by Villoutreix et al. (377). A highly rele- activity is present, to improve MICs. Throughout this optimi- .
o
vant recent review by Simmons et al. focuses on rational zation process, it is important to ascertain whether antibacte- r
g
discovery of antibacterials by SBDD (341). Antibacterial rialactivitytrackswithenzyme-inhibitorypotency.Suchtrack- /

o
SBDD is based on the extensive and growing number of se- ing may not be seen for perfectly legitimate reasons, as the
n
quenced bacterial genomes and solved crystallographic and parameters for net bacterial accumulation are not likely to M
nuclear magnetic resonance (NMR) structures of bacterial trackwithenzymeinhibition.Regardlessoftheproportionality a
proteins and their bound ligands. Through a variety of algo- ofMICtoinhibitorpotencyattheenzymelevel,antibacterial rc
h
rithms,itmakesinsilicopredictionsfordockingofnewligands activity should be shown to be dependent upon enzyme inhi-
3
(compounds and fragments, both real and theoretical), which bitionthroughouttheoptimizationprocess.Often,indiscovery 0
canthenbeprioritizedandtestedforenzymeinhibition,struc- programs,datawillbegeneratedthatshowageneraloreven ,
2
tural interactions, and other biological readouts (such as good structure-activity relationship (SAR) between enzyme 0
1
whole-cellactivity,solubility,orbioavailability).Theprocessis inhibitionandtheMIC.Thisissupportiveevidence(atbest), 9
iterativeandheuristic.TwopotentinhibitorsofHIVprotease, butitdoesnotshowcausality.Ademonstrationofcausalityis b
y
nelfinivir(182)andamprenavir(369),weredevelopedthrough especially critical with a target for which there have not been
g
suchiterativeSBDD. any antibacterial inhibitors described, where the process of u
Thenumberofpotentialtargetenzymes(selectedasessen- linkingwhole-cellactivity(MIC)toinhibitionoftheenzymeis e
s
tial in bacteria but not humans, with a broad or useful spec- criticalfortargetvalidation.Thiscanbedoneinanumberof t
trum) has been estimated to be (cid:8)160 by Payne et al. (293). ways, as noted below and reviewed by O’Neill and Chopra
Lange and coauthors (204) list 16 enzyme classes that are (281).
targets of commercialized antibacterials, in addition to the Phenotypic profiling. In order to prove that antibacterial
nonenzyme targets rRNA, lipid II, membranes, and DNA. activityisduetospecifictargetinhibition,severalavenuesare
Thus,thereareasignificantnumberof“new”targetsthathave possible. With targeted screening, there is already a starting
been nominated for screening and/or inhibitor design. It hypothesisforthemechanismofactionandmoleculartarget.
shouldbenoted,however,thatwhilethese“new”targetsmay Thus, initial work may be directed toward demonstration of
nothavebeenscreenedexplicitlyforinhibitionpreviously(al- phenotypes that should be associated with inhibition of that
though most were, in the pregenomic era), empirical screens target, such as morphological changes (e.g., filamentation for
for whole-cell growth inhibition should have implicitly inhibitors of FtsZ [81, 277]), stress responses (stringent re-
screenedforthem. sponse for inhibitors of tRNA synthetases [38]), and specific
Howdidscreeninganddesignwiththenewtargetsturnout? promoter induction (gyrase inhibition leads to homeostatic
Aninvestigationofthisquestionwillbenefitfromdiscussionof upregulation of gyrase promoters as well as SOS promoters
the process of analyzing hits that arise from targeted screens [7]).Withthesestudies,itisnecessarytouseawidevarietyof
VOL.24,2011 CHALLENGES OF ANTIBACTERIAL DISCOVERY 79
negative controls to show that the tested phenotypes are not (enoylreductase)ofE.coliinvitro,anditsMICagainstE.coli
causedbyotherclassesofinhibitors.Animportantmethodof wasraised(cid:6)7-foldbyoverexpressionofFabI.However,other
ascertaining the pathway of inhibition is measurement of ef- investigators showed that curcumin bound to FtsZ and inhib-
fects on macromolecular synthesis (MMS). This is done most ited Z-ring assembly in Bacillus subtilis (307). Curcumin has
straightforwardlybyuseofradiolabeledtracersofDNA,RNA, also been shown to inhibit the human enzymes glyoxylase I
protein,cellwall,andfattyacid/lipidsynthesis,wherethedose- (319) and monoamine oxidase B (308), as well as HIV inte-
response relationship for the test compound (and control in- grase (246). This type of pattern indicates that curcumin is a
hibitors)ismeasuredatafixedtimeofincubation(4,381).For promiscuousinhibitor(andprobablebinder)andthatcaution
specific inhibitors of one of the pathways of macromolecular shouldbeexercisedintargetattributionbyoverexpression.
synthesis, incorporation of precursors of the end product of Thus,thesemethodsalonearenotdefinitiveindemonstrat-
thatpathwaywillbeinhibitedpreferentially.Ifallpathwaysare ingthatagivenenzymeistheantibacterialtargetoftheinhib-
inhibitedwithinanarrowconcentrationrange,thenanonspe- itor. Nevertheless, these methods will generally implicate a
cific mechanism of inhibition, such as membrane lysis or en- certainpathwayorfunctionasplayingaroleinthemechanism
ergypoisoning,islikely. ofaction.
D
Under- and overexpression of the putative target. Fre- Anarrayofarrays:expression,sensitization,resistance,and o
quently used genetic methods implicating inhibition of a par- synergy. Recently, a variety of methods based on the use of w
n
ticular enzyme as the antibacterial mechanism of action of a arrays(transcriptional[121],translational[23],hypersensitiza- lo
compoundareunderexpressionoftheexpectedtargetprotein, tion [96], overexpression [394], stress response [24], and syn- a
d
inordertosensitizetheorganismtoaninhibitor,andoverex- ergy/antagonism[397]arrays)havebeendescribedthatmaybe
e
pression to yield resistance. Hypersensitization by target un- useful in identifying the antibacterial mechanism of action of d

derexpressionhasbeendemonstratedbyvariousmethods,in- aninhibitor.Thesemethodshavealsobeenusedforevaluation fr
o
cludinguseoftightlydownregulatablepromotersdirectingthe of leads from enzyme inhibitor programs or with activities m
synthesisofreducedamountsofthetargetprotein(viareduced discoveredthroughphenotypicorempiricalwhole-cellscreen-
h
transcription) (95) and upregulated production of antisense ing.Ineachofthesemethods,patternsoftheeffectsofknown tt
p
RNA, which leads to reduced protein expression (118, 398). antibacterial activities on the components of the array are :
/
Buttherearecaveats. established, and unknowns are then compared to these pat- /c
m
IftheMICofagiveninhibitorisreducedwhenitsputative terns to identify inhibitors with previously described mecha-
r
targetisdownregulated,thentheenzymeinquestionislikely nisms of action. With a hypersensitization array (96), for ex- .a
tobeessential,anditmayindeedberesponsiblefortheanti- ample,asetofstrains,eachunderproducingasingletarget,is s
m
bacterialactivityofthecompound.However,reductionofone exposedtotheunknowninhibitor,andthosestrainswhichare .
o
enzymecansensitizeanotherenzyme(perhapsthetruetarget) hypersensitivetotheinhibitorarenoted.Inafewcases,onlya r
g
to inhibition (for example, if they are both members of the single strain will be hypersensitive, indicating the probability /

o
samepathway)(96).Furthermore,eveniftheunderproduced that the underproduced enzyme is the target. However, most
n
enzymeisatargetoftheinhibitor,thatdoesnotprecludethe oftenseveralstrainswillbesensitizedtovariousextents,which M
existence of other, less-sensitive targets, nor does it even es- may indicate inhibition of a step in a particular pathway. In a
tablish that the underproduced enzyme is responsible for set- each type of array, complex patterns are often seen that are rc
h
ting the MIC when it is produced at its normal level. For difficulttointerpret,necessitatingfurtherdissectionbypheno-
3
example, underexpression of FabI sensitized S. aureus to a typicandothergeneticmeans. 0
thiopyridineinhibitorofFabI,butoverexpressiondidnotraise Target alteration. The strongest demonstration that a par- ,
2
thewild-typeMIC(214).Furthermore,MMSanalysisshowed ticularcellularmoleculeistheproximaltargetofaninhibitor 0
1
preferentialinhibitionofRNAandDNAsynthesisoverthatof is to change that putative target in such a way as to prevent 9
fattyacids.Thus,underexpressionaloneisinadequatetoprove interaction with the inhibitor and to show that this blocks b
y
thattheunderexpressedenzymeistheMIC-determiningtarget further downstream sequelae. With single-enzyme-targeted
g
ofinhibition. agents,thismaybeaccomplishedbydirectselectionandmap- u
Interestingly,sometargets,whenunderproduced,mayactu- ping and/or sequencing of mutations which increase the MIC e
s
allyreducesensitivity.ThisisseenwithFQs,wheretheinhib- significantly,asdiscussedabove.Itmayalsobedemonstrated t
itedenzyme-DNAcomplexformsa“poison”suchthatlower- byreplacingthegenefortheputativelytargetedenzymewith
ingenzymelevelsreducesthenumberofcomplexes(172). one known to encode an insensitive enzyme, as shown with
Overexpressionofatargetmaybeaccomplishedbycloning LpxC inhibitors (26). If the target enzyme is known to be
oftheexpectedtargetbehindaregulatablepromoterorbyuse present or essential in only specific species, then a lack of
ofanoverexpressionlibraryofrandompotentialtargets(which activity of an inhibitor against other species is supportive evi-
arethenscreenedforresistancetotheinhibitor)(211).Arise denceofthespecificityofaction,asseenwithClpXP(70),FabI
intheMICofaninhibitorofanoverproducedputativetarget (152), and LpxC (283). With a strong target hypothesis (as
indicates that the overproduced enzyme can indeed bind the when the agent is selected as an enzyme inhibitor), interpre-
inhibitorandloweritseffectiveintracellularconcentration,but tation of resistance selection results is relatively straightfor-
it does not ensure that the candidate enzyme is the cause of ward.Thebestevidence,asnotedabove,istheisolationofthe
growth inhibition, only that the inhibitor can bind to that en- mutant target enzyme and demonstration that the resistance
zyme: another MIC-determining target may be present. For mutation leads to reduced inhibition of (or binding to) the
example, curcumin, a spice component with a long history of alteredtargetinvitro.
dietary and medical use in Asia, was shown to inhibit FabI It is an irony of the antibacterial discovery process that
80 SILVER CLIN.MICROBIOL.REV.
finding resistance to a lead due to a single base change in a MurBtoMurFEnzymesasAntibacterialTargets
targetcanvalidatethemechanismofantibacterialactionofthe
Many attempts have been made to find inhibitors of cyto-
compoundbutmaywellsignalthattheleadisunfitfordevel-
plasmic enzymes of the peptidoglycan synthesis pathway, in-
opment,oratleastformonotherapy.
cluding MurB (UDP-N-acetylenolpyruvylglucosamine reduc-
tase)andtheUDP-N-acetylmuramyl-aminoacidligasesMurC,
RECENTRECORDOFSINGLE-ENZYME- -D, -E, and -F, which sequentially add L-Ala, D-Glu, meso-
TARGETEDAGENTS diaminopimelicacid(m-DAP)(inmostrods)orL-Lys(inmost
cocci), and D-Ala-D-Ala, respectively, to UDP-muramic acid.
TheOpacityoftheDiscoveryProcess Thesehavebeenconsideredgoodantibacterialtargetsbecause
theyarepartofthesyntheticpathwayoftheessentialmacro-
What has been the demonstrable record of antibacterial
molecule peptidoglycan and were themselves shown to be es-
discovery of novel agents in the past decade or so? Since the
sentialbyconditionalmutations(223–226).Itwouldhavebeen
clinicalpipelineofnovelagentshasbeensosmall,theoverall expected that inhibitors of these enzymes would be found in
record is clearly not good. But what avenues have been pur- the many phenotypic screens for inhibitors of the cell wall D
o
sued, and why have they failed? In general, the reasons for pathway (126, 278, 333, 336), but they were not. Merck and w
failure, or even the fact of failure, have not been revealed in Versicor (later Vicuron, and then Pfizer) reported using a n
the literature. While this could be attributed to the general “one-pot”invitroscreenforinhibitorsofMurAtoMurF,but loa
opacity of the industrial drug discovery process due to com- noinhibitorsarisingfromthemhavebeenreported(67,387). d
e
mercial concerns of intellectual property and restriction of In fact, while some inhibitors of the enzymes MurB through d
informationthatmightmovestockprices,recountingoffailed MurF have antibacterial activity, in no case has that activity f
r
o
initiativesisinfrequentintheacademicliteratureaswell.The beenshowntobeduetoinhibitionoftheMurenzymesinvitro. m
patent literature may reveal the industrial seriousness with ManyofthereportedinhibitorsofMurBtoMurFhavebeen
h
which projects are regarded. In fact, while reasons for failure described in earlier reviews (105, 196, 334, 336). Phosphinate t
t
p
maybeveiled,itshouldbenotedthatsuccessisalsooftenkept transition-stateanalogsoftheMurCto-Fenzymesweresyn- :
/
from view. In biotech and academe, early advances are often thesized by various groups in the 1990s, and some of these /c
m
published: the former to secure investor financing, the latter showedpotentenzymeinhibition(atlownanomolarlevelsfor
r
for intellectual pursuit but also to validate grant support. In some, illustrating druggability of the targets) but no antibac- .a
BigPharma,however,itisoftenthecasethatresultsofactive terialactivity,presumablyduetoalackofcellentry(127,254, s
m
programsarenotpublicizedorpublisheduntiltheyapproach 310,362,403).AnumberofinhibitorsofMurB,-C,-D,and-F, .
o
the clinic. Thus, publications may recount many dead ends foundviascreensforenzymeinhibitionorbinding,hadmicro- r
g
(without advertising them as such or explaining why they did molar(insomecases,lowmicromolar)50%inhibitoryconcen- /
not go forward), as there is little publication on actual leads trations(IC50s)andno(orweakinonecase[370])antibacte- on
rialactivity(11,30,103,141,364,370).
untiltheyaredroppedoruntiltheyentertheclinic. M
We must often rely on retrospectives of internal programs, ThethiazolidinonecoreoftheMurBinhibitorsreportedby a
suchasthatofPayneandcolleagues,whosummarizedlessons workers at Bristol-Myers-Squibb was postulated to be a rc
diphosphate mimic (11). These inhibitors were not antibacte- h
learnedfrom70targetedscreeningcampaignsatGlaxoSmith- 3
Kline(GSK)(293),oronexternalsubjectiveanalysessuchas rial, but replacement of the thiazolidinone with an imidazoli- 0
none led to gain of antibacterial activity (48). There was a ,
this and other reviews (111, 204, 209, 280, 302, 335). In the 2
correlation between MIC and enzyme-inhibitory activity in a 0
following sections, which review novel discovery efforts, it is 1
generallyunknowniforwhytheprojectsweredropped.Wasit small series, but no further proof of causality was demon- 9
strated. This is one of many cases in the literature where the b
due to high resistance frequency, insufficient potency, an in- projectwasleft(asfarasreported)atthepreproofstage. y
ability to overcome a high protein binding level, a lack of g
A number of synthetic projects undertaken by the Wyeth u
efficacy in animal models due to instability, metabolism, or group(12,119,201,212,234,395)weredirectedtowardfind- e
otherwisepoorpharmacokinetics,anonspecificmechanismof s
inginhibitorsofmultipleenzymaticstepsintheMurpathway, t
action, undisclosed toxicities, or a poor spectrum making de-
withtheideathathittingmultipletargetswouldlessenultimate
velopment clinically and/or commercially infeasible, or did
resistance selection. For many of these, the lead compound
leadoptimizationstopearlywiththerecognitionofpoorlead
was discovered in a “one-pot” MurA-to-MurF screen (234),
structures?Itwouldcertainlybeinstructivetoknowmore.
probably similar to those of Merck and Versicor. Hits from
Apossiblyinstructivesurveyinvolvestheoutputoftargeted that screen were assayed against the individual enzymes, and
discoveryprogramsorprogramsthatproducedinhibitorswith leads were chosen for expansion. Where tested, these inhibi-
purportedly identified targets. This survey should serve to ex- torswerereportedtobesubjecttoabrogationofantibacterial
emplify some of the problems of discovery and development activityby4%bovineserumalbumin(BSA).Thesecampaigns
and,furthermore,illustratethatinhibitorsofantibacterialtar- all yielded sets of compounds with various spectra of target
gets are discoverable but that few have been optimized suffi- inhibition, from inhibiting a single target (among MurA to
cientlytobechosenfordevelopment.Programsattemptingto MurF) to inhibiting sets of one, two, three, or four enzymes.
exploit(cid:8)35targetsarenotedinthetextandinTable3,among Somewereshowntoinhibitsynthesisofsolublepeptidoglycan
whichonly3(MurA,RNApolymerase,andDNAgyrase)are in whole cells, but no other MMS inhibition was measured
targetedbydrugsalreadyusedinhumantherapy. (119,395).Thelackofthisnegativecontrollowersthevalueof