STUDY OF AN ACTIVE-STATE CB1 RECEPTOR MODEL AND JWH … · 2018-02-10 · recreational use...

STUDYOFANACTIVE-STATECB1RECEPTORMODELANDJWH

COMPOUNDINTERACTIONSTOPREDICTNEWEMERGINGSYNTHETICCANNABINOIDS

by

KelseyLeighPettus

AthesissubmittedtothefacultyoftheUniversityofMississippiinpartialfulfillmentoftherequirementsoftheSallyMcDonnellBarksdaleHonorsCollege

OxfordMay2016

ApprovedBy

________________________________________________

Advisor:Dr.MurrellGodfrey

________________________________________________

Advisor:Dr.RobertJ.Doerksen

________________________________________________

Reader:Dr.ChristopherR.McCurdy

ii

©2016KelseyLeighPettus

ALLRIGHTSRESERVED

iii

ACKNOWLEDGEMENTS

IwouldliketothankDr.MurrellGodfreyforhisguidanceinadvisingthisproject.ThankstoDr.RobertDoerksenforservingasasecondreaderandsecondadvisorforthisproject,andthankstoDr.DoerksenandDr.KuldeepRoyandtheirgroupforaccesstotheirMaestrolicenseandactive-stateCB1model.IwouldalsoliketothankDr.KuldeepRoyandDr.PankajPandeyfortheirtutorialstoMaestroandtheirinvaluableassistanceinthelab.IwouldalsoliketothankDr.DouglassSullivan-González,Dr.JohnSamonds,andDr.DebraYoungfortheiradvisingandsupportduringmytenureintheSallyMcDonnellBarksdaleHonorsCollege.ThisworkwasmadepossibleinpartbyGrantNumbersP20GM104932andR15GM119061fromtheNationalInstituteofGeneralMedicalSciences(NIGMS),acomponentoftheNationalInstitutesofHealth(NIH).ItscontentsaresolelytheresponsibilityoftheauthorsanddonotnecessarilyrepresenttheofficialviewofNIGMSorNIH.ThisinvestigationwasconductedinpartinafacilityconstructedwithsupportfromtheResearchFacilitiesImprovementsProgram(C06RR14503)fromtheNationalInstitutesofHealth(NIH)NationalCenterforResearchResources.Also,thankstotheDepartmentofBioMolecularSciencesforcomputationalresources

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ABSTRACT

KELSEYPETTUS:StudyofAnActive-StateCB1ReceptorModelandJWHCompoundInteractionstoPredictNewEmergingSyntheticCannabinoids

(UnderthedirectionofDr.MurrellGodfreyandDr.RobertJ.Doerksen)

Inrecentyears,anewclassofcompounds,calledsyntheticcannabinoids,

havemadetheirappearanceinthemarketasasubstituteforillegalmarijuanaorits

constituentnaturalcannabinoids.Thesecompounds,whichareactiveatthesameG-

proteincoupledreceptors(GPCRS)ascannabinoids,arecontinuingtogain

popularitybecauseofthesamecannabinoid-likeeffectstheycreate.Thoughtwo

cannabinoidreceptorshavebeenidentifiedinhumans,knownasCB1andCB2,

syntheticcannabinoidactivityattheCB1receptorhasbeenthefocusofmuch

researchregardingsyntheticcannabinoidligandbinding.Inthisstudy,thestructure

oftheCB1receptorisfurtheranalyzedinthebindingofaclassofsynthetic

cannabinoidligands,knownasJWHcompounds,totheCB1receptor.Anactive-state

CB1modelthatwaspreviouslycreatedbyDoerksenR.etal.andmodeledfrom

BovineRhodopsinandotherGPCRsisusedinthisstudy.Adatasetoftwenty-one

activeCB1agonistsJWHcompoundsfromthenaphthoylindolefamilyweredocked

tothemodelinordertofurtheranalyzekeyinteractingresiduesontheCB1

receptor.Ofthesetwenty-onecompounds,alltwenty-onewereabletobindtothe

v

CB1active-statemodel.TheGlidedockingscoreofeachligandgeneratedfrom

Maestrocomputationalmodelingsoftwarewascollectedandcomparedtothatof

Delta-9-tetrahydrocannabinol,themainpsychoactivecomponentofmarijuana.

vi

TABLEOFCONTENTS

LISTOFFIGURESANDTABLES...............................................................................vii

INTRODUCTION................................................................................................................1

EXPERIMENTALMATERIALSANDMETHODS...................................................7

RESULTSANDDISCUSSION......................................................................................14

CONCLUSION...................................................................................................................35

REFERENCES...................................................................................................................36

vii

LISTOFFIGURESANDTABLES

Figure1:StructureofΔ9-Tetrahydrocannabinol(THC)............................................................3

Figure2:ThemodeloftheCB1receptorusedinthisstudywithboundTHCligand showningreen........................................................................................................................5

Figure3:Structuresofthetwenty-oneJWHligandsandTHCusedinthisstudy...7-11

Figure4:JWH-149andCB12Dand3DLigandInteractionDiagrams............................23


Figure6:JWH-073andCB12Dand3DLigandInteractionDiagrams.H-bondis shownasyellow-coloreddashes....................................................................................24



Figure9:JWH-015andCB12Dand3DLigandInteractionDiagrams.H-bondis shownasyellow-coloreddashes.....................................................26

Figure10:JWH-122andCB12Dand3DLigandInteractionDiagrams.........................26





viii


Figure16:JWH-424andCB12Dand3DLigandInteractionDiagrams........................29

Figure17:JWH-019andCB12Dand3DLigandInteractionDiagrams.H-bondis shownasyellow-coloreddashes.............................................................................30



Figure20:JWH-018andCB12Dand3DLigandInteractionDiagrams.H-bondis shownasyellow-coloreddashes..............................................................................31


Figure22:JWH-047andCB12Dand3DLigandInteractionDiagrams.H-bondis shownasyellow-coloreddashes...............................................................................32



Figure25:THCandCB12Dand3DLigandInteractionDiagrams.....................................34

Table1:DockingScoreandGlideEmodelscoreofeachJWHligandandTHC.............15

Table2:DistancesofHydrogenBondsFormedBetweenLigandsandInteractingCB1 Residues.....................................................................................................................................17Table3:JWHLigandsandTHCandTheirInteractingCB1Residues................................19

Table4:TheInteractionsBetweentheindoleornaphthaleneSubstructuresofeachLigand andCB1residue....................................................................................................................................21

1

INTRODUCTION

IntheUnitedStates,marijuanaisrankedasthesecondmostpervasive

recreationaldrug.ThefirstmostpervasiverecreationaldrugintheUnitedStatesis

alcohol.Thoughthepossession,use,anddistributionofmarijuanaarestillunder

federalcontrol,thelegalstatusandusagepatternsofthisdrugarechangingrapidly.

Currently,twenty-threestatesandtheDistrictofColumbiahavepassedlawsthat

permitandregulatetheusageofmarijuanaformedicinalpurposes,andfourstates

plustheDistrictofColumbiahavepassedlawsthatallowformarijuanatobeused

forrecreationalandmedicalpurposes.1

Becausethepossession,distribution,anduseofmarijuanacontinuetobe

consideredoffensesinfederaljurisdictions,drugdistributershavebeenmotivated

tocreatediverseclassesofsyntheticanalogsasalegalalternativetotraditional

marijuana.Thesevariouscompoundsaremarketedtothepublicforrecreational

usageandhavebeenfoundtopossessthesamecannabinoid-likeeffectsas

marijuanasuchaseuphoria,relaxation,increaseinsensoryawareness,andincrease

increativethinking.1Until2012,manufacturerssoldthesesyntheticcannabinoid

2

mixtures,alsocalled"newpsychoactivesubstances",ingasstations,throughthe

Internet,anddrugparaphernaliastores.Theyweresoldintheformofshredded

plantmaterialcoatedincannabinoidsprayliquidtobesmokedasherbalincenseor

liquidstobevaporizedasliquidincenseandmarked"notforhumanconsumption".

8,9Inrecentyears,thesecompoundshavegainedmuchattentionfromtheforensic

communitybecauseoftheiradverseconsequencesonhumanhealth.1Manyofthese

syntheticcompoundshavebeenfoundtobe100timesmorepotentthanthemain

psychoactivecomponentfoundinmarijuana,Δ9-Tetrahydrocannabinol(THC),and

cancauselife-threateningproblemssuchashighbloodpressure,vomiting,and

seizures.2

Thespecificclassesofcompoundsthathavegainedmuchattentionfrom

researchersincludethosecompoundsofthefollowingseries:HU,CP,JWH,AM,RCS,

WIN,andinrecentyears,URandXLR2.Thesecompoundsmimictheeffectscaused

byTHC,whichwasdiscoveredin1964byresearchersYechielGaoniandRaphael

Mechoulam.Becausethecomponentsofcannabishadbeenstudied,butnostructure

ofthemajorpsychoactivecomponenthadbeendetermined,thediscoveryofthe

structureofTHCwasamomentousbreakthroughforthescientificcommunity.2The

classificationofthestructureofTHCledtonewinsightsintothestudyof

cannabinoids.ThestructureofTHCisshowninFigure1.

3

Figure1:StructureofΔ9-Tetrahydrocannabinol(THC)

BecausesyntheticcannabinoidsarefunctionallysimilartoTHC,theybindto

thesamecannabinoidreceptorsintheperipheralorgansandthebrain.3

ResearchershaveidentifiedtwoknowntypesofcannabinoidreceptorsthatareG-

coupledproteinreceptors(GPCRs).ThesereceptorsareknownasCB1,located

primarilyinthehumanbrain,andCB2,locatedprimarilyintheimmunesystemand

peripheralorgans.3Theexistenceofcannabinoidreceptorswasfirstdiscoveredin

ratbrainin1988inwhichtheexperimentaldatadescribedaG-proteincoupled

receptorintheratbrainthatboundnaturalcannabinoids.2Shortlyafterthe

discoveryofthecannabinoidreceptorsinratbrain,researcherswereabletomap

thedistributionofcannabinoidreceptorsinhumanperipheralorgans(CB2)and

brain(CB1).1ThoughthebindingaffinitiesofcompoundsforbothCB1andCB2have

beenstudiedinthepast,ariseinstudiesonthebindingaffinitiesofcompounds

4

withtheCB1receptorhasincreasedwiththerisingpopularityofsynthetic

cannabinoids.1ResearchshowsthatitistheCB1receptorthatisresponsibleforthe

psychotropiceffectscausedbycannabis.Becauseofthis,aligand'sabilitytobindto

theCB1receptorandactasanagonistcouldindicateitspotentialtobea

recreationalusesubstituteformarijuana.1

Thoughtheprecisethree-dimensionalstructureoftheCB1receptoris

unknown,itisknownthattheCB1andCB2receptorsareG-proteincoupled

receptors(GPCRs),andtheyareembeddedwithinthecellmembrane.AsaGPCR,

theCB1receptorcontainsseven-helicaltransmembranedomainsconnectedby

threeextracellularandthreeintracellularloops.7,11Ligandsarethemoleculesthat

bindtotheGPCRs,controltheiractivity,andmodifytheirbiologicalfunctions.When

anagonistligandbindstotheCB1receptor,aconformationalchangecausesthe

receptortointeractwithG-proteinscontainedwithinthecell.10Becauseofthese

conformationalmovementsandcorrespondingG-proteininteractions,knowledge

aboutnovelligandbindingtotheCB1receptorcouldbebeneficial.6Usingthe

BovineRhodopsinX-raycrystalstructureasatemplate,severalCB1model

structureshavebeenreported.Thesemodelstructureshavefurtherbeenusedto

discovernovelCB1ligands,butnoneofthesemodelsprovidefortheprecisethree-

dimensionalstructureoftheCB1receptorandcannabinoidligandbinding.4,Studies

haveproposedthatthereisahydrophobicbindingpocketthatinteractswiththeC3

alkylchainofcannabinoids.SincethepreciseexperimentalstructureoftheCB1

receptorhasyettobereported,thechosenbestactive-stateCB1receptormodel

5

utilizedinthisstudywasamodelproposedbyDoerksenetal.5Theactive-stateCB1

modelusedinthisstudyinshowninFigure2:

Figure2:ThemodeloftheCB1receptorusedinthisstudywithboundTHCligandshowningreen.

In1984whileresearchinganddevelopingcannabinoidcompoundstoaidin

multiplesclerosisandchemotherapyresearch,JohnW.Huffmanandateamof

researchersdevelopedagroupofsyntheticcannabinoidligandsthataretoday

arguablythemostpopularsyntheticcannabinoids.Thesecompounds,calledthe

JWHseriescompounds,wereevolvedfromthecomputationalcombinationof

6

aminoalkylindolesandfeaturesofTHC.TheJWHcompoundsusedinthisstudy

comefromthenaphthoylindoleclassofsyntheticcannabinoids,namedforthe

naphthalenegrouptheycontain.Naphthalene'sstructureismadeoftwo-fused

benzenerings.12

Inthisstudy,thereceptor-ligandinteractionsofanactive-statemodelofthe

humanCB1receptor,proposedbyDoerksenR.etal.,andtwenty-oneJWH

cannabinoidligandsfromthenaphthoylindolefamilyandtheTHCmoleculeare

compared.5TheMaestromolecularmodelingcomputerprogramwasusedtostudy

theusefulnessoftheCB1modelandtoinvestigatetheimportantinteractions

betweeneachligandandCB1residues.13Theinformationfoundinthisstudycanbe

usefulindiscoveringamoreaccurateCB1modelandinpredictingthepropertiesof

uncharacterizedillicitsyntheticcannabinoidsbeforetheybecomeproductsthatare

illegallysoldtothepublic.

7

EXPERIMENTALMATERIALSANDMETHODS

LigandsSelection.

Adatasetoftwenty-oneJWHligandsfromthenaphthoylindolefamilyplustheTHC

moleculewerechosen.AlloftheseligandshaveanaffinityfortheCB1receptor.

Themolecularstructuresofthetwenty-oneligandsarelistedbelowinFigure3in

orderofhighesttolowestdockingscore.THCisshownattheend.

JWH-149 JWH-184

NN

O

8

JWH-073 JWH-164

JWH-007 JWH-015

JWH-122 JWH-120

O

N

N

O

O

N

O

N

O

N

O

N

O

9

JWH-098 JWH-081

JWH-196 JWH-148

JWH-424 JWH-019

N

O

ON

O

O

N

N

O

N

O

Br

O

N

10

JWH-210 JWH-116

JWH-018 JWH-185

JWH-047 JWH-398

N

O

N

O

O

N

O

N

N

O

N

O

Cl

11

JWH-048 THC

Figure3:Structuresofthetwenty-oneJWHligandsandTHCusedinthisstudy

LigandsPreparation

TheligandsselectedforthisstudywerepreparedusingMaestromodelingsoftware

createdbySchrödinger,LLC.13Thetwenty-oneligandswereenteredintoMaestro

usingthe2Dsketcherandthenconvertedto3Dstructuresfordocking.Usingthe

preparationwizard,theligandsweredesaltedandtautomersweregeneratedusing

OPLS3forcefield.AtargetPHof7.0+/-2.0wassetandonelowenergyring

conformationperligandwasgenerated.

N

O

12

ProteinSelectionandPreparation

ThemodelselectedforthisstudywasaCB1modelcreatedandpreparedby

DoerksenR.etal.Thismodelwaschosenasthebestmodelforexperimentation

becauseitshowedthebestoverallperformanceamongthecreatedmodels.5

DoerksenRetal.previouslypreparedtheproteinusedinthisstudy.Theprotein

preparationwizardinMaestrowasusedtocorrectanyproblemsrelatedtosteric

hindrances,distance,andhydrogenatoms.Afternoproblemsweredetectedpost

review,theorientationoftheproteinwasoptimizedusingOPLS3forcefield.

GridGeneration

TwogridsweregeneratedtotelltheMaestroprogramwheretolookontheCB1

proteinwhendocking.ThegridsweregeneratedusinginformationabouttheCB1

receptor-ligandbindingfoundinpreviousstudies.Thefirstgridwasgeneratedwith

ahydrogenbondconstraintonresidueLys192.ResiduesSer173,Tyr275,Cys355,

Ser383werechosenasrotatablegroups.Previousstudieshaveshownthatthese

fourresiduesselectedasrotatablegroupsareimportantinteractingresiduesforthe

CB1receptorinitsactivestate.Asecondgridwasalsogeneratedwithnohydrogen

bondconstraintsandnoresiduesselectedasrotatablegroups.

13

GlideDocking

Thestandardprecision(SP)moduleinGlidewasusedtodockthetwenty-one

ligandscreated.TheDoerksenR.grouphadalreadypreparedandvalidatedtheCB1-

THCcomplex,andIutilizedthiscomplexforgeneratingthegridsconsideringTHC

asanactiveligandsite.Post-dockingminimizationwasperformed,andthetoppose

ofeachligandwasselectedforstudy.Oncealloftheligandshadbeendocked,the

dockingscoreofeachligandwasreviewedinordertodeterminewhichligandshad

thebetteraffinityfortheCB1receptor.Thebindingsitewasalsostudiedtosee

whichresiduesoftheCB1proteinreceptorandwhatpartsoftheligand,theindole

ornaphthalenesubstructures,wereinteracting.Theshortestdistance(in

Angstroms)betweeneithertheindolesubstructureorthenaphthalenesubstructure

ofeachligandandtheinteractingresidueswasmeasured.

14

RESULTSANDDISCUSSION

Oncetheligandandproteinpreparationandgridgenerationhadbeen

completed,thetwenty-oneJWHligandsandtheTHCmoleculeweredockedtothe

CB1model.Thedockingresultsshowedthatalltwenty-oneligandsandtheTHC

moleculeweresuccessfullydockedintotheCB1model.Themostfavorableposefor

eachligandandtheTHCmoleculewerechosenandanalyzed.Thedockingscores

variedfrom-7.198to-9.334.ThedockingscoresoftheJWHligandsandtheTHC

moleculearelistedinTable1.

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LigandsDockingScore

GlideEmodel Ki

JWH-149 -9.334 -64.9995.0nM±2.1

nMJWH-184 -9.268 -67.039 23nMJWH-164 -9.178 -68.191 6.6nM±0.7JWH-007 -9.088 -68.246 9.5nM±4.5JWH-015 -9.044 -60.755 383nMJWH-122 -8.895 -64.202 0.69nM±0.5JWH-120 -8.872 -61.182 1054nMJWH-098 -8.845 -40.368 4.5nM±0.1JWH-081 -8.84 -63.183 1.2nM±0.03JWH-196 -8.784 -62.894 151nM±18JWH-148 -8.756 -64.212 123nMJWH-424 -8.725 -56.65 20.9nMJWH-073 -8.706 -61.424 8.9nMTHC

Molecule -8.686 -63.34 10nMJWH-019 -8.595 -42.71 9.8nM±2JWH-210 -8.536 -54.984 .46nMJWH-116 -8.5 -54.951 52nM±5.0JWH-018 -8.424 -58.632 9nM±5.0JWH-185 -8.074 -68.61 17nMJWH-047 -7.559 -56.017 59nM±3.0JWH-398 -7.452 -61.263 2.3nMJWH-048 -7.198 -36.605 10.7nM±1.0

Table1:DockingScoreandGlideEmodelscoreofeachJWHligandandTHC

Figure4andFigure24showthetwoligandsandtheirinteractionswiththe

CB1modelthathadthebestdockingscoreandthelowestdockingscore,

respectively.JWH-149wasshowntohavethemostfavorabledockingscoreof-

9.334,andJWH-048wasshowntohavetheleastfavorabledockingscoreof-7.198.

Figure4andtable3showthatJWH-149hasΠ-Πstackinginteractionswith

residuesTryptophan279(Trp279),Tryptophan356(Trp356),Phenylalanine170

(Phe170),andPhenylalanine200(Phe200).Mostligandsthatresultedinhigh

16

dockingscoresshowedprimarilythefollowinginteractionswiththeCB1protein:

indolesubstituentinteractionswithTrp279andtoalesserdegreewithPhe170

andTrp356;naphthaleneinteractionswithPhe170,Trp356,andPhe200.

PreviousstudieshaveshownthatresiduesSerine383(Ser383)andLys192

arekeyresiduesinforminghydrogenbondswithcannabinoidligandspossessing

OHandCOOHgroups,respectively.10Theresultsofthisstudyshowedthatonlytwo

dockedligandshadaninteractionwithLys192.Ahydrogenbondwasformed

betweenLys192andtheketo-oxygenportionofligandJWH-073,andLys192hada

pi-cationinteractionwiththenaphthaleneportionofligandJWH-048.Interestingly,

JWH-073hadadockingscoreof-8.706,andJWH-048asstatedpreviouslyhadthe

lowestdockingscoreof-7.198.Ser383alsoformedahydrogenbondwiththeketo-

oxygenofligandsJWH-098andJWH-424.Thedockingscoresofthesetwoligands

were-8.845and-8.725,respectively.AnalysisoftheinteractionsbetweentheCB1

modelandtheligandsalsoshowedthatanotherresidueformedahydrogenbondin

thesamewaywiththeketo-oxygenoftheligands.SimilartoLys192andSer383,

Trp279formedahydrogenbondwiththeketo-oxygenofligandsJWH-015,JWH-

019,JWH-018,andJWH-047.Thedockingscoresofeachoftheseligandswere-

9.044,-8.595,-8.424,and-7.559,respectively.

Thestrengthofeachhydrogenbondformedandcation-piinteractionwas

analyzedthroughmeasuringthedistanceinAngstroms.Thehydrogenbond

distancebetweenLys192andJWH-073wasmeasuredtobe2.40Åandthecation-pi

17

interactiondistancebetweenLys192andJWH-048wasmeasuredtobe3.77Å.The

hydrogenbonddistancesbetweenTrp279andJWH-015,JWH-019,JWH-018,and

JWH-047weremeasuredtobe2.17Å,1.90Å,2.43Å,and2.23Å,respectively.The

hydrogenbonddistancebetweenSer383andJWH-098was1.98Å,andthe

hydrogenbonddistancebetweenSer383andJWH-424was2.04Å.Theshorter

distanceindicatesthestrongerinteraction.Thedistanceandstrengthofeach

hydrogenbondcanbeseenintable2.

Ligands Lys192 Trp279 Ser383JWH-073 2.4Å JWH-015 2.17Å JWH-019 1.9Å JWH-018 2.43Å JWH-047 2.23Å JWH-098 1.98ÅJWH-424 2.04Å

Table2:DistancesofHydrogenBondsFormedBetweenLigandsandInteractingCB1Residues

AnalysisshowsthatthehydrogenbonddistancesbetweenbothTrp279and

Ser383andtheirrespectiveinteractingligandswereshorterandthereforestronger

thanthehydrogenbonddistancebetweenLys192anditsrespectiveinteracting

ligand.ThisindicatesthatLys192doesnotinteractassignificantlywiththeJWH

ligandsasotherresiduessuchasTrp279andSer383.Moreresearchonthese

residuescouldindicatetheirsignificanceinforminghydrogenbondswithnewand

undiscoveredcannabinoidligands.

18

PreviousstudieshaveindicatedthatresiduesPhe170,Phe200,Phe208,Tyr

215,Phe289,Tyr292,Tyr296,Trp356,andPhe379areessentialCB1residuesfor

aromaticstacking.PreviousstudieshavealsoshownthatresiduesPhe170andTrp

279arepresentinthedeepbindingpocketoftheCB1receptor.Thisbindingpocket

isessentialforeffectiveligandbinding.10TheresultsofthisstudyshowthatTrp

279,Trp356,Phe200,andPhe170arekeyresiduesinJWHandCB1binding.Trp

279interactedwitheighteenofthetwenty-oneligandsandtheTHCmolecule.This

isthemostinteractionsofalloftheresidues.Trp279wasalsochosenasthecentral

pointfordocking,soitmakessensethatithadthemostinteractions.Trp356

interactedwithfifteenoftheligands,andPhe200interactedwithtenoftheligands,

andPhe170interactedwithelevenligands.InteractionswithresiduesPhe177and

Phe174alsooccurred,butonlywiththelowestscoringligand,JWH-048.THCand

theJWHligandsusedinthisstudyarelistedinTable3alongwiththeCB1residues

withwhichtheyinteract.

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CB1ReceptorResidues

Ligands Trp279 Trp356 Phe170 Phe200 Phe177 Phe174 Lys192 Ser383THC

Molecule ✓ JWH-149 ✓ ✓ ✓ ✓ JWH-184 ✓ ✓ ✓ JWH-073 ✓ ✓ JWH-164 ✓ ✓ ✓ JWH-007 ✓ ✓ ✓ JWH-015 ✓ ✓ ✓ JWH-122 ✓ ✓ ✓ JWH-120 ✓ ✓ ✓ JWH-098 ✓ ✓ ✓ ✓

JWH-081 ✓ ✓ ✓ ✓ JWH-196 ✓ ✓ ✓ JWH-148 ✓ JWH-424 ✓ ✓ ✓ ✓ ✓

JWH-019 ✓ JWH-210 ✓ ✓ JWH-116 ✓ ✓ ✓ JWH-018 ✓ ✓ ✓ JWH-185 ✓ ✓ ✓ JWH-047 ✓ ✓ ✓ JWH-398 JWH-048 ✓ ✓ ✓ ✓

Table3:JWHLigandsandTHCandTheirInteractingCB1Residues

20

TheinteractingresiduesshowedΠ-Πorcation-pi,onlyLys192andJWH-

048,interactionswitheithertheindolesubstructureoftheligandorthe

naphthalenesubstructureoftheligand.Theshortestcarbon-carboninteraction

distancebetweentheCB1residuesandtheligandswasmeasured.Thesedistances

canbeseeninTable4.TheTHCmoleculeonlyshowedaninteraction

withTrp279.ThebenzeneringcontaininganOHsubstituentgroupexhibitedpi-pi

stackingwithadistanceof3.92ÅwithTrp279.

21

Ligands Trp279

and

indole

Phe170

and

indole

Trp356

and

indole

Phe170and

nap.

Trp356and

nap.

Phe200

andnap.

Trp279

andnap.

Phe174

andnap.

Phe177and

nap.

JWH-149 3.53Å 3.32Å 3.51Å 4.09Å

JWH-184 3.62Å 3.59Å 3.57Å

JWH-073 3.30Å

JWH-164 3.59Å 3.84Å 3.43Å

JWH-007 3.59Å 3.50Å 3.41Å

JWH-015 3.62Å 3.34Å 3.53Å

JWH-122 3.45Å 3.29Å 3.47Å

JWH-120 3.58Å 3.65Å 3.51Å

JWH-098 3.61Å 3.53Å 3.35Å

JWH-081 3.44Å 3.26Å 3.32Å 3.59Å

JWH-196 3.60Å 3.79Å 3.46Å

JWH-148 3.53Å

JWH-424 3.78Å 3.55Å 3.66Å 3.32Å

JWH-019 3.50Å

JWH-210 3.21Å 3.47Å

JWH-116 3.27Å 3.80Å 3.39Å

JWH-018 3.45Å 3.43Å 3.67Å

JWH-185 3.41Å 3.65Å 3.58Å

JWH-047 3.28Å 3.45Å

JWH-398

JWH-048 3.67Å 4.53Å 3.41Å

Table4:TheInteractionsBetweentheIndoleorNaphthaleneSubstructuresofeachLigandandCB1residue

*Andindoledenotesapipistacking(Π-Π)interactionbetweentheCB1substituentandtheindoleportionoftheligand.**Andnap.denotesapipistacking(Π-Π)interactionbetweentheCB1substituentandthenaphthaleneportionoftheligand

22

Asseenintable4,mostdistanceswereinthe3to4Årangewithonly2

interactiondistancesgreaterthan4Å.Thestrongestinteraction,judgedbythe

shortestdistance,occurredbetweenPhe170andthenaphthalenesubstructureof

JWH-210.ThisresultseemsinconsistentwiththedockingscoressinceJWH-210

producedadockingscoreonthebottomhalfofthedockingscoretablewithascore

of-8.536.ThoughJWH-210producedadockingscoreinthebottomhalfofthe

dockingscorerange,JWH-210possessesthehighestexperimentalbindingaffinity

(lowestKivalue=0.46nM)atCB1ofalltheligandstestedinthisstudy.Thishigh

bindingaffinityforCB1shouldbeinvestigatedfurthertoexplainwhyJWH-210and

Phe170havethestrongestinteraction.Theweakestinteraction,orlongestdistance,

wasseenbetweenPhe174andthenaphthaleneportionofJWH-048.Thisfindingis

consistentwiththeresultingdockingscoresasJWH-048producedthelowest

dockingscore.JWH-048wasalsotheonlyligandtointeractwithPhe174.Because

Phe170formedthestrongestinteractionandinteractedwithelevenligands,further

studiesshouldbedoneinordertodetermineifPhe170isalsoakeyresidueinall

cannabinoidligandbindingwithCB1.

BecauseTHCisthemainpsychoactivecomponentofmarijuana,itcouldbe

assumedthatTHCwouldproducethestrongestinteractionwithCB1,interactwith

themostCB1residues,andhavethehighestdockingscore.Asseenintable1,THC

producedadockingscoreof-8.686withthisactive-stateCB1model.Thisdocking

scoreisinthemiddletobottomhalfrangeoftheresultingJWHliganddocking

scores.ThebestposeoftheTHCmoleculeselectedandanalyzedalsoonly

23

interactedwithTrp279throughΠ-Πstackingatadistanceof3.92Å.Anexplanation

forwhyTHCdoesnotpossessthebestdockingscorecouldbeitsbindingaffinityfor

CB1.ThoughTHCpossessesabindingaffinityof10nMforCB1,elevenofthetwenty-

oneligandsstudiedpossessahigherbindingaffinity,lowerKivalue,forCB1than

THC.ThesehigherbindingaffinitiescausetheligandstobindtoCB1morestrongly

thanTHCandpotentiallycausemoreharmfuleffects.1Theinteractionsbetweenthe

CB1modelandeachofthetwenty-oneligandsandTHCareshownin2Dand3D

interactiondiagramsinfigures4-25.

Figure4:JWH-149andCB12Dand3DLigandInteractionDiagrams

Phe170 Trp279

Phe200

Trp356

24


Figure6:JWH-073andCB12Dand3DLigandInteractionDiagrams.H-bondis

shownasyellow-coloreddashes.

Phe170Trp279

Trp356

Lys192Trp279

25



Trp279

Trp356

Phe200

Trp279

Trp356

Phe200

26

Figure9:JWH-015andCB12Dand3DLigandInteractionDiagrams.H-bondisshownasyellow-coloreddashes


Trp279

Phe170

Trp356

Trp279

Phe170

Trp356

27



Trp279

Trp356

Phe200

Trp279

Trp356

Phe200

28



Trp279

Phe200

Trp356

Phe170

Trp279

Trp356

Phe200

29



Trp279

Trp279

Phe170

Trp356

Phe200

30

Figure17:JWH-019andCB12Dand3DLigandInteractionDiagrams.H-bondisshownasyellow-coloreddashes.


Trp279

Trp356

Phe170

31


Figure20:JWH-018andCB12Dand3DLigandInteractionDiagrams.H-bondis

shownasyellow-coloreddashes.

Trp356

Phe200

Phe170

Trp279

Phe170

Trp356

32


Figure22:JWH-047andCB12Dand3DLigandInteractionDiagrams.H-bondisshownasyellow-coloreddashes.

Trp279

Trp356

Phe170

Trp279

Phe170

Phe200

33

Figure23:JWH-398andCB12Dand3DLigandInteractionDiagrams.


Phe174Trp279

Phe177

Lys192

34

Figure25:THCandCB12Dand3DLigandInteractionDiagrams.

Trp279

35

CONCLUSION

ThisstudyrevealedthatTrp279,Trp356,Phe200,andPhe170arekey

residuesintheinteractionbetweenCB1andJWHligands.ItappearsthatTrp279

couldalsobeakeyresidueinforminghydrogenbondswithothercannabinoid

classes.FurtherresearchoninteractionsbetweenJWHmetabolitesandother

classesofcannabinoidswiththisCB1modelcouldrevealadditionalimportantCB1

residuesinvolvedinthebindingofnewanduncharacterizedcannabinoids.More

studiesshouldbeperformedontheligand-receptorinteractiontoultimatelycreate

anewmassspecdatabaseforthedetectionandpredictionofavarietyofillicit

syntheticcannabinoids.

36

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STUDY OF AN ACTIVE-STATE CB1 RECEPTOR MODEL AND JWH … · 2018-02-10 · recreational use...

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