Bioenergetics of Mycobacterium

30 Pages·2017·15.04 MB·English

pathogens
Review
Bioenergetics of Mycobacterium: An Emerging
Landscape for Drug Discovery
IramKhanIqbal†,SapnaBajeli†,AjitKumarAkelaandAshwaniKumar* ID
CouncilofScientificandIndustrialResearch,InstituteofMicrobialTechnology,Chandigarh160036,India;
[email protected](I.K.I.);[email protected](S.B.);[email protected](A.K.A.)
* Correspondence:[email protected]
† Theseauthorscontributedequallytothiswork.
Received:11January2018;Accepted:31January2018;Published:23February2018
Abstract: Mycobacteriumtuberculosis(Mtb)exhibitsremarkablemetabolicflexibilitythatenablesit
to survive a plethora of host environments during its life cycle. With the advent of bedaquiline
for treatment of multidrug-resistant tuberculosis, oxidative phosphorylation has been validated
asanimportanttargetandavulnerablecomponentofmycobacterialmetabolism. Exploitingthe
dependenceofMtbonoxidativephosphorylationforenergyproduction,severalcomponentsofthis
pathwayhavebeentargetedforthedevelopmentofnewantimycobacterialagents. Thisincludes
targeting NADH dehydrogenase by phenothiazine derivatives, menaquinone biosynthesis by
DG70andothercompounds, terminaloxidasebyimidazopyridineamidesandATPsynthaseby
diarylquinolines. Importantly,oxidativephosphorylationalsoplaysacriticalroleinthesurvivalof
persisters. Thus,inhibitorsofoxidativephosphorylationcansynergizewithfrontlineTBdrugsto
shortenthecourseoftreatment. Inthisreview,wediscusstheoxidativephosphorylationpathway
anddevelopmentofitsinhibitorsindetail.
Keywords:Mycobacteriumtuberculosis;bioenergetics;oxidativephosphorylation;antimycobacterials;
drugs;bedaquiline;Q203;SQ109;electrontransportchain
1. Introduction
Tuberculosis(TB)remainsaleadingcauseofdeathworldwide, withanestimated1.3million
mortalitiesin2016. Thesynergyofhumanimmunodeficiencyvirus(HIV)-TBco-infectionhasfurther
aggravatedthisasamajorpublichealthproblem. TBtreatmentisriddledwiththeuseofmultiple
drugsforatleast6months. Suchlengthytreatmenthasresultedintheriseofmultidrugresistant
(MDR)andextremelydrugresistant(XDR)strainsofMycobacteriumtuberculosis(Mtb). Treatmentof
MDR TB requires administration of a multitude of second-line TB drugs for 18–24 months; this
leads to cure rates of 60–70%. Of even more concern, cure rates for XDR-TB range from 40–50%.
Theselowcureratescouldbefurtherconfoundedbyotherfactors,suchasco-infectionsandpoor
nutrition. Giventheseobservations,newdrugswithnovelmechanismsofactionareurgentlyrequired.
Suchinnovativedrugscouldsynergizewithcurrenttreatmentregimenstoimproverecoveryratesand
shortentreatmenttimes. Extensiveresearcheffortshavebeenmadeinthisdirection,andtheU.S.Food
andDrugAdministrationhasrecentlyapprovedtwonewdrugs. Thesedrugsarebedaquiline(BDQ),
adiarylquinolineATPsynthaseinhibitor,anddelamanid,anitro-dihydro-imidazooxazolederivative
thatinhibitsmycolicacidbiosynthesis. Acceleratedapprovalofthesedrugshasraisedhopeforanew
regimenthatcouldimprovetheoutcomeoftreatmentandreducedailydoseburden.
The myth that targeting Mtb bioenergetics would be a poor therapeutic strategy due to the
presenceofparallelandalternativemetabolicpathwayshasbeendispelledbythediscoveryofBDQ.
Given the success of this compound, several research groups have focused on targeting oxidative
Pathogens2018,7,24;doi:10.3390/pathogens7010024 www.mdpi.com/journal/pathogens
Pathogens2018,7,24 2of30
phosphorylationforthediscoveryofnewdrugs. Itisimportanttonotethat,unlikemanybacteriathat
canutilizesubstrate-levelphosphorylationforATPsynthesisandgrowth,Mtbreliesuponoxidative
phosphorylation for its viability. During oxidative phosphorylation, electrons are harvested from
centralmetabolicpathways,andthenfedintotheelectrontransportchain(ETC)throughreduction
ofmenaquinone. Lateron,thequinonesarere-oxidizedbyeitherthecytochromebc -aa complexor
1 3
bythecytochromebd-typeterminaloxidase. Thetransportofelectronsbetweenmembrane-bound
primarydehydrogenaseandterminalreductaseiscoupledtothegenerationoftheprotonmotiveforce
(PMF).TheenergygeneratedasPMFfromthisprocessisutilizedbyATPsynthaseintheproduction
of ATP. In this review, we have provided an overview of the machinery involved in oxidative
phosphorylation and the development of inhibitors (listed in Table 1) targeting the components
ofoxidativephosphorylation.
2. FeedingtheElectronsintotheElectronTransportChain: ReductionofMenaquinone
Duringtheinfectioncycle,Mtbcellsharvestreductiveenergyfromhost-derivedcarbohydrates
andfattyacidsusingNADH/NAD+and/orFADH /FADredoxpairs[1,2]. NAD+actsastheprimary
2
electronsinkandisconvertedintoNADH,whichfeedselectronsintotheETCthroughtheNADH
dehydrogenasecomplex. BesidesNADHdehydrogenase,anumberofotherprimarydehydrogenases
pumpelectronsdirectlyintotheETC,suchassuccinatedehydrogenase(SDH),prolinedehydrogenase,
and L-lactate cytochrome c oxidoreductase. In this review, we have primarily focused on NADH
dehydrogenaseandSDH.
2.1. NADH/MenaquinoneOxidoreductase
NADH/menaquinone oxidoreductases are the primary port of entry for electrons into
the ETC. There are three types of these oxidoreductases in bacteria: (i) the highly-complex,
multi-subunit-proton-pumping type I NADH dehydrogenase (NDH-1), (ii) a simple, single
subunit flavoenzyme-non-proton-pumping type II NADH dehydrogenase (NDH-2), and (iii) a
sodium-pumping NADH dehydrogenase (NQR) that is unique to bacteria [3]. In 2005, using a
genome mining approach, Weinstein et al. identified NDH-1 and NDH-2 in Mtb [4]. NDH-1 is
encodedbytheNuoABCDEFGHIJKLMN operonandispredictedtobetheenergyefficientNADH
dehydrogenasethattranslocatesprotons,whiletransferringelectronstomenaquinonetogenerate
PMF.Interestingly,genesencodingNDH-1arenotessentialinMtb[5],asexemplifiedbytheirloss
from Mycobacterium leprae through genome reductive evolution [4]. NDH-1 is downregulated by
acidicpH[6],nutrientstarvation[7],andinmurinelungs[8]. Thisexpressionprofilesuggeststhat
NDH-1playsaminorroleinMtbrespiration,andthus,isnotagoodtargetforthedevelopmentof
antimycobacterials. Thisviewisfurtherstrengthenedbytheobservationthatrotenone(anNDH-1
inhibitor)doesnotkillhypoxia-adaptedMtbcells[9]. However,recentlydevelopedreporterstrains
forimagingcellularNADH/NAD+levels[10]hasdemonstratedthatexposureofaerobicMtbcultures
torotenoneleadstoaccumulationofcellularNADHlevels,suggestingaroleforNDH-1intheETC
during aerobic conditions. Furthermore, infection of macrophages with an Mtb strain that has a
deletionofnuoG(whichencodesanNDH-1subunit)acceleratestheirapoptoticdeath. Moreover,this
mutanthasreducedsurvivalinamurinemodelofTBinfection[11].
Thenon-protonpumpingNDH-2isoformsareencodedbyndh(Rv1854c)andndhA(Rv0392c).
Both the two isoforms were found to be functional in biochemical assays. NDH-2 has a stringent
catalytic function, thereby minimizing leakage of electrons to oxygen during their transfer from
NADH to menaquinone. Importantly, Mtb NDH-2 utilizes only NADH, in contrast to NDH-2 of
Saccharomyces and Corynebacterium, which uses NAD(P)H [12]. Mtb NDH-2 transfers electrons to
the quinone pool by a ping-pong mechanism [13]. Sena et al. demonstrated that NDH-2 has two
distinct substrate binding sites (i.e., NADH and quinone). NDH-2 first interacts with NADH to
receiveelectrons,andthenreleasesNAD+beforeinteractingwiththequinone[14]. Itisassumedthat
thereductionofquinone/menaquinoneisarate-limitingstepintheoverallreaction[15]. Boththe
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isoforms of NDH-2 are conserved in slow-growing mycobacterial species, with the exception of
M.leprae,inwhichndhAisabsent[4]. ndhisessentialforMtbsurvival[5],whereasndhAdisruption
usingtransposonmutagenesisistolerated[16]. Importantly,disruptionofNDH-2throughinhibitors,
suchasphenothiazineanalogues,leadstomycobacterialdeathinaerobicculturesandinanimals[4].
In agreement with these findings, the NDH-2 inhibitor thioridazine (TZ) kills Mtb that are in a
hypoxia-inducednon-replicatingstate[9]. ndhisalsoinduceduponMtbinfectionofmacrophages[17].
Interestingly,mammalianmitochondrionlacksanNDH-2orthologue,whichprovidesasignificant
therapeuticwindow. EarlierstudieshavealsovalidatedNDH-2asatargettocontrolPlasmodium
growth[18].
2.2. InhibitorsofNADHDehydrogenase
TheabovecitedliteraturestronglysuggeststhatNDH-2isanattractivetargetwhoseinhibition
would elicit a choke point in the mycobacterial respiratory chain. In an effort to discover
additionalrespiratoryinhibitors,Harveyandcolleaguesfoundthatphenothiazineanalogueshave
antimycobacterial activity and demonstrated that these compounds inhibit NDH-2 [4]. They also
demonstratedthatclassicalinhibitorsofNDH-1(piericidinA,rotenone,andpyridaben)donotinhibit
Mtbrespiration. PhenothiazineswerealsoeffectiveagainstMDRstrains,suggestinganewmodeof
action[19,20]. Trifluoperazine(TPZ),aphenothiazineanalogue,caneffectivelyinhibitMtbgrowth,
andsynergizeswithrifampicin(RIF)tokillintracellularMtbresidinginmacrophages[21,22]. Arecent
report indicates that TPZ significantly inhibits ATP synthesis in M. leprae [23]. Phenothiazines
alsoinhibiteffluxpumpsandcalcium-bindingprotein. Similarantimycobacterialactivity(bothin
drug-sensitiveanddrug-resistantstrains)wasobservedwiththeNDH-2specificinhibitor,thioridazine
(TZ),inMtb-infectedmice[24]. TZhasbeenusedfor60yearstocontrolpsychosisandisconsidered
therapeuticallysafe. ThedrugtargetsNDH-2toblocktheETCofmycobacteriaandiseffectiveagainst
latentTB[13,25]. GiventhecriticalroleofNDH-2andthepotentactivityofTPZandTZagainstMtb
cells,highthroughputscreening(HTS)wasperformedwith11,000compoundsrationallyselected
fromacommerciallibrary(Biofocus,DPI).Thiseffortresultedintheidentificationofleadcompound
42a(MTC420)thathadaminimuminhibitoryconcentration(MIC)inthenanomolarrange. Thislead
compoundiscapableofkillingdrug-sensitiveand-resistantstrainsofMtbinaerobicandhypoxic
cultures. Furthermore,42aalsohasfavorablepharmacokineticandtoxicologicalprofiles[26].
Clofazimine (CFZ) is derived from phenazines, and has potent anti-tuberculosis activity [27].
ThedrugeffectivelykillsM.leprae,andisthereforeusedforthetreatmentofleprosy[28].Themainside
effectofthisdrugisskinpigmentation. RecentlydevelopedCFZderivativesnamely,B746andB4157,
haveincreasedanti-mycobacterialactivityandelicitlesspigmentation,andarethereforesuitablefor
testingfortreatmentofmycobacterialinfectioninanimalmodels[29,30]. Aneminentstudyfromthe
HarveygroupdemonstratedthatthebactericidalactionofCFZdependsuponitsreductionbyNDH-2.
ThereducedformofCFZishighlyunstable,andspontaneouslyreactswithoxygentogeneratereactive
oxygenspecies(ROS)thatleadstothekillingofmycobacterialcells[31]. ThiscycleofCFZ-mediated
ROSgenerationcontinuesunderaerobicconditions. Importantly,theNDH-1presentinmitochondrial
andbacterialrespiratorychainsdoesnotreduceCFZ.Therefore,Gram-negativebacteria,including
Escherichia coli, Pseudomonas denitrificans, and P. aeruginosa do not produce bactericidal ROS upon
exposuretoCFZ[32,33]. CFZisalsoactiveagainstMDRTBstrains, ismetabolizedslowly, andis
associatedwithalowfrequencyofresistance[34]. CFZsynergizeswithBDQandQ203torapidlykill
Mtbinvitroandinsidemacrophages[35].
SinceNDH-2isarespiratorychokepointofMtb,AstraZeneca(India)performedanHTSagainst
MtbNDH-2with100,000compounds. Thiseffortidentifiedquinolinylpyrimidinesasanewclass
of NDH-2 inhibitors. A good correlation was observed between enzyme inhibition (nanomolar)
andanti-mycobacterialactivity(micromolar)ofquinolinylpyrimidines[36]. Thesecompoundsare
non-toxicandhavedesirableabsorption,distribution,metabolism,andexcretion(ADME)profiles,
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makingthemsuitableforfurtherdevelopment. Inlinewiththesefindings,compoundsbelongingto
7-phenylbenzoxaboroleseriesexhibitedpotentanti-Mtbactivity[37].
2.3. SuccinateDehydrogenase
Thesuccinatedehydrogenase(SDH)orsuccinate/menaquinoneoxidoreductaseformscomplexII
oftherespiratorychain. Importantly,thisenzymecomplexisalsoanintegralpartofthetricarboxylic
acid (TCA) cycle. SDH links the respiratory chain with central metabolism [38]. The enzyme
oxidizessuccinatetofumarateinthecytoplasm,andduringthisprocess,itsimultaneouslyreduces
menaquinonetomenaquinolinthemembrane. Thisenzymecomplexiscomprisedoffoursubunits:
SdhA, SdhB, SdhC, and SdhD. SdhA is a flavoprotein (flavin is bound here covalently, and not
as a cofactor) that catalyzes the conversion of succinate into fumarate. SdhB possesses three Fe–S
clustersthatfacilitatethetransferofelectronsfromsuccinatetomenaquinone. TheSdhCandSdhD
subunitsanchorSdhAandSdhBtothemembrane,facilitatingthetransferofelectronsfromsuccinate
to menaquinone using haem. Notably, fumarate reductase (FRD) is a paralogue of SDH that can
catalyze the reverse reaction. FRD (encoded by the FrdA-Rv1556 operon) is primarily functional
in anaerobic conditions. Mtb utilizes menaquinone (MQH2 E(cid:48) = −74 mV) to reduce fumarate in
0
thelaststepoftheanaerobicETC[39–41]. Interestingly,MtbpossessestwoisoformsofSDH;Sdh1
(encodedbyRv0247c–Rv0249c)andSdh2(encodedbyRv3316–Rv3319). Itiscrucialtonotethatunder
hypoxicconditions,MtbcellsutilizethereverseTCAcycletoaccumulateandsecretesuccinateinto
theculturemedium[36,42]. Theexcretionofsuccinatehasbeenassociatedwiththeupregulationof
genesinvolvedinthereverseTCAcycle. Additionofsuccinatetotheculturemediumresultedin
membranepotentialdepolarizationandcelldeath,suggestingthatsecretionofsuccinateiscritical
for surviving hypoxic stress [42,43]. A role for SDH in this adaption had been predicted, since
inhibitionofSDHfunctionusing3-nitropropionate(3NP)resultsindecreasedsurvivalofMtb[42].
IndividualSdh1andSdh2deletionmutantsweregeneratedinordertodissecttheroleofbothSdh1
andSdh2duringhypoxicadaptation. ThisrevealedthatsurvivaloftheSdh1mutantwasimpaired
in stationary phase, while the survival of the Sdh2 mutant was not affected [44]. The function of
Sdh1asthecatalyticcenterduringaerobicconditionswasdemonstratedthroughtheuseofstable
isotopelabellingandmassspectroscopy[44]. Sdh2wasnotrequiredforthisactivityduringaerobic
conditions. Furthermore, depletion of Sdh1 resulted in increased levels of the reduced lipogenic
electroncarrier,menaquinol,whichwasassociatedwithanincreasedrateofrespiration,suggesting
thatSDHisaregulatorofrespiration[44]. Importantly,survivaloftheSdh1mutantwascompromised
intheMtb-infectedC3HeB/FeJmousemodelsystem,whichproduceslesionssimilartothehypoxic
granulomasfoundinhumansuponMtbinfection[44]. Theseobservationssuggestthatinhibitionof
SDHcanhelpintheeliminationofhypoxia-inducedpersistersintheinfectedlungs. Sdh1is,thus,a
potentialdrugtarget,however,stringentselectivitywillberequiredtoavoidageneralinhibitionof
mitochondrialrespiration.
2.4. MenaquinoneBiosynthesisandInhibitorsofMenaquinoneBiosynthesisPathwayinMtb
Ubiquinone (Q) and menaquinone (MK) are the primary lipid-soluble electron carriers that
transportelectronsintheETC[1]. InmostGram-positivebacteriaandinmycobacteria,MKisthe
primaryelectroncarrier,whileGram-negativebacteriapossessbothQandMK[45]. Mycobacterial
species,includingMtb,possessMK-9(H )(hereafterMK)[46,47].Besidesplayingtheroleasanelectron
2
carrier,MKassistsintheproperfoldingofsecretedproteinsthroughvitaminKepoxidereductase
or VKOR [48]. The ratio of oxidized to reduced MK is sensed by the DosRST system to control
mycobacterialmetabolism[49,50]. SinceMKistheonlyelectroncarrierinmycobacteria,andbecause
humansdonotsynthesizethisvitamin,itisbelievedtobeanexcellenttargetforthedevelopmentof
drugsagainstmycobacterialpathogens. MKissynthesizedinbacteriathroughaclassicalpathway
andasecond,alternatepathway. Bothpathwaysbeginwithachorismatemolecule,whichisthen
metabolizedtoadifferentMKprecursor,dependingonwhethertheclassicaloralternatepathwayis
Pathogens2018,7,24 5of30
engaged. AlthoughthealternatepathwayisoperationalinevolutionarilyrelatedStreptomyces,ithas
notbeenreportedinmycobacteria. Inregardtotheclassicalpathway,chorismateisconvertedinto
MKthroughaseriesofreactionscarriedoutbyenzymesencodedbytheMenFDHCEBAcluster[5],
Pathogens 2018, 7, x FOR PEER REVIEW 5 of 29
as summarized in Figure 1. Some of these enzymes are essential for mycobacterial growth [5,51]
enzymes are essential for mycobacterial growth [5,51] suggesting that MK biosynthesis is a valid
suggestingthatMKbiosynthesisisavaliddrugtarget.
drug target.

Figure 1. Mycobacterial menaquinone biosynthesis pathway and its inhibitors. (A–H) represent
Figure 1. Mycobacterial menaquinone biosynthesis pathway and its inhibitors. (A–H)
chorismate, isochorismate, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate, o-succinylbenzoate,
represent chorismate, isochorismate, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate,
o-succinylbenzoyl-CoA, 1,4-dihydroxy-2-naphthoyl CoA, demethylmenaquinone, and menaquinone
O-succinylrbeespnezcotiavetely,. OD-rsuugcs ctihnayt ltbaregnezt othyel -mCeonAaq,u1in,4o-ndei hbiyodsyrnotxhyes-i2s -ennazpymhtehs oayrel CshooAwn, dbeym reedt hflyatlhmeaedn aquinone,
andmenaqaruroinwos.n erespectively.Drugsthattargetthemenaquinonebiosynthesisenzymesareshown
byredflatheadarrows.
Since cholesterol metabolism is important for mycobacterial survival within the host, the effect
of exposure of Ro 48-8071 (an inhibitor of cholesterol biosynthesis) on mycobacterial growth was
Since cholesterol metabolism is important for mycobacterial survival within the host, the
analyzed. This compound killed Mycobacterium bovis Bacillus Calmette Guerin (BCG) cells at low
effectofexmpicorsoumroelaor fcoRncoen4t8ra-t8io0n7s1 [5(2a]n. Fiunrthhiebr imtoertaoboflcich laobleelslitnegr oexlpbeiroimseynntst hideesnitsif)ieodn MmK ybcioosbynatchteesrisi algrowth
was analytzoe bde .theT tharigsetc oofm Rop 4o8u-8n07d1. kTihlelseed finMdiyncgosb waecrtee rsiuupmporbteodv ibsy Bthaec oibllsuersvaCtioanlm theatt tReo G48u-8e0r7i1n w(aBs CG) cells
at low miccarpoambleo olaf rredcuocnincge onxtyrgaetnio cnonssu[5m2p]t.ionF duurrtinhge grromwethta. Fbuorlthicer lsacbruetlinliyn lgede tox ptheer iidmenetnifitcsatiiodne onft ified MK
MenA (Rv0534c) as the target of Ro 48-8071. The same group utilized a rational drug design
biosynthesistobethetargetofRo48-8071. Thesefindingsweresupportedbytheobservationthat
approach to target MenA. They screened around 100 molecules, and established that an
Ro 48-8071allwylaamsincoampaetbhloenoonfe rceldasus coifn gcomoxpyougnedns caoren psuotmenpt tiniohnibidtoursr ionf gMgtbro swtratihn.s, Finucrluthdienrg stchrouset iny led to
theidentifiwchaictiho nareo fdMruge-nreAsis(tRanvt 0[5533]4. cT)haiss tchlaesst aorf gMetenoAf Rinohi4b8it-o8rs0 7w1a.sT ahlseo shaimghelyg erfofeucptivue tialgiaziendst arational
drugdesighnypaopxipar-ionadcuhcedto dtraurgg-teotleMranetn pAer.siTstheresy [5s4c].r eInetnereedstianrgolyu, anudra1c0hi0n mREo, lae nceuwle qsu,iannoldinee satnatibbliiosthice dthatan
isolated from Rhodococcus erythropolis JCM 6824 is highly effective against Gram-positive bacteria
allylaminomethononeclassofcompoundsarepotentinhibitorsofMtbstrains,includingthosewhich
[55]. The aurachin RE molecules were modified to specifically target MenA, and validated as MenA
aredrug-reinshisibtaitnorts[ i5n3 f]u.nTcthioisnacll ians vsitorfo MasseanyAs. Tinhehsieb ciotomrpsowunadss awlseore hloiwgh mlyicreofmfeocltairv ienhaigbaitionrss tofh ayeproobxici a-induced
drug-tolergarnowttphe arnsdis theyrpsox[5ic4 ]s.urIvnivtaelr eofs tMintbg l[y5,6]a. uArdadcihtiionnaRllyE, ,7a-mneethwoxyq-u2-innaoplhitnheol-abnasteibd iomtoicleciusloelsa ted from
Rhodococcuhsaevrey bteherno puotilliiszeJdC aMs l6ea8d2 4striuschtuigrehs lfyore tfhfee cdteivveeloapgmaeinnts otfG nroanm-tr-apdoitisointiavl einbhaibcittoerrsi aof[ 5M5e]n.AT hofe aurachin
Mtb [57]. These efforts led to the development of bicyclic inhibitors that exert potent growth-
REmoleculesweremodifiedtospecificallytargetMenA,andvalidatedasMenAinhibitorsinfunctional
suppressing activity against Mtb [57]
invitroassays. Thesecompoundswerelowmicromolarinhibitorsofaerobicgrowthandhypoxic

survivalofMtb[56]. Additionally,7-methoxy-2-naphthol-basedmoleculeshavebeenutilizedaslead
structuresforthedevelopmentofnon-traditionalinhibitorsofMenAofMtb[57]. Theseeffortsledto
thedevelopmentofbicyclicinhibitorsthatexertpotentgrowth-suppressingactivityagainstMtb[57].
Pathogens2018,7,24 6of30
High-densityphenotypicprofilinghasidentifiedmenB(Rv0548c)asessentialformycobacterial
growth[58]. MenBcatalyzestheformationof1,4-dihydroxy-2-naphthoyl-CoAfromo-succinylbenzoyl
CoA through a Claisen condensation reaction. Li and coworkers performed an HTS with more
than 100,000 compounds, which identified a few hundred leads. Subsequent elaboration yielded
multiple1,4-benzoxazinecompoundsthatwerepotentinhibitorsofMenBactivity,andconsequently
blocked mycobacterial growth [59]. These leads were then utilized for the development of
4-oxo-4-chlorophenylbutenoylmethylesterderivativeswithpotentmycobactericidalactivityagainst
replicatingandnon-replicatingMtb[60].
MenE (Rv0542c) is the fifth enzyme in the MK biosynthesis pathway, and catalyzes the
formationofOSB-CoAfromO-succinyl-1-benzoate(OSB)throughtwomechanisticsteps. Thefirst
is the ATP-dependent adenylation of OSB, and the second step is thioesterification with CoA.
Given the structural and functional similarity of MenE with adenylate-forming enzymes, it is a
promisingtargetofacyl-AMPintermediates. Consideringthemechanisticdetails,Tanandcoworkers
employed OSB-AMP analogues to inhibit MenE in Mtb [61]. The IC of OSB-AMP analogues
50
MeOSB-AMS, MeOSB-AMSN, and MeOSB-AVSN for MenE are in the low micromolar range [61].
FurtherdevelopmentoftheseanaloguesresultedintheidentificationofOSB-AMSwithanIC for
50
MenEinthenanomolarrange;thesecompoundsarecompetitiveinhibitorsofthebindingofATPand
OSB[62]. Paradoxically,theseinhibitorshadpoormycobacterialgrowthinhibitoryactivity,perhaps
duetotheirinstability,andtheOSB-AMPanalogueswere,therefore,furthermodified. Thisresultedin
thedevelopmentofOSB-AMS.Importantly,thedifluoroindanediolanalogue11ofOSB-AMSwasan
effectivemycobactericidalcompound[63].
Inanefforttoidentifyinhibitorsofoxidativephosphorylation,Allandandcoworkerscreated
a cell-based screen [64]. In this screen, the mWasabi reporter was fused to the promoter of the
cydAB operon (coding for part of cytochrome bd oxidase) to monitor inhibition of respiration in
thepresenceof168knowninhibitorsofmycobacterialgrowth. ThisledtotheidentificationofDG70
asaninhibitorofbacterialrespiration. DG70isabiphenylbenzamidethatkillsbothdrug-sensitive
and -resistant Mtb cells [64]. Interestingly, this novel inhibitor is highly specific against Mtb, and
does not kill non-tuberculous mycobacteria (NTMs), Gram-positive, Gram-negative, or ESKAPE
(Enterococcusfaecium,Staphylococcusaureus,Klebsiellapneumonia,Acinetobacterbaumannii,P.aeruginosa,
andEnterobacter)pathogens. SequencingoftwoDG70-resistantspontaneousmutantstrainssuggested
the presence of a unique single nucleotide polymorphism (SNP) in menG. MenG (Rv0558, also
known as MenH) catalyzes the conversion of demethylmenaquinol to menaquinol. Importantly
MK4, an analogue of MK9, rescued the DG70-mediated killing of Mtb cells, thus validating that
MK biosynthesis is the target of DG70. Importantly, DG70 synergizes with BDQ, isoniazid (INH),
andPA824tokillMtb[64]. Insummary,alltheseobservationsindicatethatsmallmoleculetargeting
ofMKbiosynthesis(summarizedinFigure1)isavalidtherapeuticstrategy.
3. OxidationofMenaquinone: ATaleofTwoTerminalOxidases
Mycobacteria possess a branched ETC. During aerobic respiration, electrons are fed into the
menaquinone pool of respiratory chains. Electrons are then transferred to oxygen
(reduced/oxidized)
through two branches, each of which possesses a terminal oxidase. One is cytochrome bd-type
menaquinol oxidase (encoded by cydABDC), wherein electrons are transferred directly from MK
(reduced)tooxygen. Theotherbranchrunsthroughasupercomplexofmenaquinol-cytochromec
oxidoreductase or the bc complex and aa type cytochrome oxidase [1,65–67]. The bc -aa branch
1 3 1 3
pumpsprotonsoutofthe cellduringtransfer ofelectronsto oxygen, whereasthe bd-typeoxidase
doesnotpumpprotons,buthasahigheraffinityforoxygen[68]. ItisassumedthatsimilartoE.coli,
thetwoterminaloxidasesofmycobacteriahavedifferentaffinitiesforoxygen;however,thisawaits
experimentalvalidation.
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3.1. bc -aa Pathway
1 3
Thebc complexisakeycomponentofthebc -aa pathway,andisrequiredforthebulkofthe
1 1 3
electrontransfertooxygenduringnormoxia[69]. bc isencodedbytheqcrCABoperoninMtb. It
1
consistsofredoxgroupscomprisinga2Fe/2Scentre,locatedonaRieskeprotein(QcrA),twob-type
haems(lowandhighpotential)locatedonasinglepolypeptide(QcrB),andthehaemofcytochromec
1
(QcrC).QcrAofMtbhasthreetransmembranehelicesandcharacteristicsequencemotifs(CSHLGC
and CPCH) of 2Fe–2S Rieske iron-sulfur proteins, QcrB has a 120 amino acid extension at the
C-terminus, andtheQcrCsubunitconsistsoftwohaembindingmotifs(CVSCHandCASCH)for
c-type cytochromes, suggesting a covalent di-haem (bcc) configuration [70]. According to Q-cycle
model,oxidationofquinolmoleculesoccursattheinterfaceofcytochromebandthe2Fe–2Scluster
carryingdomainoftheRieskeprotein,whichformsthecatalyticcenterPatthepositivelycharged
membrane side of the enzyme [71]. Each quinol molecule oxidizes at the center P, liberating two
electrons that move towards different acceptors. One electron proceeds to the iron-sulfur Rieske
protein,andisthentransferredtocytochromec,whiletheothermovestoanotherquinone-binding
site on the opposite side of the membrane through haem molecules of variable redox potentials.
ComplexIIIreleasesfourprotonsforeverytwoelectronstransferredfrommenaquinoltocytochrome
cintotheperiplasmicsideofthemembrane[69].
The Mtb aa -type cytochrome c oxidase is comprised of four subunits: CtaB (a cytochrome c
3
oxidaseassemblyfactor),CtaD(cytochromecoxidasesubunitIcontaininghaema,a ,andcopper
3 B
(Cu ),CtaC(cytochromecoxidasesubunitIIcontainingcopper (Cu )andCtaE(subunitIII).Theaa
B A A 3
cytochromecoxidaseofMtbisencodedbyctaBCDE,whichisdispersedoverdifferentlocationsofthe
Mtbgenome[72]. Interestingly,thectaEgeneislocatedimmediatelyupstreamoftheqcrCABoperon.
The putative fourth subunit is encoded by ctaF (Rv2199c). This subunit is co-purified with other
subunitsinCorynebacterium. Importantly,thedeletionmutantsdisplaythephenotypesimilartoCtaC
mutant[73]. Fourprotonsaretakenup,whiletwoprotonsarereleasedintotheperiplasmforevery
twoelectronspassingthroughcomplexIV[72]. TherearethreectaDallelesinMycobacteriumsmegmatis,
while only one in Mtb, indicating the presence of multiple isoforms of cytochrome c oxidase in
M.smegmatis[65]. Theproton-pumpingtype(andenergeticallymoreefficient)bc -aa branchofETCis
1 3
essentialformycobacteria,asitsdeletionbyhomologousrecombinationislethal[66]. Thisobservation
indicatesthatthebc -aa branchofETCisanimportantdrugtarget.
1 3
UnlikeE.coli,whichutilizesmembrane-solublecytochromeCfortransferringelectronsbetween
bc complexandaa cytochromecoxidase,Mycobacterium,Corynebacteriumglutamicum,andRhodococcus
1 3
donotpossesscytochromeC[70,74,75]. Importantly, inC.glutamicum, theQcrCcontainsanextra
bindingsiteforanotherhaemcthatmayfunctionasamergedcytochromeC[76]. Moreover,subunit
IIoftheoxidasecontainsanadditional30aminoacidsthatcouldparticipateinthedirectinteraction
betweencomplexesbc andaa ,tocreatearespiratory“supercomplex”[66],obviatingtherequirement
1 3
for free cytochrome c [74,76]. Indeed, in C. glutamicum, bc and aa are isolated as a complex that
1 3
is resistant to detergent treatment [73]. A similar scenario was predicted for mycobacteria [74],
andwasvalidatedfollowingtheisolationofadetergent-resistantsupercomplexinM.smegmatis[77].
Theinteractionbetweenthebc andaa complexesisguidedbyhydrophobicinteractions,whileionic
1 3
interactionsfacilitatethealignmentbetweenthetwocomplexesforefficientelectrontransferfrom
menaquinoltooxygen[77].Recently,overexpressionoftheMtbcomplexIIIinM.smegmatiswasshown
toyieldafunctional,stable,hybridsupercomplexinthepresenceofdodecylmaltosidedetergent[78].
Theseobservationsstronglysuggestthat,inmycobacterialcells,thebc complexandaa cytochrome
1 3
coxidaseinteractwitheachothertofacilitatetheflowofelectronsfromMKtooxygen,withoutthe
requirementofcytochromeC.
3.2. Cytochromebd-Oxidase
Cytochrome bd-type menaquinol oxidase (bd-oxidase) in Mtb is non-proton pumping, and is
thereforealessenergeticallyefficientterminaloxidase. E.colibd-oxidaseistheprototypicalmember
Pathogens2018,7,24 8of30
of this enzyme class and is encoded by two separate operons, cydAB and cydDC. This is contrary
to Mtb, in which all the genes are transcribed from a single cydABDC operon. cydAB encodes the
functional cytochromes, while the products of cydDC contribute to cytochrome bd assembly [79].
Furthermore,inE.colimutantsofcydDC,theperiplasmicspaceismoreoxidizedthaninthewildtype
bacteria[79]. ThefunctionofcydDCinmycobacteriaislargelyunknown,butitplaysanimportant
role in mycobacterial persistence invivo. cydDC mutation reduces the ability of Mtb to survive
the transition from acute to chronic infection in mice [12]. Another report suggests that cydDC
supportsmycobacterialpersistenceinINH-treatedmice[13].Comparedtotheirwildtypecounterparts,
cytochromebdmutantsofE.coliaresensitivetostressinducedbytemperaturealterations,nitricoxide,
H O ,andiron(III)chelators. Additionally,theyareunabletoresumegrowthuponenteringintothe
2 2
stationaryphase[80–83]. Importantly,thegrowthofcydAmutantM.smegmatisisnormalatambient
oxygenlevels,butisseverelyimpairedduringhypoxia(0.5–1%airsaturation). Thesemutantsare
also sensitive to cyanide, and are outcompeted when co-cultured with wild type M. smegmatis in
itspresence[65]. InE.coli,bd-oxidasehasahigheraffinityforoxygen,andisinducedinresponse
to low oxygen tension [84]. Although the affinity of the Mtb bd-oxidase for oxygen has not been
determined,itsexpressionisalsoinducedduringhypoxia[65]. TheexpressionofcydABisregulated
byaSenX3-RegX3two-componentsystem[85],whichalsoactsasanoxygen-controlledreplication
switchinMtb[86]. ItwasrecentlydemonstratedthatMtbcydABmutantsaresensitivetoH O and
2 2
antibioticstress[87]. Importantly,drugsinhibitingmycobacterialrespirationenhancetheexpression
ofcydAB[88]. InterestinglyinM.smegmatis,inactivationofbc complexresultsintheupregulationof
1
thebd-typeterminaloxidase;however,thebc complexdoesnotcompensateforthelossofthebd-type
1
oxidase[87]. Geneticinactivationofcytochromebd-oxidaseinvariouspathogenicmicroorganismslike
Shigellaflexneri,Brucellaabortus,andSalmonellaentericaserovarTyphimurium,impairstheirintracellular
survivalandvirulence[89–91].
PPPPPaaaaattttthhhhhooooogggggeeeeennnnnsssss 22222000001111188888,,,,, 77777,,,,, xxxxx FFFFFOOOOORRRRR PPPPPEEEEEEEEEERRRRR RRRRREEEEEVVVVVIIIIIEEEEEWWWWW 88888 ooooofffff 2222299999
tttttyyyyypppppeeeee bbbbbaaaaacccccttttteeeeerrrrriiiiiaaaaa [[[[[7777799999]]]]]..... TTTTThhhhheeeee fffffuuuuunnnnnccccctttttiiiiiooooonnnnn ooooofffff cccccyyyyydddddDDDDDCCCCC iiiiinnnnn mmmmmyyyyycccccooooobbbbbaaaaacccccttttteeeeerrrrriiiiiaaaaa iiiiisssss lllllaaaaarrrrrgggggeeeeelllllyyyyy uuuuunnnnnkkkkknnnnnooooowwwwwnnnnn,,,,, bbbbbuuuuuttttt iiiiittttt ppppplllllaaaaayyyyysssss aaaaannnnn
iiiiimmmmmpppppooooorrrrrtttttaaaaannnnnttttt rrrrrooooollllleeeee iiiiinnnnn mmmmmyyyyycccccooooobbbbbaaaaacccccttttteeeeerrrrriiiiiaaaaalllll pppppeeeeerrrrrsssssiiiiisssssttttteeeeennnnnccccceeeee iiiiinnnnn vvvvviiiiivvvvvooooo..... cccccyyyyydddddDDDDDCCCCC mmmmmuuuuutttttaaaaatttttiiiiiooooonnnnn rrrrreeeeeddddduuuuuccccceeeeesssss ttttthhhhheeeee aaaaabbbbbiiiiillllliiiiitttttyyyyy ooooofffff MMMMMtttttbbbbb tttttooooo
sssssuuuuurrrrrvvvvviiiiivvvvveeeee ttttthhhhheeeee tttttrrrrraaaaannnnnsssssiiiiitttttiiiiiooooonnnnn fffffrrrrrooooommmmm aaaaacccccuuuuuttttteeeee tttttooooo ccccchhhhhrrrrrooooonnnnniiiiiccccc iiiiinnnnnfffffeeeeeccccctttttiiiiiooooonnnnn iiiiinnnnn mmmmmiiiiiccccceeeee [[[[[1111122222]]]]]..... AAAAAnnnnnooooottttthhhhheeeeerrrrr rrrrreeeeepppppooooorrrrrttttt sssssuuuuuggggggggggeeeeessssstttttsssss ttttthhhhhaaaaattttt
cccccyyyyydddddDDDDDCCCCC sssssuuuuuppppppppppooooorrrrrtttttsssss mmmmmyyyyycccccooooobbbbbaaaaacccccttttteeeeerrrrriiiiiaaaaalllll pppppeeeeerrrrrsssssiiiiisssssttttteeeeennnnnccccceeeee iiiiinnnnn IIIIINNNNNHHHHH-----tttttrrrrreeeeeaaaaattttteeeeeddddd mmmmmiiiiiccccceeeee [[[[[1111133333]]]]]..... CCCCCooooommmmmpppppaaaaarrrrreeeeeddddd tttttooooo ttttthhhhheeeeeiiiiirrrrr wwwwwiiiiilllllddddd tttttyyyyypppppeeeee
cccccooooouuuuunnnnnttttteeeeerrrrrpppppaaaaarrrrrtttttsssss,,,,, cccccyyyyytttttoooooccccchhhhhrrrrrooooommmmmeeeee bbbbbddddd mmmmmuuuuutttttaaaaannnnntttttsssss ooooofffff EEEEE..... cccccooooollllliiiii aaaaarrrrreeeee ssssseeeeennnnnsssssiiiiitttttiiiiivvvvveeeee tttttooooo ssssstttttrrrrreeeeessssssssss iiiiinnnnnddddduuuuuccccceeeeeddddd bbbbbyyyyy ttttteeeeemmmmmpppppeeeeerrrrraaaaatttttuuuuurrrrreeeee
aaaaalllllttttteeeeerrrrraaaaatttttiiiiiooooonnnnnsssss,,,,, nnnnniiiiitttttrrrrriiiiiccccc oooooxxxxxiiiiidddddeeeee,,,,, HHHHH22222OOOOO22222,,,,, aaaaannnnnddddd iiiiirrrrrooooonnnnn (((((IIIIIIIIIIIIIII))))) ccccchhhhheeeeelllllaaaaatttttooooorrrrrsssss..... AAAAAddddddddddiiiiitttttiiiiiooooonnnnnaaaaallllllllllyyyyy,,,,, ttttthhhhheeeeeyyyyy aaaaarrrrreeeee uuuuunnnnnaaaaabbbbbllllleeeee tttttooooo rrrrreeeeesssssuuuuummmmmeeeee
gggggrrrrrooooowwwwwttttthhhhh uuuuupppppooooonnnnn eeeeennnnnttttteeeeerrrrriiiiinnnnnggggg iiiiinnnnntttttooooo ttttthhhhheeeee ssssstttttaaaaatttttiiiiiooooonnnnnaaaaarrrrryyyyy ppppphhhhhaaaaassssseeeee [[[[[8888800000–––––8888833333]]]]]..... IIIIImmmmmpppppooooorrrrrtttttaaaaannnnntttttlllllyyyyy,,,,, ttttthhhhheeeee gggggrrrrrooooowwwwwttttthhhhh ooooofffff cccccyyyyydddddAAAAA mmmmmuuuuutttttaaaaannnnnttttt MMMMM.....
sssssmmmmmeeeeegggggmmmmmaaaaatttttiiiiisssss iiiiisssss nnnnnooooorrrrrmmmmmaaaaalllll aaaaattttt aaaaammmmmbbbbbiiiiieeeeennnnnttttt oooooxxxxxyyyyygggggeeeeennnnn llllleeeeevvvvveeeeelllllsssss,,,,, bbbbbuuuuuttttt iiiiisssss ssssseeeeevvvvveeeeerrrrreeeeelllllyyyyy iiiiimmmmmpppppaaaaaiiiiirrrrreeeeeddddd ddddduuuuurrrrriiiiinnnnnggggg hhhhhyyyyypppppoooooxxxxxiiiiiaaaaa (((((00000.....55555–––––11111%%%%% aaaaaiiiiirrrrr
sssssaaaaatttttuuuuurrrrraaaaatttttiiiiiooooonnnnn)))))..... TTTTThhhhheeeeessssseeeee mmmmmuuuuutttttaaaaannnnntttttsssss aaaaarrrrreeeee aaaaalllllsssssooooo ssssseeeeennnnnsssssiiiiitttttiiiiivvvvveeeee tttttooooo cccccyyyyyaaaaannnnniiiiidddddeeeee,,,,, aaaaannnnnddddd aaaaarrrrreeeee ooooouuuuutttttcccccooooommmmmpppppeeeeettttteeeeeddddd wwwwwhhhhheeeeennnnn cccccooooo-----cccccuuuuullllltttttuuuuurrrrreeeeeddddd
wwwwwiiiiittttthhhhh wwwwwiiiiilllllddddd tttttyyyyypppppeeeee MMMMM..... sssssmmmmmeeeeegggggmmmmmaaaaatttttiiiiisssss iiiiinnnnn iiiiitttttsssss ppppprrrrreeeeessssseeeeennnnnccccceeeee [[[[[6666655555]]]]]..... IIIIInnnnn EEEEE..... cccccooooollllliiiii,,,,, bbbbbddddd-----oooooxxxxxiiiiidddddaaaaassssseeeee hhhhhaaaaasssss aaaaa hhhhhiiiiiggggghhhhheeeeerrrrr aaaaaffffffffffiiiiinnnnniiiiitttttyyyyy fffffooooorrrrr oooooxxxxxyyyyygggggeeeeennnnn,,,,,
aaaaannnnnddddd iiiiisssss iiiiinnnnnddddduuuuuccccceeeeeddddd iiiiinnnnn rrrrreeeeessssspppppooooonnnnnssssseeeee tttttooooo lllllooooowwwww oooooxxxxxyyyyygggggeeeeennnnn ttttteeeeennnnnsssssiiiiiooooonnnnn [[[[[8888844444]]]]]..... AAAAAlllllttttthhhhhooooouuuuuggggghhhhh ttttthhhhheeeee aaaaaffffffffffiiiiinnnnniiiiitttttyyyyy ooooofffff ttttthhhhheeeee MMMMMtttttbbbbb bbbbbddddd-----oooooxxxxxiiiiidddddaaaaassssseeeee
fffffooooorrrrr oooooxxxxxyyyyygggggeeeeennnnn hhhhhaaaaasssss nnnnnooooottttt bbbbbeeeeeeeeeennnnn dddddeeeeettttteeeeerrrrrmmmmmiiiiinnnnneeeeeddddd,,,,, iiiiitttttsssss eeeeexxxxxppppprrrrreeeeessssssssssiiiiiooooonnnnn iiiiisssss aaaaalllllsssssooooo iiiiinnnnnddddduuuuuccccceeeeeddddd ddddduuuuurrrrriiiiinnnnnggggg hhhhhyyyyypppppoooooxxxxxiiiiiaaaaa [[[[[6666655555]]]]]..... TTTTThhhhheeeee
eeeeexxxxxppppprrrrreeeeessssssssssiiiiiooooonnnnn ooooofffff cccccyyyyydddddAAAAABBBBB iiiiisssss rrrrreeeeeggggguuuuulllllaaaaattttteeeeeddddd bbbbbyyyyy aaaaa SSSSSeeeeennnnnXXXXX33333-----RRRRReeeeegggggXXXXX33333 tttttwwwwwooooo-----cccccooooommmmmpppppooooonnnnneeeeennnnnttttt sssssyyyyysssssttttteeeeemmmmm [[[[[8888855555]]]]],,,,, wwwwwhhhhhiiiiiccccchhhhh aaaaalllllsssssooooo aaaaaccccctttttsssss aaaaasssss
aaaaannnnn oooooxxxxxyyyyygggggeeeeennnnn-----cccccooooonnnnntttttrrrrrooooolllllllllleeeeeddddd rrrrreeeeepppppllllliiiiicccccaaaaatttttiiiiiooooonnnnn ssssswwwwwiiiiitttttccccchhhhh iiiiinnnnn MMMMMtttttbbbbb [[[[[8888866666]]]]]..... IIIIIttttt wwwwwaaaaasssss rrrrreeeeeccccceeeeennnnntttttlllllyyyyy dddddeeeeemmmmmooooonnnnnssssstttttrrrrraaaaattttteeeeeddddd ttttthhhhhaaaaattttt MMMMMtttttbbbbb cccccyyyyydddddAAAAABBBBB
mmmmmuuuuutttttaaaaannnnntttttsssss aaaaarrrrreeeee ssssseeeeennnnnsssssiiiiitttttiiiiivvvvveeeee tttttooooo HHHHH22222OOOOO22222 aaaaannnnnddddd aaaaannnnntttttiiiiibbbbbiiiiioooootttttiiiiiccccc ssssstttttrrrrreeeeessssssssss [[[[[8888877777]]]]]..... IIIIImmmmmpppppooooorrrrrtttttaaaaannnnntttttlllllyyyyy,,,,, dddddrrrrruuuuugggggsssss iiiiinnnnnhhhhhiiiiibbbbbiiiiitttttiiiiinnnnnggggg mmmmmyyyyycccccooooobbbbbaaaaacccccttttteeeeerrrrriiiiiaaaaalllll
rrrrreeeeessssspppppiiiiirrrrraaaaatttttiiiiiooooonnnnn eeeeennnnnhhhhhaaaaannnnnccccceeeee ttttthhhhheeeee eeeeexxxxxppppprrrrreeeeessssssssssiiiiiooooonnnnn ooooofffff cccccyyyyydddddAAAAABBBBB [[[[[8888888888]]]]]..... IIIIInnnnnttttteeeeerrrrreeeeessssstttttiiiiinnnnnggggglllllyyyyy iiiiinnnnn MMMMM..... sssssmmmmmeeeeegggggmmmmmaaaaatttttiiiiisssss,,,,, iiiiinnnnnaaaaaccccctttttiiiiivvvvvaaaaatttttiiiiiooooonnnnn ooooofffff bbbbbccccc11111
cccccooooommmmmpppppllllleeeeexxxxx rrrrreeeeesssssuuuuullllltttttsssss iiiiinnnnn ttttthhhhheeeee uuuuuppppprrrrreeeeeggggguuuuulllllaaaaatttttiiiiiooooonnnnn ooooofffff ttttthhhhheeeee bbbbbddddd-----tttttyyyyypppppeeeee ttttteeeeerrrrrmmmmmiiiiinnnnnaaaaalllll oooooxxxxxiiiiidddddaaaaassssseeeee;;;;; hhhhhooooowwwwweeeeevvvvveeeeerrrrr,,,,, ttttthhhhheeeee bbbbbccccc11111 cccccooooommmmmpppppllllleeeeexxxxx dddddoooooeeeeesssss
Pathogens2018,7,24nnnnnooooottttt cccccooooommmmmpppppeeeeennnnnsssssaaaaattttteeeee fffffooooorrrrr ttttthhhhheeeee lllllooooossssssssss ooooofffff ttttthhhhheeeee bbbbbddddd-----tttttyyyyypppppeeeee oooooxxxxxiiiiidddddaaaaassssseeeee [[[[[8888877777]]]]]..... GGGGGeeeeennnnneeeeetttttiiiiiccccc iiiiinnnnnaaaaaccccctttttiiiiivvvvvaaaaatttttiiiiiooooonnnnn ooooofffff cccccyyyyytttttoooooccccchhhhhrrrrrooooo9mmmmmoeeeeef 30
bbbbbddddd-----oooooxxxxxiiiiidddddaaaaassssseeeee iiiiinnnnn vvvvvaaaaarrrrriiiiiooooouuuuusssss pppppaaaaattttthhhhhooooogggggeeeeennnnniiiiiccccc mmmmmiiiiicccccrrrrroooooooooorrrrrgggggaaaaannnnniiiiisssssmmmmmsssss llllliiiiikkkkkeeeee SSSSShhhhhiiiiigggggeeeeellllllllllaaaaa fffffllllleeeeexxxxxnnnnneeeeerrrrriiiii,,,,, BBBBBrrrrruuuuuccccceeeeellllllllllaaaaa aaaaabbbbbooooorrrrrtttttuuuuusssss,,,,, aaaaannnnnddddd
SSSSSaaaaalllllmmmmmooooonnnnneeeeellllllllllaaaaa eeeeennnnnttttteeeeerrrrriiiiicccccaaaaa ssssseeeeerrrrrooooovvvvvaaaaarrrrr TTTTTyyyyyppppphhhhhiiiiimmmmmuuuuurrrrriiiiiuuuuummmmm,,,,, iiiiimmmmmpppppaaaaaiiiiirrrrrsssss ttttthhhhheeeeeiiiiirrrrr iiiiinnnnntttttrrrrraaaaaccccceeeeelllllllllluuuuulllllaaaaarrrrr sssssuuuuurrrrrvvvvviiiiivvvvvaaaaalllll aaaaannnnnddddd vvvvviiiiirrrrruuuuullllleeeeennnnnccccceeeee [[[[[8888899999–––––9999911111]]]]].....
Table1. Summaryofpotentialdrugmoleculeswiththeirrespectivetargets,bactericidalproperties,
andstructures. TTTTTaaaaabbbbbllllleeeee 11111..... SSSSSuuuuummmmmmmmmmaaaaarrrrryyyyy ooooofffff pppppooooottttteeeeennnnntttttiiiiiaaaaalllll dddddrrrrruuuuuggggg mmmmmooooollllleeeeecccccuuuuullllleeeeesssss wwwwwiiiiittttthhhhh ttttthhhhheeeeeiiiiirrrrr rrrrreeeeessssspppppeeeeeccccctttttiiiiivvvvveeeee tttttaaaaarrrrrgggggeeeeetttttsssss,,,,, bbbbbaaaaacccccttttteeeeerrrrriiiiiccccciiiiidddddaaaaalllll ppppprrrrrooooopppppeeeeerrrrrtttttiiiiieeeeesssss,,,,,
aaaaannnnnddddd ssssstttttrrrrruuuuuccccctttttuuuuurrrrreeeeesssss.....
Drug DDDDDrrrrruuuuuggggg Target TTTTTaaaaarrrrrgggggeeeeettttt PrPPPPPorrrrrpoooooppepppereeeetrrrrrittttteiiiiieeeeessssss SSSSSSttttttrrrrrruuuuuuccccccttttttuuuuuurrrrrreeeeee
Thioridazine TTTTThhhhhiiiiiooooorrrrriiiiidddddaaaaazzzzziiiiinnnnneeeee NDH-2 NNNNNDDDDDHHHHH-----22222 MIMMMMMCI9IAIIICCC0CCAAp-AAA99999800000pp-p-p-p-p-–88888prpppp1–––––orrrr5r11111oovooo55555vvµvvev µµµeµeµeedegddgdgddggg/ / / / / /mmmmmmLLLLLL

Trifluoperazine TTTTTrrrrriiiiifffffllllluuuuuooooopppppeeeeerrrrraaaaazzzzziiiiinnnnneeeee NDH-2 NNNNNDDDDDHHHHH-----22222 MIMMMMMC9IIIIICCC0CC-99999100000-----911111.999992.....22222µ µµµµµgggggg//////mmmmmmLLLLLL

Clofazimine CCCCClllllooooofffffaaaaazzzzziiiiimmmmmiiiiinnnnneeeee NDH-2 NNNNNDDDDDHHHHH-----22222 PhMPPPPPahhIhhhsMMMMMCaaeaaassssIs9IIIIIeeCCeeCeC0CI -III99I9I9I900I0I0I0I0Ic--II-I-I-I.00 00 0l2 ccccci.....l5l22nll22l2iiiii55nn55n5nniµ c i iiiµµiµµµcccgaccaaggaaggagl/lll/l/l/// t mm mmmtmtttrtrrrrriiiLLiLiLiLLaaaaaa l lllllssssss

QuinolinylpyrimiQQQdQQiuuuuuniiiiinnennnooooollllliiiiinnnnnyyyyylllll pppppyyyyyrrrrriiiiimmmmmiiiiidddddiiiiinnnnneeeeeN DH-2 NNNNNDDDDDHHHHH-----22222 MIMMMMMC9IIIIICCC0CC-99999800000------888883-----23333322222µ µµµµµgggggg//////mmmmmmLLLLLL

3-Nitropropionate33333(-----3NNNNNNiiiiitttttPrrrrrooooo)ppppprrrrrooooopppppiiiiiooooonnnnnaaaaattttteeeee (((((33333NNNNNPPPPP))))) S DH SSSSSDDDDDHHHHH AMcAAAMAMAIMMMtCciccccIIIvIIttt9ttCCCCCiiiii0evvvvv99999-0e0e0e0e0ei3----- n 333i3i3.iiinn3nn.n....33v333 vv µ vv v iµµµµµviiiiiMvvMvMvvMMMoooooo
Pathogens2018,7,24 10of30
Table1.Cont.
PPPPPPPPaaaaaaaatttttttthhhhhhhhooooooooggggggggeeeeeeeennnnnnnnssssssss 2 2222222000000001111111188888888,,,,,,, , 7 7777777,,,,,,, , x xxxxxxx F FFFFFFFOOOOOOOORRRRRRRR P PPPPPPPEEEEEEEEEEEEEEEERRRRRRRR R RRRRRRREEEEEEEEVVVVVVVVIIIIIIIIEEEEEEEEWWWWWWWW 99999999 o oooooooffffffff 2 222222299999999
PPPPaaaatttthhhhooooggggeeeennnnssss 2222000011118888,,,, 7777,,,, xxxx FFFFOOOORRRR PPPPEEEEEEEERRRR RRRREEEEVVVVIIIIEEEEWWWW 9999 ooooffff 22229999
Drug Target Properties Structure
DG70 DDDDDDDDDDDDGGGGGGGGGGGG777777777777000000000000 MenG MMMMMMMMMMMMeeeeeeeeeeeennnnnnnnnnnnGGGGGGGGGGGG MIMMMMMMMMMMCMMII9IIIIIIIIIICCCCCCCCCC0CC-9999999999994000000000000-----------.-4444444444844...........8.88888888888µ µ µµµµµµµµµµgµgggggggggg/gg////////////mmmmmmmmmmmmmLLLLLLLLLLLLL

AurachinRE AAAAAAAAAAAAuuuuuuuuuuuurrrrrrrrrrrraaaaaaaaaaaacccccccccccchhhhhhhhhhhhiiiiiiiiiiiinnnnnnnnnnnn R RRRRRRRRRRREEEEEEEEEEEE MenA MMMMMMMMMMMMeeeeeeeeeeeennnnnnnnnnnnAAAAAAAAAAAA MIMMMCMMMMMMMMM9IIIIIIIIIIIIC0CCCCCCCCCCC99999<9999999000000000000 < <<<<<<<<<1<< 21 1111111111122.22222222225...........5.55555555555µ µ µµµµµµµµµµµggggggggggggg/////////////mmmmmmmmmmmmmLLLLLLLLLLLLL

Ro48-8071 RRRRRRRRRRRRoooooooooooo 4 44444444444888888888888------------888888888888000000000000777777777777111111111111 MenA MMMMMMMMMMMMeeeeeeeeeeeennnnnnnnnnnnAAAAAAAAAAAA MIMMMMMMMMMMMCMIIIII9IIIIIIICCCCCCCCCC0CC-9999999999990030000000000------------33333333333µ3 µ µµµµµµµµµgµµgggggggggg/gg////////////mmmmmmmmmmmmmLLLLLLLLLLLLL

7-Methoxy-2-naph77777777777t7--------h----MMMMMMMMMMMMoeeeeeeeleeeeetttttttttttthhhhhhhhhhhhooooooooooooxxxxxxxxxxxxyyyyyyyyyyyy------------222222222222------------nnnnnnnnnnnnaaaaaaaaaaaapppppppppppphhhhhhhhhhhhtttttttttttthhhhhhhhhhhhoooooooooooollllllllllll MenA MMMMMMMMMMMMeeeeeeeeeeeennnnnnnnnnnnAAAAAAAAAAAA MIMMMMMMMMMMMCMIIIII9IIIIIIICCCCCCCCCC0CC-9999999999990030000000000------------33333333333µ3 µ µµµµµµµµµgµµgggggggggg/gg////////////mmmmmmmmmmmmmLLLLLLLLLLLLL

AllylaminomethanAAAAAAAAAAAAolllllllllllnlllllllllllllyyyyyyyyyyyyellllllllllllaaaaaaaaaaaammmmmmmmmmmmiiiiiiiiiiiinnnnnnnnnnnnoooooooooooommmmmmmmmmmmeeeeeeeeeeeetttttttttttthhhhhhhhhhhhaaaaaaaaaaaannnnnnnnnnnnoooooooooooonnnnnnnnnnnneeeeeeeeeeee M enA MMMMMMMMMMMMeeeeeeeeeeeennnnnnnnnnnnAAAAAAAAAAAA MIMMMMMMMMMMMCMIIIII9IIIIIIICCCCCCCCCC0CC-9999999999990010000000000------------11111111111µ1 µ µµµµµµµµµgµµgggggggggg/gg////////////mmmmmmmmmmmmmLLLLLLLLLLLLL

AurachinD AAAAAAAAAAAAuuuuuuuuuuuurrrrrrrrrrrraaaaaaaaaaaacccccccccccchhhhhhhhhhhhiiiiiiiiiiiCinnnnnnnnnnnn y D DDDDDDDDDDDt o chromebdCCCCCCCCCCCoCbbbbbbbbbbbbyyyyyyyyyyxdyddydddddddddttttttttttit t o o oooooooo do oooooooooooooccccccccccxxcxxxcxxxxxaxxhhhhhhhhhhhihiiiiiiiiisiiddrddrddddrddrrrrrrrddrreooooooooooaaoaaaaaoaaaaammmmmmmmmsmssssssmsssmsseeeeeeeeeeeeeeeeeeeeee e e MMMMMIMMMMMMMMCIIIIIIIIIIII9CCCCCCCCCCCC09999999999-990000000000080------------8888888888588555555555555 µ µ µµµµµµµµµµµMMMMMMMMMMMMM

(PhenoxyalkyPlAbBenSz((((((((((((iPPPPPPPPPPPPmhhhhhhhhhhhheeeeieeeeeeeednnnnnnnnnnnnoaoooooooooooxzxxxxxxxxxxxyyoyyyyyyyyyyaaaaaaalaaaaaellllllllllllkkkkkkkkkkskkyPyPy)yyPyyPyPyyPPPPPyPyPllllAllllAllAAAAAAAAllAbbAbbbbbbbbbbeBeBeBeBeBeeBBeBeeBBeBeBnnnnnnnnnnSSSSnSSSSSSnSSzzzz zzzz zz zz iiiiiiiiiiiimmmmmmmmmmmmiiiiiiiiiiiiddddddddddddaaaaaaaaaaaazzzzzzzzzzzzoooooooooooolllllllllllQleeeeeeeeeeeessssssssssssc)))))))))))) r B QQQQQQQQQQQQccccccccccccrrrrrrrrrrrrBBBBBBBBBBBB MMIMMMMMMMMMMMCIIIIIIIIIII9ICCCCCCCCCCCC09999999999-990000000000000------------0000000.000000...........0.0000000005005555555555565666666666666 µ µ µµµµµµµµµµµMMMMMMMMMMMMM

Q203(imidazopyriQQQQQQQQQQdQQ2i22222222222n000000000000e333333333333 ( a(((((((((((iiiiiiiiiiimimmmmmmmmmmmmiiiiiiiiiiiiidddddddddddddaaaaaaaaaaeaazzzzzzzzzz)zzooooooooooooppppppppppppyyyyyyyyyyyyrrrrrrrrrrrriiiiiiiiiiiiddddddddddddiiiiiiiiiiiinnnnnnnnnnnneeeeeeeeeeee a aaaaaaaaaaammmmmmmmmmmmiiiiiiiiiiiiddddddddddQddeeeeeeeeeeeec)))))))))))) r B QQQQQQQQQQQQccccccccccccrrrrrrrrrrrrBBBBBBBBBBBB MMPIMMhMMMIMMMMMCMMMMMMMMMMMMMPCPMPPPPPPPPPaPII5IIIIIIhIIhhhhhhhIhhIIIII5IshIIICIIhCCCCCCCICC0ICCCCCCCCCCCaCa0aaaaaCaeaaCaa-5555555555ss-s5ss5ssss5s550550555505s000s000005500eI020e00e0-e00e0ee0-0eee--------0e0e--.-------0---00000 00-00 - . 2 0202c22 2222I22 IIIIIII2I7I2..I.....I... .. 8 2. l. 2 ..2.2..2.22..222 c .c27c.cc27ccccc777777i77cc7788n88888888llnlllll8lll 8 ln lii iini iiinii nn n nn n nn iinMn nnn nnnnnnnnnninnnnnnnnnnMnMMMMcMMMMMMMiiMMiiMiiiiMiiMMMMMMMiiMcMcccacccccccc a ia a a a aa a aal ai a i iie i ii in ii i ile lenlenlelelleenlenllenennnnnlenle tn x x xt xxtxxxxtxtx tttt ttr x txvt rv rvrrvrvrrvrvvrvrvv r v i r vv vi vivivivivviivivviiiaiiviiiiviiiiiiaaaaaiaaaaaitttatttattitttiiiiiiiiititlirlrilrrvlrlrlrrvllrlrrvllvvvvvvvvrlrlsvsvssssssosssooooooooososooooooooo oooo oo

Pyrrolo[3,4-c]pyridPPPPPPPPiyyyyyyynyrrrrrrrrerrrrrrrroooo-oooo1llllllllooooooo,o3[[[[[[[[3333-3333d,,,,,,,4,4444444i-------o-cccccccc]n]]]]]]]ppppppppeyyyyyyyyrrrrrrrriiiiiiiiddddddddiiiiiiiinnnnnnnneeeeeeee--------11111111,,,,,,,3,3333333--------ddddddddiiiiiiiiooQoooooonnnnnnnnceeeeeeeer B QQQQQQQQccccccccrrrrrrrrBBBBBBBB
PPPPyyyyrrrrrrrroooolllloooo[[[[3333,,,,4444----cccc]]]]ppppyyyyrrrriiiiddddiiiinnnneeee----1111,,,,3333----ddddiiiioooonnnneeee QQQQccccrrrrBBBB

Lansoprazole LLLLLLLLLLLLaaaaaaaaaaaannnnnnnnnnnnssssssssssssoooooooooooopppppppppppprrrrrrrrrrrraaaaaaaaaaaazzzzzzzzzzzzoooooooooooolllllllllllleeeeeeeeeeee QcrB QQQQQQQQQQQQccccccccccccrrrrrrrrrrrrBBBBBBBBBBBB MAIMMMMMMMMMMCMMcAAAAAAAAAAIAI9AIIIIIIIIItICCCCCCCCCC0CcCicccccccccccv-t9t9ttt9tt9t9tt99999t9t9i10i0ii0i0ii0i00ii000ie0i0vv-vv-vvvv-v-v------v-v0-1111111111e1ee1eeeeeeeiee40000000000 n 0 0 i iii4iiii4ii44444444iin4n4nnnnnµnnnnn v µ µ µµ µµµµ µ µ µg µv vvvvvvvvvvivggggggggggvig/igiiiiiiiiiivv/vvv/vvvvv////////vv//ommmmmmmmmmmomooomoooooooo LLLL LLLLLL LLL

Bedaquiline BBBBBBBBBBBBeeeeeeeeeeeeddddddddddddaaaaaaaaaaaaqqqqqqqqqqqquuuuuuuuuuuuiiiiiiiiiiiilllllllllllliiiiiiiiiiiinnnnnnnnnnnneeeeeeeeeeee ATPsynthasssssssssssessyyyyyyyyyyyyAnAnAAAnAAnAnAAnnnnnAnAnttttttttttTTtTTTTTtTTThhThhhhhThhhhhPPPPPPPPPPaaaaaPaaaaaPaa s s s s ssssss s seeeeeeeeeeee PMAhIpaMCMMMMMMMMMAAAAAAMAAAAMAAPsPPPPpPPPPPPP9eppIppIppppIpphIIIIhIIIhhhhhphhhpIrIh0hCCCCCCCCCCCppCpppppaopppaaaaaaIapaa-paa9999I9999sr990srsrsrsrssrr9srsrvsr90sr0sr00000000Io0eoeo0ooeooeoeooee-eee--.--o----e-oe-e-000 0000 v0000 v fc vv f v vvDvfvfv0fff0 vI IvIIIfIIIIIdoo.Io.o.Io.0oo......lIeIeI.IeoIeIIeeIe0Ie.Iee0000000I00eIei0I0IrIrISIIIrIrIIrrrd4dIddddddIdd0nr00 0000d 000 df 0 0 c ccc-ccDcc ccD-DD 4DDoD c4 4 c4 4 44 4 44iD 4f l4Tflf0fllfflfllflffllfcl-fl-i--io----io--iiriioioioiooSoooS-SSi-SSSoi0on0n.0n0n0n00nnS0n00nBan0n0nrr1rr-rrrr-rr-----.rM.r...i.....i-iiilii iTii. T1 T .T 1 Ti T1T1111i111c3 cMc cTc1McccccMM1MMMMMMccMM3t3B33a3B33a333BBaaaBaaBaBaa33aBDarµ l l lDllllDl llD D DDD Dµ DD µ l µiµµlµµµDµµ D µ µ t atgttRttttttttgRgRrggRgrRggRgRRgrgRrRrRrrrrrgRgrRlr/iiiiiiii-/ii/s/////i///-i-a---a/----a-a/aaaaaam-mTm-ammmmmmmaTTmTTmTTTTTTmTllTllllllfllllsssssBsssBssBBBoBBBBsBBLsLLLLBLLLLLB L L L r
ffffoooorrrr DDDDSSSS----TTTTBBBB

SquaramiPPdaaetthhooggeennssS SSSS SSSSSSS22qqqqqqqqqqq0q0uuuuuuuuuu1u1uaaaaa8aaaaa8aarrrrrr,rrrr,rr a aaaaaaaaa7aa7mmmmmmmmmm,mm, xxiiiiiiiiiiiidd ddddd dddddFFeeeeeeeeeeeOeO RRA PPTEEPEEsRRy RRnEEthVVaIIsEEssssssssssessWWyyyyyyyyyyyyAnAnAAAnAAnAnAAnnnnnAnAn ttttttttttTTt TTTTTtTTT hhThhhhhThhhhhPPPPPPPPPPaaaaaPaaaaaPaa s s s s ssssss s seeeeeeeeeeee PAMrPPPPPPPPPPePPcArArA-rArArMAArrArAArrMMIMMMMMMMArArMMeecteeeeeeeeCeeciccccccc-cc-l------c--cII-IIIIIvI-IIctcItictctIctccttct9ccttCCctCCCCCcCtCCiiCniiiCiiiilillllllil0ellivvlvvvlvvvvviiiiiiiviii9v9999i99999iin-n9nnnnn9nnn0e0en0e0e0e00ee0en0e0eci0e00e--------i--ii iinii -iii - i0a0i 00c0i00 0c00icci.cicciciicciii00cicinn5nnnnnnnn.a.a..a.lan..a.aa..aaan.a5.a5v55555555 5 5l l llllllll tv lv µlv vvvv v vv vi vµ µtrµ µµtµµµµµttttttttµµvtiitiiiiiriiirrrrMrrrirriivrvvrvMvvvvMvvMMMMMMMMivivaiiMiiiiiioMiiaaaoaaaaoaaaooooooooaaolol l lll ll l ll s l l ss sss sssssss 110 0 ooff 2299

SQ109 SSQQ110099 PMF PPMMFF PMhPPaIhhMMsCaaeIs9IsCCIe0eI - 99II000cII-- . l00cc7i.l.nl877iinn8i8µci i µcµcagaaglg/ll/ t/ tmtmmrrriiiaLLaLall l sss
Pyrazinamide PPyyrraazziinnaammiiddee PMF PPMMFF MIMMCAI9ICC0ApA-99p10p0p--0p1rp1o000rro0vo0µ v veµµgeedggd/d// mmmLLL

3.3. Supercomplex Inhibitors
3.3. Supercomplex Inhibitors
TThhee eesssseennttiiaalliittyy ooff MMttbb ccyyttoocchhrroommee bbcc11,, aalloonngg wwiitthh tthhee llaarrggee ddiiffffeerreenncceess iinn ssttrruuccttuurree aanndd
function from its mammalian counterpart, makes it a good target for therapeutic intervention [78]. A
function from its mammalian counterpart, makes it a good target for therapeutic intervention [78]. A
vvaarriieettyy ooff aannttiibbiioottiiccss aanndd cchheemmiiccaall ccoommppoouunnddss hhaavvee lloonngg bbeeeenn kknnoowwnn ttoo iinnhhiibbiitt tthhee bbcc11--aaaa33 ccoommpplleexx..
The aurachin class of compounds contains quinone analogues, which are reported to inhibit a
The aurachin class of compounds contains quinone analogues, which are reported to inhibit a
variety of cytochrome oxidases [56,92,93]. Aurachin D is well-known among this class of compounds
variety of cytochrome oxidases [56,92,93]. Aurachin D is well-known among this class of compounds
that inhibit E. coli cytochrome bd-oxidase [92]. It inhibits oxygen consumption in M. smegmatis in a
that inhibit E. coli cytochrome bd-oxidase [92]. It inhibits oxygen consumption in M. smegmatis in a
dose-dependent manner [87]. These observations demonstrate the importance of cytochrome bd
dose-dependent manner [87]. These observations demonstrate the importance of cytochrome bd
inhibitors as drug molecules. Optimized derivatives of aurachin D, with better ability to penetrate
inhibitors as drug molecules. Optimized derivatives of aurachin D, with better ability to penetrate
the mycobacterial cell wall, have a great potential as a new class of antitubercular drugs [87].
the mycobacterial cell wall, have a great potential as a new class of antitubercular drugs [87].
Similar to aurachin D, myxothiazol-based compounds, which are isolated from Myxococcus
Similar to aurachin D, myxothiazol-based compounds, which are isolated from Myxococcus
fulvus, target mitochondrial cytochrome b. Myxothiazol interacts both with cytochrome b and an
fulvus, target mitochondrial cytochrome b. Myxothiazol interacts both with cytochrome b and an
iron-sulfur protein of the complex; this displaces quinone from the high-affinity binding site of the
iron-sulfur protein of the complex; this displaces quinone from the high-affinity binding site of the
iron-sulfur protein [94]. Importantly, myxothiazol inhibits the growth of Mycobacterium sp. GBF 3 at
iron-sulfur protein [94]. Importantly, myxothiazol inhibits the growth of Mycobacterium sp. GBF 3 at
an MIC value of 6.3 µg/mL [95], indicating that respiratory inhibitors can successfully target
an MIC value of 6.3 µg/mL [95], indicating that respiratory inhibitors can successfully target
mycobacterial species. Interestingly, myxothiazol does not kill Gram-negative or -positive bacteria,
mycobacterial species. Interestingly, myxothiazol does not kill Gram-negative or -positive bacteria,
but does exhibit strong antifungal activity [95]. A few other antifungal antibiotics, such as mucidin
but does exhibit strong antifungal activity [95]. A few other antifungal antibiotics, such as mucidin
(from Basidiomycetes Oudemansiella mucida) and strobilurin A (from Strobilurin stenacellus) also inhibit
(from Basidiomycetes Oudemansiella mucida) and strobilurin A (from Strobilurin stenacellus) also inhibit
tthhee bbcc11 ccoommpplleexx bbyy bbiinnddiinngg ttoo tthhee ssaammee ssiittee aass mmyyxxootthhiiaazzooll [[9966]].. HHoowweevveerr,, aannttiimmyycciinn AA ((aannootthheerr
antibiotic) binds to a different location, inhibiting the oxidation of cytochrome b subunit [97].
antibiotic) binds to a different location, inhibiting the oxidation of cytochrome b subunit [97].
A screen in search of new antibiotics identified a novel compound, stigmatellin, from Stigmatella
A screen in search of new antibiotics identified a novel compound, stigmatellin, from Stigmatella
aurantiaca strain Sg a15, that showed activity against various Gram-positive bacteria, yeasts, and
aurantiaca strain Sg a15, that showed activity against various Gram-positive bacteria, yeasts, and
filamentous fungi [98]. Subsequently, it was found to block mitochondrial electron transport by
filamentous fungi [98]. Subsequently, it was found to block mitochondrial electron transport by
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The capability of a number of small molecules to specifically target energy production in Mtb
The capability of a number of small molecules to specifically target energy production in Mtb
has validated oxidative phosphorylation as a viable drug target. Since energy production through
has validated oxidative phosphorylation as a viable drug target. Since energy production through
oxidative phosphorylation plays an important role in Mtb survival during hypoxia-induced
oxidative phosphorylation plays an important role in Mtb survival during hypoxia-induced
non-replicating persistence [9,100], blockade of oxidative phosphorylation could be pursued,
non-replicating persistence [9,100], blockade of oxidative phosphorylation could be pursued,
therapeutically, to kill persisters. A number of small molecule inhibitors that specifically target the
therapeutically, to kill persisters. A number of small molecule inhibitors that specifically target the
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aarree ssttrruuccttuurraall aannaalloogguueess ooff qquuiinnoonnee,, ttaarrggeett tthhee qquuiinnoonnee bbiinnddiinngg ccaattaallyyttiicc ddoommaaiinnss ooff tthhee bbcc11 ccoommpplleexx
[101]; prominent examples are the imidazo[1,2-a]pyridines (IPs). Using HTS, IPs were identified as
[101]; prominent examples are the imidazo[1,2-a]pyridines (IPs). Using HTS, IPs were identified as
potent inhibitors of Mtb and BCG [101]. Importantly, four IP inhibitors were shown to specifically
potent inhibitors of Mtb and BCG [101]. Importantly, four IP inhibitors were shown to specifically
target Mtb strains and BCG, but were not able to kill a number of other Gram-positive and
target Mtb strains and BCG, but were not able to kill a number of other Gram-positive and
Gram-negative bacteria, or human cell lines, including HepG2 and Neuro2A. Whole genome
Gram-negative bacteria, or human cell lines, including HepG2 and Neuro2A. Whole genome
sequencing of the spontaneous resistant mutants established that these IPs targeted QcrB. These
sequencing of the spontaneous resistant mutants established that these IPs targeted QcrB. These
findings were further supported by induction of drug resistance upon overexpression of qcrB [101].
findings were further supported by induction of drug resistance upon overexpression of qcrB [101].
HTS of 100,997 compounds led to the discovery of phenoxyalkylbenzimidazole (PAB) class of
HTS of 100,997 compounds led to the discovery of phenoxyalkylbenzimidazole (PAB) class of
compounds showing activity against Mtb with MICs in the nanomolar range, and low cytotoxicity
compounds showing activity against Mtb with MICs in the nanomolar range, and low cytotoxicity
against eukaryotic cells [102,103]. A recent study identified the probable target of PABS to be the
against eukaryotic cells [102,103]. A recent study identified the probable target of PABS to be the
QQccrrBB ssuubbuunniitt ooff ccyyttoocchhrroommee bbcc11 ooxxiiddaassee,, ffuurrtthheerr ccoonnffiirrmmiinngg iitt ttoo bbee aann iimmppoorrttaanntt ddrruugg ttaarrggeett [[110044]]..
An independent screen of more than 100,000 compounds to identify inhibitors of macrophage-
An independent screen of more than 100,000 compounds to identify inhibitors of macrophage-
resident bacterial growth led to the isolation of two highly active IP compounds with less toxicity
resident bacterial growth led to the isolation of two highly active IP compounds with less toxicity