Zika virus and drug discovery chemEd · 2017-04-17 · 1 Zika virus and drug discovery: a...
11
1 Zika virus and drug discovery: a classroom-based bioinformatics research project and general method for repurposing existing drugs Sandra Porter, Austin Community College and Digital World Biology LLC, Seattle, WA Abstract The most engaging student research projects combine relevant topics with the opportunity to develop and practice skills that can be applied outside of class. Cases of emerging viruses and outbreaks of infectious agents capture student attention. The Zika virus outbreak has been devastating to young families in Latin America and the Caribbean. Pictures of affected babies with unusually small heads and severe birth defects have alarmed people throughout the western hemisphere. Although few cases of Zika virus infection have been reported in the United States, the mosquito that carries Zika virus, Aedes aegypti, is endemic in the Southern U.S., and could transmit Zika virus in states like Florida, and Texas. In this project, students use the bioinformatics and molecular modeling skills they’re learning in class to identify drugs that might be able to treat people infected by Zika virus. Introduction I teach an on-line bioinformatics course through Austin Community College that focuses on common applications for bioinformatics tools in biotechnology. Students learn how to search databases, use molecular models, analyze molecular sequences, and characterize genetic variation. Although bioinformatics provides us with interesting real-life examples to study, our abilities are limited when it comes to finding student projects that include opportunities to make new discoveries. Last spring, I developed a research project designed to engage students by having them apply their skills and knowledge to the problem of finding drugs to treat Zika virus. It seemed that the devastating effects of Zika infection combined with the possibility (albeit low) that students taking our class might be impacted, would have a positive effect on student engagement. Students begin the project by looking at Health Map to see where Zika virus has been reported and read about the outcomes of Zika virus infections. Through their reading, they learn that the NCBI structure database contains structures of proteins bound to inhibitors and drugs and that it might be possible to repurpose an existing drug to treat Zika virus. Students read about the Zika virus genome and life cycle and decide whether to look for a drug that inhibits the RNA polymerase or the protease. They find the protein sequence for one of these proteins and use it to search the structure database to find similar proteins that are bound to drugs / inhibitors. After that, they use the Molecule World™ iPad app (1) to identify amino acids that contact the drug and determine if those same residues are present in the Zika virus protein. In developing an example to show students how this process would work, I determined that the Zika virus polymerase would be likely to bind to Gilead’s anti-Hepatitis C drug Sovaldi ® (Sofosbuvir). This prediction was borne out by two recently published studies showing that Sofosbuvir does inhibit Zika virus replication (2, 3). This general process for repurposing existing drugs is summarized below. Using this
Transcript of Zika virus and drug discovery chemEd · 2017-04-17 · 1 Zika virus and drug discovery: a...
1
Zikavirusanddrugdiscovery:aclassroom-basedbioinformaticsresearchprojectandgeneralmethodforrepurposingexistingdrugs
SandraPorter,AustinCommunityCollegeandDigitalWorldBiologyLLC,Seattle,WAAbstractThemostengagingstudentresearchprojectscombinerelevanttopicswiththeopportunitytodevelopandpracticeskillsthatcanbeappliedoutsideofclass.Casesofemergingvirusesandoutbreaksofinfectiousagentscapturestudentattention.TheZikavirusoutbreakhasbeendevastatingtoyoungfamiliesinLatinAmericaandtheCaribbean.Picturesofaffectedbabieswithunusuallysmallheadsandseverebirthdefectshavealarmedpeoplethroughoutthewesternhemisphere.AlthoughfewcasesofZikavirusinfectionhavebeenreportedintheUnitedStates,themosquitothatcarriesZikavirus,Aedesaegypti,isendemicintheSouthernU.S.,andcouldtransmitZikavirusinstateslikeFlorida,andTexas.Inthisproject,studentsusethebioinformaticsandmolecularmodelingskillsthey’relearninginclasstoidentifydrugsthatmightbeabletotreatpeopleinfectedbyZikavirus.
IntroductionIteachanon-linebioinformaticscoursethroughAustinCommunityCollegethatfocusesoncommonapplicationsforbioinformaticstoolsinbiotechnology.Studentslearnhowtosearchdatabases,usemolecularmodels,analyzemolecularsequences,andcharacterizegeneticvariation.Althoughbioinformaticsprovidesuswithinterestingreal-lifeexamplestostudy,ourabilitiesarelimitedwhenitcomestofindingstudentprojectsthatincludeopportunitiestomakenewdiscoveries.Lastspring,IdevelopedaresearchprojectdesignedtoengagestudentsbyhavingthemapplytheirskillsandknowledgetotheproblemoffindingdrugstotreatZikavirus.ItseemedthatthedevastatingeffectsofZikainfectioncombinedwiththepossibility(albeitlow)thatstudentstakingourclassmightbeimpacted,wouldhaveapositiveeffectonstudentengagement.StudentsbegintheprojectbylookingatHealthMaptoseewhereZikavirushasbeenreportedandreadabouttheoutcomesofZikavirusinfections.Throughtheirreading,theylearnthattheNCBIstructuredatabasecontainsstructuresofproteinsboundtoinhibitorsanddrugsandthatitmightbepossibletorepurposeanexistingdrugtotreatZikavirus.StudentsreadabouttheZikavirusgenomeandlifecycleanddecidewhethertolookforadrugthatinhibitstheRNApolymeraseortheprotease.Theyfindtheproteinsequenceforoneoftheseproteinsanduseittosearchthestructuredatabasetofindsimilarproteinsthatareboundtodrugs/inhibitors.Afterthat,theyusetheMoleculeWorld™iPadapp(1)toidentifyaminoacidsthatcontactthedruganddetermineifthosesameresiduesarepresentintheZikavirusprotein.Indevelopinganexampletoshowstudentshowthisprocesswouldwork,IdeterminedthattheZikaviruspolymerasewouldbelikelytobindtoGilead’santi-HepatitisCdrugSovaldi®(Sofosbuvir).ThispredictionwasborneoutbytworecentlypublishedstudiesshowingthatSofosbuvirdoesinhibitZikavirusreplication(2,3).Thisgeneralprocessforrepurposingexistingdrugsissummarizedbelow.Usingthis
2
processinaclass-basedresearchprojectprovidesanewavenueforstudentstoapplybioinformaticstoolstoreal-lifeproblemsandpotentially,makediscoveries.Themethodforidentifyingpotentialdrugs1.Decideonadrugtarget.2.Findtheaminoacidsequencefortheproteinyouwishtotarget.3.UseproteinblasttoquerytheProteinDatabaseProteins(pdb)databaseattheNCBI.4.Scantheresultstofindproteinsboundtodrugs(orinhibitors).5.UseMoleculeWorld™toidentifyaminoacidsinthedrugbindingsite.6.Determineifaminoacidsinthedrugbindingsitearepresentinbothproteins.Howdoweapplythismethod?Studentsinourbioinformaticscoursehaveusedthisprocessforthepastthreesemesters.Inthissection,IdescribetheprocessinmoredetailandpresentanexamplethatIdevelopedwithsofosbuvir(Sovaldi®).1.Decideonadrugtarget.Thefirststepistodecidewhichproteintotarget.Althoughmanydifferentproteinscanbeusedasdrugtargets,ourstudentschoosebetweentwokindsofproteins:theviralRNApolymeraseandviralprotease.Polymerasesplayanimportantroleinthelifecycleofavirusbymakingcopiesoftheviralgenome.Thesecopiesgetpackagedintonewviralparticlesthatcangoonandinfectnewcells.Proteasesplayanimportantroleinvirusesthatmakepolyproteins.Thesepolyproteinsmustbecutintosmallerpartsbyaproteaseinordertofunction.BothpolymeraseandproteaseinhibitorsareusedtotreatinfectionswithHIV.Sofosbuvir,fromGilead,isanexampleofanFDA-approveddrugthatinhibitstheRNApolymerasefromHepatitisCvirus.2.Findtheaminoacidsequencefortheproteinyouwishtotarget.TheNCBI(NationalCenterforBiotechnologyInformation)wasestablishedin1988asadivisionoftheNationalLibraryofMedicine,attheNationalInstitutesofHealth(NIH).Theiroverallmissionistocreatesystemsforstoringandanalyzingknowledgeaboutmolecularbiology,biochemistry,andgeneticsandtofacilitatetheuseofsuchdatabasesandsoftwarebytheresearchandmedicalcommunity(http://ncbi.nlm.nih.gov).Aspartofthismission,theNCBIhassetupresourcepagesforspecificviralgenomes,inparticularpathogenicvirusessuchasZikavirus,MERs,andEbola(Fig1https://www.ncbi.nlm.nih.gov/genome/viruses/)
3
Figure1.TheViralGenomeportalattheNCBI.StudentsnavigatetotheviralgenomespageandchooseZikavirustoaccessacollectionofZikavirusresources(Fig.2https://www.ncbi.nlm.nih.gov/genome/viruses/variation/Zika/).
Figure2.ZikaVirusResourcesattheNCBI.TheZikaVirusResourcepagehaslinkstoHealthMap,theCDC(CentersforDiseaseControl),WHO(theWorldHealthOrganization),andpublications.Fromhere,weusethe“NCBIZikavirusreferencegenome”linkfollowedbytheGenBankrecordlinktolocatetheaccession
4
numberforproteinsequencesfromeithertheproteaseortheZikavirusRNApolymerase(Fig3).
Figure3.AccessionnumbersfortheZikavirusproteaseandZikavirusRNApolymerase.3.UseproteinblasttoquerytheProteinDatabaseProteins(pdb)databaseattheNCBI.TheaccessionnumberfortheRNApolymeraseisYP_009227205.1.Accessionnumbersactlikecatalognumbers.Theyallowustofindspecificsequencesinadatabase.ClickingthelinkedaccessionnumberfromtheGenBankrecordtakesustotheproteinrecord(Fig4).
5
Figure4.TheproteinrecordfortheZikavirusRNApolymerasewithalinktorunBLASTontheright.Ontherighthandsideoftheproteinrecord(Fig.4)isalinktoRunBLAST.Studentsselectthislinktoaccesstheproteinblastsearchalgorithm.Proteinblastisanalgorithmthatsearchesdatabasesofproteinsequencestofindsimilarproteins.FromtheBLASTsearchform,wechoosetheProteinDataBankdatabase(Fig.5).Alltheproteinsinthisdatabasecanbefoundinfilesofmolecularstructures.
6
Figure5.TheBLASTform.Next,weenterZikaintheorganismfieldandclickExcludesothatwecanavoidgettingresultsfromZikavirusproteins.It’snotnecessarytoknowthetaxidforthisorganism.Thisinformationisenteredbyautocompleteafterwetype“Zika”intheorganismfield(Fig.6).
Figure6.ExcludingZikavirusfromtheBLASTsearch.Now,weclickBLASTtostartthesearch.
7
OurBLASTresultsappearshortly.Thetopgraphshowsthepolymerasedomainintanontherightside(Fig.7).ThegraphbelowshowswhererelatedproteinsaligntotheZikaproteinsequencethatweusedasaquery.
Figure7.BLASTresultsshowingconserveddomainsintheZikavirusproteinsequence4.Scantheresultstofindproteinsboundtodrugs(orinhibitors).Nowthatwehaveasetofsimilarproteins,thenextstepisfindproteinsthatareboundtopotentialdrugs.Wescrolldownthepageandreadthetitlestoidentifystructureswhereaproteinisboundtosomekindofinhibitororpotentialdrug.InFigure8,wecanseeonestructurewheretheRNApolymerasefromDenguevirusisboundtocompound29andanotherwhereit’sboundtocompound27.Bothcompounds27and29mightbedrugcandidates.Theseareshowninthesamealignmentbecausetheproteinsequencesareidentical.
Figure8.Proteinsequencesfrommolecularstructurefilesshowingthattheproteinmaybeboundtoaninhibitor.Wecanconfirmthatthesecompoundsmightbedrugcandidatesorinhibitorsbyfollowingthelinkstothestructurerecordandskimmingthetitleofthepaperorabstractifthere’soneaccompanyingthestructure.BothofthesecompoundsaredescribedinapaperdiscussingpotentinhibitorsofDengue
8
virus.Thetitleandtheabstracttellustheauthorswerelookingforpotentialdrugs.Asmentionedearlier,Iwantedtoprovidestudentswithanexampletoshowhowthisprocessmightwork.SinceSofosbuvirhasbeenapprovedbytheFDAtotreatHepatitisCinfectionsandtheHepatitisCvirusbelongstothesameclassofvirusesasZikavirus,IdecidedtocomparetheZikavirusRNApolymerasetotheHepatitisCRNApolymerasetoseeiftheZikavirusproteinmightcontainalikelybindingsiteforSofosbuvir.IusedblastptocomparethesetwoproteinsandfoundaregionwithasignificantEvalue(Fig.9).AanEvalueof0.001showsthereisa1in1000chancethattwoproteinswouldmatchtothisextent.Oftheaminoacids,25%areidenticaland43%areeitheridenticalorconserved.
Figure9.AstatisticallysignificantalignmentbetweenaregionoftheZikavirusRNApolymerasequerysequenceandtheRNApolymerasefromHepatitisCvirus.5.UseMoleculeWorld™toidentifyaminoacidsinthedrug-bindingsite.Tolookatthebindingsite,Idownloaded4WTGinMoleculeWorldfromtheMMDB(MolecularModelingDatabase).ThisstructurecontainstheRNApolymerase,RNAintheprocessofbeingcopied,andSofosbuvir.SofosbuvirisanucleotideanaloguethatblocksRNAsynthesisbypreventingRNApolymerasefromaddingnewresiduestothechainafterthedrughasbeenincorporated.LookingatthestructureshowsmethatSofosbuvirhasbeenincorporatedintotheRNAchain(Fig.10).Toidentifytheaminoacidsinthebindingsite,ItouchtheShowsequencebuttontoopenthesequenceviewerandtouchthenameofthedrugtoselectit.Twomanganeseatomsappearedtobeboundtothedrug,soIselectedthoseaswell.
9
Figure10.HepatitisCvirusRNApolymeraseboundtoRNAandSofosbuvirfromstructure4WTGinMoleculeWorld.ThenextstepistotouchtheSelectionbuttonandchooseSelectnearby.Thishighlightseverythingwithin5-6angstromsofthedrug.Ingeneral,anythingwithin5angstromsofasubstratewouldbeconsideredtobelocatedinthebindingsite.Next,IopentheShow/Hidemenuandchoose“Allatomsinresidue”toseetheaminoacidsidechains.Viewingthesidechainsshowsmeifanyaminoacidresiduesformbondstothedrug.Icanalsohideeverythingthat’snotselectedtomaketheinteractioneasiertosee.SincesomeofthehighlightedobjectsinmystructureareRNA,Ideselectthoseandhidethemaswell.Figure11showssomeoftheaminoacidsinthebindingsiteforsofosbuvirwiththeaminoacidsequencebelow.
10
Figure11.AminoacidsintheSofosbuvirbindingsitearecoloredbyresidueinboththestructureandthesequence.6.Determineifaminoacidsinthedrug-bindingsitearepresentinbothproteins.Onceweknowwhichaminoacidsbindtothedrugandwhicharelocatedinthebindingsite,wecanaddthesedatatothesequencealignmenttoseeifthesesameaminoacidsarepresentinbothproteins.Acommonwaytoshowthesimilaritybetweentwosequencesistoalignthemwithonesequenceontopandtherelatedsequencebelow.Figure12showsthisalignment,withtheRNApolymerasesequencefromZikavirusontop(theQuery)andtheHepatitisCvirusRNApolymeraseisshownonthebottom.Whenthetwosequencesmatch,theaminoacidisshowninthemiddle.Iftheaminoacidsaresimilar,butnotidentical,a+signisshown.Thishelpsustovisualizethesimilaritybetweentwoproteinsequences.WecopythealignmentfromtheBLASTresultsandpasteitintoaWordfiletomakeiteasytoannotate.Thisoftenrequiresabitofformattingworkaswell.IalsofindithelpfultotouchtheMoleculeiconandselecttheoptiontocolortheaminoacidsbyresidue.Then,Iscrollthroughthesequencetofindhighlightedaminoacids.WhenIfindthem,Ihighlightthecorrespondingaminoacidsinthealignedsequences.
11
InFigure11,Icanseethatarginine158mayformasaltbridgewithoxygensinthenearbyphosphategroup.Thisaminoacidisinbothproteins(Fig.12).Asparticacid225islikelytoformahydrogenbondtothedrugandAsparticacid220formsametalbondtooneofthemanganeseatoms.
Figure12.AlignedsequencesfromtheZikavirusRNApolymerase(Query)andtheHepatitisCvirusRNApolymerase.AminoacidsintheSofosbuvirbindingsitethatarefoundinbothproteinsarehighlighted.Basedonthenumberofsharedaminoacidsinthebindingsiteandthetypesofinteractionsbetweensharedaminoacids,andthedrug,IpredictedthatsofosbuvirwouldinhibittheZikavirusRNApolymerase.Thispredictionwasrecentlyconfirmedbyinvivoexperimentsincellcultureandinmice(2,3).ConclusionThemethoddescribedinthisarticlehasbeensuccessfullyusedwithstudentsinthreesemesters.Studentshaveenjoyeditandcommentedthattheylikedoingthingsthatrelatetoreallife.Thistechniqueissuitableforstudentprojectsforanumberofreasons.Studentscancanapplythistechniquetomultiplekindsofdrugtargetsandcarryoutthestepsinvolvedusingpubliclyavailabledataandlow-cost,user-friendlysoftware.Inaddition,thisgivesstudentsanopportunitytolearnandpracticeskillsthatareusedinthebiotechindustry.Furthermore,thissamemethodwasusedinthetwopapersdiscussedearlierandpredictionsmadethroughthismethodwereshowntobecorrectwhenexperimentswereperformedwithcellculturesandmice.References:1.MoleculeWorld™isauser-friendlyiPadappforworkingwithmolecularstructuresandisavailableintheiTunesappstore:https://itunes.apple.com/us/app/molecule-world/id863565223?mt=8Formoreinformation,seeDigitalWorldBiology.com2.Sacramento,C.et.al.TheclinicallyapprovedantiviraldrugsofosbuvirinhibitsZikavirusreplication.SciRep.2017Jan18;7:40920.3.Bullard-Feibelman,K.M.,Govero,J.,Zhu,Z.,Salazar,V.,Veselinovic,M.,Diamond,M.S.,Geiss,B.J.,TheFDA-approveddrugsofosbuvirinhibitsZikavirusinfection,AntiviralResearch(2016),doi:10.1016/j.antiviral.2016.11.023
![Reviewed outbreaks of Zika virus in Thailand · Nigeria_AEN75265.1|:3063-3307 polyprotein, partial [Zika virus] Malaysia_AEN75264.1|:3069-3313 polyprotein, partial [Zika virus] Cambodia_AFD30972.1|:3069-3313](https://static.fdocuments.in/doc/165x107/605f37d6ae2e93483277f4e8/reviewed-outbreaks-of-zika-virus-in-thailand-nigeriaaen7526513063-3307-polyprotein.jpg)
