Impact of GPCR Structures on Drug Discovery

Structures of 70 unique G protein-coupled receptors (GPCRs) have been determined, with over 370 structures in total bound to different ligands and the receptors in various conformational states. Structure-based drug design has been applied to an increasing number of GPCR targets over the past decade and now a few of these drug candidates have entered clinical trials. Given the length of time required for a drug to reach the market, there are no documented examples of licensed drugs being developed with the aid of a structure, but this is likely to change as current efforts come to fruition. Structures of 70 unique G protein-coupled receptors (GPCRs) have been determined, with over 370 structures in total bound to different ligands and the receptors in various conformational states. Structure-based drug design has been applied to an increasing number of GPCR targets over the past decade and now a few of these drug candidates have entered clinical trials. Given the length of time required for a drug to reach the market, there are no documented examples of licensed drugs being developed with the aid of a structure, but this is likely to change as current efforts come to fruition. G protein-coupled receptors (GPCRs) are integral membrane proteins that transduce chemical signals from the extracellular matrix into the cell (Flock et al., 2015Flock T. Ravarani C.N.J. Sun D. Venkatakrishnan A.J. Kayikci M. Tate C.G. Veprintsev D.B. Babu M.M. Universal allosteric mechanism for Gα activation by GPCRs.Nature. 2015; 524: 173-179Crossref PubMed Scopus (152) Google Scholar, Venkatakrishnan et al., 2013Venkatakrishnan A.J. Deupi X. Lebon G. Tate C.G. Schertler G.F. Babu M.M. Molecular signatures of G-protein-coupled receptors.Nature. 2013; 494: 185-194Crossref PubMed Scopus (952) Google Scholar). There are about 800 GPCRs encoded by the human genome and they respond to a wide variety of signals that range in size from photons to small proteins (Foord et al., 2005Foord S.M. Bonner T.I. Neubig R.R. Rosser E.M. Pin J.P. Davenport A.P. Spedding M. Harmar A.J. International Union of Pharmacology. XLVI. G protein-coupled receptor list.Pharmacol. Rev. 2005; 57: 279-288Crossref PubMed Scopus (380) Google Scholar). They are divided into six classes based on amino acid sequence similarities, but only four of the classes (A, B, C, and F) are found in humans. Class A (rhodopsin-like) contains the largest number of GPCRs (719 in humans). Roughly half of class A GPCRs are sensory receptors involved in smell (pheromone receptors) or vision (rhodopsins). The ∼350 non-sensory receptors are activated by diffusible ligands such as hormones or neurotransmitters and contain many well-characterized drug targets. For example, antagonists of the β1-adrenoceptor (β1AR) are used in cardiovascular disease (beta blockers) (Bristow, 2011Bristow M.R. Treatment of chronic heart failure with β-adrenergic receptor antagonists: a convergence of receptor pharmacology and clinical cardiology.Circ. Res. 2011; 109: 1176-1194Crossref PubMed Scopus (113) Google Scholar), whereas agonists of β2AR are treatments for asthma (Cazzola et al., 2011Cazzola M. Calzetta L. Matera M.G. β(2) -adrenoceptor agonists: current and future direction.Br. J. Pharmacol. 2011; 163: 4-17Crossref PubMed Scopus (125) Google Scholar). Another well-studied receptor is the adenosine A2A receptor that is currently a target in immune-oncology (Allard et al., 2016Allard B. Beavis P.A. Darcy P.K. Stagg J. Immunosuppressive activities of adenosine in cancer.Curr. Opin. Pharmacol. 2016; 29: 7-16Crossref PubMed Scopus (129) Google Scholar) and Parkinson’s disease (Schwarzschild et al., 2006Schwarzschild M.A. Agnati L. Fuxe K. Chen J.F. Morelli M. Targeting adenosine A2A receptors in Parkinson’s disease.Trends Neurosci. 2006; 29: 647-654Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). Class A receptors typically contain short disordered N-terminal regions (Figure 1A), although a few receptors such as the glycoprotein hormone receptors contain a large and well-ordered N-terminal domain. Class B receptors (48 in humans) are divided into two subclasses, the secretin receptor and adhesion receptors. The secretin sub-class contain a large extracellular domain that is involved in binding peptide ligands, with only a small portion of the ligand interacting with the transmembrane region (Figure 1B); the glucagon-like peptide receptor GLP1R is a well-characterized target for the treatment of diabetes (Andersen et al., 2018Andersen A. Lund A. Knop F.K. Vilsbøll T. Glucagon-like peptide 1 in health and disease.Nat. Rev. Endocrinol. 2018; 14: 390-403Crossref PubMed Scopus (128) Google Scholar). Class C receptors (22 in humans) form stable dimers that are essential for receptor signaling (Pin et al., 2005Pin J.P. Kniazeff J. Liu J. Binet V. Goudet C. Rondard P. Prézeau L. Allosteric functioning of dimeric class C G-protein-coupled receptors.FEBS J. 2005; 272: 2947-2955Crossref PubMed Scopus (133) Google Scholar), whereas receptors from other classes are believed to be signaling-competent as monomers (Whorton et al., 2007Whorton M.R. Bokoch M.P. Rasmussen S.G. Huang B. Zare R.N. Kobilka B. Sunahara R.K. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein.Proc. Natl. Acad. Sci. USA. 2007; 104: 7682-7687Crossref PubMed Scopus (539) Google Scholar), although transient dimers may be important in signaling in vivo (Milligan et al., 2019Milligan G. Ward R.J. Marsango S. GPCR homo-oligomerization.Curr. Opin. Cell Biol. 2019; 57: 40-47Crossref PubMed Scopus (36) Google Scholar). The metabotropic glutamate receptors (mGluRs) are archetypal members of class C with a large extracellular domain that binds agonists such as glutamate that activate the receptor (Figure 1C). Inhibition of mGluRs has been targeted for various disease settings including schizophrenia, depression and movement disorders (Niswender and Conn, 2010Niswender C.M. Conn P.J. Metabotropic glutamate receptors: physiology, pharmacology, and disease.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 295-322Crossref PubMed Scopus (1103) Google Scholar). Class F receptors (11 members in humans) contain the frizzled and smoothened receptors. Smoothened can be activated by steroids (Figure 1D) and small molecules have been successfully progressed for the treatment of cancers (Ruat et al., 2014Ruat M. Hoch L. Faure H. Rognan D. Targeting of Smoothened for therapeutic gain.Trends Pharmacol. Sci. 2014; 35: 237-246Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, Zhang et al., 2018Zhang X. Dong S. Xu F. Structural and Druggability Landscape of Frizzled G Protein-Coupled Receptors.Trends Biochem. Sci. 2018; 43: 1033-1046Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). GPCRs are considered ideal drug targets from many perspectives, and currently, 34% of small molecule drugs bind to GPCRs (Santos et al., 2017Santos R. Ursu O. Gaulton A. Bento A.P. Donadi R.S. Bologa C.G. Karlsson A. Al-Lazikani B. Hersey A. Oprea T.I. Overington J.P. A comprehensive map of molecular drug targets.Nat. Rev. Drug Discov. 2017; 16: 19-34Crossref PubMed Scopus (776) Google Scholar). Non-sensory GPCRs have evolved over millennia to efficiently bind signaling molecules in the orthosteric binding site (Figure 1), which is typically a deep cleft in the extracellular face of the receptor. The size, shape, and amino acid composition of the orthosteric binding site is thus also very well suited to designing small synthetic molecules that bind there (Shoichet and Kobilka, 2012Shoichet B.K. Kobilka B.K. Structure-based drug screening for G-protein-coupled receptors.Trends Pharmacol. Sci. 2012; 33: 268-272Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), either inhibiting GPCR function (antagonists) or activating the receptor (agonists). The position of the orthosteric binding site on the outside of the cell also makes it easier to develop drugs because they do not need to be engineered to cross the plasma membrane. GPCRs are also good drug targets because they are highly dynamic, and during activation, there are considerable changes in the receptor shape (Hilger et al., 2018Hilger D. Masureel M. Kobilka B.K. Structure and dynamics of GPCR signaling complexes.Nat. Struct. Mol. Biol. 2018; 25: 4-12Crossref PubMed Scopus (273) Google Scholar, Kobilka and Deupi, 2007Kobilka B.K. Deupi X. Conformational complexity of G-protein-coupled receptors.Trends Pharmacol. Sci. 2007; 28: 397-406Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar). Even in the absence of agonists, GPCRs undergo transitions to an active state that is capable of coupling to intracellular G proteins (known as basal activity), but in the presence of agonists, there is a higher probability of GPCRs being in an active state and coupling to a G protein. During activation, the intracellular ends of transmembrane helices H5 and H6 move outward to form a cleft in the receptor where the C terminus of the G protein α-subunit binds and stabilizes the active state of the receptor (García-Nafría and Tate, 2019García-Nafría J. Tate C.G. Cryo-EM structures of GPCRs coupled to Gs, Gi and Go.Mol. Cell. Endocrinol. 2019; 488: 1-13Crossref PubMed Scopus (50) Google Scholar, Rosenbaum et al., 2009Rosenbaum D.M. Rasmussen S.G. Kobilka B.K. The structure and function of G-protein-coupled receptors.Nature. 2009; 459: 356-363Crossref PubMed Scopus (1406) Google Scholar). Upon G protein coupling, there is a concomitant closure of the loops over the orthosteric binding site, resulting in both an increase in affinity (Warne et al., 2019Warne T. Edwards P.C. Doré A.S. Leslie A.G.W. Tate C.G. Molecular basis for high-affinity agonist binding in GPCRs.Science. 2019; 364: 775-778Crossref PubMed Scopus (29) Google Scholar) of the agonist for the receptor and a decrease in its off-rate (DeVree et al., 2016DeVree B.T. Mahoney J.P. Vélez-Ruiz G.A. Rasmussen S.G. Kuszak A.J. Edwald E. Fung J.J. Manglik A. Masureel M. Du Y. et al.Allosteric coupling from G protein to the agonist-binding pocket in GPCRs.Nature. 2016; 535: 182-186Crossref PubMed Scopus (126) Google Scholar). These changes in receptor shape can be affected by binding of molecules to allosteric sites distinct from the orthosteric binding site (Thal et al., 2018Thal D.M. Glukhova A. Sexton P.M. Christopoulos A. Structural insights into G-protein-coupled receptor allostery.Nature. 2018; 559: 45-53Crossref PubMed Scopus (114) Google Scholar) and may either reduce the probability of activation (negative-allosteric modulator [NAM]) or enhance receptor signaling by an agonist (positive-allosteric modulator [PAM]). Allosteric modulators have been found that bind to the lipid face of transmembrane α helices, to intracellular sites, and also to extracellular sites (Figure 2). There is considerable complexity and diversity in how small molecules affect GPCR signaling, with some molecules (partial agonists) activating GPCRs less than the native ligand and both PAMs and NAMs also potentially having some agonist activity (May et al., 2007May L.T. Leach K. Sexton P.M. Christopoulos A. Allosteric modulation of G protein-coupled receptors.Annu. Rev. Pharmacol. Toxicol. 2007; 47: 1-51Crossref PubMed Scopus (522) Google Scholar, Wootten et al., 2013Wootten D. Christopoulos A. Sexton P.M. Emerging paradigms in GPCR allostery: implications for drug discovery.Nat. Rev. Drug Discov. 2013; 12: 630-644Crossref PubMed Scopus (282) Google Scholar). Finally, it is known that there are multiple signaling pathways for GPCRs, and it is sometimes possible to bias the signaling of a given GPCR through either a specific G protein or through β-arrestin (Smith et al., 2018Smith J.S. Lefkowitz R.J. Rajagopal S. Biased signalling: from simple switches to allosteric microprocessors.Nat. Rev. Drug Discov. 2018; 17: 243-260Crossref PubMed Scopus (231) Google Scholar), which could reduce the side effects of some drugs (Whalen et al., 2011Whalen E.J. Rajagopal S. Lefkowitz R.J. Therapeutic potential of β-arrestin- and G protein-biased agonists.Trends Mol. Med. 2011; 17: 126-139Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). Despite the long history and obvious desirability of developing drugs targeting GPCRs, there are several problems associated with their development. For example, the muscarinic M1 receptor is a well-validated target for agonists that could alleviate cognitive decline during neurodegeneration (Moran et al., 2019Moran S.P. Maksymetz J. Conn P.J. Targeting Muscarinic Acetylcholine Receptors for the Treatment of Psychiatric and Neurological Disorders.Trends Pharmacol. Sci. 2019; 40: 1006-1020Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). However, the orthosteric binding site of M1 is virtually identical to those of the related receptors M2, M3, M4, and M5 as they all bind the native ligand acetylcholine (Thal et al., 2016Thal D.M. Sun B. Feng D. Nawaratne V. Leach K. Felder C.C. Bures M.G. Evans D.A. Weis W.I. Bachhawat P. et al.Crystal structures of the M1 and M4 muscarinic acetylcholine receptors.Nature. 2016; 531: 335-340Crossref PubMed Scopus (172) Google Scholar), and activation of M2 and M3 in particular gives rise to dose-limiting side effects (gastrointestinal [GI] disturbances, cardiovascular effects). Another potential source of side effects when targeting other receptors could arise due to signaling through multiple different pathways (Whalen et al., 2011Whalen E.J. Rajagopal S. Lefkowitz R.J. Therapeutic potential of β-arrestin- and G protein-biased agonists.Trends Mol. Med. 2011; 17: 126-139Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). There are thus ample opportunities for improved specificity and efficacy in existing validated targets and in developing drugs to new receptors as existing drugs only target approximately one third of potential druggable GPCRs (Santos et al., 2017Santos R. Ursu O. Gaulton A. Bento A.P. Donadi R.S. Bologa C.G. Karlsson A. Al-Lazikani B. Hersey A. Oprea T.I. Overington J.P. A comprehensive map of molecular drug targets.Nat. Rev. Drug Discov. 2017; 16: 19-34Crossref PubMed Scopus (776) Google Scholar). These challenges are ideally suited to approaches based on structure-based drug design (SBDD), and the on-going revolution in structure determination of GPCRs is well placed to drive a similar revolution in SBDD applied to GPCRs. The aim of this review is to introduce the structural biology approaches to determining GPCR structures, their utility in facilitating SBDD, and the impact that they have had and will have on drug development. For the past 20 years, X-ray crystallography has been the method of choice for determining GPCR structures (Figure 2; Table S1). This has been facilitated by the development of several generic complementary techniques (Tate and Schertler, 2009Tate C.G. Schertler G.F. Engineering G protein-coupled receptors to facilitate their structure determination.Curr. Opin. Struct. Biol. 2009; 19: 386-395Crossref PubMed Scopus (147) Google Scholar) to stabilize receptors in detergent solution during purification. These include receptor thermostabilization (Magnani et al., 2016Magnani F. Serrano-Vega M.J. Shibata Y. Abdul-Hussein S. Lebon G. Miller-Gallacher J. Singhal A. Strege A. Thomas J.A. Tate C.G. A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies.Nat. Protoc. 2016; 11: 1554-1571Crossref PubMed Scopus (54) Google Scholar), the use of high-affinity ligands (Zhang et al., 2015Zhang X. Stevens R.C. Xu F. The importance of ligands for G protein-coupled receptor stability.Trends Biochem. Sci. 2015; 40: 79-87Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and the application of fusion proteins to facilitate the formation of crystal contacts during crystallization using lipidic cubic phase (Chun et al., 2012Chun E. Thompson A.A. Liu W. Roth C.B. Griffith M.T. Katritch V. Kunken J. Xu F. Cherezov V. Hanson M.A. Stevens R.C. Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors.Structure. 2012; 20: 967-976Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, Rosenbaum et al., 2007Rosenbaum D.M. Cherezov V. Hanson M.A. Rasmussen S.G. Thian F.S. Kobilka T.S. Choi H.J. Yao X.J. Weis W.I. Stevens R.C. Kobilka B.K. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function.Science. 2007; 318: 1266-1273Crossref PubMed Scopus (1117) Google Scholar). The inactive state of GPCRs bound to inverse agonists tends to be the most stable state of a receptor and, on the whole, this tends to be the most tractable state to crystallize, although many structures have also now been determined bound to high-affinity agonists (Figure 2; Table S1). However, very often structures of agonist-bound receptors are in an intermediate state (Lebon et al., 2012Lebon G. Warne T. Tate C.G. Agonist-bound structures of G protein-coupled receptors.Curr. Opin. Struct. Biol. 2012; 22: 482-490Crossref PubMed Scopus (79) Google Scholar), because the fully active state needs to be stabilized by an intracellular binding partner, either a G protein or a G protein mimetic such as a conformation-specific nanobody that binds on the intracellular face of an activated receptor (Rasmussen et al., 2011aRasmussen S.G. Choi H.J. Fung J.J. Pardon E. Casarosa P. Chae P.S. Devree B.T. Rosenbaum D.M. Thian F.S. Kobilka T.S. et al.Structure of a nanobody-stabilized active state of the β(2) adrenoceptor.Nature. 2011; 469: 175-180Crossref PubMed Scopus (1181) Google Scholar, Rasmussen et al., 2011bRasmussen S.G. DeVree B.T. Zou Y. Kruse A.C. Chung K.Y. Kobilka T.S. Thian F.S. Chae P.S. Pardon E. Calinski D. et al.Crystal structure of the β2 adrenergic receptor-Gs protein complex.Nature. 2011; 477: 549-555Crossref PubMed Scopus (2030) Google Scholar). X-ray crystallography has also been useful in determining GPCR structures in the fully active state (Figure 2B). The first active state structure determined was that of rhodopsin, either at low pH (Park et al., 2008Park J.H. Scheerer P. Hofmann K.P. Choe H.W. Ernst O.P. Crystal structure of the ligand-free G-protein-coupled receptor opsin.Nature. 2008; 454: 183-187Crossref PubMed Scopus (781) Google Scholar) or with a peptide from the C terminus of the G protein bound (Scheerer et al., 2008Scheerer P. Park J.H. Hildebrand P.W. Kim Y.J. Krauss N. Choe H.W. Hofmann K.P. Ernst O.P. Crystal structure of opsin in its G-protein-interacting conformation.Nature. 2008; 455: 497-502Crossref PubMed Scopus (890) Google Scholar). At the time, it was unclear whether this was indeed the fully active state, but subsequent structures of rhodopsin coupled to either a heterotrimeric G protein (Gao et al., 2019Gao Y. Hu H. Ramachandran S. Erickson J.W. Cerione R.A. Skiniotis G. Structures of the Rhodopsin-Transducin Complex: Insights into G-Protein Activation.Mol. Cell. 2019; 75: 781-790Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, Kang et al., 2018Kang Y. Kuybeda O. de Waal P.W. Mukherjee S. Van Eps N. Dutka P. Zhou X.E. Bartesaghi A. Erramilli S. Morizumi T. et al.Cryo-EM structure of human rhodopsin bound to an inhibitory G protein.Nature. 2018; 558: 553-558Crossref PubMed Scopus (116) Google Scholar) or a mini-G protein (Tsai et al., 2018Tsai C.J. Pamula F. Nehme R. Muhle J. Weinert T. Flock T. Nogly P. Edwards P.C. Carpenter B. Gruhl T. et al.Crystal structure of rhodopsin in complex with a mini-Go sheds light on the principles of G protein selectivity.Sci. Adv. 2018; 4: eaat7052Crossref PubMed Scopus (25) Google Scholar) now support this. Attempts to use G protein peptides to determine the active state structures of other receptors have been unsuccessful, except for one case where the peptide was tethered to the C terminus of the receptor, although the peptide did not bind in the expected manner (Liu et al., 2019Liu X. Xu X. Hilger D. Aschauer P. Tiemann J.K.S. Du Y. Liu H. Hirata K. Sun X. Guixa-Gonzalez R. et al.Structural Insights into the Process of GPCR-G Protein Complex Formation.Cell. 2019; 177: 1243-1251Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Crystallization of a receptor coupled to a heterotrimeric G protein has only been successful in one instance (Rasmussen et al., 2011bRasmussen S.G. DeVree B.T. Zou Y. Kruse A.C. Chung K.Y. Kobilka T.S. Thian F.S. Chae P.S. Pardon E. Calinski D. et al.Crystal structure of the β2 adrenergic receptor-Gs protein complex.Nature. 2011; 477: 549-555Crossref PubMed Scopus (2030) Google Scholar). However, this galvanized the engineering of mini-G proteins, which are composed of the GTPase domain from the α-subunit of the heterotrimeric G protein (Carpenter and Tate, 2016Carpenter B. Tate C.G. Engineering a minimal G protein to facilitate crystallisation of G protein-coupled receptors in their active conformation.Protein Eng. Des. Sel. 2016; 29: 583-594PubMed Google Scholar, Nehmé et al., 2017Nehmé R. Carpenter B. Singhal A. Strege A. Edwards P.C. White C.F. Du H. Grisshammer R. Tate C.G. Mini-G proteins: Novel tools for studying GPCRs in their active conformation.PLoS ONE. 2017; 12: e0175642Crossref PubMed Scopus (59) Google Scholar). These recapitulate the pharmacology of heterotrimeric G proteins and have been used to determine crystal structures of the active state of A2AR (Carpenter et al., 2016Carpenter B. Nehmé R. Warne T. Leslie A.G. Tate C.G. Structure of the adenosine A(2A) receptor bound to an engineered G protein.Nature. 2016; 536: 104-107Crossref PubMed Scopus (234) Google Scholar) and rhodopsin (Tsai et al., 2018Tsai C.J. Pamula F. Nehme R. Muhle J. Weinert T. Flock T. Nogly P. Edwards P.C. Carpenter B. Gruhl T. et al.Crystal structure of rhodopsin in complex with a mini-Go sheds light on the principles of G protein selectivity.Sci. Adv. 2018; 4: eaat7052Crossref PubMed Scopus (25) Google Scholar). The impact of X-ray crystallography on understanding the molecular basis for GPCR pharmacology has been enormous. A single structure of a GPCR will of course give a detailed molecular snapshot of how a ligand interacts with a receptor and the shape of the orthosteric binding pocket. In some cases, particularly if the receptor is thermostabilized, multiple structures can be determined bound to ligands of different affinity, giving an understanding in how different ligands bind. More importantly, if the ligands are of different efficacy, then their different effects on receptor structure can be monitored, suggesting a molecular basis for efficacy (Warne et al., 2011Warne T. Moukhametzianov R. Baker J.G. Nehmé R. Edwards P.C. Leslie A.G. Schertler G.F. Tate C.G. The structural basis for agonist and partial agonist action on a β(1)-adrenergic receptor.Nature. 2011; 469: 241-244Crossref PubMed Scopus (491) Google Scholar, Warne et al., 2019Warne T. Edwards P.C. Doré A.S. Leslie A.G.W. Tate C.G. Molecular basis for high-affinity agonist binding in GPCRs.Science. 2019; 364: 775-778Crossref PubMed Scopus (29) Google Scholar). Comparison between structures in an active state and an inactive state leads to an understanding of how GPCRs are activated by agonists (Weis and Kobilka, 2018Weis W.I. Kobilka B.K. The Molecular Basis of G Protein-Coupled Receptor Activation.Annu. Rev. Biochem. 2018; 87: 897-919Crossref PubMed Scopus (258) Google Scholar). The diversity of receptor structures available has led to a unified mechanism for receptor activation (Venkatakrishnan et al., 2016Venkatakrishnan A.J. Deupi X. Lebon G. Heydenreich F.M. Flock T. Miljus T. Balaji S. Bouvier M. Veprintsev D.B. Tate C.G. et al.Diverse activation pathways in class A GPCRs converge near the G-protein-coupling region.Nature. 2016; 536: 484-487Crossref PubMed Scopus (145) Google Scholar) and how the receptors in turn activate G proteins (Flock et al., 2015Flock T. Ravarani C.N.J. Sun D. Venkatakrishnan A.J. Kayikci M. Tate C.G. Veprintsev D.B. Babu M.M. Universal allosteric mechanism for Gα activation by GPCRs.Nature. 2015; 524: 173-179Crossref PubMed Scopus (152) Google Scholar). In terms of SBDD, crystallography also allows the attractive possibility of determining multiple structures of receptors bound to many different ligands. For example, multiple structures of β1AR, β2AR, and A2AR have been determined bound to 14, 14, and 19 different ligands, respectively (Table S1). In a few cases, it was also possible to soak existing crystals with ligands to obtain structures more rapidly (Rucktooa et al., 2018Rucktooa P. Cheng R.K.Y. Segala E. Geng T. Errey J.C. Brown G.A. Cooke R.M. Marshall F.H. Doré A.S. Towards high throughput GPCR crystallography: In Meso soaking of Adenosine A2A Receptor crystals.Sci. Rep. 2018; 8: 41Crossref PubMed Scopus (46) Google Scholar). However, it must be said that the throughput of structure determination of GPCRs (after the first structure has been completed) is of the order of a few per month, which is two or three orders of magnitude slower than in the most successful cases for soluble proteins (Muench et al., 2019Muench S.P. Antonyuk S.V. Hasnain S.S. The expanding toolkit for structural biology: synchrotrons, X-ray lasers and cryoEM.IUCrJ. 2019; 6: 167-177Crossref PubMed Scopus (12) Google Scholar). Although the first 20 years of structure determination of GPCRs has revolutionized our understanding of their structure and function, a parallel revolution (Vinothkumar and Henderson, 2016Vinothkumar K.R. Henderson R. Single particle electron cryomicroscopy: trends, issues and future perspective.Q. Rev. Biophys. 2016; 49: e13Crossref PubMed Google Scholar) in electron cryo-microscopy (cryo-EM) is set to galvanize the GPCR field in future years and will eventually rival X-ray crystallography in its ability to determine novel GPCR structures and drive SBDD (García-Nafría and Tate, 2020García-Nafría J. Tate C.G. Cryo-Electron Microscopy: Moving Beyond X-Ray Crystal Structures for Drug Receptors and Drug Development.Annu. Rev. Pharmacol. Toxicol. 2020; 60: 51-71Crossref PubMed Scopus (32) Google Scholar). Since 2017, more structures of GPCRs in the active state coupled to heterotrimeric G proteins have been determined by cryo-EM than by X-ray crystallography (Figure 2). This is because cryo-EM can now determine structures of smaller proteins than previously, due to developments in direct electron detectors (McMullan et al., 2016McMullan G. Faruqi A.R. Henderson R. Direct Electron Detectors.Methods Enzymol. 2016; 579: 1-17Crossref PubMed Scopus (94) Google Scholar) and computer programs for structure determination (Zivanov et al., 2018Zivanov J. Nakane T. Forsberg B.O. Kimanius D. Hagen W.J. Lindahl E. Scheres S.H. New tools for automated high-resolution cryo-EM structure determination in RELION-3.eLife. 2018; 7: e42166Crossref PubMed Scopus (1114) Google Scholar). The resolution of the cryo-EM structures of GPCRs is typically between 3.0–3.5 Å resolution, although often it is worse, especially around the orthosteric binding pocket, and structure determination and model building takes weeks, compared to hours for structure determination of X-ray crystallographic data by molecular replacement. Thus currently using cryo-EM to determine GPCR structures is best suited to the first structure determination of a novel receptor, rather than high-throughput structure determination for SBDD, although some targets that are more stable and rigid may be more amenable for high-throughput approaches (e.g., Masiulis et al., 2019Masiulis S. Desai R. Uchański T. Serna Martin I. Laverty D. Karia D. Malinauskas T. Zivanov J. Pardon E. Kotecha A. et al.GABAA receptor signalling mechanisms revealed by structural pharmacology.Nature. 2019; 565: 454-459Crossref PubMed Scopus (169) Google Scholar). There is, however, no doubt that cryo-EM will continue to improve and will be able to solve the structures of smaller proteins at higher resolution in the foreseeable future. Current therapeutics target only ∼25% of potentially druggable GPCRs expressed in humans. There are now 103 GPCR targets (out of a possible 403) for which there is at least one marketed agent in clinical practice (Table S2). Of these, 41 are agonists, 27 antagonists, and both modalities are used for 35 targets (Figure 3). However, because there are 403 GPCR targets of possible therapeutic utility, there is the potential total of ∼800 targetable profiles in either agonist (or positive modulator) or antagonist (or negative modulator) mode. Therefore, it might be argued that in fact less than 13% of the potentially useful therapeutic modalities of the family have been “drugged” to date. This leaves enormous potential for further discovery of therapeutic agents, now that we have entered an era in which structure determination of GPCRs is tractable. Furthermore, the outline above does not acknowledge the complexity of GPCR biology and the opportunities for much more subtle modulation of each system via partial or biased agonism or by physiological isolation of effects by topical administration (e.g., to the lung or skin). The pedigree of GPCRs as a drug discovery target class is generally well accepted, and new drugs have continued to come to the market year on year from this target class (Christopher et al., 2017Christopher J.A. Congreve M. Deflorian F. Marshall F.H. Advances and Insights from CNS G Protein-Coupled Receptor Crystallography.in: Bronson J.L. Medicinal Chemistry Reviews. American Chemical Society-Medicinal Chemistry Division, 2017: 69-87Google Scholar). In the last 5 years, 41 drugs that target GPCRs have been approved by the Food and Drug Administration (FDA) (excluding diagnostic agents), which represents 19% percent of the total drugs approved during that period