Speculative strategies for new antibacterials

16 Pages·2013·0.99 MB·English

by  Stuart Shapiro

TheJournalofAntibiotics(2013)66,371–386
&2013JapanAntibioticsResearchAssociation Allrightsreserved0021-8820/13
www.nature.com/ja
REVIEW ARTICLE
Speculative strategies for new antibacterials:
all roads should not lead to Rome
Stuart Shapiro
In concert with improvements in personal hygiene and public sanitation, the discovery and development of antibiotics during
the latter half of the last century has reduced substantially the morbidity and mortality associated with bacterial diseases.
However, the past decade has witnessed a sharp reduction in interest in antibacterial drug development by ‘big pharma’,
compounded by a decline in the breadth of chemical space for new antibacterial molecules and a failure to exploit the plethora
of cellular processes potentially targetable by novel classes of antibacterial molecules. This review focuses on some strategies
relating to antibacterial chemotherapy, paths less trodden, which the author considers worthy of further exploration.
TheJournalof Antibiotics(2013)66, 371–386; doi:10.1038/ja.2013.27; published online24 April 2013
Keywords: antimetabolites;hybrid antibiotics;methyltransferaseinhibitors; polymyxins; toxin–antitoxins
Theneedfornewantibacterialstocountertherisingtideofpathogen antibiotic biosynthesis. Thereafter, his interests migrated from phy-
resistance,andthelackofprogresstomeetthiscrucialmedicalneed, siological control of actinomycete antibiotogenesis8 to antibiotic
have been bewailed and discussed at length in the biomedical discovery and preclinical development, pursued in both academic
literature and will not be reiterated here; interested readers are and industrial milieux. Over the past two decades he had the good
referred to recent reports from the IDSA,1 ECDC/EMEA,2 Center fortunetoworkonabroadarrayofantimicrobialtypes,rangingfrom
for Global Development3 and WHO.4 Resistance is an inevitable botanicalandsyntheticorochemoprophylactica9to‘classic’antibiotic
outcome of the evolutionary dynamic set in motion by exposure of familiessuch asb-lactams, topoisomerase inhibitors,macrolidesand
microorganismstoantibiotics;whiledrugmakersmaystandathwart dihydrofolate reductase inhibitors. During these years of ‘active
their prey declaring ‘Resistance is futile!’, in fact antibiotic service’ in the anti-infectives industry, he witnessed the transfer of
introductions constitute a classic case of nascentes morimur, where responsibility for antibiotic discovery and development from big
thebestwecandoiskeepourcurrentantimicrobialarmamentarium pharma to small-to-medium-size pharmaceutical and biotechnology
asteportwoaheadofthepathogensuntilsuccessorantibioticsreach firms often lacking the confluence of experience, creativity and
the market. Antibiotic stewardship and prudent prescribing financing to initiate, or to sustain, programs aimed at designing,
strategies,5 antibiotic cycling and rational combination therapy can identifying and commercializing innovative products for the
retard the rate at which resistance emerges and disseminates but, antimicrobial market.
given their genetic plasticity and physiologic resourcefulness, In light of the above, the author seeks to share some personal
microorganisms will always outwit us; to the microbe’s credit but thoughtsaboutavenuesbeingexplored,orremainingtobeexplored,
our misfortune, reentrenchment of resistance usually occurs sooner vis-a`-visantibacterial chemotherapy.
ratherthanlater.Thus,continuousidentificationanddevelopmentof
new, more efficacious products with acceptable safety profiles HYBRIDANTIBIOTICS: ANTIBACTERIAL OPPORTUNITIES
to combat agents of infection are required if we are to avoid sliding ORANTIBACTERIALTOURISM?
into a post-antibiotic era6,7 with its ensuant toll in morbidity and Theterm‘hybridantibiotic’canrefereithertounnaturalantibiotics,
mortality. usually of polyketide origin, produced by microorganisms as a
The author’s professional interests in microbiology extend back consequence of mixing or altering biosynthetic genes;10,11 or to
some forty years. They focused initially on comparative bacterial synthetic molecules where two (structurally diverse) antibacterial
physiology and enzymology though, during the 1980s, he was pharmacophores are coupled to form a single molecular entity.12
privileged to work in the laboratory of Professor Dr Leo C Vining The following focusesexclusivelyon the latter.
(Biology Department, Dalhousie University, Halifax, Nova Scotia, Hybrid antibiotics represent a type of combination therapy in
Canada),wherehestudiedthemetabolicregulationofstreptomycete which the drug pair, rather than being independent entities, are
HarryLimeInstituteforPenicillinResearch,Schlettstadterstrasse21,Basel,Switzerland
Correspondence:SShapiro,HarryLimeInstituteforPenicillinResearch,Schlettstadterstrasse21,CH-4055Basel,Switzerland.
E-mail:[email protected]
Dedicatedtothememoryofmybelovedwife,CorineMiche`leShapiro-Bloch(1956–2013).
Received22January2013;revised7March2013;accepted11March2013;publishedonline24April2013
Speculativestrategiesfornewantibacterials
SShapiro
372
covalently and stably linked. An antibiotic hybrid should at least (iii) CBR-2092 (Figure 3), a fluoroquinolone–rifamycin hybrid
mimic,ifnotimproveupon,thesynergismobtainablewhenthetwo invented by the now-defunct Cumbre Pharmaceuticals, Inc.
differentpharmacophoresareadministeredsimultaneouslyasseparate (Dallas, TX, USA); and
drugs. Additionally, it is projected that independent mutations (iv) TD-1792 (Figure 4), a cephalosporin–glycopeptide hybrid
affecting each of the hybrid molecule’s targets will be required to (Theravance,Inc., South San Francisco, CA, USA).
compromise completely its clinical value. A further point for
considerationisthepharmacodynamicsofthehybridmoleculewhen AsdiscussedbyPokrovskayaandBaasov,12mosthybridantibiotics
the poles of the hybrid consist of moieties with dissimilar pharma- synthesized to date have a fluoroquinolone at one pole.
codynamicdrivers (vide infra). Fluoroquinolones, exemplified by ciprofloxacin, are bactericidal
A hybrid antibiotic ought to have the advantage of relatively drugs that target bacterial topoisomerases II and IV, and typically
straightforward pharmacokinetic and safety profiles, obviating the have a broad spectrum of activity that includes pseudomonads and
need to ‘mix-and-match’ pharmacological and toxicological proper- other recalcitrant Gram-negative pathogens. When combined with
ties that can complicate conventional combination therapy. None- oxazolidinones or rifamycins, however, the anti-Gram-negative
theless,‘mix-and-match’problemsoftencanberesolvedonthebasis activity of the fluoroquinolone component is compromised,
of a good understanding of the properties of antibiotic pairs, rendering the hybrid predominantly anti-Gram-positive.12 Loss of
supplemented by knowledge of the relevant physiological status of activity against Gram-negative pathogens likely is due, in large
the patient (renal sufficiency, hepatic sufficiency, etc.) and prior measure, to the bulk of the hybrids (mw MCB-3681, 627; mw
experiencewiththesameorsimilardrugcombinationsasreportedin
the biomedicalliterature.13
Inconventionalcombinationtherapy,twoindependentlyadminis-
tereddrugsarefreetoseektheirpreferredtargets.Wherethepolesof
a hybrid antibiotic have to interact with spatially disparate sites, the
amount of drug availabletoreachbothtargets is reduced, evenina
working volume as small as that of a bacterial pathogen, when
bindingofthehybridantibiotictoonetargetprecludessimultaneous
binding to its alternative target. This ‘bilocation dilemma’ can be
addressedeitherbyadministeringthehybridathigherconcentrations
tofacilitatepartitioningofthedrugbetweencompetingtargets;orby
designingahybrid whosepolestargetindependentbutnearbydrug-
binding sites(vide infra).
One of the earliest attempts to create a hybrid antibiotic was the
synthesis by Chu and Bardos14 of 2(4)-imino-4(2)-amino-2,4-
dideoxyriboflavin (Figure 1), a Januskopf structure (lacking a spacer
moiety), containing structural features of 2,4-diaminopyrimidine
antifolatesandriboflavinantagonists.Theinvitroantibacterialactivity
ofthiscompoundwasreversedbyexogenousfolinicacidorriboflavin.
Mostexperimentalhybridantibioticsconsistofapairofmostlyintact
antibiotic molecules, tethered by a linker or spacer, in which each
antibiotic’s binding site for its respective targetis preserved.12,15,16 At
least foursuchproducts have entered clinical trials:
(i) MCB-3837 (Figure 2), the phosphate ester prodrug of a
fluoroquinolone–oxazolidinone hybrid, MCB-3681, invented Figure 2 MCB-3837 (upper structure) and cadazolid (ACT-179811) (lower
by thenow-defunctMorphochemAG (Basel, Switzerland); structure).
(ii) cadazolid (ACT-179811; Actelion Pharmaceuticals, Ltd.,
Allschwil (BL), Switzerland) (Figure 2), a fluoroquinolone–
oxazolidinone hybrid structurally very similar toMCB-3681;
Figure1 2(4)-Imino-4(2)-amino2,4-dideoxyriboflavin. Figure3 CBR-2092.
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373
Figure4 TD-1792.
cadazolid,586;mwCBR-2092,1205),whichimpedespassagethrough hybrid is the inhibition of protein synthesis rather than of DNA
theoutermembraneofGram-negativepathogens,thougheffluxmay synthesis,26 though different oxazolidinone–fluoroquinolone hybrids
havesomerole,aslinezolid,17fluoroquinolones18andrifampicin19,20 reportedly can target preferentially either topoisomerase or the
aresubstratesforeffluxpumpsinGram-negativebacteria.Antibiotics ribosome.27 Cadazolid has MIC s towards methicillin-resistant S.
90
belonging to the fluoroquinolone and rifamycin families have a aureus(MRSA)andvancomycin-resistantenterococciof0.25mgml(cid:2)1
proclivity for rapid development of endogenous resistance during and2mgml(cid:2)1,respectively,23makingitofprospectiveclinicalinterest
serialpassageinthepresenceofsubinhibitoryconcentrationsofeither for treatment of systemic infections caused by these pathogens.
oftheseantibiotics,anditwasnotedbyRobertsonetal.21thatpassage However, the product seems to be in development exclusively for
oftheCBR-2092-susceptiblestrainStaphylococcusaureusATCC29213 C.difficileinfections,invitingquestionsaboutthesystemicstabilityor
(broth MIC, 0.015mgml(cid:2)1) in the presence of CBR-2092 led to toxicity of the drug, especially in the light of oxazolidinones being
elevatedMICstowardsthehybridthroughaccumulationofmutations well-known mitotoxicants,28 and fluoroquinolones being associated
in RNA polymerase (Rpo) and topoisomerases II (Gyr) and IV with a broad array of adverse effects29–32 and increased risk of C.
(Par): 0.125mgml(cid:2)1 by passage 2 (rpoBR484H), 0.5mgml(cid:2)1 by difficile infection.33 Unfortunately, issues relating to safety
passage 5 (rpoBR484HgyrADL520), culminating at 64mgml(cid:2)1 pharmacology and toxicology of parenteral cadazolid have not been
by passage 26(rpoBR484HgyrADL520,S84LparCR236(duplication),H103Y). reported. Insofar as cadazolid, scheduled to commence a Phase III
Cadazolid was supposed to have been introduced at the 22nd trial this year (http://www1.actelion.com/en/our-company/news-and-
European Congress of Clinical Microbiology and Infectious Diseases events/index.page?newsId=1666815), is being positioned narrowly as
in April 2012,22 but actually debuted some months later during an oral treatment for C. difficile infection, it may be expected to
the 52nd Interscience Conference on Antimicrobial Agents and encounter fierce competition, not only from the current generic
Chemotherapy (ICAAC). The compound presumably is devoid of standards of care, metronidazole and vancomycin, but also from a
clinically useful anti-Gram-negative activity (except perhaps towards recentlyapproveddrug,fidaxomicin(lipiarmycin,amacrocycle),and
some Bacteroides spp., for which an MIC of 4mgml(cid:2)1 was numerous products in development addressing the same niche,
90
reported23), and it is in development for treatment of infections including ramoplanin (a lipoglycodepsipeptide), surotomycin
caused by Clostridium difficile, for which an MIC of 0.25mgml(cid:2)1 (formerly CB-183,315, a lipopeptide), rifaximin (an ansamycin),
90
was obtained.23,24 Oxazolidinones (for example, linezolid) and NVB302 (a lantibiotic) and LFF571 (a thiopeptide). Moreover,
fluoroquinolones generally have good oral bioavailability, but that antibiotic treatment of C. difficile infection eventually may be
ofcadazolidisnegligible.25Cadazolidisactiveagainstbothlinezolid- supplanted by fecaltransplanttherapy.34
and moxifloxacin-resistant strains of C. difficile, and population In 2009, Long and Marquess15 published a table of hybrid
analyses suggest that the hybrid molecule has a low frequency of antibiotics gleaned from the patent literature, to which may be
resistance towards this pathogen. Serial passage on subinhibitory added the aminoglycoside–fluoroquinolone hybrids of Pokrovskaya
concentrations of cadazolid produced strains with MICs 1 log et al.,35 the mutilin–fluoroquinolone hybrid of Asahina et al.,36 and
2
dilution step higher towards this compound than those of the the cephalosporin–triclosan hybrid of Li et al.37 This plethora of
parents,23 though with o10 serial passages having been performed preclinical structures conveys the impression that efforts to create
itisprematuretoreachfirmconclusionsaboutthelackofproclivity hybrid antibiotics oftenamount to little more than random pairing,
forendogenousresistanceemergenceofC.difficiletowardscadazolid. where compounds with known antibacterial activity are combined
The principalmode ofaction ofthis oxazolidinone–fluoroquinolone arbitrarily in the hope of obtaining hits that will lead to clinical
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Speculativestrategiesfornewantibacterials
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374
candidates.Withoutdenigratingtheingenuityandresourcefulnessof
the medicinal chemists tasked with synthesizing hybrid antibiotics,
oneisleftwithanaggingfeelingthatthiseffortcouldbedirectedboth
more rationallyand more successfully.
Onestrategyistocircumventthe‘bilocationdilemma’byselecting
pairsofantibioticsbindingtoindependenttargetsincloseproximity
to one another. Such an approachwas pursued by Theravance, Inc.,
whosescientistsreasonedthattetheringcephalosporins,whichtarget
the D,D-transpeptidase activity of penicillin-binding proteins in the
bacterial periplasm (or ‘inner wall zone’), to glycopeptides, which
bind the terminal D-alanyl-D-alanine moiety of a lipid-bound
periplasmic peptide involved in cell wall crosslinking, would yield
hybrids where both poles can bind simultaneously to their targets.
Synergism has been documented for combinations of b-lactam
Figure5 Sparsophenicol.
antibiotics with glycopeptides towards glycopeptide-resistant enter-
ococci,38,39 and whenvancomycin and cephalosporins were attached
at different positions, all of the hybrids exhibited synergistic
antibacterial activity. That the specifics of attachment of
vancomycin to cephalosporin had little impact on activity implies
that the two poles of a single dimer do not bind simultaneously to
their different targets,40 the observed synergism arising from
inhibition at successive steps in the same pathway, as occurs when
sulfamethoxazole,adihydropteroatesynthetaseinhibitor,iscombined
with trimethoprim, a dihydrofolate reductase inhibitor41 (also see
Osborne etal.42).The compound which Theravance progressed into
clinicaldevelopment,TD-1792,isaheterodimerofvancomycinanda
ceftazidime-like b-lactam, bactericidal towards MRSA and
heterogeneous vancomycin-intermediate S. aureus (hVISA), with an
MICtowardsthesestaphylococciof0.03mgml(cid:2)1,43aswellasanMIC
towards C. difficile of 1mgml(cid:2)1.44 The pharmacodynamic driver for
efficacyofvancomycinisAUC/MIC,whereasthatofcephalsoporinsis
t4MIC.45InamurineneutropenicthighmodelofMRSAinfection,
Hegde et al.46 found that AUC/MIC was a better predictor of TD-
1792efficacythant4MIC.Ofcourse,thespectrumofamoleculeof Figure 6 Chloramlincomycin (upper structure) and lincophenicol (lower
structure).
this size (mw TD-1792, 1983) is restricted to Gram-positive
pathogens. Results of a Phase II trial of TD-1792 in complicated
skinand soft tissueinfectionshave beenpublished.47
Besidesperiplasmicconstituents,anothertargetofpotentialinterest molecule is linked to the pyrrolidine of clindamycin. Neither hybrid
for rationally designed hybrid antibiotics is the ribosome. High- molecule inhibited polyphenylalanine biosynthesis in an in vitro
resolutionX-raystructuresofantibioticsboundtothe50Sribosomal system prepared from Escherichia coli though, surprisingly,
subunit of bacteria48–52 showed overlap between the binding sites of chloramlincomycin had an MIC of 6.25mgml(cid:2)1 vs a strain of
erythromycin,clindamycinandchloramphenicol,anditislikelythat, Streptococcus pyogenes towards which chloramphenicol had an MIC
for most eubacteria, conservation of ribosome structure across of 3.13mgml(cid:2)1.
species and genera translates into similarities in modes of binding Guided by precise information about antibiotic binding to ribo-
oftheseantibioticstothepeptidyltransferasecenterorexittunnelof somesaffordedbyX-rayanalysis,scientistsatRib-XPharmaceuticals,
the ribosome. Inc.(NewHaven,CT,USA)synthesizedRx-2102(Figure7),ahybrid
Evenbeforehigh-qualitycrystallographicdataforantibioticsbound of florfenicol and azithromycin connected by an 4-(n-propyl)-1,2,3-
to bacterial ribosomes were available, biochemical studies53,54 had triazolespacer,withlowMICs(p1mgml(cid:2)1)towardsnotonlyawild-
indicated that erythromycin, clindamycin and chloramphenicol typepneumococcus,butalsotowardspneumococcalstrainsresistant
shared nearby, probably overlapping, binding sites, which led some toazithromycinduetoanA2058G(E.colinumbering)transitionand
groups, such as that of Jirˇı´ Zemlicˇka at the Wayne State University toA2058monomethylationþan L4 mutation.52
School of Medicine (Detroit, MI, USA) to rationally design hybrid While hybrid antibiotics are, by definition, heterodimers, some
antibiotics with improved binding affinities for ribosomes. In the effort also has been directed towards the rational design of homo-
early 1980s, Zemlicˇka and Bhuta55 reported synthesis of dimers.Vancomycin,forexample,formsadimerinaqueoussolution,
sparsophenicol (Figure 5), derived from chloramphenicol and spar- anddimerizationofthisglycopeptide isenhancedinthepresenceof
somycin (a product of Streptomyces sparsogenes), which strongly the cell wall analog di-N-acetyl-L-Lys-D-Ala-D-Ala. On the presump-
inhibited protein synthesis in vitro, but was devoid of antibacterial tion that vancomycin dimerization has an important role in its
activity. A decade later, Zemlicˇka et al.56 reported synthesis of antibioticactivity,57medicinalchemistshavesynthesizedvancomycin
chloramlincomycin (Figure 6), in which the aliphatic arm of dimersasprospectiveantibiotics.Griffinetal.58systematicallyprobed
chloramphenicol is linked to the pyranoside of clindamycin; and the effect of linkage orientation and linker length on antibacterial
lincophenicol (Figure 6), in which most of the chloramphenicol potency towards Gram-positive cocci, and observed that both
TheJournalofAntibiotics
Speculativestrategiesfornewantibacterials
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375
considered to be equivalent, though Elemam et al.61 identified
KPC-producing Klebsiella pneumoniae strains more susceptible
(up to 4 log dilution steps) to colistin than to polymyxin B.
2
Moreover, polymyxin B and colistin may have different clearance
mechanisms.62
During the past decade, efforts have been made to modulate the
renotoxicityassociatedwithpolymyxinswithoutcompromisingtheir
pharmacodynamicpropertiesbyusing‘rational’dosingregimens.63–66
However,internationallyacceptedguidelinesforuseofpolymyxinsto
treatseriousGram-negativeinfectionshaveyettoemergefromthese
studies.
An alternative to improved dosing is the discovery and develop-
ment of new polymyxins unencumbered by the toxicities associated
with colistin and polymyxin B. At the 50th ICAAC in 2010, Cubist
(Lexington, MA, USA) and BioSource Pharm, Inc. (Spring Valley,
NY, USA) presented a dozen posters about a novel polymyxin,
CB-182,804 (Figure 8), which has been followed up by a single
article devoted to the product.67 Towards a collection of over 5400
clinical isolates of Enterobacteriaceae (E. coli, K. pneumoniae,
Enterobacter spp.) and non-fermentative bacilli (Acinetobacter
baumannii, Pseudomonas aeruginosa), MIC s for CB-182,804 were
90
higher than those for polymyxin B by 2 log dilution steps;67
2
moreover, the data presented at ICAAC failed to demonstrate
convincingly that the nephrotoxic potential of CB-182,804 was
diminished significantly compared with that of polymyxin B. Since
2010 there has been no update on the status of CB-182,804 on
Figure7 Rx-2102. Cubist’swebsiteand,absentevidencetothecontrary,itislikely that
the development program for thisproduct hasbeenshelved.
Others have pursued a more rational and systematic approach to
the discovery of safer polymyxin derivatives, predicated on the
parameters influence the in vitro activity of vancomycin dimers, assumption that cationicity is an important determinant of amino-
thoughnosingledimerproveduniformlymorepotentthanallother glycosidenephrotoxicity,68,69thoughBosmansandDeBroe70remark
dimers (or vancomycin, for that matter) against pneumococci, thataminoglycosidenephrotoxicpotentialdoesnotcoincide(strictly)
enterococciand S.aureus displaying variousresistotypes. with cationic load. Northern Antibiotics Oy (Helsinki, Finland) has
The considerable investment ofintellectual and material resources created synthetic polymyxins, or ‘neopolymyxins’, modeled after
to date has generated few hybrid antibiotics that can be considered polymyxin B and colistin, bearing net charges over the range þ2
‘clinical trial-worthy’. It is likely that the success rate could be to þ5. When a subset of these compounds was assayed
improved substantially if laboratories would forego the temptation for binding affinity to rat renal cortex brush border membranes,71
to pressgang into hybrids whichever antibiotics pass their way, and an approximately linear relationship between cationic load and
instead focus on innovative products that try to address the membrane affinity was obtained (Figure 9) (M Vaara and T Vaara,
‘bilocationdilemma’,asZemlicˇka,TheravanceandRib-Xhavesought personalcommunication).72
to do. Moreover, given the particularly urgent need for new drugs Most of Northern Antibiotic’s efforts have focused on NAB739
targetingGram-negativepathogens,medicinalchemistswoulddowell (Figure8),aneopolymyxinmodeledafterpolymyxinBbutwithanet
toapplyprinciplesofrationaldrugdesigntotetherpharmacophores charge of þ3. The relatively modest affinity of this synthetic
whosecombinedsize(and/ormembranotropicproperties)enablethe lipocyclopeptide for rat renal brush border membranes suggests a
hybrids to surmount the barrier posedby the outer membrane. reduced potential for inducing acute kidney injury, consistent
with pharmacokinetic results, where bolus administration in
POLYMYXINSANDNEOPOLYMYXINS:BACKTOTHEFUTURE? Sprague-Dawley rats led to a much higher renal clearance
The alarming increase in incidence of infections attributable to (0.53±0.30mlmin(cid:2)1kg(cid:2)1 vs 0.010±0.008mlmin(cid:2)1kg(cid:2)1) and
multidrug-resistantGram-negativepathogenshasledtoaresurgence urinary recovery (19.40±7.38% of dose vs 0.18±0.14% of dose)
in the use of an old antibiotic class, the polymyxins. These cationic for NAB739 thanforcolistin.73
lipocyclopeptides, products of Bacillus spp., were discovered during No cytotoxicty towards Chinese hamster lung fibroblasts was
the 1940s and introduced into the clinic during the 1950s, but observed for NAB739 at concentrations up to 128mgml(cid:2)1.72
superseded by other drugs with anti-Gram-negative activity due to Studies with porcine renal proximal tubule epithelial cells showed
toxicityissues,includingallergenicity,neurotoxicity,andmostnotably thattheconcentrationofNAB739requiredtoinducenecrosisinthis
nephrotoxicity.59 Two generic polymyxin products are available, cell linewasnearlyan orderof magnitude higherthan thatrequired
polymyxin B and colistin (polymyxin E). While generally thought for polymyxin B.74 More recently, Vaara and Vaara75 found that the
to be interchangeable, there are some differences between these IC of NAB739 towards HK-2, an immortalized human proximal
50
products. Polymyxin B is administered intravenously as the active tubulecelllinethatdoesnotrequireelectroporationforsusceptibility
principle, whereas colistin is administered as a sulfomethylated topolymyxins,was337mgml(cid:2)1,whereasIC sforpolymyxinBand
50
prodrug to reduce its toxicity.60 The two antibiotics often are colistinwere 13mgml(cid:2)1 and 45mgml(cid:2)1, respectively.
TheJournalofAntibiotics
Speculativestrategiesfornewantibacterials
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376
Ms1.2
B
B 1
or r0.8
nity f0.6
aff0.4
e
v0.2
ati
el 0
r 0 1 2 3 4 5 6
cationic load
Figure 9 Cationic load vs (neo)polymyxin affinity for renal brush border
membranes (rBBMs), normalized to polymyxin B (affinity¼1). A full color
versionofthisfigureisavailableatTheJournalofAntibioticsjournalonline.
antibacterial activity of NAB739 towards E. coli and K. pneumoniae
approximatesthatofpolymyxinB,itsactivitytowardsA.baumannii
and P. aeruginosa is noticeably less. However, if the safety profile of
NAB739 proves disproportionately better than that of colistin or
polymyxinB,thenthetherapeuticwindowforNAB739maybewide
enough to enable its use for treatment of serious infections
attributable to non-fermentative Gram-negative bacilli, especially in
renally compromised patients unable to tolerate an additional
nephrotoxic burden. The outcome of a thorough preclinical toxicol-
ogystudyofNAB739(andits‘colistinanalog’,whichmaybeevenless
nephrotoxic than NAB739) in rodent and non-rodent species,
encompassing the latest information about renal biomarkers,
isawaitedeagerly.
ANTIMETABOLITES: TIMEFOR A RENAISSANCE?
Anantimetaboliteisacompoundwhosestructuralresemblancetoan
essential metabolite (biosynthetic intermediate or end product)
enables it to interfere with one or more essential physiological
processes, leading to bacteriostasis or bactericidality. The first
synthetic antibiotic, Prontosil, was an antimetabolite prodrug whose
active principle, sulfanilamide, resembles p-aminobenzoic acid
and is a competitive inhibitor of dihydropteroate synthetase.78
While nucleotide analogs have been introduced as antivirals
(forexample,azidothymidine)andasingleantifungalantimetabolite
(5-fluorocytosine) has been approved for therapeutic use, the only
antimetabolites used to treat bacterial infections are sulfa drugs and
trimethoprim. As these interfere with different steps in the pathway
leading to folic acid (‘vitamin B ’), sulfa drugs, especially
9
sulfamethoxazole, and trimethoprim often are combined into
a single orally availablemedication.
During the past 70 years, hundreds of antimetabolites (analogs of
nucleosides,aminoacids,carbohydrates,lipids,vitaminsandsoforth)
have been synthesized or isolated from natural sources, but few of
thesehaveachievedmarketingapprovalbyhealthregulatoryagencies,
Figure8 CB-182,804(upperstructure)andNAB739(lowerstructure). sometimes for reasons of toxicity, and sometimes due to lack of
efficacy during clinical trials. However, an urgent need for new
antibiotics to treat refractory multidrug-resistant infections warrants
MIC s(mgml(cid:2)1)forNAB739were2,2,8and16towardsE.coli, reconsideration of the antimetabolite approach as a source of new
90
K. pneumoniae, Acinetobacter spp., and P. aeruginosa, respectively, antibacterials. The success of sulfonamides and trimethoprim as
comparedwith2,1,2and2forpolymyxinB.76Inamousemodelof inhibitors of the biosynthesis of folic acid raises the question
intra-abdominal infection with E. coli IH3080 (O18:K1:H7),77 ofwhetherthisstrategycanbeappliedsuccessfullytoothervitamins.
NAB739 dosed at 4mgkg(cid:2)1 proved approximately as effective, in Consider, for example, biotin (‘vitamin H’, ‘vitamin B ’), a small
7
terms of change in log CFU per peritoneum 6h after initial sulfur-containing molecule that acts as a carboxyl shuttle in physio-
10
treatment,as polymyxinB dosedat2mgkg(cid:2)1. logical carboxylations, biosynthesized by microorganisms and plants
The neopolymyxin congeners invented by Northern Antibiotics butnotmammals.79Duringtheearly1950s,Grundyetal.80isolated
remain at an early stage of preclinical development. While the from the spent medium of Streptomyces virginiae a small molecular
TheJournalofAntibiotics
Speculativestrategiesfornewantibacterials
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377
Figure 10 Actithiazic acid (acidomycin) (upper structure) and
amiclenomycin(lowerstructure). Figure11 50-Amino-50-N-(biotinyl)sulfamoyl-50-deoxyadenosine.
weight compound, actithiazic acid (acidomycin) (Figure 10), with bisubstratehasanMICp0.5mgml(cid:2)1towardsM.tuberculosisstrains,
invitroantimycobacterialactivity.Twentyyearslater,scientistsatthe withvery low toxicities towards Vero (African green monkey kidney
Institute of Microbial Chemistry in Tokyo81,82 isolated from culture epithelial) cells and DU145 (human prostate cancer) cells, but was
filtratesofStreptomyceslavendulaesubsp.amiclenomycinianL-amino inactive (MICs 4256mgml(cid:2)1) towards Gram-positive cocci,
acid, amiclenomycin (Figure 10), whose in vitro inhibitory activity, Gram-negative bacilli and yeasts, possibly due to restricted uptake
restricted to mycobacteria, was reversible by exogenous biotin and/orefflux.
(whereas amiclenomycin originally was proposed to have a trans In contrast to biotin, mammalian cells possess the genetic
structure,Mannetal.83showedthatithasacisstructure).Eisenberg machinery (localized to their mitochondria) for synthesis of
andHsuing84attributedtheactionofactithiazicacidtoinhibitionof R-(þ)-a-lipoic acid, a coenzyme involved in decarboxylation
biotinformationfromdethiobiotinbybiotinsynthase(BioB),though reactions. Lipoic acid biosynthesis proceeds through attachment of
just how actithiazic acid blocks the formation of the tetrathiophene octanoate to the lipoyl domain (the E2 subunit of a-ketoacid
ringofbiotinremainsobscure.In2005,Mannetal.83describedhow dehydrogenases or the H subunit of glycine decarboxylase) of a
amiclenomycinreactswithpyridoxal-50-phosphateintheactivesiteof lipoate-dependent enzyme, followed by sulfurylation to generate
BioA (7,8-diaminopelargonic acid aminotransferase), an enzyme adithiolanering.Endogenouslipoatesynthesisisessentialformouse
involved in an early step of biotin biosynthesis, to form an embryonic survival,89 though the extent to which mammalian cells
aromatic adduct that remains tightly bound to the active site, generallycansatisfytheirneedforlipoatethroughdenovosynthesisis
thereby aborting biotin synthesis. A study by Shi and Aldrich,85 unclear.Bacteriaandmammaliancellspossesslipoateproteinligases,
using synthetic analogs of amiclenomycin, indicated that inhibitory thatrecyclelipoatereleasedduringproteindegradationbyattachingit
activity is dependent upon the ring moiety being planar rather than to empty lipoyl domains, either directly or in concert with an
puckered. amidotransferase.90 Analogous to the reaction catalyzed by biotin
Thus,already40yearsago,therewasevidencethatsomemicrobial protein ligase, lipoate attachment through the action of lipoate
natural products affecting biotin biosynthesis selectively inhibited protein ligaseproceedsthrough a lipoyl-AMPintermediate.91
mycobacteria.Thepastdecadehaswitnessedarenewalofinterestin Certainbacterialpathogens,suchasS.aureus,Enterococcusfaecalis,
antibiotins as a potential means of treating multidrug-resistant S. pyogenes, Chlamydia trachomatis and Listeria monocytogenes,
and extremely drug-resistant tuberculosis. Park et al.86 reported that are lipoate auxotrophs,92–94 whereas Burkholderia pseudomallei, the
deletionofthegeneencodingBioAmakesM.tuberculosisdependent causative agent of melioidosis, seems to have a complete pathway
on exogenous biotin, with little or no growth at o0.006mgml(cid:2)1 of forlipoatebiosynthesisbutrequiresexogenouslipoateforvirulencein
biotinandwild-typegrowthat40.06mgml(cid:2)1.Asthebiotintiterin mice and for optimal intercellular spreading.95 Inhibition of lipoate
human serum is B0.0005mgml(cid:2)1, M. tuberculosis cannot acquire protein ligase by a bisubstrate analog of lipoyl-AMP might afford a
sufficient biotin from a human host to establish and maintain an potent, possibly narrow-spectrum chemotherapeutic agent for
active infection, so the disease state is dependent upon de novo treating infections attributable to certain pathogens. As of this
synthesis of biotin by the pathogen. The authors remark that the writing, nosuchinhibitors have beendescribed.
conclusion of Kitahara et al.82 that the ability of M. tuberculosis to Incontrasttobiotinanda-lipoic acid, which intheircatalytically
establishand maintainaninfectionisindependentofde novobiotin active form always are covalently bound to their apoenzymes, the
synthesis is an artifact of amiclenomycin’s poor pharmacokinetic riboflavin (vitamin B , vitamin G)-derived coenzymes flavin mono-
2
behavior, including spontaneousaromatization. nucleotide(FMN)andflavinadeninedinucleotide(FAD)usuallyare
Rather than targeting biotin biosynthesis per se (for example, notcovalentlyattachedtotheirapoenzymes.96Whereflavinylationof
SoaresdaCostaetal.87),someresearchgroupshavefocusedonbiotin an amino acid residue occurs, it will have proceeded without the
protein ligase, which catalyzes attachment of biotin to the e-amino intercessionofadedicatedflavinproteinligase.97,98Freeriboflavinis
group of a conserved lysyl residue in biotin-dependent enzymes. devoidofbioactivity; itmustbe50-phosphorylatedbyflavokinaseto
Towards this end, Duckworth et al.88 synthesized a bisubstrate, FMN or further adenylylated by FAD synthetase to generate
50-amino-50-N-(biotinyl)sulfamoyl-50-deoxyadenosine (Figure 11), enzymically-active species, which participate in diverse biochemical
modeled after biotinyl-AMP formed by biotin protein ligase. The processes, mostly redox reactions.
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Figure 14 5-(4-Chlorophenyl)-5-hydroxy-3-(trifluoromethyl)-4,5-dihydro-1H-
pyrazol-1-yl](m-tolyl)methanone.
dihydroxy-2-butanone-4-phosphatewith6-(1-D-ribitylamino)-5-ami-
nouracil to form 6,7-dimethyl-8-ribityllumazine; and riboflavin
synthase, which combines two molecules of 6,7-dimethyl-8-ribityllu-
mazine to yield one molecule of riboflavin and one molecule of
6-(1-D-ribitylamino)-5-aminouracil. In the course of a longstanding
Figure 12 Roseoflavin (upper structure) and 8-demethyl-8-aminoriboflavin
research effort aimed at deciphering the mechanisms of these
(lowerstructure).
enzymes, Cushman et al.108 obtained a bienzyme competitive
inhibitor, 5-(1,5,6,7-tetrahydro-6,7-dioxo-8-D-ribityllumazin-5-yl-)
pentane 1-phosphate (Figure 13), with K values of 13mgml(cid:2)1 for
i
Bacillus subtilis lumazine synthase, 0.006mgml(cid:2)1 for M. tuberculosis
lumazine synthase, and 0.07mgml(cid:2)1 for E. coli riboflavin synthase.
Alumazinesynthase/riboflavinsynthasedualinhibitorshouldconfer
a therapeutic advantage over a monoenzyme inhibitor, withvitamin
synthesis constricted at two bottlenecks, requiring mutations in two
enzymes to achieve full resistance. Results of whole-cell assays with
this compound were not presented, and such compounds might
encounter difficulties gaining access to bacterial cytoplasm. In a
follow-upstudy,Cushmanetal.109usedhigh-throughputscreeningof
a commercial chemical library to identify [5-(4-chlorophenyl)-5-
Figure 13 5-(1,5,6,7-Tetrahydro-6,7-dioxo-8-D-ribityllumazin-5-yl-)pentane hydroxy-3-(trifluoromethyl)-4,5-dihydro-1H-pyrazol-1-yl](m-tolyl)
1-phosphate.
methanone(Figure14),anoncompetitiveinhibitorofM.tuberculosis
riboflavin synthase (K, 8.4mgml(cid:2)1), with an MIC (microplate
i
Alamar Blue assay) towards this pathogen (strain H Rv, replicating
37
Theriboflavinbiosyntheticpathwaysinmicroorganismsandplants phenotype)of14mgml(cid:2)1.
are well established.99 Riboflavin auxotrophy is rare among bacteria, Besides the examples given here, other coenzymes, such as
but riboflavin is an essential growth factor for some Gram-positive thiamine,110–113 CoA114–116 and menaquinone,117,118 have been
pathogens, including E.faecalis, S.pyogenesand L. monocytogenes;100 examined as potential targets for antibacterial therapy, and it
whereas some Gram-negative species, such as E. coli and Salmonella remains to be seen which, if any, of these avenues will attract
spp., lack flavin uptake systems and are dependent on endogenous adequate interest, manpower, and financing to support a sustained
riboflavinsynthesis.101Morgunovaetal.102 havearguedthatdenovo researcheffort.
biosynthesis of riboflavin probably is essential for survival of
pathogenicmycobacteria. RIBOSOMALMETHYLTRANSFERASE INHIBITORS:
Streptomyces davawensis produces roseoflavin (Figure 12),103 the EMBRACING EPIGENICITY?
only known natural riboflavin analog with antibiotic activity, which Target modification by methylation of rRNA constitutes an efficient
targets the FMN riboswitch in some bacteria.104,105 Roseoflavin is a means by which bacteria achieve resistance to ribosomotropic
substrate for human flavokinase and human FAD synthetase, antibiotics. Gram-positive and Gram-negative species can harbor
whereas its biosynthetic precursor, 8-demethyl-8-aminoriboflavin differentfamiliesofenzymescatalyzingtransferofmethylmoietiesto
(Figure 12),106 is a substrate only for human flavokinase. As most strategic sites on bacterial ribosomes, resulting in high levels of
flavoproteinsuseFADratherthanFMNastheircoenzyme,96Pedrolli resistance towards a broadspectrum ofantibiotics.
et al.107 suggested that 8-demethyl-8-aminoriboflavin might be a Aminoglycoside-modifying enzymes remain the predominant
better lead structure for development of novel flavin antibacterials, mechanism of resistance to this class of antibiotics, though the past
thoughtargetingthebiosyntheticpathwayforriboflavinmightprove decade has seen the emergence in Asia and global dissemination of
a better strategy, asthis pathway islacking inmammals. 16S ribosomal methyltransferases (16S-rMTases), principally among
Mostworkonantiriboflavinshasfocusedonthelasttwoenzymes Enterobacteriaceae, but also among P. aeruginosa and A. baumannii,
of the riboflavin pathway: lumazine synthase, which condenses 3,4- which pose serious problems for aminoglycoside therapy.
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Zhouetal.119reportedthat26%of741consecutivestrainsofGram- resistant that inducible producers,138 though this can be species-
negative bacilli collected at a Shanghai hospital contained a 16S- specific139 and may depend upon whether A2058 is mono- or
rMTase; and 490% of nearly 200 multidrug-resistant E. coli and K. dimethylated.140
pneumoniaeisolates collected inPeking by Yang etal.120 containeda During the past decade, still another 23S-rMTase of prospective
16S-rMTase (also see Li et al.121). SENTRY data for 2007–2008 clinicalimportancehasbeendescribed.Cfr(‘chloramphenicol-florfe-
indicatedprevalenceratesfor16S-rMTasesamongEnterobacteriaceae nicol resistance’) methylates C8-A2503 of bacterial 23S rRNA, con-
of10.5%inIndia,6.9%inChina,6.1%inKorea,5%inTaiwanand ferring resistance to phenicols, lincosamides, oxazolidionones,
3.1%inHongKong.122Whiletheprevalenceof16S-rMTasesoutside pleuromutilins, A-type streptogramins and 16-member macro-
Asia remains low, it is just a matter of time before this resistance lides.141,142 Though this enzyme is encountered infrequently, it has
mechanism migrates westward and becomes established in Europe been responsible for several outbreaks of linezolid resistance among
andNorth America. staphylococci,143–145(alsoseePournarasetal.146)andthecfrgenehas
Mostclinicallyrelevant16S-rMTases,belongingtotheRmtorArm beendetectedina strain of Proteus vulgarisofporcineorigin.147
families, methylate N7-G1405 and confer resistance to 4,6-disubsti- By restricting propagation of ribosomes with low avidities for
tuted2-deoxystreptamineaminoglycosides(foeexample,gentamicin, antibiotics,administrationofasuitablemethyltransferaseinhibitorto
tobramycin, amikacin and plazomicin (ACHN-490)122) but not ArmorRmtproducersoughttoenhancesusceptibility toaminogly-
4,5-disubstituted 2-deoxystreptamine aminoglycosides (for example, cosidesinapathogenpopulationwheretheseenzymesaresignificant
neomycin), 4-monosubstituted 2-deoxystreptamine aminoglycosides determinants of antibiotic resistance. Similarly, administration of an
(for example, apramycin), or those lacking a 2-deoxystreptamine ErmorCfrinhibitoroughttopromoteashiftinMICtowardslower
moiety (for example, streptomycin). Much less commonly values among pathogens where one or the other methyltransferase
encountered is NpmA, whose methylation of N1-A1408 confers a is the main determinant of drug resistance. To date, discovery of
broadrangeofresistance,encompassingneomycinandapramycinas selectivemethyltransferaseinhibitorstoovercomebacterialresistance
wellas4,6-disubstituted2-deoxystreptamineaminoglycosides,butnot hasfocusedlargelyonErms,thoughthereisnoreasonwhyanalogous
streptomycin.123,124 Distribution of these 16S-rMTases remains programs to identify Cfr or 16S-rMTase inhibitors could not be
confined to Gram-negative species, though their ability to function pursued.
in Gram-positive pathogens such as S. aureus is worrisome.125 Most methyltransferases use S-adenosyl-L-methionine (SAM) as a
Moreover, 16S-rMTases often are harbored on the same plasmid methyl donor, and the catalytic domain of SAM-dependent methy-
encoding metallo-b-lactamase NDM-1 and other resistance lasesiswellconserved.148Therefore,competitiveinhibitorsofSAMor
mechanisms,121,126 contributing to the threat of panresistant its demethylation product S-adenosyl-L-homocysteine, such as those
Gram-negative pathogens. describedbyHadjuketal.149orHanessianandSgarbi,150couldprove
KsgA is another 16S-rMTase, responsible for dimethylations of unduly toxic towards the human Erm orthologues DIM1 and
N6-A1518andN6-A1519,whosedistributionincludesGram-positive mt-TFB,151 as well as other essential mammalian SAM-dependent
species and mycobacteria as well as Gram-negative microorganisms. enzymes152,153(cf.Luetal.154).Thestreptomyceteproductsinefungin
ContrarytothesituationwithRmtorArmmethyltransferases,high- (Figure 15), a structural analog of SAM, is too toxic to be of either
level resistance towards kasugamycin is manifested in the absence of veterinary155orhumanclinicaluse.
KsgA activity, when A1518 and A1519 are not methylated.127 Nearly two decades ago, Clancy et al.156 screened 160000
Of greater relevance than its effect on kasugamycin resistance is the substances in the Pfizer chemical library and identified several
influence on ribosome biosynthesis of KsgA, where it appears to compounds belonging to different structural types that enhanced
function asa late-stage ribosome biogenesis factor, inwhich context theactivityofazithromycininvitrotowardsS.aureus,E.faecalisand
KsgAhasbeenproposedasan attractive antibacterial drugtarget.128 groupBstreptococciwithanMLS resistotypeandthatwerebelieved
B
The Erm (‘erythromycin ribosomal methylase’) enzymes comprise not to be competitive with SAM. When several hits were tested in
anotherfamily of structurally homologousmethyltransferases, which mice with lethal systemic infections caused by an MLS -resistant
B
methylate N6-A2058 in the peptidyl transferase center of 23S rRNA. S. aureus or a macrolide-susceptible S. pyogenes, none of them were
Overlap of binding sites for antibiotics to the bacterial ribosome abletorescuestaphylococcal-infectedanimals,andonlyoneofthem,
around A2058 leads to multidrug resistance towards macrolides, containing a penem moiety, was able to protect some streptococcal-
lincosamidesandclassBstreptogramins,129,130hencetheterm‘MLS ’ infected animals. However, no animal experiments involving
B
inconnectionwithresistanceduetoErms.FarmoreErmsthan16S- coadministration ofa macrolide orclindamycinwith anyofthe hits
rMTases have been described so far (http://faculty.washington.edu/ were described explicitly, and the project appears to have been
marilynr/ermweb1.pdf). discontinued. (It stands to reason that Clancy et al.156 would have
Erm-type 23S-rMTases occur in Enterobacteriaceae, Haemophilus
spp., Gram-positive and Gram-negative anaerobes and mycobac-
teria,131,132 but the clinical impact of MLS resistance is probably
B
greatest among Gram-positive cocci. ErmA is commonly found
among methicillin-resistant S. aureus and tends to be produced
constitutively, whereas ErmC is encountered more often among
methicillin-susceptible S. aureus and tends to be inducible, though
there may be regional differences; ca. 10% of clinical isolates of S.
aureus harbor both ErmA and ErmC133–135 (also see Spiliopoulou
etal.136).ErmBisfoundmostlyinstreptococciandenterococci;some
95% of clinical isolates of Enterococcus faecium are resistant to
erythromycin, primarily due to constitutive production of ErmB133
(cf.Portilloetal.137).ConstitutiveproducersofErmtendtobemore Figure15 Sinefungin.
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380
performed experiments in which mice given lethal doses of MLS - reported.Theauthorssuggestedthatasuperiorselectiveinhibitorof
B
resistant Gram-positive cocci were adminstered a purported Erm Erms mightbeobtainedbylinking SAMoran SAManalogwith an
inhibitor to assess its impact on rescuing animals cotreated with a rRNA analog, as the two substrates bind in adjacent clefts separated
macrolide or clindamycin, and Feder et al.157 assume that this was by a peptide ridge.
indeedthecase.However,Clancyetal.156donotstateexplicitlythat Recently Baker et al.163 described a scintillation proximity assay,
such experiments were done.) Though there is no indication of adaptabletohigh-throughputformat,forquantifyingtransferof[3H-
bindingexositesforErms,158GiannattasioandWeisblum159reported CH]SAMinto16SrRNAbyRmtAorKsgA,andinto23SrRNAby
3
several peptide inhibitors of ErmC0 (a soluble form of ErmC ErmC0. Baker and Rife161 used this assay to screen compounds for
produced by B. subtilis), which they proposed acted at sites other their ability to inhibit reactions catalyzed by ErmC0 and KsgA,
thanthe active siteofthe enzyme. supplemented by cocrystallization studies with ErmC0 to determine
The specificity of 16S- and 23S-rMTases for only one or two inwhichsubstratepockethitswerebinding.Followingonthereport
adjacent ribosomal nucleotides, and the inherent toxicological ofClancyetal.156thattheIC forErmC0ofN6-cyclopentyladenosine
50
risk of blocking the SAM binding site, clearly point towards the (‘UK-80882’) (Figure 18) was not sensitive to increasing concentra-
RNA binding site as the preferred target for design of ribosomal tionsofSAM,BakerandRife161preparedandtestedthiscompound
methyltransferase inhibitors. The three-dimensional structures of as a reference inhibitor, plus several new N6-substituted adenosine
ErmC0 and ErmAM (a B-type Erm from Streptococcus pneumoniae) derivatives as prospective ErmC’ or KsgA inhibitors. In their first
have been solved. Although no Erm-RNA cocrystals have been library, they found N6-octylamine adenosine to be the most active
obtained to date, the RNA binding pocket of ErmC0 has been compoundtowardsErmC0 (51%inhibition,comparedwith32%for
characterized,148 which can serve as a starting point for in silico anequimolarconcentrationofN6-cyclopentyladenosine),whereasthe
dockingofcombinatorialchemistryscaffolds.Moreover,Schluckebier most active compound towards KsgA was N6-octyladenosine
et al.160 obtained the structure of M.TaqI, a restriction-modification (39% inhibition, compared with 16% for an equimolar
enzymefromthethermophileThermusaquaticus,cocrystalizedwitha concentration of N6-cyclopentyladenosine). Cocrystallization with
10-base pair duplex oligodeoxynucleotide and the nonreactive SAM ErmC0 showed that N6-octylamine adenosine bound to the SAM
analog 50-[2-(amino)ethylthio]-50-deoxyadenosine. The catalytic pocket rather than the rRNA pocket, but careful analysis of this
domain of M.TaqI, an N6-adenine-specific DNA monomethylase, costructure suggested additional strategies for obtaining inhibitors
alignswith those ofErms andKsgA.161 that would bind preferentially to the rRNA site. When such
Using an in silico approach, Alvesalo et al.162 identified a pair of compounds were prepared during a second round of synthesis,
2,1,3-benzoxadiazole derivatives, ‘MB12’ and ‘MB13’ (Figure 16), improved inhibition towards ErmC0 was observed for several of
inhibitory towards Chlamydia pneumoniae, a cause of atypical them; the most active compound in this second library, 50-deoxy-
community-acquired pneumonia, to the extent of 56.1% and 50-butylphenylamine-N6-cyclopentyladenosine, inhibited ErmC0
85.5%, respectively, at 15–17mgml(cid:2)1. At that concentration, the (93%) nearly as much as an equimolar concentration of sinefungin
compounds had no cytotoxicity or viability effects on human lung (98%).KineticstudiesofErmC0 suggested thattheseinhibitorswere
cells.Federetal.157remarkthatthesecompoundsboundintheSAM bindinginthe rRNApocketratherthanintheSAM pocket, though
pocket of ErmC0 rather than in the rRNA pocket. Using their own confirmationbyX-rayanalysisofcocrystalswith‘second-generation’
docking approach, Feder et al.157 identified a compound, 4-methyl- inhibitors was not successful at showing a bound ligand in any site.
2,6-di[(4-methylphenyl)thio]nicotinonitrile (‘RF00667’) (Figure 17), Most‘second-generation’inhibitorswerepoorlyactivetowardsKsgA,
predictedtobindexclusivelyintherRNApocketofErmC0,whichwas indicating that, despite their overall structural and biochemical
a noncompetitive inhibitor of ErmC’, with an IC of 65mgml(cid:2)1. similarities, the two enzymes can behave differently towards
50
However,macrolideorclindamycinMICs,asafunctionofinhibitor inhibitors, and that optimizing the inhibitory power of a
concentrationinpathogensexpressingtheMLS resistotype,werenot compound towards one enzyme may compromise its inhibitory
B
Figure16 MB12(R¼H)andMB13(R¼Cl).
Figure17 4-Methyl-2,6-di[(4-methylphenyl)thio]nicotinonitrile(‘RF00667’). Figure18 N6-Cyclopentyladenosine(‘UK-80882’).
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