interactionwin10能玩lol吗

Liuetal.
CellDeathDiscovery(2018)4:28DOI10.
1038/s41420-018-0029-6CellDeathDiscoveryARTICLEOpenAccessMagnololrestorestheactivityofmeropenemagainstNDM-1-producingEscherichiacolibyinhibitingtheactivityofmetallo-beta-lactamaseShuiLiu1,YonglinZhou1,XiaodiNiu2,TingtingWang1,JiyunLi3,ZhongjieLiu3,JianfengWang1,ShushengTang3,YangWang3andXumingDeng1AbstractTheemergenceofplasmid-mediatedNewDelhimetallo-β-lactamase-1(NDM-1)incarbapenem-resistantGram-negativepathogensisanincreasingclinicalthreat.
HerewereportthediscoveryofanNDM-1inhibitor,magnolol,throughenzymeinhibitionscreening.
WeshowedthatmagnololsignicantlyinhibitedNDMenzymeactivity(IC50=6.
47g/mL),anditrestoredtheactivityofmeropenemagainstEscherichiacoliZC-YN3,anNDM-1-producingE.
coliisolate,ininvitroantibacterialactivityassays.
Magnolollackeddirectantibacterialactivity,butcomparedwithmeropenemalone,itreducedtheMICsofmeropenemagainstE.
coliZC-YN3by4-foldandkilledalmostallthebacteriawithin3h.
Molecularmodelingandamutationalanalysisdemonstratedthatmagnololbindsdirectlytothecatalyticpocket(residues110to200)ofNDM-1,therebyblockingthebindingofthesubstratetoNDM-1andleadingtoitsinactivation.
OurresultsdemonstratethatthecombinationofmagnololandmeropenemmayhavethepotentialtotreatinfectionscausedbyNDM-1-positive,carbapenem-resistantGram-negativepathogens.
IntroductionTheemergenceanddisseminationofmultidrug-resistantpathogens,especiallyGram-negativebacteriathatencodeextended-spectrumβ-lactamasesandareresistanttoalmostallcurrentlyavailableβ-lactamantibiotics,isaworldwidepublichealthproblem1,2.
Potentcarbapenems,suchasmeropenemandimipenem,wereonceregardedasthelastlineofdefenseagainstmultidrug-resistantGram-negativebacteria3astheyarerelativelystableinthepre-senceofmostbacterialβ-lactamases,includingextended-spectrumβ-lactamases4.
However,theincreasinguseofcarbapenemscreatedaviciouscyclethatgaverisetocarbapenem-resistantGram-negativepathogens5,6.
NewDelhimetallo-β-lactamase-1(NDM-1),classiedasanAmblerclassB1metallo-β-lactamase(MBL)7andrstidentiedinKlebsiellapneumoniae8,isacarbapenemasethatwasspreadgloballyamongmulti-drugresistantbac-terialpathogens9.
NDM-1givesentericbacteriatheabilitytoinactivatealmostallβ-lactams,includingcarbapenems10,andthisMBLisnotinhibitedbyanyexistingβ-lactamaseinhibitors11–14.
Therefore,thepresenceofNDM-1anditscloselyrelatedvariantsinEnterobacteriaceaeisthreateningtodiminishtheeffectivenessofcarbapenems15,forcingtheWorldHealthOrganizationtoissueaglobalwarning16.
MBLsuseoneortwozincionsintheiractivesitetoactivateanucleophilicwatermoleculethatcleavesthelactamring7.
Althoughtheywererstidentiedhalfacenturyago17,manyMBLs,suchastheVeronaintegron-2018TheAuthor(s).
OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.
0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproductioninanymediumorformat,aslongasyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.
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Ifmaterialisnotincludedinthearticle'sCreativeCommonslicenseandyourintendeduseisnotpermittedbystatutoryregulationorexceedsthepermitteduse,youwillneedtoobtainpermissiondirectlyfromthecopyrightholder.
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org/licenses/by/4.
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Correspondence:YangWang(wangyang@cau.
edu.
cn)orXumingDeng(dengxm@jlu.
edu.
cn)1KeyLaboratoryofZoonosis,MinistryofEducation,InstituteofZoonosis,CollegeofVeterinaryMedicine,JilinUniversity,Changchun,China2DepartmentofFoodQualityandSafety,JilinUniversity,Changchun,ChinaFulllistofauthorinformationisavailableattheendofthearticleTheseauthorscontributedequally:ShuiLiuandYonglinZhou.
EditedbyARuniOfcialjournaloftheCellDeathDifferentiationAssociation1234567890():,;1234567890():,;encodedMBL(VIM)andimipenemase(IMP)variants,wereonlypresentinlesspathogenicspecies,andgenesencodingtheseMBLswerechromosomallylocatedinmostisolates.
Asaresult,theyhavelongbeenneglectedinclinicalsettings17.
Inrecentyears,theblaNDM-1genewasrstidentiedinatransferableplasmid,andsoonafter,varioushorizontalgenetransferelementspermittedNMD-1andotherresistancegenestospreadrapidlyandglob-ally10.
AsblaNDM-1isoneofthemajordrivingforcesfortherapidspreadofcarbapenemresistancethathasresultedinagreatcrisisthatthreatenstheuseofβ-lactamantibioticstotreatinfectionscausedbyNDM-1-producingpathogens,itisconsideredtobethemaintargetfornoveleffectiveinhibitors18.
Despitethehighunmetmedicalnecessity,feweffectiveclinicalinhibitorsofNDM-1andotherMBLshavebeenreportedsofar19,20.
HereweidentiedapotentinhibitoroftheNDM-1enzyme,magnolol(Fig.
1a),anaturalcompoundisolatedfromthebarkofmagnolia(MagnoliaofcinalisRehderandEHWilson)trees,usingapuriedNDM-1proteinandtheβ-lactamasesubstratenitrocenscreeningapproach.
BybindingtotheactivesiteofNDM-1,mag-nololeffectivelyinhibitedthebiologicalactivityofNDM-1andsuccessfullyrescuedtheeffectivenessofmeropeneminvitroagainstNDM-1-expressingE.
coli.
Thecombi-nationofmagnololandmeropenemmayhavethepotentialtotreatinfectionscausedbyNDM-1-positive,carbapenem-resistantGram-negativepathogens.
ResultsMagnolol-mediatedinhibitionofEscherichiacoliZC-YN3invitroEnzymeinhibitionassaysusingpuriedrecombinantNDM-1enzymesdemonstratedthatamong75naturalcompounds,onlymagnololhadasignicantimpactonMBLenzymeactivityinvitro(Fig.
1b).
NDM-1activityFig.
1Magnolol-mediatedinhibitionofE.
coliZC-YN3invitro.
aChemicalstructureofmagnolol.
bMagnololinhibitsNDM-1activity.
Thevaluesaretheaveragesofthreeindependentexperiments.
**indicatesP1,024μg/mL),anditalsohadnoinuenceonthegrowthofNDM-1-producingE.
coliZC-YN3(Fig.
1c).
Inagreementwiththesynergymentionedabove,time-killcurvesdemonstratedthatthecombinationofmag-nololplusmeropenemexertedkillingeffectsonE.
coliZC-YN3,thoroughlykillingthebacteriaby3hafterco-incubation(Fig.
1d).
OurresultsindicatethatmagnololrestoredtheactivityofmeropenemagainstE.
coliZC-YN3invitro.
Moleculardynamics(MD)simulationfortheNDM-1-magnololcomplexUsingacomputationalbiologymethod,weexploredthepotentialbindingmodeofmagnololtotheactivesiteofNDM-1(Fig.
2a).
ItisobviousthatmagnololcanbindtoNDM-1viahydrogenbondingandhydrophobicinterac-tions.
Duringthetimecourseofthesimulation,magnolollocalizedtothecatalyticpocketofNDM-1(residues110to200).
Indetail,thebindingmodelofmagnololtoNDM-1revealedthatthesidechainofmagnololcanformonehydrogenbondwithSer217.
Thecomplexreachedequilibriumat100nsbasedonananalysisoftheroot-mean-squaredeviationsofthebackboneCαatoms(Fig.
2b).
Moreover,thenumberofhydrogenbondswascalculatedduringthesimulation.
Thenumberofhydro-genbondsuctuatedbetweenoneandtwoduringthesimulation,furtherconrmingthatthereisonehydrogenbondbetweenmagnololandNDM-1(Fig.
2c).
ToexploretheenergycontributionsfromtheresiduesofthebindingsitesintheNDM-1-magnololcomplex,theenergydecompositionwascalculatedfortheNDM-1-magnololcomplex.
Fiveresidues,Val73,Lys211,Leu218,Gly219,andHis250,madeastrongtotalbindingenergycontribution,withaΔEtotalof≤2.
0kcal/mol(Fig.
3a).
Inaddition,residuesHis189,Cys208,Asp212,andSer217alsocontributedappreciablytothetotalbindingenergy,withaΔEtotalof≤1.
0kcal/mol.
TheseresultssuggestFig.
3ThepredictedinteractionmechanismbetweenmagnololandNDM-1.
Decompositionofthebindingenergyonaper-residuebasisintheWT-NDM-1-magnololcomplex.
Thehistogramchartshowsthetotal(a),vanderWaals(b),electrostatic(c),andsolvation(d)contributionsforthecomplexes.
Thehighestoccupiedmolecularorbital(e)andlowestunoccupiedmolecularorbital(f)ofmagnolol.
gInteractionsbetweenmagnololandtheresiduesofthebindingsitesinNDM-1areshownusingatwo-dimensionaldiagrambyLigplusLiuetal.
CellDeathDiscovery(2018)4:28Page4of8OfcialjournaloftheCellDeathDifferentiationAssociationthattheseveresidues(Val73,Lys211,Leu218,Gly219,andHis250)arekeyresiduesformagnololbinding.
AsshowninFig.
3b–d,mostofthedecomposedenergyinteractionoriginatedfromvanderWaalsinteractions,apparentlythroughhydrophobicinteractions,whiletheelectrostaticcontributionappearedtohaveanunfavor-ableinuenceonthesekeyresiduesduringcomplexfor-mation.
ThemagnololconformationwasoptimizedwiththeB3LYP/6-311G*setwithGaussian09software.
AsshowninFig.
3e,f,thehighestoccupiedmolecularorbitalandthelowestunoccupiedmolecularorbitalofmagnololindicatedthatthebenzeneringistheactivebindingcenterofmagnolol(Fig.
3g).
Amongtheseabove-mentionedvekeyresidues,sincetheVal73islocatedoutsidethecatalyticpocketandirrelevantfortheactivityofNDM-1,andtheLeu218andHis250mutantmayinduceaconformationchangeinNDM-1andfurtheraffectthebindingabilityofmagnololtootherresidues;therefore,weselectedtheLys211AlaandGly219Alamutantstoconrmthesetheoreticalresults.
ThetotalbindingfreeenergyfortheNDM-1-magnololcomplexandtheirdetailedenergycontributionswerecalculatedaccordingtothemolecularmechanicsPoisson–Boltzmannsurfaceareaapproach,andtheyaresummarizedinTable2.
Thebindingfreeenergy,ΔGbind,oftheinteractionbetweenmagnololandwild-type(WT)NDM-1wasgreaterthanthatoftheLys211AlaandGly219Alamutants,whichmeansthatWT-NDM-1hasthestrongestabilitytobindmagnolol.
Viauorescencespectroscopyquenching,wemeasuredΔGbindandthenumberofbindingsitesbetweenmagnololandtheLys211AlaandGly219Alamutants,andtheseresultswerehighlyconsistentwiththoseobtainedbycomputationalmethods(Table2),furtherconformingtheinformationgeneratedbytheMDsimulationoftheNDM-1-magnololcomplex,namely,thatbecauseofthebindingoftheinhibitormagnololtotheactivesiteregion(residuesVal73,Lys211,Leu218,His189,Cys208,Asp212,Ser217,Gly219,andHis250),thebiologicalactivityofNDM-1waslargelyinhibited(Fig.
4a).
MagnololhasnoeffectonthestabilityofNDM-1AsshowninFig.
4b,thestabilityofNDM-1wasnotaffectedafter0,2,4,or8hofincubationwithdifferentconcentrationsofmagnolol.
ThesedataindicatethatmagnololhasnoimpactonthestabilityofNDM-1.
DiscussionMBLsarethemajortargetsfordevelopingefcientinhibitorsagainstcarbapenem-resistantEnterobacter-iaceae18.
Throughunremittingefforts,varioustypesofMBLinhibitorswithdifferentmechanismshavebeendescribed.
In2014,Kingetal.
identiedafungalnaturalproduct,aspergillomarasmineA(AMA),whichisasaneffectiveinhibitorofNDM-1andVIM-2,throughacell-basedscreeningapproach21.
AMAovercametheresis-tancemediatedbythesetwoMBLsbyaffectingNDM-1/VIM-2-boundzinc,anditfullyrestoredtheantibacterialactivityofmeropenem.
Inamousemodel,AMAwasshowntobeapotentialtherapeuticagent,butoneofthemajorchallengestoitsclinicalapplicationisthedifcultyassociatedwithitschemicalsynthesis.
Fortunately,LiaoTable2Thebindingfreeenergy(kcal/mol)oftheWT-NDM-1-magnolol,Lys211Ala-magnolol,andGly219Ala-magnololcomplexesbasedoncomputationalmethodsandthevaluesofthebindingconstants(KA)basedonuorescencespectroscopyquenchingWT-NDM-1K211AG219AThebindingfreeenergy13.
6±2.
17.
9±1.
28.
6±1.
6KA(1*104)L/mol8.
6±1.
74.
8±1.
15.
5±1.
3AlltestsweredeterminedintriplicateFig.
4aTheeffectsofWT-NDM-1anditsmutantsonNDM-1activity.
Thevaluesaretheaveragesofthreeindependentexperiments.
*indicates0.
0105,and**indicatesP<0.
01.
bTheeffectsofmagnololonthestabilityofNDM-1Liuetal.
CellDeathDiscovery(2018)4:28Page5of8OfcialjournaloftheCellDeathDifferentiationAssociationetal.
reportedthersttotalsynthesisandstereochemicalcongurationreassignmentofAMAthatisamenabletotheefcientpreparationofAMA20.
Inaddition,researchbyChiouetal.
demonstratedthatebselen,whichisananti-oxidantdrugalreadysafelyusedinhumanstudies,mightbeapromisinginhibitorofNDM-1bytargetingtheCysresidueattheactivesite.
However,itsanti-oxidantactivityandtoxicitymightlimititspotential23.
Klingleretal.
tested11approveddrugscontainingathiolmoiety,andtheyfoundfourapproveddrugs(captopril,thiorphan,dimercaprol,andtiopronin)possessedinhibitoryactivityforNDM-1,VIM-1,andIMP-7.
However,thecon-centrationsrequiredtorestoretheirantibacterialactivitycouldnotbereachedinpathogens24.
Forpracticalandtechnicalreasons,todate,therearefewinhibitorswithestablishedpotentialforclinicalapplication.
Thus,thedevelopmentofMBLinhibitorstorestoretheactivityofβ-lactamantibioticsisofgreatnecessity.
Naturalcompoundshaveplayedanimportantroleinthediscoveryofantibiotics.
Magnolol,anabundantnat-uralcompoundisolatedfromM.
ofcinalis,hasbeenusedwidelyintraditionalChinesemedicine25.
HereweshowedthatmagnololinhibitedtheactivityofNDM-1,whichwasconrmedbyinvitroexperiments.
Comparedwiththeaboveinhibitors,magnololhastheadvantagesofabun-dantsourcesandeasypreparation26,27.
Thehighhydro-phobicityandlowsolubilityofmagnololmaybethemajorobstaclestoitsbioavailabilityandclinicalefcacy28,29.
However,werevealedthattheconcentrationsrequiredtorestoreantimicrobialactivitycouldbeachievedinbac-teria.
Moreover,magnololinhibitedNDM-1activitywithoutimpactingNDM-boundzinc,whichdiffersfromthemetal-depletionmechanismsofAMA.
Thetoxicityassociatedwithcrossreactivitywithhumanmetallo-enzymesisamajorchallengeforthedevelopmentofMBLinhibitors21.
Knownchelators,suchasEDTA,havebeengreatlyrestrictedinclinicalusebecauseofthesesideeffects.
Forinstance,themedianlethaldoseofEDTAwascalculatedtobe28.
5mg/kgwhenadministeredintrave-nouslyinmice30.
Magnololhaslittletoxicityinvivobecauseofthedifferencesinitsmodeofactioncomparedwiththoseofothermetal-ion-chelatingagents.
Animalstudiesdemonstratedthatmagnololshowednoclinicalsignsoftoxicityinmiceandrats31,32.
Notably,magnololhasverylowtoxicityindogs(nomortalityat1g/kgwhenadministeredintravenouslytodogs)33.
Thus,thesedataindicatethatmagnololmaybeapotentiallysafeinhibitorofNDM-1.
Inconclusion,ourdatademonstratethatmagnololinhibitedβ-lactamaseenzymaticactivitybybindingtotheactivesiteofNDM-1,anditrestoredtheactivityofmeropenemagainstNDM-1-positiveE.
coliisolates.
Invitro,synergisticactivitywasobservedwiththecombi-nationofmagnololplusmeropenem.
Takentogether,theseresultsidentiedapotentialclinicallyefcaciousdoseusinginvitro,whichwillcontributetothefuturedevelopmentofaneffectiveNDM-1inhibitor.
MaterialsandmethodsBacterialstrainsandchemicalsTheNDM-1-producingE.
coliisolateswasoriginatedfromourpreviousstudy34.
Magnolol(≥98%pure)andmeropenem(≥87%pure)werepurchasedfromtheNationalInstitutesforFoodandDrugControl(Beijing,China).
Stocksolutionsofmagnololwerepreparedindimethylsulfoxide(DMSO,Sigma-Aldrich,St.
Louis,MO,USA).
Meropenemwasdissolvedinsterilewater.
PlasmidconstructionandproteinpuricationToproducerecombinantNDM-1inE.
coli,apET28a-NDM-1plasmidwiththerestrictionsitesBamHIandXhoIwasconstructed.
AblaNDM-1genewithoutthesignalpeptidewasampliedfromstrainE.
coliZC-YN3withtheprimersNDM-1-F/NDM-1-R(TableS1).
ThisvectorencodestheintactNDM-1sequencefusedtoanamino-terminalhistidinetag.
TheLys211AlaandGly219AlamutationsofNDM-1wereintroducedintopET28a-NDM-1usingtheQuickChangeSite-directedMutagen-esisKit(Stratagene,SanDiego,CA,USA)withthepri-mersK211A-F/K211A-RandG219A-F/G219A-R,respectively(TableS1).
Allconstructedstrainswerever-iedbyPCRandsequencing.
ProteinexpressionwasperformedaccordingtoLiaoetal20.
EnzymeinhibitionassaysTheassaywasconductedaccordingtoLiaoetal.
'smethod20withminormodications.
TondNDM-1inhibitors,weselected75naturalcompoundsasscreeningcompounds(TableS2).
Assayswerereadin96-wellplatesatanabsorbanceof492nmusingamicroplatereader(TecanAustriaGmbH,Grdig,Austria)atroomtem-perature.
Positivecontrolswereperformedinthepre-senceofenzymeandintheabsenceofinhibitors,whereasnegativecontrolswereperformedintheabsenceofenzyme.
Residualactivity=AA0/A100A0*100%,whereArepresentstheabsorbanceofinhibitorgroupsat492nm,andA0andA100represent0%and100%activityasdeterminedinthenegativecontrolsandpositivecon-trols,respectively.
AntibacterialactivityassaysinvitroMICsofmagnolol,meropenem,andcombinationsofmagnololplusmeropenemagainstE.
coliisolatesweredeterminedusingthebrothmicrodilutionmethodfol-lowingtheguidelinesoftheClinicalandLaboratoryStandardsInstitute.
Thecombinationswereevaluatedbycalculatingthefractionalinhibitoryconcentration(FIC)indexvalues.
ToevaluatetheeffectofmagnololontheLiuetal.
CellDeathDiscovery(2018)4:28Page6of8OfcialjournaloftheCellDeathDifferentiationAssociationgrowthofthetestedstrains,agrowthcurveassaywasperformed.
Specically,E.
coliZC-YN3wasculturedinLuria–Bertanimediumat37°Cwithshaking(180rpm)toanopticaldensityat600nmof0.
3andthenaliquotedintove50-mLconicalasks.
Magnolol(ortheDMSOcon-trol)wasaddedtotheveculturesat0,16,32,64,and128μg/mL.
Thebacteriawereculturedat37°Cwithconstantshaking,andcellgrowthwasestimatedbymeasuringtheOD600every30min.
Inaddition,thepotentialbactericidaleffectofmagnololcombinedwithmeropenemwasevaluatedbytime-killingassays35.
MolecularmodelingTheinitialstructureofNDM-1wasobtainedfromthethree-dimensionalX-raystructure(PDBcode:4EXS).
Toobtainthestartingstructureofthemagnolol/NDM-1complexfortheMDsimulation,astandarddockingprocedureforarigidproteinandaexibleligandwasperformedwithAutoDock436,37.
Subsequently,theMDsimulationofthecomplexwasperformed.
Theprocessesofthecomputationalbiologymethodhavebeendescribedindetailinpreviousreports38,39.
DeterminationofthebindingafnityofmagnololtomutantNDM-1proteinsTheuorescence-quenchingmethodwasusedtomea-surethebindingconstants(KA)ofmagnololwiththeNDM-1mutants(Lys211AlaandGly219Ala).
A280-nmexcitationwavelengthwitha5-nmbandpassanda345-nmemissionwavelengthwitha10-nmbandpasswereusedforthemeasurements.
Detailsofthemeasurementshavebeendescribedpreviously40,41.
NDM-1stabilityassaysForNDM-1stabilityassays,puriedNDM-1wasincubatedwithoutmagnololorwith8and32g/mLmagnololfor0,2,4,and8hat37°C.
WesternblottingwasperformedtoinvestigatethestabilityofNDM-1treatedwithmagnolol.
Anti-histidine-tagantibodies(1:4,000dilution,ProteintechGroup,Inc.
,Rosemont,IL,USA)andhorseradishperoxidase-conjugatedgoatanti-mouseantibodies(1:2,000dilution,ProteintechGroup,Inc.
)wereusedastheprimaryandsecondaryantibodies,respectively.
StatisticalanalysisDataarepresentedasthemean±standarddeviationfromthreeindependentexperiments,andtheywereanalyzedusingSPSSStatisticsforWindows,version19.
0(IBMCorp.
Armonk,NY,USA).
SignicantdifferencesweredeterminedusinganindependentStudent'st-testasindicated.
Differenceswereconsideredstatisticallysig-nicantwhenPvalueswerelessthan0.
05.
AcknowledgementsThisworkwassupportedbytheNationalKeyTechnologyR&DProgram(No.
2016YFD05013)andtheNationalNaturalScienceFoundationofChina(31422055and81661138002).
Authordetails1KeyLaboratoryofZoonosis,MinistryofEducation,InstituteofZoonosis,CollegeofVeterinaryMedicine,JilinUniversity,Changchun,China.
2DepartmentofFoodQualityandSafety,JilinUniversity,Changchun,China.
3CollegeofVeterinaryMedicine,ChinaAgriculturalUniversity,Beijing,ChinaConictofinterestTheauthorsdeclarethattheyhavenoconictofinterest.
Publisher'snoteSpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalafliations.
SupplementaryInformationaccompaniesthispaperathttps://doi.
org/10.
1038/s41420-018-0029-6Received:22November2017Accepted:22December2017References1.
Spellberg,B.
etal.
Theepidemicofantibiotic-resistantinfections:acalltoactionforthemedicalcommunityfromtheInfectiousDiseasesSocietyofAmerica.
Clin.
Infect.
Dis.
46,155–164(2008).
2.
Brown,E.
D.
&Wright,G.
D.
Antibacterialdrugdiscoveryintheresistanceera.
Nature529,336–343(2016).
3.
Alm,R.
A.
,Johnstone,M.
R.
&Lahiri,S.
D.
CharacterizationofEscherichiacoliNDMisolateswithdecreasedsusceptibilitytoaztreonam/avibactam:roleofanovelinsertioninPBP3.
J.
Antimicrob.
Chemother.
70,1420–1428(2015).
4.
Schneider,K.
D.
,Karpen,M.
E.
,Bonomo,R.
A.
,Leonard,D.
A.
&Powers,R.
A.
The1.
4AcrystalstructureoftheclassDbeta-lactamaseOXA-1complexedwithdoripenem.
Biochemistry48,11840–11847(2009).
5.
Karaiskos,I.
&Giamarellou,H.
Multidrug-resistantandextensivelydrug-resistantGram-negativepathogens:currentandemergingtherapeuticapproaches.
ExpertOpin.
Pharmacother.
15,1351–1370(2014).
6.
Livermore,D.
M.
HastheeraofuntreatableinfectionsarrivedJ.
Antimicrob.
Chemother.
64(Suppl1),i29–i36(2009).
7.
Bush,K.
&Jacoby,G.
A.
Updatedfunctionalclassicationofbeta-lactamases.
Antimicrob.
AgentsChemother.
54,969–976(2010).
8.
Yong,D.
etal.
Characterizationofanewmetallo-beta-lactamasegene,bla(NDM-1),andanovelerythromycinesterasegenecarriedonauniquegeneticstructureinKlebsiellapneumoniaesequencetype14fromIndia.
Antimicrob.
AgentsChemother.
53,5046–5054(2009).
9.
Berrazeg,M.
etal.
NewDelhiMetallo-beta-lactamasearoundtheworld:aneReviewusingGoogleMaps.
Eur.
Surveill.
19,20(2014).
10.
Kumarasamy,K.
K.
etal.
EmergenceofanewantibioticresistancemechanisminIndia,Pakistan,andtheUK:amolecular,biological,andepidemiologicalstudy.
LancetInfect.
Dis.
10,597–602(2010).
11.
StruelensM.
J.
,MonnetD.
L.
,MagiorakosA.
P.
,SantosO'ConnorF.
,GieseckeJ.
EuropeanNDMSP.
NewDelhimetallo-beta-lactamase1-producingEnter-obacteriaceae:emergenceandresponseinEurope.
Eur.
Surveill.
15,9–16(2010).
12.
Nordmann,P.
,Poirel,L.
,Walsh,T.
R.
&Livermore,D.
M.
TheemergingNDMcarbapenemases.
TrendsMicrobiol.
19,588–595(2011).
13.
Cornaglia,G.
,Giamarellou,H.
&Rossolini,G.
M.
Metallo-beta-lactamases:alastfrontierforbeta-lactamsLancetInfect.
Dis.
11,381–393(2011).
14.
Nordmann,P.
,Poirel,L.
,Toleman,M.
A.
&Walsh,T.
R.
Doesbroad-spectrumbeta-lactamresistanceduetoNDM-1heraldtheendoftheantibioticerafortreatmentofinfectionscausedbyGram-negativebacteriaJ.
Antimicrob.
Chemother.
66,689–692(2011).
15.
Walsh,T.
R.
Emergingcarbapenemases:aglobalperspective.
Int.
J.
Antimicrob.
Agents36(Suppl3),S8–S14(2010).
Liuetal.
CellDeathDiscovery(2018)4:28Page7of8OfcialjournaloftheCellDeathDifferentiationAssociation16.
Hsu,L.
Y.
&Koh,T.
H.
WorldHealthDay2011:combatingantimicrobialresistance.
Singap.
Med.
J.
52,230–231(2011).
17.
Ellar,D.
J.
&Lundgren,D.
G.
FinestructureofsporulationinBacilluscereusgrowninachemicallydenedmedium.
J.
Bacteriol.
92,1748–1764(1966).
18.
Drawz,S.
M.
&Bonomo,R.
A.
Threedecadesofbeta-lactamaseinhibitors.
Clin.
Microbiol.
Rev.
23,160–201(2010).
19.
Albu,S.
A.
etal.
TotalsynthesisofAspergillomarasmineAandrelatedcom-pounds:asulfamidateapproachenablesexplorationofstructure-activityrelationships.
Angew.
Chem.
55,13259–13262(2016).
20.
Liao,D.
etal.
TotalsynthesisandstructuralreassignmentofAspergillomar-asmineA.
Angew.
Chem.
55,4291–4295(2016).
21.
King,A.
M.
etal.
AspergillomarasmineAovercomesmetallo-beta-lactamaseantibioticresistance.
Nature510,503–506(2014).
22.
King,D.
T.
,Worrall,L.
J.
,Gruninger,R.
&Strynadka,N.
C.
NewDelhimetallo-beta-lactamase:structuralinsightsintobeta-lactamrecognitionandinhibition.
J.
Am.
Chem.
Soc.
134,11362–11365(2012).
23.
Chiou,J.
etal.
EbselenasapotentcovalentinhibitorofNewDelhimetallo-beta-lactamase(NDM-1).
Chem.
Commun.
51,9543–9546(2015).
24.
Klingler,F.
M.
etal.
Approveddrugscontainingthiolsasinhibitorsofmetallo-beta-lactamases:strategytocombatmultidrug-resistantbacteria.
J.
Med.
Chem.
58,3626–3630(2015).
25.
Alexeev,M.
,Grosenbaugh,D.
K.
,Mott,D.
D.
&Fisher,J.
L.
Thenaturalproductsmagnololandhonokiolarepositiveallostericmodulatorsofbothsynapticandextra-synapticGABA(A)receptors.
Neuropharmacology62,2507–2514(2012).
26.
Chen,L.
etal.
Rapidpuricationandscale-upofhonokiolandmagnololusinghigh-capacityhigh-speedcounter-currentchromatography.
J.
Chromatogr.
A1142,115–122(2007).
27.
Lee,Y.
J.
etal.
TherapeuticapplicationsofcompoundsintheMagnoliafamily.
Pharmacol.
Ther.
130,157–176(2011).
28.
Wang,Y.
J.
,Chien,Y.
C.
,Wu,C.
H.
&Liu,D.
M.
Magnolol-loadedcore-shellhydrogelnanoparticles:drugrelease,intracellularuptake,andcontrolledcytotoxicityfortheinhibitionofmigrationofvascularsmoothmusclecells.
Mol.
Pharm.
8,2339–2349(2011).
29.
He,S.
,Zhang,Z.
,Xu,F.
,Zhang,S.
&Lei,Z.
Micronizationofmagnoliabarkextractwithenhanceddissolutionbehaviorbyrapidexpansionofsupercriticalsolution.
Chem.
Pharm.
Bull.
58,154–159(2010).
30.
Matsuura,A.
etal.
Pharmacologicalprolesofaspergillomarasminesasendothelinconvertingenzymeinhibitors.
Jpn.
J.
Pharmacol.
63,187–193(1993).
31.
Li,N.
etal.
Evaluationoftheinvitroandinvivogenotoxicityofmagnoliabarkextract.
Regul.
Toxicol.
Pharmacol.
RTP49,154–159(2007).
32.
Liu,Z.
etal.
Evaluationofshort-termandsubchronictoxicityofmagnoliabarkextractinrats.
Regul.
Toxicol.
Pharmacol.
RTP49,160–171(2007).
33.
Poivre,M.
&Duez,P.
BiologicalactivityandtoxicityoftheChineseherbMagnoliaofcinalisRehder&E.
Wilson(Houpo)anditsconstituents.
J.
Zhe-jiangUniv.
Sci.
B18,194–214(2017).
34.
Wang,Y.
etal.
ComprehensiveresistomeanalysisrevealstheprevalenceofNDMandMCR-1inChinesepoultryproduction.
Nat.
Microbiol.
2,16260(2017).
35.
Kang,W.
etal.
EffectiveantimicrobialactivityofapeptidemutantCbf-14-2againstpenicillin-resistantbacteriabasedonitsunnaturalaminoacids.
Eur.
J.
Pharm.
Sci.
105,169–177(2017).
36.
Morris,G.
M.
etal.
AutoDock4andAutoDockTools4:automateddockingwithselectivereceptorexibility.
J.
Comput.
Chem.
30,2785–2791(2009).
37.
Hu,R.
,Barbault,F.
,Maurel,F.
,Delamar,M.
&Zhang,R.
Moleculardynamicssimulationsof2-amino-6-arylsulphonylbenzonitrilesanaloguesasHIVinhibi-tors:interactionmodesandbindingfreeenergies.
Chem.
Biol.
DrugDes.
76,518–526(2010).
38.
Dong,J.
etal.
OroxylinAinhibitshemolysisviahinderingtheself-assemblyofalpha-hemolysinheptamerictransmembranepore.
PLoSComput.
Biol.
9,e1002869(2013).
39.
Niu,X.
etal.
Molecularinsightintotheinhibitionmechanismofcyrtominetintoalpha-hemolysinbymoleculardynamicssimulation.
Eur.
J.
Med.
Chem.
62,320–328(2013).
40.
Jurasekova,Z.
,Marconi,G.
,Sanchez-Cortes,S.
&Torreggiani,A.
Spectroscopicandmolecularmodelingstudiesonthebindingoftheavonoidluteolinandhumanserumalbumin.
Biopolymers91,917–927(2009).
41.
Bandyopadhyay,S.
,Valder,C.
R.
,Huynh,H.
G.
,Ren,H.
&Allison,W.
S.
ThebetaG156CsubstitutionintheF1-ATPasefromthethermophilicBacillusPS3affectscatalyticsitecooperativitybydestabilizingtheclosedconformationofthecatalyticsite.
Biochemistry41,14421–14429(2002).
Liuetal.
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