New Insights to Resistance of a Novel Drug Bedaquiline using in-vitro Mutants of ATP Synthase in Mycobacterium Tuberculosis Ameeruddin Nusrath Unissa 1* ; Appisetty Ramya Lakshmi 1 ; Swathi Sukumar 1 ; Luke Elizabeth Hanna 2 1 Centre for Biomedical Informatics, National Institute for Research in Tuberculosis, India 2 Division of Clinical Research, National Institute for Research in Tuberculosis, India *Corresponding to :A. Nusrath Unissa, Department of Clinical Research, National Institute for Research in Tuberculosis (NIRT), Indian Council of Medical Research (ICMR), No. 1, Mayor Sathyamoorthy Road, Chet- put, Chennai 600 031, Tamil Nadu, India. Phone: 91-044 -2836 9597; Fax: +91-(044)-2836 2528; E-mail: [email protected]Chapter 2 Diagnosis and Management of Tuberculosis Abstract Bedaquiline (BDQ) is the new first-in-class anti-tuberculosis (TB) compound belonging to the class of diarylquinolone with activity against drug-sensitive and drug-resistant Mycobacterium tuberculosis (M. tuberculosis). This novel drug has the immense potential to shorten TB treatment duration and has been advocated for multi-drug resistant (MDR)-TB treatment. Therefore, BDQ resistance can be con- sidered as a major public health problem and molecular investigation of the same is the utmost need of the hour. The target based concept of resistance to BDQ is caused by mutation in c-ring of adenosine tri phosphate (ATP) synthase, a critical enzyme in the synthesis of ATP in M. tuberculosis coded by atpE gene. BDQ inhibits the proton pump of mycobacterialATP synthase. To understand the molecular basis of BDQ re- sistance using mutants (MTs) as the emergence of strains resistant to BDQ may pose a potential threat to the TB control program, we undertook a initiative to study seven in vitro mutants of AtpE viz., Asp28Gly, Asp28Ala, Asp28Pro, Asp28Val, Glu61- Asp, Ala63Pro and Ile66Met (D28G, D28A, D28P D28V, E61D, A63P, and I66M) which involves in resistance against BDQ. Molecular modeling and docking was performed to understand the interacting behaviour of mutant (MT) enzymes with BDQ in comparison to wild-type (WT). These results indicate that the substitutions in AtpE except D28G showed high affinity towards the drug in comparison to the WT. This could be due to the favourable interactions in mutants compared to WT.
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New Insights to Resistance of a Novel Drug Bedaquiline using in-vitro Mutants of ATP Synthase in Mycobacterium Tuberculosis
Ameeruddin Nusrath Unissa1*; Appisetty Ramya Lakshmi1; Swathi Sukumar 1; Luke Elizabeth
Hanna2
1 Centre for Biomedical Informatics, National Institute for Research in Tuberculosis, India2 Division of Clinical Research, National Institute for Research in Tuberculosis, India
*Corresponding to :A. Nusrath Unissa, Department of Clinical Research, National Institute for Research in
Tuberculosis (NIRT), Indian Council of Medical Research (ICMR), No. 1, Mayor Sathyamoorthy Road, Chet-
Abstract Bedaquiline(BDQ)isthenewfirst-in-classanti-tuberculosis(TB)compoundbelonging to theclassofdiarylquinolonewithactivityagainstdrug-sensitiveanddrug-resistantMycobacterium tuberculosis (M. tuberculosis).ThisnoveldrughastheimmensepotentialtoshortenTBtreatmentdurationandhasbeenadvocatedformulti-drugresistant(MDR)-TBtreatment.Therefore,BDQresistancecanbecon-sideredasamajorpublichealthproblemandmolecularinvestigationofthesameistheutmostneedofthehour.ThetargetbasedconceptofresistancetoBDQiscausedbymutationinc-ringofadenosinetriphosphate(ATP)synthase,acriticalenzymeinthesynthesisofATPinM.tuberculosiscodedbyatpEgene.BDQinhibitstheprotonpumpofmycobacterialATPsynthase.TounderstandthemolecularbasisofBDQre-sistanceusingmutants(MTs)astheemergenceofstrainsresistanttoBDQmayposeapotentialthreattotheTBcontrolprogram,weundertookainitiativetostudysevenin vitromutantsofAtpEviz.,Asp28Gly,Asp28Ala,Asp28Pro,Asp28Val,Glu61-Asp,Ala63ProandIle66Met(D28G,D28A,D28PD28V,E61D,A63P,andI66M)which involves in resistanceagainstBDQ.Molecularmodelinganddockingwasperformed tounderstand the interactingbehaviourofmutant (MT)enzymeswithBDQincomparisontowild-type(WT).TheseresultsindicatethatthesubstitutionsinAtpEexceptD28GshowedhighaffinitytowardsthedrugincomparisontotheWT.ThiscouldbeduetothefavourableinteractionsinmutantscomparedtoWT.
Despitebeingacontrollable,preventableandcurabledisease,tuberculosis(TB)stillre-mainasamajorpublichealthprobleminmanypartsoftheworld.Theincreaseinmulti-drugresistant(MDR-TB)isdefinedasresistancetothetwomosteffectivefirstlineTBdrugs:ri-fampicin(RIF)andisoniazid(INH)hasintensifiedthemagnitudeofthesituation.Extensivelydrug-resistantTB(XDR-TB) isemergingasanevenmoreominous threat, that is resistanttoanyfluoroquinolone,andatleastoneofthreeinjectablesecond-linedrugs(capreomycin,kanamycin,andamikacin),inadditiontoINHandRIF.DrugresistanceinTBisessentiallyapotentialthreattotheTBcontrolprograms.Themostrecentdrug-resistancesurveillancedataissuedbytheWorldhealthorganization(WHO)estimatesthatanaverageofroughly9%ofMDR-TBcasesareXDR-TB[1].
1.2. Properties of Bedaquiline (BDQ)
Bedaquiline(BDQ)isalsoknownasTMC207orR207910,isadrugbelongingtotheclassofdiarylquinolineandfoundtobeeffectiveagainstbothdrugsusceptibleanddrugresis-tanceTB[2].ItisaverypromisingandrelativelynewcandidatedrugfortreatmentofTBandmycobacterial infections[2].TheactivityspectrumofTMC207includes themycobacterialspecies thatarepathogenic tohumans, suchasMycobacterium tuberculosis (M. tuberculo-sis),butalsoatypicalpathogenicspecies,suchasM. avium complex, M kansasii,andthefastgrowersM. fortuitum and M. abscessus[2].Itisfoundtobeactivewithinmacrophages,andanimportantagentinshorteningthedurationofanti-TBtreatment[3].
BDQ exhibits a novelmechanism of actionwhich efficiently inhibits the adenosine5-triphosphate(ATP)synthaseofmycobacteriasuchasM. tuberculosis[2].IttargetssubunitcoftheATPsynthaseofM. tuberculosis,leadingtoinhibitionoftheactivityofprotonpumpintheenzyme[8].ThestructureofATPsynthasewasdescribedintermsoftwosectors,amem-branousF0(ab2c10–15)andamembrane-extrinsicF1(α3β3γδε)[9].BindingofBDQatthelevelof theproton-bindingsite totheoligomericandproteolipicsubunitc inF0domainofATPsynthaseblocksrotationofdiscsofATPsynthase,whichculminatesintheinhibitionofATPsynthesisandeventuallytothedeathofmycobacteria[10,11].
1.5. Mechanism of resistance of BDQ
Targetbasedresistancewasfoundtooccurinresistantisolateswithmutationsinsubunitcatpositions28(Asp→Gly/Ala/Val/Pro),61(Glu→Asp),63(Ala→Pro),and66(Ile→Met)basedontheM. tuberculosisaminoacidnumberingsystem[12,13].Theassumptionwassup-portedbythefactthatthemycobacterialspeciesnaturallyresistanttoBDQ,i.e.,M. xenopi, M. novacastrense, and M. shimoidei,displayaMetatposition63insubunitcinplaceofaconservedAlainthespeciessusceptibletothedrug[12,13].
TheinteractionprofileofWTandMT-AtpEswithBDQatitsbindingsiteareillustratedin Figure 7.Ingeneral,theinhibitorandenzymemakeapatternofcomplementaryhydrogen(H)bondsbetween their respectivebackbone atoms. In caseofWT-AtpEcomplexedwithBDQ,singlecarbonHbondwasseenfollowedbytwoPi-pistackedinteractionswithPhe65,andotherresiduesinvanderWaalscontactdistanceasshowninFigure 7.IncaseofMT-D28G,threecarbonHbondswereobservedbetweenBDQandresiduesGlu61,Phe62andPhe65,respectively,followedbyweakPi-alkylinteractions.IncontrasttoWTinMT-D28A,severalinteractionsprincipallyofalkyl,Pi-alkyl,Pi-PistackedwerefoundandPi-sigmabe-tweenthedrugandPhe66wasalsofound.InMT-D28Pcomplexedwiththedrugmolecule,twocarbonHbondswereformedwithresiduesPhe54andGly58,followedbyalkyl,Pi-alkylPi-pistackedinteractions.Surprisingly,inMT-D28VcomplexedwithBDQnoHbondswerefound.Notably,bromineatomoftheBDQwasinvolvedinalkylinteractionwiththeresidueLeu68.Inaddition,alkyl,Pi-alkylandtwoamidePi-stackedtypesofinteractionswereob-served.Interestingly,bromineatomoftheBDQwasinvolvedinalkylinteractionwiththeresi-dueLeu68(Figure 7).IncaseofMT-E61D,acarbonHbondwithAsp142wasformedandPi-alkyl,Pi-pistackedinteractionswerefoundasshowninFigure 7.InMT-A63PcomplexedwithBDQ,anamide-PistackedandPialkylinteractionswerefoundwithGlu61andAla62residues.InMT-I66MwithBDQ,interestingly,sulphurbasedinteractionswithMet66itselfwasfound.Then,api-SigmawithLeu59andPi-PiT-shapedwithPhe146werealsofound(Figure 7).
The reason for thehigh score in allmutants (D28A,D28PD28V,E61D,A63P, andI66MexceptD28G)withBDQcouldbeattributedtothepresenceoffavourableinteractionsthatlacksinWT.ThiscouldinturnbeduetothesubstitutionofAla/Pro/ValintheMTproteinsinplaceofAspinWTatposition28.Inthesemutants(D28A,D28PD28V),Alacontainsoneextramethylgroup,ProcontainsaheterocyclicgroupandValcontainstwomethylgroupsinsteadofAspwhichisanacidicaminoacid,mighthaveinducedmorestructuralchangesintheprotein’ssidechain,whichwasobviousinchangesinthepatternofinteractions(manyalkyl, Pi-alkyl, Pi-Pi stacked andPi-sigma inD28A; two carbonHbonds, alkyl, Pi-alkyl,Pi-pi stacked interactions inD28Pandalkyl,Pi-alkyl and twoamidePi-stacked inD28V)
Figure 7:DifferencesinnetworkofinteractionsbetweenWTandmutantsofAtpEfromM. tuberculosis with BDQ
AcarbonHbond,Pi-alkyl,Pi-pistackedinteractionsinE61Dandamide-PistackedandPialkylinteractionsinA63P;interactionssuchassulphurbasedwithMet66itselfwasfound.Api-SigmawithLeu59andPi-PiT-shapedwithPhe146werefoundinMT-I66M.ThesetypesofinteractionsinallmutantsconsequentlyreflectedinhighscorethantheWT.IncontrastincaseMT-D28G,asGlydoesnotcontainsafunctionalgrouporsidechain,resultedinlowerscore(onlyPi-alkyl)thantheWT(twoPi-pistackedinteractions).Thus,basedonthesefind-ingsitcanbeassumedthatthemutants-D28A,D28P,D28V,I66M,A63P,andE61Dcouldlead tohigh level resistancecompared toMT-D28G thatmay lead to low level resistance.Therefore,inthispilotstudy,aprimaryeffortwastakentounderstandtheeffectofbindingaffinityofWTandMTproteinsofAtpEwithBDQ,whichshowedmoreaffinitytowardsthemutantscomparedtotheWT.Therefore,itcanbesuggestedthatthemutantsdisplayedmoreaffinitywithBDQ,becauseofthesubstitutionthatinducesstructuralchangesandBDQbindsvery tightly leading to thesloworno releaseof thedrug tomediate its inhibitoryactivity,therebyleadstoBDQresistance.
DiagnosisandManagementofTuberculosis
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4. Conclusions
Thefindingsinthisconcisereporthaveprovidedsomeusefulinsightstowardsunder-standingthebasisofin vitro-BDQresistanceinM. tuberculosis.Although,theeffectofdock-ingcouldbebetterexplainedafterperformingmoleculardynamics forunderstanding theirfunctionprecisely,yet, the informationprovidedoverherecanbeuseful tounderstand theimpactofsuchsubstitutionandconsequentchangesinbindingability.However,furtherstruc-turalstudiesareneededtogetadeeperunderstandingofthemechanismofATPresistancetoBDQthatwillaidindevelopmentofinhibitorsthatareselectiveagainstATPsynthasewhichcancircumvent theproblemofBDQresistance. In addition, tounderstandmoreabout thebindingaspectsofBDQwithothernon-targetbased resistance i.e., efflux-based resistancetoBDQwhichwasidentifiedinpairedisolatesfrompatientstreatedwithBDQ,aswellasinmice,showingcross-resistancetoclofazimine[24].Thus,structuralstudiesrelatedtoefflux-basedresistancemutantsleadingtocauseofBDQresistancearealsoneeded.