Novel Allosteric Modulators of GProtein-coupled Receptors*Published, JBC Papers in Press, June 22, 2015, DOI 10.1074/jbc.R115.662759
Patrick R. Gentry1, Patrick M. Sexton2,and Arthur Christopoulos2,3
From Drug Discovery Biology, Monash Institute of PharmaceuticalSciences, Monash University, 399 Royal Parade,Parkville, Victoria 3052, Australia
G protein-coupled receptors (GPCRs) are allosteric proteins,because their signal transduction relies on interactions betweentopographically distinct, yet conformationally linked, domains.Much of the focus on GPCR allostery in the new millennium,however, has been on modes of targeting GPCR allosteric siteswith chemical probes due to the potential for novel therapeutics.It is now apparent that some GPCRs possess more than onetargetable allosteric site, in addition to a growing list of putativeendogenous modulators. Advances in structural biology are alsoshedding new insights into mechanisms of allostery, althoughthe complexities of candidate allosteric drugs necessitate rigor-ous biological characterization.
G protein-coupled receptors (GPCRs),4 also known as7-transmembrane (7TM) receptors, are the largest superfamilyof cell surface receptor proteins encoded by the human genome(1). These integral membrane proteins are highly dynamic andexist in an equilibrium between various functionally distinctconformational states (2). GPCRs fulfil the vital biological func-tion of transducing a wide range of extracellular signals(e.g. photons, lipids, neurotransmitters, hormones, peptides,enzymes, ions, odorants) across the cell membrane into thecytosolic space. Physiologically, the process begins when anendogenous extracellular signal interacts with the primary(“orthosteric”) binding site of GPCR, resulting in a conforma-tional rearrangement that conveys the signal through theplasma membrane-spanning 7TM region and subsequentlytriggering intracellular signaling cascades via heterotrimeric G
proteins and other accessory proteins (3). Because GPCR-me-diated signaling systems are involved in regulating a multitudeof physiological and pathophysiological processes, it is not sur-prising that the GPCR superfamily encompasses the targets ofmore actual and potential drugs than any other family of pro-teins (4, 5).
To date, the majority of probe compounds and marketeddrugs that target GPCRs are small molecules, but it is notewor-thy that there is a growing interest in utilizing biologics andantibodies to target these receptors as well (6). In addition,although the mode of action of the bulk of GPCR-targetingagents remains orthosteric, the turn of the millennium has wit-nessed substantial efforts in alternative methods of modulatingGPCR activity, specifically by targeting topographically distinctallosteric sites. This minireview discusses some of the key char-acteristics associated with GPCR allostery and ongoing chal-lenges and opportunities in understanding and exploiting thephenomenon.
Characteristics of GPCR Allostery
Allostery is a widespread biological phenomenon thatdescribes the ability of interactions occurring at one site of amacromolecule to modulate interactions at a spatially distinctbinding site on the same macromolecule in a reciprocal man-ner. Since allosteric effects were first described in archetypalexamples, such as the heme-heme interactions of hemoglobin,allostery has been acknowledged as a means by which proteinsand other molecules (e.g. DNA) may amplify, attenuate, bias,and otherwise fine-tune their physiological functions (2, 7–9).Initial observations of allosteric phenomena in enzymes weremechanistically summarized first in the Monod-Wyman-Changeux (MWC) and subsequently in the Koshland-Nem-ethy-Filmer (KNF) models (10, 11). Although the MWC modeldepicts allostery as a concerted process (i.e. conformationalselection), and the KNF model describes it as a sequential pro-cess (i.e. conformational induction), each model reflects validkey aspects of the nature of allostery, which involves ligand-mediated shifts in the population of pre-existing macromolec-ular conformational ensembles and resulting changes in theinteractive properties of the new ensembles (12). In addition toenzymes, it became apparent that other protein classes, includ-ing GPCRs, possess many of the characteristics associated withallosteric proteins (13). GPCRs are conformationally dynamicproteins that act as conduits for the transfer of energy over adistance. Indeed, GPCR signal transduction is intrinsicallyallosteric as it involves the binding of an extracellular stimulusand subsequent propagation of the signal through the proteinto a topographically distinct (e.g. �50 Å) intracellular site rec-ognized by G proteins, �-arrestins, and others. Moreover,because of the broad diversity of endogenous activators ofGPCRs, an orthosteric region on one type of receptor (e.g. classA biogenic amine receptor) may represent an allosteric domainin another type of receptor (e.g. class B secretin family or class Cglutamate family receptors) (14) (Fig. 1).
* This work was supported by Program Grant APP1055134 of the NationalHealth and Medical Research Council (NHMRC) of Australia. This is the sec-ond article in the Thematic Minireview series “New Directions in G Protein-coupled Receptor Pharmacology.” The authors declare that they have noconflicts of interest with the contents of this article.
1 A 2015 Sir Keith Murdoch Fellow of the American Australian Association.2 Principal Research Fellows of the NHMRC.3 To whom correspondence should be addressed. Tel.: 03-9903-9067; Fax:
03-9903–9581; E-mail: [email protected] The abbreviations used are: GPCR, G protein-coupled receptor; 7TM,
Perhaps not surprisingly, therefore, the attractiveness andtractability of GPCRs as drug targets, coupled with advances indrug screening at these receptors, have uncovered differenttypes of allosteric GPCR modulators (Table 1) that are generallyclassified operationally based on their modes of pharmacology.An allosteric ligand that potentiates an agonist-mediatedreceptor response is referred to as a “positive allosteric modu-lator” (PAM), whereas one that attenuates activity is known as a“negative allosteric modulator” (NAM) (15). Mechanistically,these effects can be achieved through modulation of the bind-ing affinity of orthosteric ligands and/or through changes in theability of the orthosteric ligand-occupied receptor complex tointeract with intracellular transducer proteins. In contrast aligand that binds at the allosteric site without affecting receptoror orthosteric ligand activity (at equilibrium) is classed as “neu-tral allosteric ligand” (NAL). “Allosteric agonists” are ligandsthat are capable of directly activating the receptor from anallosteric site even in the absence of an orthosteric agonist (15),whereas “PAM agonists” and “NAM agonists” display mixedmodes of modulation and direct GPCR activation depending onthe cellular context (15). More recently, “bitopic ligands” havealso been described, which are defined as hybrid molecules pos-sessing separate orthosteric and allosteric pharmacophoresthat concomitantly engage with their respective sites on a singleGPCR to mediate novel pharmacology; several molecules ini-tially classified as allosteric agonists in the literature have sincebeen reclassified as bitopic ligands (15–18).
There are a number of general characteristics associated withallosteric GPCR modulators that present both unique advan-tages over orthosteric ligands as well as challenges to successfuldetection or validation of allosteric compounds. The first char-acteristic is the potential for allosteric ligands to exhibit greaterreceptor subtype selectivity. This property has two potential
origins: i) a decreased evolutionary pressure for sequence con-servation within allosteric sites relative to the orthosteric sitebetween GPCR subtypes (assuming there is no endogenousallosteric ligand for such a site, but see the next section) and/orii) selective cooperativity with an orthosteric site at one recep-tor subtype while exhibiting neutral cooperativity at other sub-types of that receptor family (19). The nature of the cooperativ-ity between orthosteric and allosteric sites on a GPCR alsorepresents a second important characteristic of allostery thathas practical and therapeutic implications. If the modulatordisplays minimal direct allosteric agonism in its own right, thenit will act as a PAM or NAM only when and where the endog-enous ligand is released, thus maintaining the natural spatio-temporal “rhythms” of the endogenous orthosteric ligand.Furthermore, very subtle degrees of positive or negative coop-erativity (which may be all that is necessary for certain GPCRsand disease states) result in an allosteric “effect ceiling” thatincreases the likelihood of on-target safety in overdose situa-tions, although this also poses a challenge for the screening ofmodulators with low degrees of cooperativity (20). The abilityto achieve unprecedented modes of on-target selectivity and/orfine-tune endogenous responses as a consequence of pure PAMor NAM activity may prove particularly important in diseaseswhere tight physiological regulation is vital, such as neurode-generation, schizophrenia, diabetes, and endocrine disorders.
A particularly interesting phenomenon associated with allos-tery that has been most noted at GPCRs is the property of“probe dependence,” wherein the magnitude and direction ofan allosteric effect can change depending on the nature of theinteracting ligands (15, 21). Probe dependence has substantialimplications for GPCR drug discovery and GPCR biology. Forinstance, many GPCRs have more than one endogenousorthosteric agonist, and totally different effects can be observed
FIGURE 1. Potential allosteric ligand-binding regions on GPCRs; some representative allosteric modulators recognizing each region are also listed.RAMP, receptor activity-modifying protein; MRAP, melanocortin receptor accessory protein; Cmpd, compound; AZ, AstraZeneca; Fz4, frizzled4 receptor; CaSR,calcium-sensing receptor; PCEP, 3-amino-3-carboxypropyl-2�-carboxyethyl phosphinic acid.
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(or missed altogether) depending on which agonist is used toactivate the receptor in the presence of a given allosteric mod-ulator (22). In addition, the allosteric nature of GPCR signaltransduction means that the ability of a ligand-bound receptorto recognize cellular effector molecules is also subject to probedependence, this time directed intracellularly rather thanbetween two ligands. Specifically, the ability of orthosteric orallosteric GPCR ligands to stabilize different functionally rele-vant conformations of the same GPCR can give rise to the phe-nomenon of “biased agonism,” whereby only a subset of thepossible signaling repertoire of the receptor is recruited at therelative expense of other pathways (21, 23, 24).
Although beyond the scope of the current minireview, theallosteric pharmacology of bitopic ligands also presents aunique set of characteristics. Because of their dual pharmaco-phore nature, bitopic ligands can provide both greater selectiv-ity through interaction with an allosteric site and higher affinitythrough concomitant engagement of the orthosteric site. Ajudicious choice of orthosteric and allosteric building blockscan also yield bitopic ligands that display novel biased agonism(25). Although the spatiotemporal control of endogenous sig-naling afforded by pure allosteric modulator ligands is lost withbitopic ligands, the latter may prove particularly useful in situ-ations where endogenous agonist tone is progressively lost (e.g.neurodegenerative disorders) (18).
Endogenous Allosteric Modulators
Although most studies of GPCR allostery have traditionallyfocused on the actions of exogenous allosteric modulatorsbecause of the implications for novel drug discovery (seebelow), these receptors can also be modulated by a variety ofendogenous substances (26). As mentioned above, the bestcharacterized allosteric interaction at GPCRs is the positivecooperativity exhibited between the intracellular G protein-binding site with the orthosteric site (3). The GPCR intracellu-lar face can also interact with �-arrestins: endogenous GPCRaccessory proteins that were originally characterized as scaf-folding proteins involved in the termination of GPCR signaling,internalization, and recycling of receptors. It is now recognized
that �-arrestins can be involved in G protein-independent sig-nal transduction (27–30). These unique signaling propertiesemerge as a result of specific receptor conformations that havebeen shown to interact more readily with �-arrestins, leading toincreased �-arrestin binding and biased signaling through non-canonical signaling pathways (31–33). Although beyond thescope of the current review, it is also widely acknowledged thatdifferent types of GPCRs have the potential to associate withother proteins, such as receptor activity-modifying proteins(RAMPs) or melanocortin receptor accessory proteins(MRAPs), or with each other in the form of homo- or hetero-dimers (or higher order oligomers) with novel pharmacologicalproperties (26). In a number of such instances, cooperativeinteractions have been noted between orthosteric ligandswithin such complexes (34).
In addition to the ubiquitous allosteric sites utilized by Gproteins and �-arrestins, individual GPCR classes or subclassespossess more specific allosteric sites that may be targeted byendogenous allosteric modulators. For example, pharmacolog-ical and crystallographic evidence has shown that sodium isvital for stabilizing the inactive state of many class A GPCRs viaan allosteric site centered on a highly conserved aspartate resi-due, Asp2.50 (26). Additionally, aromatic amino acids (e.g.L-phenylalanine, L-tryptophan, and L-tyrosine) bind at a sitenear to, but spatially distinct from, the orthosteric site withinthe “Venus flytrap” (VFT) N-terminal domain of the calcium-sensing class C GPCR to potentiate the actions of extracellularcalcium at a number of intracellular signaling pathways(35–37). A similar allosteric site has been shown to be located inthe Venus flytrap of the metabotropic glutamate (mGlu) class CGPCRs. This site was initially reported as an allosteric chloride-binding site in the mGlu1 subtype, but some synthetic smallmolecule agonists have since been reported to bind this site inmGlu4 (38 – 40).
As summarized in Table 1, a growing number of substancesencompassing not only amino acids and ions, but also lipids,peptides, and proteins, have been proposed to act as putativeendogenous allosteric modulators of different types of GPCRs
(26). It should be noted that in many of these instances, conclu-sive validation of an allosteric mechanism remains to be estab-lished, but the study of endogenous GPCR modulators mayprove to be a fertile ground for uncovering novel biology inhealth and disease. For instance, the nature and composition ofendogenous lipidic substances and numerous types of peptidesand proteins can vary dramatically in inflammation; if it can beshown that some of these substances are bona fide allostericmodulators of specific GPCRs, then this may represent a novelavenue for understanding and targeting GPCR functionality ininflammatory disease (26). In addition, GPCR-directed autoan-tibodies have been identified as potential endogenous allostericligands related to a number of chronic disease states. In partic-ular, autoantibodies have been identified in patients with a vari-ety of cardiovascular diseases, including autoantibodies againstthe AT1 receptor, the �1-adrenergic receptor, the M2 musca-rinic acetylcholine receptor (mAChR), the �1-adrenergicreceptor, the ETA receptor, and the 5-HT4 receptor (26). Addi-tionally, the central nervous system can be affected by autoan-tibody activity. For instance, autoantibodies against GABABhave been found in the cerebrospinal fluid of limbic encephali-tis patients, whereas patients with basal ganglia encephalitiswith dominant movement and psychiatric disease present withautoantibodies against the dopamine D2 receptor (26).
Exogenous Allosteric Modulators
Numerous synthetic small molecules with an allosteric modeof action have been reported for GPCRs (Table 1). The mAChRfamily is arguably the most well studied class A GPCR system inthis regard (41). Indeed, the first examples of GPCR allostericmodulators, the alkane bis-ammonium family of ligands, wereidentified at the mAChRs (42). Since that time, allosteric mod-ulators of nearly every mode of action have been found to targetthe mAChRs, including PAMs, NAMs, PAM and NAM ago-nists, and bitopic ligands (17, 43– 48). Interestingly, most ofthese ligands bind to the mAChRs at a shared site (albeit withdifferent affinities depending on the subtype), often referred toas the “common” allosteric site and highlighting how the tar-geting of a common allosteric domain can yield markedly dif-ferent biological behaviors (49, 50). However, evidence hasbeen provided for the existence of a second allosteric site on themAChRs, recognized by indolocarbazole analogues of stauro-sporine (e.g. KT5720 and KT5823) and the benzimidazolederivatives WIN63577 and WIN51708. As noted with the“common site” modulators, the second-site compounds alsodemonstrated positive, negative, or neutral interactions withacetylcholine depending on the mAChR subtype, but showedlargely neutral interactions with the binding of common sitemodulators, further supporting the presence of at least twoallosteric sites on a single GPCR (51–54).
The notion that a single GPCR may possess more than oneallosteric site for exogenous small molecules is likely morewidespread. For instance, a number of allosteric modulatorshave been described for the gonadotropin-releasing hormonereceptor, including Furan-1 and its derivative, FD-1 (55), as wellas a series of amiloride derivatives (56). Interestingly, interac-tion studies between the two different classes of allostericligand revealed neutral cooperativity, despite each class indi-
vidually modulating orthosteric ligands (56), again suggestive ofmore than one allosteric site on this receptor family. The intra-cellular face of GPCRs may also harbor allosteric sites for selec-tive small molecules. For instance, binding sites for allostericantagonists of the CXCR2, CCR4, and CCR5 chemokine recep-tors have been identified among the intracellular loops (57–59).Intracellular allosteric sites are also utilized by pepducins: cell-penetrating, lipidated peptides that target the intracellularloops. By tailoring the peptide’s design to the intracellulardomain of a particular GPCR, researchers have been successfulin producing pepducins selectively targeting a number ofGPCRs involved in inflammatory diseases, including protease-activated receptors (PAR1, -2, -4) and chemokine receptors(CXCR1, -2, -4) (60, 61).
Class B GPCRs, such as the secretin, glucagon, and gluca-gon-like peptide 1 receptors, have proven notoriously intrac-table to small molecule discovery and have remained an areaof intense research with regard to allosteric drugs. One com-mon observation is the discovery of direct-acting allostericagonists, in addition to PAM agonists or NAMs (62). Pureclass B GPCR PAMs have thus far remained relatively moreelusive, but whether this reflects a fundamental property ofthis class of GPCR or the relative immaturity of detailedstudies of Class B allostery remains to be determined. Incommon with the preceding examples, however, there isclear evidence suggesting the presence of more than oneallosteric site on Class B GPCRs (63, 64).
Within the class C GPCRs, the mGlu receptors have a veryrich allosteric pharmacology. In particular, allosteric modu-lation of the mGlu5 receptor has been of interest as a targetfor the treatment of schizophrenia and Alzheimer disease(65, 66). As such, multiple small molecule PAMs have beendeveloped for mGlu5 (67–72), with the bulk acting at a com-mon site in the upper region of the 7TM domain oftentermed the “MPEP-binding site” after the prototypicalmGlu5 NAM, MPEP (65, 66). However, several ligands havebeen identified that are non-competitive with the MPEP site,including the PAMs VU357121 and CPPHA, indicating thepresence of at least two distinct allosteric sites (67, 69, 73,74). Further investigations have shown that PAMs acting oneach of these sites may have varied effects on different sig-naling pathways, indicating that allosteric modulation of dis-crete allosteric sites may have significant effects on theresponse of the GPCR (75, 76).
Finally, the Class F GPCR family, which includes theSmoothened and Frizzled receptors, has also been considereddifficult to target with small molecules, but recent break-throughs have revealed that these receptors possess allostericbinding sites for exogenous ligands. For example, a series ofsmall molecules that were originally designed to act as pharma-cological chaperones for a misfolded mutant of Frizzled4 weresubsequently identified as novel allosteric modulators of thewild-type form of Frizzled4; the binding site for these com-pounds was proposed to be located in the vicinity of intracellu-lar loop 3 of the receptor (77). The recent crystal structure ofthe Smoothened receptor bound to the allosteric modulator,Sant1, also revealed an allosteric pocket that is located deepwithin the transmembrane-spanning cavity of the receptor,
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toward the cytosolic end; this is in contrast to the binding sitethat is closer to the extracellular entrance and utilized bycanonical ligands of this receptor (78).
It is important to note that although small molecules havemade an undeniable impact on the study of allostery, there isgrowing interest in utilizing biologics to target allosteric sites.Thus far, allosteric antibodies have shown the most promise intargeting receptor tyrosine kinases, such as the insulin receptor,where the monoclonal antibody XMetA allosterically binds andactivates the receptor (79, 80). A subcategory of allosteric anti-bodies known as “allosteric ligand-modifying antibodies”(ALMA) acts by binding the endogenous ligand prior to inter-acting with the receptor (15). One example is gevokizumab, ananti-interleukin-1� antibody that binds interleukin and altersthe conformation of the endogenous ligand. Subsequently, thegevokizumab-interleukin-1� complex binds in a characteristicternary complex with the receptor (81, 82). Although examplesof rationally designed allosteric antibodies targeting GPCRshave yet to be described, the novel mechanisms of allostericantibodies and allosteric ligand-modifying antibodies offer avaluable new strategy for the study of GPCR allostery in thefuture.
Chemical Biology Challenges in Designing AllostericModulators of GPCRs
The production of highly site-specific allosteric probe mole-cules is a crucial factor in the study of allosteric interactions.Structure-based drug design is still a relatively nascent fieldwith respect to GPCRs, although substantial progress hasoccurred in recent years (see below). Within both industry andacademia, a large number of resources have been dedicated tothe generation, collection, and curation of large libraries of nat-ural and synthetic small molecules from which to screen for andoptimize novel allosteric ligands, and this remains the majorsource of such compounds (83, 84). Given this significantinvestment, the methodologies with which these novel ligandsare interrogated are central to any research campaign. The fieldof chemical biology has provided a number of tools to probeallosteric mechanisms of GPCR structure and function. Thesetechniques may provide the biochemical and biophysicalinformation necessary to garner a clear understanding of thestructural dynamics and signaling behavior of allostericinteractions on a chemical level. Techniques such as biolu-minescent and Förster resonance energy transfer as well assingle-molecule detection fluorescence have gained popu-larity for the study of GPCRs and validated binding partners.However, there are limitations for using these techniques inthe process of screening for novel ligands, not least of whichis the challenge of applying these methods to a high through-put screening format (85, 86). Measurement of second mes-sengers of G protein signaling (e.g. calcium and cAMP) or�-arrestin recruitment has proven more successful in thisrespect; nevertheless, it remains challenging to developselective small molecule allosteric modulators.
For example, allosteric ligands often possess delicate struc-ture-activity relationships. That is, in a given chemical series, aseemingly minor modification to a molecule’s steric or elec-tronic properties often leads to the complete ablation of its
activity. Similarly, certain chemical series demonstrate “modeswitching” whereby small modifications to the structure canresult in dramatically changed pharmacological profiles (87).These concerns are particularly relevant when further modifi-cations are applied to the probes, for instance in the generationof irreversible or photoactivatable allosteric molecules (88, 89),or through the use of these chemical probes in vivo, where bio-chemical transformations undertaken by metabolic processesmay alter a modulator’s potency, cooperativity, receptor selec-tivity, or mode of action.
Novel chemotypes of allosteric modulators also pose chal-lenges to pharmacological characterization. For instance, manyallosteric modulators display phenomena such as probe depen-dence, and can impose biased signaling on the actions oforthosteric ligands (Table 2). These may go unnoticed with theuse of a single screening methodology. Only through the mea-surement of an allosteric ligand’s effect on multiple down-stream pathways in the presence of all relevant endogenousorthosteric ligands can a compound’s properties be fully eluci-dated, and this must be factored into all allosteric discoveryprograms (90). Further complexities in characterizing a novelallosteric ligand may be caused by differences between species’receptor isoforms, differential effects on orthosteric ligandaffinity and efficacy, varied kinetics between different signalingpathways, and the possibility of undetected endogenous allos-teric interactions. Therefore efforts must be made to fully char-acterize and validate novel chemical probes in as many experi-mental paradigms as possible to compose the clearest picture ofthe compound’s properties.
What Can Structural Biology Reveal About AllostericMechanisms?
Although the use of selective allosteric probe molecules andfunctional assays can reveal much about allosteric pharmacol-ogy, the information provided by crystal structures and otherhigh resolution approaches is invaluable for more direct,molecular level insights into GPCR allostery. Since the turn ofthe millennium, when the first high resolution GPCR crystalstructure was solved for bovine rhodopsin (91), there has been aseemingly exponential growth in the number of crystal struc-tures solved for a range of class A, B, and C GPCRs (Fig. 2),although the majority of these rely on some form of proteinengineering to improve stability and crystal formation. This hasallowed the observation of conformational snapshots adoptedby structures co-bound to ligands and/or interacting proteins,offering an unprecedented view into the fundamental struc-tural basis for receptor function.
Of note, there have been a number of inactive state crystalstructures of GPCRs in binary complexes with small mole-cule NAMs. These include the chemokine receptor CCR5bound to maraviroc, the corticotropin-releasing factorreceptor CRF1 bound to CP-376395, the mGlu1 bound toFITM, the mGlu5 bound to mavoglurant, and the aforemen-tioned Smoothened receptor bound to Sant1 (78, 92–95).Despite providing new insights into the binding behavior ofthese NAMs, the inactive and binary complex natures ofthese structures do not capture the complete structuralmechanisms that underlie cooperativity between the allos-
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teric and orthosteric sites. Indeed, the CRF1 and mGlu struc-tures lack the key N-terminal domains that constitute much(or all) of the orthosteric binding site.
A major breakthrough in recent years has been the crystalstructure depicting the classic ternary complex of anorthosteric agonist binding the �2-adrenergic receptor coupledto a Gs protein (96). This structure offered the first observationof how a GPCR orthosteric site is allosterically coupled to Gprotein activation. In terms of small molecule allostery, therecent structure of an active M2 mAChR in complex with thehigh efficacy orthosteric agonist, iperoxo, and the PAM,LY02119620 (97), also represented a major structural biologyadvance in understanding allostery at a GPCR. For example, thestructure is consistent with the predictions of the MWC modelof allostery in that the PAM preferentially recognizes and sta-bilizes a preformed active state of the receptor. Nonetheless,these structures still represent first steps in our molecular levelunderstanding of mechanisms underlying GPCR allostery. Phe-nomena such as biased allosteric modulation, probe depen-dence, and the actual mechanisms underlying transmission ofcooperativity remain challenging as they require the ability tocapture multiple states in the absence and presence of multipleligands. In the meantime, additional insights into the structuraland dynamic mechanisms of allostery are being obtained viaother methods. For example, the inactive M2 mAChR structurehas been subjected to long timescale molecular dynamics sim-ulations with a diverse range of small molecule allosteric mod-
ulators, in the absence or presence of an orthosteric ligand, toidentify binding poses and mechanisms underlying cooperativ-ity for a broad set of NAMs (98). The use of NMR has alsoallowed researchers to study the conformational flexibility ofthe receptor as a whole, providing more information on recep-tor dynamics than can be obtained through static crystal struc-tures, although no study has directly applied this approach toGPCR allosteric modulators to date (99, 100).
Conclusions
Allosteric modulation of GPCRs is now a widely acceptedphenomenon with substantial implications for novel drug dis-covery, yet many fundamental issues remain to be addressed.For example, it is now clear that a single GPCR can possessmore than one allosteric site, but whether the targeting of suchsites can lead to differential behaviors remains unknown. Theprevalence of endogenous allosteric modulators remains to bedetermined, but they may prove to be a mechanism of tissue-specific regulation in normal physiology or disease that may beamenable to chemical manipulation. The ascendance of biolog-ics as therapeutics opens new vistas for targeting of GPCRs witha greater degree of specificity than previously possible, yet theextent with which such substances can interact allostericallywith GPCRs is largely unexplored. As more detailed GPCRstructural information becomes available, it too will profoundlyaffect our understanding of allostery in GPCRs as well as themanner by which allosteric molecules are designed. In prepar-
ing for such an eventuality, the rigorous biological character-ization of each new allosteric site and allosteric ligand discov-ered remains paramount.
Author Contributions—All authors wrote the manuscript.
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