Bryan
L
Roth
ChemogenetictechnologiessuchasDesignerReceptors ExclusivelyActivatedbyDesignerDrugs(DREADDs)arewidely usedtoremotelycontrolneuronalandnon-neuronalsignaling. DREADDsexistformostofthecanonicalGprotein-coupled receptorsignalingpathways,andprovideasyntheticbiology platformusefulforelucidatingtheroleofneuronalsignalingfor brainfunction.Here,afocusedreviewisprovidedthatshows howrecentinsightsobtainedfromGPCRstructuralstudies informourunderstandingofthesechemogenetictoolsfroma structuralperspective.
Address
DepartmentofPharmacologyandDivisionofMedicinalChemistryand
ChemicalBiology,UniversityofNorthCarolina,ChapelHillSchoolof
Medicine,ChapelHill,NC27514,UnitedStates
Correspondingauthor:Roth,BryanL([email protected])
CurrentOpinioninStructuralBiology2019,57:9–16
ThisreviewcomesfromathemedissueonEngineeringanddesign:
syntheticsignaling
EditedbyAndreasMo¨glichandHaraldJanovjak
ForacompleteoverviewseetheIssue andtheEditorial
Availableonline25thFebruary2019
https://doi.org/10.1016/j.sbi.2019.01.016
Introduction
Gprotein-coupledreceptors(GPCRs)—withmorethan 800 members — represent the largest family of mem-brane proteins in the human genome [1]. GPCRs also represent the single largest family of therapeutic drug targets in the druggable genome [2], with 20–30% of approveddrugshaving GPCRsastheirmajormolecular target [3,4]. GPCRs are expressed in essentially every majororganinthebody,withapproximately80%being expressed in the brain [5]; many GPCRs have their highestlevelofexpressioninnervoustissue[5].GPCRs transduce their signals via binding to and activating hetereotrimericGproteins;Gproteinscoupletovarious downstream effectors to modulate second messengers including cAMP, inositol trisphosphate (IP3), calcium (Ca2+)andothers[3].GPCRsalsocansignalviaarrestin proteins [6] which bind to activated GPCRs following phosphorylation by G protein receptor kinases (GRKs) [7].ArrestinsalsoarrestG-proteinsignalingandinteract with clathrintopromote endocytosis[8].
Withinneurons, GPCRs,viaGproteinactivation, regu-late neuronal excitability [9], vesicle release [10], ion channel activity [11,12], and a variety of intracellular secondmessengers[seeRef.[13]forreview].Howthese alterations in neuronal signaling and firing ultimately result in various brain processes such as perception, cognition,emotion,motivation,andsoonisyetunclear. Someyearsago, FrancisCrickproposedthatinorderto ultimately understandthe neuronalbasisof thesebrain activities[14,15]wewouldneed:
“ ...to identifythemanydifferenttypesof neu-rons in the cerebral cortex and other partsof the brain. One of the next requirements...is to be abletoturnthefiringofoneormoretypesofneuron on and off in the alert animal ...One way-out suggestion is to engineer these neurons so that when one of them fires it would emit a flash of lightof aparticularwavelength...”[15]
The past decade has seen remarkable progress on this frontandessentiallyalloftheitemsinCrick’s‘wishlist’ are in place. First, RNA-seq based technologies have begun cataloging the many different types of neurons [16] and large numbersof engineered mouse lines are availabletoultimatelyprovidegeneticaccessto individ-ual types of neurons [17,18]. Second, optogenetic [19,20] and chemogenetic [21,22] technologies have providedtools‘toturnthefiringofoneormoretypesof neurononandoffinthealertanimal’.Finally,genetically encodedcalcium[23]andvoltage[24]sensorsare avail-able to image neurons that ‘emit a flash of light of a particular wavelength’ [25]and bevisualized with fiber optics [26]andminiaturemicroscopes [27].
signaling cascade for a particular process (Figure 1). Although these pioneering chemogenetic technologies were useful for archetypical proof-of-concept studies [31,32], they were notwidely adopted.To circumvent problemsinherentwiththefirst-generationchemogenetic tools(lackofinertligandsandhighbasalactivityof recep-tors),mylabdevelopedDREADDs(DesignerReceptors ExclusivelyActivatedbyDesignerDrugs)[21],whichare nowroutinelyusedbyneuroscientistsandothersto modu-late neuronal activity and cellular signaling [22]. DREADDsarealsousedtoclarifyhowGPCRsignaling processesareimportantformediatingnormalandabnormal physiology[33,34,35,36].
DREADDs[22]areGPCRsthathavebeenengineeredto: (a)lackappreciableresponsestotheirendogenousligand;(b) haveminimalbasal (constitutive) activityand(c)beactivated by a pharmacologically inert ligand. Many types of DREADDscurrentlyexist,includingthosebasedon mus-carinic[22],k-opioid[37]andtheFFA2fattyacidGPCRs [38]. All of these DREADDs activate the canonical G proteinstowhichtheyarecoupledafterstimulationbytheir otherwise inert cognate ligand. Thus, the M2 and M4-DREADDs(hM2DiandhM4Di,respective)activate Gi-familyproteins,M1-DREADDs,M3-DREADDsand M5-DREADDs (hM1Dq,hM3Dq and hM5Dq respectively)
activateGq/11.Thek-opioidDREADD(KORD)activates Gi-familyreceptors,whiletheFFA2DREADDactivatesGi andGqfamilyGproteins.AparticularlyusefulDREADD hasbeendeveloped,inwhichthehighlyconservedarginine in the conserved ‘DRY’motif essential for receptor activation [3,39,40]ismutated.This particularmutation—R3.50L— yields an M3-(R3.50L)-DREADD that is able to recruit arrestin, but can no longer activate G protein signaling [41].ThisM3-(R3.50L)-DREADDcanbeusedto deter-minetheroleofarrestinsignalingincellularprocesses.
DREADDscanbeexpressedviavirallymediated transduc-tion[42]orcanbegeneticallyencodedinessentiallyanycell type[43,44]andactivatednon-invasivelyviadrug-likesmall molecules[45]torevealthephysiologicalconsequencesof GPCRsignalingindefinedcells.Accordingly,DREADDs provideasyntheticbiological platformforselectivelyand non-invasively modulating GPCR signaling. Here, I will summarizeemergingstructuralinsightsintotheactionsof DREADDsthatpromisetotransformandextendthe use-fulnessofthischemogeneticplatform.
Structural
insights
into
muscarinic
DREADD
actions
Thefirst DREADDswerebasedonhuman muscarinic acetylcholine receptors. These muscarinic DREADDs
Figure1
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Current Opinion in Structural Biology
DREADD-basedchemogenetictoolsmodulatecellularfunction.
ShownisahypotheticalpancreaticbetacellwhereinaGq-coupledDREADDcanbeactivatedbyCNOtoinduceinsulinrelease(orangecircles).
RedstarsshowtheapproximatelocationsoftheY3.33CandA3.56GmutationswhichrendertheM3-muscarinicreceptorinsensitiveto
created by directed molecular evolution in yeast [22,46]tobeactivatedbytheinertclozapine metabo-lite clozapine-N-oxide (CNO). To date, muscarinic DREADDs are the most widely used chemogenetic tools in the neurosciences. Two of these — hM2Di and hM4Di — inhibit neuronal activity [22] via Gb/
g-mediatedactivationofhyperpolarizingGIRKchannels and inhibition of synaptic release of neurotransmitters [47]. The Gq-coupled DREADD (hM3Dq) is most frequently used to enhance neuronal activity [48], via Gq-mediateddepolarizationandmodulationofion chan-nel activity [44]. When combined with Cre-responsive viruses and genetically encoded Cre-recombinase expressedindiscreteneurons,DREADDs canbeused toremotelycontrolneuronalactivityindefinedneurons and,consequently,modulateanumberofbehaviorsand physiologicalresponses [49,44].
Althoughwidelyused,theseDREADDsarenotwithout liabilities.Thus,forinstance,CNOmaybemetabolized toclozapineatlowlevelsinmice[50]andrats[51]andtoa greaterextentinguineapigsandhumans[52].As cloza-pineisanantipsychoticdrugwithhighaffinityforalarge number of neurotransmitter receptors [53], such a con-versionmaybeproblematicandvariouscontrolsarenow recommended when using CNO [see Ref. [44] for review].Alternatively,CNOanalogswithimproved bio-availabilityandwithoutmetabolicliabilitiessuchasC21 and perlapinecanalsobeused[45,54].Going forward, novel DREADD chemogeneticactuators having differ-ent chemotypes with enhanced bioavailabilty will be useful.
the numbering system developed by Ballesteros and Weinstein [55]. Thus, for the muscarinic DREADDs the conserved amino acids of interest are Y3.33 in TM3andA5.46inTM5(numberedaccordingto Balles-teros and Weinstein [55]). When these are mutated to Y3.33C and A5.46G they yield muscarinic receptors insensitive to the endogenous ligand acetylcholine and potently activatedbyCNO[22](Figure 2aand e).As these particular mutations were arrived at via several cycles of directed evolution and employed unusual [non-conservative]substitutions(e.g.Y!CandA!G) itwas notimmediatelyclearwhytheseparticular muta-tionswereessentialforthedesiredchemicaland biologi-calphenotype.
Several years after the invention of the muscarinic DREADDs,bothinactive[56]andactive-statestructures of the M2 muscarinic receptor were published [57]. Figure 2 also shows an active state structure of the wild-type M2 muscarinic receptor (2D [57]), a model of anactive state hM2Di-DREADD(Figure 2b) and a summaryofkeytransitions(Figure2c).Althoughitisnot entirelyevidentfromthestructurewhythesemutations transformCNOfromaweak antagonistintoanagonist, ourrecentstudiesprovideaclue.Thus,wefoundthata similarA5.46Gmutationofthe5-HT2Bserotonin recep-tortransformstheantagonistmethysergideintoapotent agonist [58]. This is accomplished by providing bulk-tolerancefortheN-methylgroupofmethysergide,which allows for the inward movement of TM5 required for stabilizingtheactive state.Presumably,asimilar transi-tion in the hM2Di-DREADD (Figure 2b–d) is key for
Figure2
hM2Di hM2Di 4MQS hM3Dq
MODEL
2 Å hM2Di
INACTIVE ACTIVE
(a) (b) (c) (d) (e)
Current Opinion in Structural Biology
StructuralinsightsintoDREADDactions.
ShownaresnakeplotsoftheM2(a)andM3(e)[72]muscarinicreceptorswiththelocationsoftheY3.33CandA3.56Gmutationshighlightedin
red.Panel(d)showstheactivestatestructureoftheM2muscarinicreceptorwhile(b)and(c)showamodeloftheM2-DREADDandthe
accommodating bulky constituents of CNO, although direct structural studies will ultimately be essential to determiningthisfor certain.
Structure-inspired
design
of
a
k
-opioid
DREADD
The first DREADDs were developed using an unbi-ased, directed evolution approach and, although suc-cessful for many applications, they suffered from the inability to ‘multiplex’. To be able to use synthetic biological approaches to separately activate distinct GPCR signaling cascades sequentially or simulta-neouslyinthe sameneuronswouldsolve the problem ofmultiplexing.Withtheexplosioninstructural infor-mation related to GPCRs [59] we set out to use this information to design a new DREADD. We choseas ourtemplate the k-opioidreceptor(KOR) forwhicha high resolution structure was available [60] and for whichNMR-basedstructuralinformationwasavailable forthe activestate[61].Wehadpreviouslydiscovered that the non-nitrogenous natural product salvinorin A wasapotentandselectiveKORagonist[62]andthatit did not rely on the conserved Asp3.32 found in the bindingpocket ofmanybiogenicaminereceptors [63] forbindingoractivation.Indeed,SalvinorinAbinding and functional potency were enhanced by a D3.38N mutation while binding of the native peptide ligand dynorphin A(1–17) wasabolished[63].
Accordingly,wecreatedaD3.38Nmutantandfoundthat notonly wasSalvinorin A’spotencyenhanced,but that therelativelyinertmetabolite SalvinorinB(SALB)was transformed into a potent agonist [37]. In a series of studies,wewereabletoshowthatSALBwasinertatthe tested doses in vivo but was able to afford potent and
efficacious neuronal silencing in vivo in genetically defined neurons [37]. We dubbed this the k-opioid receptor DREADD(KORD) and note thatit has been usedwidely in theneurosciences to silence genetically identified neurons [64–66].Recentstructural studies of theactiveKORhaverevealedapotentialmechanismfor SALB’spotencyatKOR[40].
Inaccordancewithanewactive statestructureof KOR [40], we found that the epoxymorphinan-derivative MP1104 bound to KOR at some distance (2.6A˚ ) from the highly conserved D3.38 (Figure 3b). The D3.38A mutationofthisresiduehadamodesteffectonbindingof MP1104, although it abolishes binding of all known endogenous KOR ligands [37]. The D3.38N mutation ispredictedtoresultinamovementof3A˚ outwardfrom the binding pocket due to repulsive forces with the positivelychargedgroupsofmorphinansandopioid pep-tides. Such a movement ispredicted, based on simula-tions, to enhance SALB binding by desolvation [37] thereby providing a molecular mechanism for SALB’s remarkable efficacy at KORD. The D3.38N mutation alsoprovides repulsive energyto hinder thebinding of endogenousopioidpeptides,whichareknowntointeract with this residue in opioid receptors for high affinity binding[61,67]in theactive state.
Future
potential
developments
DREADDsandotherchemogenetictools[68]alongwith optogenetictechnologies[69]havetransformed neurosci-enceandotherdisciplines.Thecurrently available che-mogenetic and optogenetic technologies provide many orthogonal approaches to manipulate cellular signaling andneuronalfiring.Withthecurrentrevolutionoccurring in membrane protein structural determination via
Figure3
D3.32
D3.32N
(a) (b) (c)
6B73
Current Opinion in Structural Biology
Structuralfeaturesofthek-opioidDREADD.
Shownin(a)istheinactivestatestructureofthek-opioidreceptorindicatingthelocationofD3.32showninsphererepresentation.(b)showsa
close-upofthebindingpocketoftheactivestatek-opioidreceptorhighlightingthechangeinrotomerofD3.32frominactive(purple)toactive
(green).(c)showsamodelofthek-opioidDREADDwhereintheD3.32Nmutatedresidueispushedoutofthebindingpocket,freeingupenergyof
crystallography and cryo-electronmicroscopy, the fields are poised to deliveranewgeneration of toolsinspired anddesignedwithhighresolutionstructuralinsights(see Refs.[37,70]forexamples).Combininghighresolution structures with automated docking, for instance, could provide novel chemogenetic actuators for engineered receptors, as has been done for native GPCRs [71] (Figure 4).
Inaddition todevelopingnovelchemogeneticactuators andnewDREADDs,itcontinuestobeusefultoengineer GPCRstoactivatedesignedsignalingnetworks.Thus,for instance, DREADDsareavailablefor Gi[22,37],Gq
[22],Gs[33]andarrestin[41]signaling,althoughnone are available that are selective for G12/13, gustducin, transducin or Golf. As well, although the Gi and Gs-selective DREADDs are currently available, they also mobilize arrestin to at least some extent, and to have thosethatdonotinteractwitharrestinatall(ashasbeen done for a Gq/11-DREADD [36]) will be useful for interrogatingarrestin-dependentandindependent path-waysincells andintactanimals.
Conclusion
Fromtheprecedingexamples,itshouldbeclearthatthe field of chemogenetics is maturing, and that, when
N H N N N
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High resolution structures identify potential selectivity motifs
Docking millions of compounds against mutant receptor to identify
chemogenetic actuators N
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Current Opinion in Structural Biology
Computationalapproachesforstructure-guideddiscoveryofnovelchemogeneticactuators.
InthetoppanelisshownacomparisonoftheD3-(purple)[73]andD4-(green)[71]dopaminereceptorstructureshighlightingtheselectivityfilter
whichprovidesatemplateforlarge-scaledockingcampaigns[71].Thelowerpanelillustrateshowasimilarprocesscouldbeusedonamutant
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Conflict
of
interest
statement
Nothingdeclared.Acknowledgements
Workintheauthor’slabissupportedbytheNIH(R37DA045657, RO1MH112205,U24DK116195)andtheMichaelHookerDistinguished ProfessorshipinPharmacology.TheauthorthanksWesKroeze,PhDfor helpfuledits.
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![Figure 2 also shows an active state structure of the wild-type M2 muscarinic receptor (2D [57]), a model of an active state hM2Di-DREADD (Figure 2b) and a summary of key transitions (Figure 2c)](https://thumb-us.123doks.com/thumbv2/123dok_us/8205849.2175825/3.918.91.838.748.1040/figure-structure-muscarinic-receptor-figure-summary-transitions-figure.webp)
