Small Molecule Allosteric Modulators of G-Protein-Coupled Receptors

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Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX

Small Molecule Allosteric Modulators of G‑Protein-Coupled Receptors: Drug−Target Interactions Shaoyong Lu and Jian Zhang* Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China

ABSTRACT: G-protein-coupled receptors (GPCRs) are the largest class of signaling receptors that are most frequently targeted by therapeutic drugs. Allosteric modulators bound to GPCRs at allosteric sites provide the potential for differential selectivity and improved safety compared with traditional orthosteric ligands. The recent breakthroughs in GPCR structural biology have made structures of GPCRs from classes A, B, C, and F complexed with small-molecule allosteric modulators available. Knowledge of the detailed receptor−modulator interactions at the allosteric sites is useful for structure-based GPCR drug design of novel therapeutics. This Perspective comprehensively summarizes the current status of structural complexes between GPCRs and their small-molecule allosteric modulators, particularly the key receptor−modulator interactions at the allosteric sites. Then, the structural diversity of allosteric sites across four GPCR subfamilies is compared. This study is expected to contribute to the design of GPCR allosteric drugs with an improved therapeutic action. of highly selective compounds for unique GPCR subtypes.17−19 Furthermore, in the class B GPCRs where endogenous ligands comprise peptides, the development of drug-like non-peptide molecules is challenging due to the flat, large orthosteric sites, which are pharmacologically intractable since a small molecule cannot bind tightly.20−22 A possible solution to this challenge is the design of selective allosteric modulators for the specific GPCR subtypes through binding to their allosteric sites,23−31 which are spatially and topographically distinct from their orthosteric sites. The sequence and structural diversity of allosteric sites in a single GPCR subfamily allow for the development of selective allosteric GPCR modulators. Moreover, allosteric modulators do not compete with endogenous ligands bound to orthosteric sites. Instead, they exert their effects on a receptor in concert with the endogenous orthosteric ligands, thereby allowing modulation of the receptor activity rather than completely shutting it off and potentially alleviating the chances of side effects.32−35 However, the discovery of allosteric modulators faces challenges, including

1. INTRODUCTION G-protein-coupled receptors (GPCRs), encoded by ∼1000 genes, constitute the largest family of membrane receptors shared by conserved seven-transmembrane (7TM) helices, which are connected by three extracellular loops (ECL1−3) and three intracellular loops (ICL1−3).1−3 GPCR-mediated signal transduction is triggered by various endogenous ligands, including neurotransmitters, hormones, peptides, chemokines, lipids, purines, ions, photons, and odorants.4,5 Dysregulation of GPCR signaling is associated with a broad spectrum of human disorders and diseases, such as central nervous system disorders, cancers, and cardiac, metabolic, and inflammatory diseases.6−9 Therefore, GPCRs are important targets in drug development.10−13 The GPCR kingdom is classified into four major subfamilies on the basis of sequence similarity:14−16 class A (the rhodopsin family), class B (the secretin and adhesin family), class C (the glutamate family), and class F (the frizzled/taste family). Class A is the prototypical family of GPCR superfamily that includes the largest number of members. Traditional GPCR drug discovery has focused on targeting the orthosteric sites, where the endogenous ligands are bound. The highly conserved orthosteric sites located in the extracellular or 7TM domains across members of a single GPCR subfamily hinder the design © XXXX American Chemical Society

Special Issue: Allosteric Modulators Received: December 14, 2017 Published: February 19, 2018 A

DOI: 10.1021/acs.jmedchem.7b01844 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Perspective

Table 1. Solved Crystal Structures of GPCRs in Complex with Small-Molecule Allosteric Modulators released year

PDB code

resolution (Å)

allosteric modulator

CCR5 CCR2 CCR9 M2 GPR40 GPR40 GPR40 P2Y1 β2AR PAR2

2013 2016 2016 2016 2014 2017 2017 2015 2017 2017

4MBS 5T1A 5LWE 4MQT 4PHU 5TZR 5TZY 4XNY 5X7D 5NDZ

2.71 2.81 2.8 3.7 2.33 2.2 3.22 2.3 2.7 3.6

maraviroc CCR2-RA-[R] vercirnon LY2119620 TAK-875 MK-8666 MK-8666, AP8 BPTU Cmp-15PA AZ3451

7TM helical bundle (I−III, V−VII), extracellular side 7TM helical bundle (I−III, VI−VIII), intracellular side 7TM helical bundle (I−III, VI−VIII), intracellular side 7TM helical bundle (II, VI, VII, ECL2, ECL3), extracellular side outside 7TM helical bundle (III−V, ECL2), extracellular side outside 7TM helical bundle (III−V, ECL2), extracellular side AP8 is outside 7TM helical bundle (II−V, ICL2), intracellular side outside 7TM helical bundle (I−III, ICL1), intracellular side 7TM helical bundle (I, II, VI−VIII, ICL1), intracellular side outside 7TM helical bundle (II−IV), extracellular side

57 58 59 18 60 61 61 62 63 64

CRF1R GCGP GLP-1R GLP-1R

2013 2015 2017 2017

4K5Y 5EE7 5VEW 5VEX

2.98 2.5 2.7 3.0

CP-376395 MK-0893 PF-06372222 NNC0640

7TM helical bundle (III, V, VI), intracellular side outside 7TM helical bundle (VI, VII), intracellular side outside 7TM helical bundle (V−VII), intracellular side outside 7TM helical bundle (V−VII), intracellular side

65 66 67 67

mGlu5 mGlu5 mGlu1

2014 2015 2014

4OO9 5CGD 4OR2

2.6 2.6 2.8

mavoglurant HTL14242 FITM

7TM helical bundle (II, III, V−VII), extracellular side 7TM helical bundle (II, III, V−VII), extracellular side 7TM helical bundle (II, III, V−VII, ECL2), extracellular side

68 69 70

SMO

2016

5L7I

3.3

vismodegib

7TM helical bundle (I, II, V−VII, LD, ECL2, ECL3), extracellular side

71

GPCR

allosteric site

refs

class A

class B

class C

class F

shallow allosteric modulator SARs (structure−activity relationships),36 low binding affinities of allosteric modulators,37 difficulty in incorporating polar and solubilizing groups (lowering log P) into allosteric modulators,36 the emergence of drug-resistant mutations,38 and the high diversity of allosteric sites in species homologs.39 Despite the challenges in the discovery of allosteric modulators, allosteric modulation is currently regarded as a unifying mechanism for receptor function and regulation owing to its considerable advantage;40 it is also a novel strategy for GPCR drug discovery.41,42 The repertoire of allosteric GPCR modulators has experienced an upsurge with an exponential growth in the number of allosteric GPCR modulators in recent years.43−49 In structure-based drug design, the identification of allosteric sites in GPCRs where allosteric modulators can bind is a prerequisite for discovering a large number of potent and selective drugs, and X-ray crystallography has been the primary way to solve the 3D structures of allosteric GPCR−smallmolecule modulator complexes. However, a long-standing conundrum in GPCR structure determination concerns the significant conformational plasticity of GPCRs that challenges receptor crystallization.50 The substantial technology breakthroughs in receptor engineering and crystallization in the past few years have facilitated the determination of many crystal structures of classes A, B, C, and F GPCRs in complex with their allosteric modulators.51−56 The structures of GPCRs solved in antagonist-bound inactive conformations and agonistbound multiple (intermediate, “active-like”, and active) conformations provide a mechanistic understanding of agonist-dependent GPCR activation. Notably, structural insight into the detailed receptor−ligand interactions at the allosteric sites is useful for structure-based GPCR drug design of novel therapeutics. In this Perspective, we summarize the current status of GPCR structures that have been solved with their allosteric small-molecule modulators. The X-ray structural complexes of

GPCRs and their allosteric small-molecule modulators are categorized in accordance with the GPCR subfamilies. We mainly focus on the key receptor−ligand interactions at the allosteric sites and will not outline the effect of allosteric modulators on the cooperativity of orthosteric ligand binding and signaling. Moreover, the structural diversity of allosteric sites across all GPCR subfamilies is analyzed. Results of this study contribute to the design of GPCR allosteric drugs with therapeutic activity.

2. SOLVED STRUCTURAL COMPLEXES OF GPCRS AND ALLOSTERIC SMALL-MOLECULE MODULATORS To date, 18 GPCR crystal structures in complex with their allosteric small-molecule modulators are solved (Table 1), including 10 class A, 4 class B, 3 class C, and 1 class F GPCRs. The corresponding small-molecule allosteric modulators are shown in Figures 1−4. The 10 crystal complexes from class A include CC chemokine receptor 5 (CCR5) bound to maraviroc (PDB code 4MBS),57 CC chemokine receptor 2 (CCR2) bound to CCR2-RA-[R] (PDB code 5T1A),58 CC chemokine receptor 9 (CCR9) bound to vercirnon,59 GPR40 bound to TAK-875 (PDB code 4PHU),60 MK-8666 (PDB code 5TZR),61 or MK-8666 and AP8 (PDB code 5TZY),61 P2Y1 purinergic receptor bound to BPTU (PDB code 4XNV),62 β2 adrenergic receptor (β2AR) bound to Cmp-15PA (PDB code 5X7D),63 protease-activated receptor 2 (PAR2) bound to AZ3451 (PDB code 5NDZ),64 and muscarinic 2 acetylcholine receptor (M2R) bound to LY2119620 (PDB code 4MQT).18 The 4 crystal complexes from class B include corticotropinreleasing factor 1 receptor (CRF1R) bound to CP-376395 (PDB code 4K5Y),65 glucagon receptor (GCGR) bound to MK-0893 (PDB code 5EE7),66 and glucagon-like peptide-1 receptor (GLP-1R) bound to PF-06372222 (PDB code 5VEW)67 or NNC0640 (PDB code 5VEX).67 The 3 crystal complexes from class C include metabotropic glutamate 5 B



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Figure 1. Chemical structures of small-molecule allosteric modulators bound to class A GPCRs.

(mGlu5) receptor bound to mavoglurant (PDB code 4OO9)68 or HTL14242 (PDB code 5CGD)69 and mGlu1 bound to FITM (PDB code 4OR2).70 The only one crystal complex from class F is smoothened receptor (SMO) bound to vismodegib (PDB code 5L7I).71

that disrupt CCR5-gp120 protein−protein interactions is a promising strategy for the treatment of HIV-1 infection.75,76 Maraviroc (Figure 1) is an anti-HIV drug available in the market.77 It can disrupt the CCR5-gp120 interaction by attaching to CCR5 through an allosteric mechanism.78 The determination of the cocrystal complex of CCR5 and maraviroc shows that maraviroc occupies an extracellular cavity on top of the 7TM helical bundle from the helices I−III and V−VII (Figure 5A)57 and does not interact with the ECL2, which is among the major binding determinants for CCR5 chemokine agonists. In the maraviroc binding site (Figure 5B), Glu2837.39 (superscript represents residue number in accordance with Ballesteros−Weinstein nomenclature79) forms a salt bridge with the protonated nitrogen of the tropane group of maraviroc. Tyr2516.51 forms a hydrogen bond with the carboxamide nitrogen of the ligand. Tyr371.39 forms a hydrogen bond with the amine of the triazole group of the ligand. Thr1955.39 and Thr2596.59 are involved in a hydrogen bond with one of the fluorines in the cyclohexane ring of the ligand. For

3. CLASS A ALLOSTERIC GPCR−SMALL-MOLECULE MODULATOR INTERACTIONS 3.1. CCR5−Maraviroc Structure. CC chemokine receptors specifically bind and respond to cytokines of the CC chemokine family.72 They contain 19 members of the CC chemokine receptor subfamily. As one of the members, CCR5 can bind and subsequently be activated by endogenous CC chemokine ligands (CCLs), such as CCL3 and CCL4.73 It forms a co-receptor with the viral envelope glycoprotein gp120; this co-receptor is required for human immunodeficiency virus type 1 (HIV-1) infectivity.74 Therefore, designing inhibitors C



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Figure 2. Chemical structures of small-molecule allosteric modulators bound to class B GPCRs.

Figure 3. Chemical structures of small-molecule allosteric modulators bound to class C GPCRs.

of CCR5 in an inactive conformation that blocks chemokine and gp120 binding. 3.2. CCR2−CCR2-RA-[R] Structure. CCR2 is a member of the CC chemokine receptor subfamily. CCR2, which is a key regulator, mediates the activation and migration of inflammatory monocytes in response to endogenous CCLs, such as CCL2.73 CCR2 is therefore a target for therapeutic intervention in a plethora of inflammatory and neurodegenerative diseases, including multiple sclerosis, atherosclerosis, pulmonary fibrosis, and diabetic kidney disease.80 CCR2-RA-[R] (Figure 1) is a NAM of CCR2 with good in vitro activity (IC50 = 0.17 μM) for CCR2, good selectivity profiles against the two most homologous CCR1 and CCR5, and an excellent DMPK profile.81 The determination of the cocrystal structure of CCR2 and its allosteric CCR2-RA-[R] antagonist, together with its orthosteric BMS-681 antagonist (Figure 6), shows that CCR2-RA-[R] occupies an allosteric site on the intracellular side, which is more than 30 Å away from the orthosteric site on the extracellular side where BMS-681 is bound (Figure 7A).58 The allosteric site of CCR2-RA-[R] is formed by residues from the intracellular ends of helices I−III and VI−VIII (Figure 7B). This site partially overlaps with the binding site of G-protein in homologous receptors, thereby suggesting that CCR2-RA-[R] binding sterically prevents the

Figure 4. Chemical structures of a small-molecule allosteric modulator bound to class F GPCR.

the hydrophobic interactions, the cyclohexane ring of the ligand is located inside the hydrophobic pocket formed by Gln1945.38, Ile1985.42, Leu2556.55, and Met2797.35; the phenyl group protrudes into a deep hydrophobic cleft formed by a cluster of aromatic residues, Tyr1083.32, Phe1093.33, Phe1123.36, Trp2486.48, and Tyr2516.51; and the triazole group is engaged in hydrophobic interactions with Trp862.60, Tyr892.63, and Met2877.43. The allosteric mechanism of maraviroc-mediated inhibition of chemokine signaling may involve the stabilization D



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Figure 5. (A) Extracellular view of the crystal structure of CCR5-maraviroc complex (PDB code 4MBS). The CCR5 is light pink, and the ECL2 is lime colored. Maraviroc is shown in stick representation. (B) Key residues in CCR5 for maraviroc binding. Salt bridge and hydrogen bonds are green and yellow dotted lines, respectively. Residues are numbered in accordance with Ballesteros−Weinstein nomenclature.

chemokine CCL2 from binding to CCR2, whereas the orthosteric antagonists directly compete with chemokine binding and indirectly prevent G-protein coupling.82 These findings are in good agreement with those of structural analysis. Thus, the existence of cooperativity between the allosteric CCR2-RA-[R] and orthosteric BMS-681 antagonists significantly stabilizes the CCR2 in its inactive conformation. 3.3. CCR9−Vercirnon Structure. CCR9, which is another member of the CC chemokine receptor subfamily, mediates leukocyte recruitment to the gut after activation by endogenous CCLs, such as CCL25.73 Thus, it is implicated in inflammatory bowel disease.83 Vercirnon (Figure 1) is a selective allosteric antagonist of CCR9 that has entered phase III clinical trials for the treatment of Crohn’s disease, but its efficacy is limited because very high doses are required to inhibit CCR9 activation.83 The cocrystal structure of CCR9 in complex with vercirnon has recently been determined.59 The structure shows that vercirnon binds to the intracellular side of the CCR9 (Figure 8A), which is approximately 33 Å away from the orthosteric site. The allosteric site of vercirnon is formed by residues from the intracellular ends of helices I−III and VI−VIII (Figure 8B); this

Figure 6. Chemical structure of BMS-681.

G-protein from binding to CCR2. In the allosteric site (Figure 7C), the hydroxyl group of CCR2-RA-[R] forms a bifurcated hydrogen bond with the backbone amines of Glu3108.48 and Lys3118.49, and the pyrrolone carbonyl group is hydrogenbonded to the backbone amide of Phe3128.50. For the hydrophobic interactions, the phenyl group contacts with Val631.53, Leu671.57, Leu812.43, Tyr3057.53, and Phe3128.50, and the cyclohexane ring interacts with Leu812.43, Leu1343.46, Ala2416.33, Val2446.36, and Ile2456.37. Previous pharmacological investigations of small-molecule CCR2 antagonists have revealed that the allosteric CCR2-RA-[R] antagonist directly hinders G-protein coupling and allosterically prevents the

Figure 7. (A) Overview of the cocrystal structure of allosteric CCR2-RA-[R] and orthosteric BMS-681 antagonist-bound CCR2 (PDB code 5T1A). (B) Intracellular view of allosteric CCR2-RA-[R] binding site. (C) Key residues in CCR2 for CCR2-RA-[R] binding. Hydrogen bonds are yellow dotted lines. E



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Figure 8. (A) Overview of the cocrystal structure of an allosteric vercirnon antagonist-bound CCR9 (PDB code 5LWE). (B) Intracellular view of allosteric vercirnon binding site. (C) Key residues in CCR9 for vercirnon binding. Hydrogen bonds are yellow dotted lines.

Figure 9. (A) Overview of the cocrystal structure of an allosteric LY2119620 agonist- and orthosteric iperoxo agonist-bound M2 receptor (PDB code 4MQT). The M2 receptor is light pink, and the ECL2 is lime colored. The ECL3 is orange colored. LY2119620 and iperoxo are shown in stick representations. (B) Intracellular view of allosteric LY2119620 binding site. (C) Key residues in M2 receptor for LY2119620 binding. Charge− charge interaction and hydrogen bonds are green and yellow dotted lines, respectively. (D) Comparison of M2 receptor structure with (light pink) and without (light cyan, PDB code 4MQS) LY2119620 bound. The extracellular vestibule, particularly for the ECL3, shows a subtle conformational change upon LY2119620 binding.

of the CCR9 and forms a bifurcated hydrogen bond with the side chains of Arg78 and Thr81 from the ICL1. The ketone group is hydrogen bonded to the side chain of Thr2566.37. For the hydrophobic interactions, the tert-butylphenyl group makes numerous contacts with the hydrophobic cleft formed by Val691.53, Val721.56, Tyr731.57, Leu872.42, Tyr3177.53, and

position of vercirnon bound to CCR9 is similar to that of CCR2-RA-[R] bound to CCR2. In the allosteric vercirnon binding site (Figure 8C), the sulfone group of vercirnon is engaged with the hydrogen bonding interactions with the backbone amine of Glu3228.48, Arg3238.49, and Phe3248.50. The pyridine-N-oxide group protrudes toward the intracellular face F



Perspective

Figure 10. (A) Extracellular view of the crystal structure of GPR40−TAK-875 complex (PDB code 4PHU). The GPR40 is light pink, and the ECL2 is in lime. TAK-875 is shown in stick representation. (B) Key residues in GPR40 for TAK-875 binding. Salt bridge and hydrogen bonds are green and yellow dotted lines, respectively.

Phe3248.50. The chlorophenyl group is located in a narrow, hydrophobic cleft surrounded by residues Leu872.43, Ile1403.46, Val2596.40, and Tyr3177.53, whereas the pyridine-N-oxide group is located in a polar cavity surrounded by residues Thr832.39, Asp842,40, Arg1443.50, Arg3238.49, and Thr81 from the ICL1. Structural analysis of the allosteric antagonism of vercirnon suggests that similar to CCR2-RA-[R], vercirnon binds to the intracellular side of CCR9 and subsequently constrains the intracellular half of the CCR9 in an inactive conformation. This condition is sterically incompatible with G-protein or arrestin binding. 3.4. M2−LY2119620 Structure. Muscarinic acetylcholine receptors consisting of five subtypes, M1−M5, are activated by endogenous acetylcholine to regulate a large body of peripheral and central physiological functions in the human body.84 The M2 muscarinic acetylcholine receptor (M2 receptor) is mainly located in the heart and plays an important role in modulating cardiac function.85 The orthosteric acetylcholine binding site of the M2 receptor is located in a small cavity formed by the 7TM helical bundles. LY2119620 (Figure 1) is a high-affinity M2/M4 receptorselective positive allosteric modulator (PAM) that modulates the muscarinic-agonist iperoxo at the M2/M4 receptor subtypes.86 Recently, X-ray crystal structures of the M2 receptor bound to a high-affinity, orthosteric agonist iperoxo alone and in combination with LY2119620 have been solved to elucidate the atomic details of the allosteric binding site and the structural basis underlying the allosteric mechanism of the M2 receptor.18 The structure of the M2−iperoxo−LY2119620 ternary complex shows that the allosteric LY2119620 agonist binds to the extracellular vestibule of the receptor, which is directly above the orthosteric iperoxo binding site (Figure 9A). The allosteric binding site of LY2119620 is formed by residues from the extracellular side of helices II, VI, and VII and the ECL2 and ECL3 (Figure 9B). In contrast to the maraviroc in the allosteric binding site (extracellular side) of CCR5 that does not come in contact with the ECL2, LY2119620 extensively interacts with the ECL2 and ECL3, thereby suggesting that LY2119620 is proximal to the lipid layer. In the allosteric LY2119620 binding site (Figure 9C), the piperidine group is engaged in a charge−charge interaction with Glu172 from the

ECL2; the amide oxygen and nitrogen make hydrogen bonds with the side chains of Tyr802.61 and Asn419 from the ECL3, respectively; the bridging oxygen accepts a hydrogen bond from the side chain of Asn4106.58. In addition to polar contacts, the hydrophobic interactions between M2 receptor and LY2119620 are mainly from the aromatic residues, including Tyr802.61, Tyr832.64, Trp4227.35, Tyr4267.39, and Tyr177 from the ECL2. Superimposition of the crystal structures between the M2− iperoxo−LY2119620 ternary complex and the M2−iperoxo binary complex demonstrates that the allosteric LY2119620 binding site shows no appreciable difference in the two structures in the presence or absence of LY2119620 except a subtle change in the extracellular vestibule in response to LY2119620 binding (Figure 9D). This comparison indicates that the allosteric binding site of M2 receptor is largely preformed in the absence of the orthosteric iperoxo agonist. Structural comparison of the two structures suggests that the allosteric agonism of LY2119620 involves the stabilization of the closed extracellular vestibule by LY2119620. This condition may in turn directly stabilize the open, active conformation of the intracellular side of helix VI. 3.5. GPR40−TAK-875, GPR40−MK-8666, and GPR40− MK-8666−AP8 Structures. Human GPR40, also named free fatty acid (FFA) receptor 1, is predominantly expressed in pancreatic β cells and intestinal enteroendocrine cells and is activated by physiological long-chain FFAs, such as linoleic and palmitic acids.87 The activation of GPR40 by FFAs enables promotion of glucose-mediated insulin secretion. Thus, development of GPR40 full or partial agonists is an important strategy for the treatment of type 2 diabetes mellitus.88,89 TAK-875 (Figure 1) or fasiglifam, an orally available, selective GPR40 partial agonist, enhances the agonistic activity of the endogenous FFAs by attaching to an allosteric site of GPR40.90 It previously reached phase III clinical trials for the treatment of type 2 diabetes mellitus, but its use was terminated in further clinical trials due to toxicity. The cocrystal structure of GPR40 bound to TAK-875 shows that TAK-875 is positioned in a noncanonical site formed by helices III−V and the ECL2 (Figure 10A);60 this site is adjacent to the exterior membrane surface. In the allosteric TAK-875 binding site (Figure 10B), the carboxylate group of TAK-875 is engaged G



Perspective

Figure 11. (A) Overview of the ternary complex structure of a full allosteric AP8 agonist-bound and partial allosteric MK-8666 agonist-bound GPR40 receptor (PDB code 5TZY). The GPR40 is light pink, and the ICL2 is lime colored. AP8 and MK-8666 are shown in stick representations. (B) Key residues in GPR40 for AP8 binding. Hydrogen bonds are yellow dotted lines. (C) Structural comparisons of the ternary GPR40−MK8666−AP8 complex structure (light pink) versus the binary GPR40−MK-8666 (PDB code 5TZR) complex structure (cyan).

Figure 12. (A) Overview of the cocrystal structure of an allosteric BPTU antagonist-bound P2Y1 receptor (PDB code 4XNV). The P2Y1 receptor is in light pink, and the ICL2 is in lime. BPTU is shown in stick representation. (B) Key residues in P2Y1 for BPTU binding. Hydrogen bonds are yellow dotted lines.

in salt bridge interactions with the side chains of Arg1835.39 and Arg2587.35 and hydrogen binding interactions with the side chains of Tyr913.37 and Tyr2406.51. The dihydrobenzofuran group is encapsulated by residues Ala833.29, Phe873.33, Leu1384.57, Phe1424.61, Leu171ECL2, and Trp174ECL2 defining a hydrophobic pocket. The benzyl group is situated between helices III and IV and forms hydrophobic interactions with residues Val843.30, Leu1354.54, and Phe1424.61. The remaining moiety of TAK-875 is exposed outside the receptor. In particular, the terminal sulfonate group is not required for the agonistic activity but is responsible for reducing lipophilicity and improving the ADMET properties of TAK-875. Unlike TAK-875 that is a partial allosteric agonist of GPR40, AP8 (Figure 1) is a full allosteric agonist (AgoPAM) of GPR40 that exhibits higher potency and stronger positive functional cooperativity with endogenous FFAs than partial allosteric agonists, such as TAK-875 and MK-8666 (Figure 1).91 Radioligand binding interaction studies have revealed the

presence of positive functional cooperativity between AgoPAMs and partial allosteric agonists and that mutagenesis of specific residues responsible for binding of partial allosteric agonists exerts no effect on AgoPAMs binding.91 These results suggest the existence of multiple ligand-binding sites in GPR40 for binding distinct allosteric agonists. Very recently, two cocrystal structures of GPR40 bound to a partial allosteric agonist MK-8666 (similar to TAK-875) and a AgoPAM AP8 and GPR40 bound to MK-8666 alone are solved.61 The GPR40−MK-8666−AP8 ternary complex structure unequivocally demonstrates that the AgoPAM AP8 binds to a welldefined lipid-facing pocket formed by helices II−V and ICL2; this AgoPAM AP8 binding site is outside the intracellular halves of the TM helical bundle (Figure 11A), and this site is completely distinct from that of the partial allosteric agonist MK-8666 located in the extracellular halves of TM helical bundle. The MK-8666 binding site is analogous to the TAK875 binding site. In the allosteric AP8 binding site (Figure H



Perspective

Figure 13. (A) Overview of the cocrystal structure of an allosteric Cmp-15PA antagonist-bound and orthosteric carazolol agonist-bound β2AR (PDB code 5X7D). The β2AR is light pink, and the ICL1 is lime. Cmp-15PA and carazolol are shown in stick representations. (B) Intracellular view of allosteric Cmp-15PA binding site. (C) Key residues in β2AR for Cmp-15PA binding. Hydrogen bonds are yellow dotted lines.

and ICL1 (Figure 12A).62 In the allosteric BPTU binding site (Figure 12B), only polar interactions are observed between the urea group of BPTU and P2Y1, in which the NHs of the BPTU’s urea group form two hydrogen bonds with the backbone carboxyl of Leu1022.55. The two hydrogen bonds are important for BPTU binding because replacement of the urea linker by two or four other atom linkers results in a significantly decreased level of binding affinity. For the hydrophobic interactions, the pyridyl group of BPTU comes in contact with residues Ala1062.59 and F119ICL1. The benzene ring within the phenoxy group of BPTU is situated in a hydrophobic subpocket formed by residues from helices II and III, including Thr1032.56, Met1233.24, Leu1263.27, and Gln1273.28; by contrast, the tert-butyl substituent on the phenoxy group engages in a hydrophobic interaction with Leu1022.55. At the opposite side of the ligand, the ureido phenyl ring is involved in aromatic− aromatic interactions with Phe621.43 and Phe661.47. To account for the mechanism of allosteric antagonism of P2Y1 receptor by BPTU, all-atom long-time-scale molecular dynamics (MD) simulations were performed.92 The simulations showed that BPTU binding to the P2Y1 receptor stabilizes the extracellular helical bundles to increase the lipid order, thereby stabilizing the ionic lock between Lys461.46 and Arg195ECL2 and restraining the receptor in an inactive state. 3.7. β2AR−Cmp-15PA Structure. The β2AR, which is an extensively characterized member of the GPCR family, is largely expressed in bronchial smooth muscle and triggers bronchodilation when activated by its endogenous ligands, such as epinephrine and norepinephrine.96 Thus, β2AR is an important target for the treatment of cardiovascular and pulmonary diseases.97 Cmp-15 (Figure 1) is a selective NAM of β2AR with low micromolar affinity, which was identified from DNA-encoded small-molecule libraries comprising 190 million distinct compounds.98 It displays negative cooperativity with an orthosteric agonist but positive cooperativity with an orthosteric inverse agonist. Pharmacological and biochemical characterizations of Cmp-15 reveal that it binds to the intracellular surface of the β2AR, which is distinct from known β2AR orthosteric ligands for binding at the orthosteric site in the extracellular side. A polyethylene glycol−carboxylic acid derivative of Cmp-15, designated as Cmp-15PA (Figure 1), has been further synthesized to improve the occupancy of Cmp-15 in crystallized β2AR. The determination of the cocrystal structure of β2AR bound to an allosteric Cmp-15PA

11B), the carboxylate group of AP8 accepts three hydrogen bonds from the side chains of Tyr442.42, Ser1234.42, and Tyr114ICL2. The methyl and cyclopropyl groups engage in hydrophobic and aromatic interactions with residues Leu1063.52, Tyr114ICL2, Phe117ICL2, and Tyr1224.41. The piperidine-chroman group makes hydrophobic contacts with residues Ala993.45, Ala1023.48, Val1264.45, and Ile1975.53. The terminal trifluoromethoxyphenyl group fits into a hydrophobic pocket formed by residues Ile1304.49, Leu1334.52, Val1344.53, Leu1905.46, and Leu1935.47. In the AP8-bound GPR40 structure, one side of AP8 interacts with the receptor, whereas the other side interacts with the lipid bilayer, which constitutes the outer surface of the AP8-binding site. AP8 is completely sequestered in a hydrophobic environment. Superimposition between the GPR40−MK-8666−AP8 ternary complex structure and the GPR40−MK-8666 binary complex structure reveals significant conformational differences in the regions of ICL2, helices IV and V, and ECL2 (Figure 11C), which are the engagement with the two agonists. The ICL2 that constitutes the AP8 binding site adopts a short helical conformation in the ternary complex structure, which is disordered in the binary complex structure. This condition indicates that the AP8-binding site of GPR40 is an “induced-fit” pocket. The conformational rearrangements of ICL2 and helices IV and V upon AgoPAM AP8 binding promote GPR40 in an active-like state and can explain cooperative allosteric regulation of GPR40 between the partial allosteric agonist MK-8666 and the AgoPAM AP8. 3.6. P2Y1−BPTU Structure. The human purinergic P2Y1 receptor is activated by the endogenous agonist adenosine 5′diphosphate (ADP) to facilitate platelet aggregation; thus, it is an important drug target for the treatment of thrombosis.92,93 Inhibition of the P2Y1 receptor decreases ADP-induced platelet aggregation; therefore, P2Y1 receptor antagonists provide a promising strategy for the development of antithrombotic drugs.94 BPTU (Figure 1), which is a non-nucleotide small-molecule P2Y1 allosteric antagonist, dramatically reduces platelet aggregation in a rat arterial thrombosis model with a minor effect on bleeding and exhibits a good selectivity profile against the highly homologous P2Y12 receptor.95 The determination of the P2Y1−BPTU binary complex structure shows that BPTU binds to the external receptor surface at the interface of receptor/lipid bilayer, which is outside the 7TM helical bundle; this BPTU binding site is distinct from the endogenous ADPbinding site and is formed largely by residues from helices I−III I



Perspective

Figure 14. (A) Overview of the cocrystal structure of an allosteric AZ3451 antagonist-bound PAR2 (PDB code 5NDZ). The PAR2 is light pink, and AZ3451 is shown in stick representation. (B) Key residues in PAR2 for AZ3451 binding. Hydrogen bonds are yellow dotted lines.

cocrystal structure of PAR2 bound to AZ3451 shows that AZ3451 binds to a remote allosteric site outside the helical bundle (Figure 14A), which is distinct from an orthosteric antagonist AZ8838 located near the extracellular surface.64 The allosteric AZ3451 binding site of PAR2 is lined by residues from helices II−IV. The only hydrogen bond in the allosteric binding site (Figure 14B) is formed between the benzimidazole nitrogen of AZ3451 and the side chain of Tyr2104.61. For the hydrophobic interactions, the central benzimidazole moiety of AZ3451 engages in a hydrophobic interaction with Leu2034.54. The 1,3-benzodioxole ring is located inside a hydrophobic pocket, which is defined by residues Ala1202.49, Leu1232.52, Phe1543.31, Ala1573.34, Cys1613.38, Trp1994.50, and Ile2024.53. The cyclohexyl ring protrudes into the binding site and is positioned between the interface of the lipid layer and the receptor, which contacts with Leu1232.52. The benzonitrile group with the cyclohexyl ring also sits at the interface of the lipid layer and the receptor and forms an aromatic−aromatic interaction with Tyr2104.61. The allosteric antagonism of PAR2 by AZ3451 is proposed based on the solved binary PAR2− AZ3451 complex structure. AZ3451 binding to the allosteric site may restrain the interhelical conformational rearrangement, which is required for receptor activation as performed by agonist binding.

antagonist and orthosteric carazolol agonist clearly shows that Cmp-15PA is located at the intracellular surface of the β2AR, which is spatially distant from the orthosteric site for binding of carazolol (Figure 13A).63 The allosteric Cmp-15PA binding site for β2AR is primarily formed by residues from the helices I, II, and VI−VIII and the ICL1 (Figure 13B), which is similar to that of the allosteric antagonist CCR2-RA-[R] for CCR2 and the allosteric antagonist vercirnon for CCR9. However, the electron density for the polyethylene glycol−carboxylic acid moiety of Cmp-15PA is invisible in the binding site. This condition reflects the disordered property of this moiety in the ternary β2AR−Cmp-15PA−carazolol complex structure. In the allosteric Cmp-15PA binding site (Figure 13C), Cmp-15PA forms hydrogen bonding interactions with the side chains of Asn692.40, Thr2746.36, Ser3298.47, and Asp3318.49 and a cation−π interaction with the Arg63ICL1 via the bromobenzyl ring. The cyclohexylmethylbenzene group is located inside a hydrophobic pocket surrounded by residues Val541.53, Ile581.57, Leu64ICL1, Ile722.43, Leu2756.37, Tyr3267.53, and Phe3328.50. A comparison of the ternary β2AR−Cmp-15PA−carazolol complex structure with the inactive- and active-state structures of β2AR reveals that Cmp-15PA binding stabilizes the helix VI in an inactive conformation and sterically prevents β2AR from coupling with the G protein Gs. 3.8. PAR2−AZ3451 Structure. Protease-activated receptors (PARs), as a unique family of GPCRs, are not activated by endogenous ligands but rather activated by the cleavage of their N terminus at a specific site by proteases such as trypsin, tryptase, and factor Xa/XVIIa.99 The newly formed N-terminus, also called a tethered ligand, subsequently binds to the receptor to initiate receptor activation. PARs have four subtypes, namely, PAR1, PAR2, PAR3, and PAR4. PAR2 is mainly expressed on epithelial and endothelial cells and plays an important role in physiological processes, including immunity, tissue metabolism, and respiratory and gastrointestinal functions.100 Therefore, it is implicated in a wealth of diseases, such as inflammation, metabolic dysfunction, multiple sclerosis, and cancer.100,101 AZ3451 (Figure 1) is a small-molecule antagonist of PAR2 that is highly lipophilic. It had an acceptable in vitro activity (IC50 < 2.5 nM) for PAR2 and sound selectivity profiles against the two highly homologous PAR1 (IC50 > 50 μM) and PAR4 (IC50 = 0.38 μM).64 Recently, the determination of the

4. CLASS B ALLOSTERIC GPCR−SMALL-MOLECULE MODULATOR INTERACTIONS 4.1. CRF1R−CP-376395 Structure. CRF1R is a member of the class B GPCR that is activated by the corticotropinreleasing factor (CRF), which is a 41 amino acid peptide and plays a key role in the regulation of the hypothalamic−pituitary axis.102 CRF1R is expressed in brain areas, including the hypothalamus, pituitary, and amygdala and cortex.103 Physiological and pathophysiological studies of the CRF system in animal models and humans indicate that CRF1R antagonists provide a therapeutic potential for the treatment of depression, anxiety, and other stress-related disorders.104−106 CP-376395 (Figure 2), as a non-peptide small-molecule CRF1R antagonist, demonstrated strong potency in several in vivo models and reduced fed-fasted food effects.105 A cocrystal structure of CRF1R−CP-376395 complex was solved to understand the detailed molecular interactions between J



Perspective

Figure 15. (A) Overview of the cocrystal structure of an allosteric CP-376395 antagonist-bound CRF1R (PDB code 4K5Y). The CRF1R is light pink, and CP-376395 is shown in stick representation. (B) Key residues in CRF1R for CP-376395 binding. Hydrogen bonds are yellow dotted lines.

Figure 16. (A) Overview of the cocrystal structure of an allosteric MK-0893 antagonist-bound GCGR (PDB code 5EE7). The GCGR is light pink, and MK-0893 is shown in stick representation. (B) Key residues in GCGR for MK-0893 binding. Salt bridge and hydrogen bonds are green and yellow dotted lines, respectively. The water molecule involved in water-mediated hydrogen bonds is represented by a red sphere.

CRF1R and CP-376395.65 In the binary complex structure, CP376395 binds to an allosteric site located in the intracellular half of the receptor (Figure 15A), which is approximately 18 Å away from the orthosteric peptide agonist-binding site located at the center of the large cavity presented to the extracellular side of the receptor. CP-376395 binds in a deep site created by residues from helices III, V, and VI. The pyridine nitrogen of CP-376395 in the allosteric binding site (Figure 15B) is involved in a hydrogen bond with the side chain of Asn2835.50. For the hydrophobic interactions, the aryloxyl moiety is located inside a hydrophobic pocket created by residues Phe2845.51, Leu2875.54, Ile2905.57, Thr3166.42, Leu3196.45, and Leu3206.46. The central pyridine ring forms hydrophobic interactions with Met2063.47 and Val2795.46. The exocyclic alkylamino group binds in a hydrophobic pocket defined by residues Phe2033.44, Leu2805.47, Leu3236.49, and Tyr3276.53. The allosteric antagonism of CRF1R by CP-376395 is still unclear owing to the unavailability of the agonist-bound CRF1R structure. However, the binding of CP-376395 to the allosteric site of CRF1R restrains the receptor in an inactive conformation through tethering the cytoplasmic half of helix VI to helix III and helix V.

4.2. GCGP−MK-0893 Structure. Glucagon, a 29 amino acid peptide, is released from the α-cells of the islets of Langerhans located in the pancreas and is an endogenous agonist for glucagon receptor (GCGR).107 Glucagon binding to GCGR stimulates hepatic glucose production through gluconeogenesis and glycogenolysis, thus increasing hepatic glucose production.108,109 GCGR is expressed in liver, intestinal smooth muscle, kidney, brain, and adipose tissue.110 The antagonism of GCGR provides a promising strategy for the potential treatment of type 2 diabetes mellitus, considering its significant role in the regulation of plasma glucose levels.111−113 MK-0893 (Figure 2), a small-molecule antagonist of GCGR, is the first clinical candidate of GCGR.114 Administration of MK-0893 to GCGR mice and rhesus monkeys decreased glucagon-stimulated glucose elevation. Moreover, MK-0893 markedly reduced blood glucose levels of GCGR mice on a high fat diet. A cocrystal structure of binary GCGP−MK-0893 complex has recently been resolved to uncover the detailed binding mode of MK-0893 to the GCGR.66 In the structure, MK-0893 binds to an allosteric site outside the 7TM helical bundle between helices VI and VII, extending into the lipid bilayer (Figure 16A). Despite belonging to the class B GPCRs, the allosteric MK-0893 binding site in the GCGR is different K



Perspective

Figure 17. (A) Overview of the cocrystal structure of an allosteric PF-06372222 antagonist-bound GLP-1R (PDB code 5VEW). (B) Overview of the cocrystal structure of an allosteric NNC0634 antagonist-bound GLP-1R (PDB code 5VEX). The GLP-1R is light pink, and PF-06372222 and NNC0634 are shown in stick representations. (C) Key residues in GLP-1R for PF-06372222 binding. (D) Key residues in GLP-1R for NNC0634 binding. Hydrogen bonds are yellow dotted lines.

amide.115 The activation of GLP-1R leads to inhibition of glucagon secretion and stimulation of insulin secretion in a glucose-dependent manner.116 As such, GLP-1R agonists are useful for the treatment of type 2 diabetes mellitus.117−120 Recently, clinical studies have elucidated that the antagonism of GLP-1R also plays an important role in the treatment of acute and chronic stress as well as anxiety.121 The 7TM domain architecture of GLP-1R is similar to that of GCGR. In fact, PF-06372222 (Figure 2) and NNC0640 (Figure 2) are small-molecule NAMs of GCGR. However, they are also effective for antagonist GLP-1R. Most recently, the cocrystal structures of complex human GLP-1R with PF06372222 and NNC0640 have been solved.67 The two crystal structures clearly show that PF-06372222 and NNC0640 bind to the same allosteric site located outside helices V−VII proximal to the intracellular half of the GLP-1R (Figure 17A and Figure 17B). The terminal anionic carboxylic acid group of PF-06372222 in the allosteric binding site (Figure 17C) is involved in hydrogen bonding interactions with the side chains of residues Ser3526.41 and Asn4067.61. The amide group of the ligand forms hydrogen bonds with the side chains of Ser3526.41 and Lys3516.40, and the aminopyridine moiety engages a hydrogen bond with the side chain of Ser3556.44. For hydrophobic interactions, the trifluoromethylpyrazole moiety of the ligand is located in a hydrophobic pocket created by residues Ile3285.58, Val3315.61, Val3325.62, Leu3355.65, and Phe3476.36. The pyridine ring creates hydrophobic contacts with the alkyl chain of Lys3516.40, and the phenyl ring establishes hydrophobic interactions with the alkyl chain of Lys3516.40 and Leu4017.56. Finally, the dimethylcyclobutane

from the allosteric CP-376395 binding site in the CRF1R; the latter is located deep within the 7TM helical bundle. For polar interactions, the terminal anionic carboxylic acid group of MK0893 forms a salt bridge with Arg3466.37, hydrogen bonds with the side chain of Asn4047.61 and the backbone amine of Lys4057.62, and a water-mediated hydrogen bond with the side chain of Ser3506.44 and the backbone carbonyl of Leu3997.56 in the allosteric MK-0893 binding site (Figure 16B). In addition, the amide group of the ligand engages in hydrogen bonding interactions with the side chains of Lys3496.40 and Ser3506.41. For hydrophobic interactions, the methoxynaphthalene moiety forms numerous hydrophobic interactions with residues Leu3295.61, Phe3456.36, Leu3526.43, Thr3536.44, and the alkyl chain of Lys3496.40. The phenylethylpyrazole core forms hydrophobic interactions with residues Thr3536.44 and Leu3997.56 and a π−cation interaction with Lys3496.40. Currently, the crystal structures of agonist-bound GCGR are unavailable. As a result, the precise allosteric antagonism of GCGR by MK-0893 is unclear. However, the allosteric antagonism can be proposed based on the solved binary GCGP−MK-0893 complex structure. The occupation of the allosteric site of GCGR by MK-0893 restrains the outward helical movement of helix VI, which is required for G-protein coupling, leading to blockade of the glucagon activation of the receptor. 4.3. GLP-1R−PF-06372222 and GLP-1R−NNC0640 Structures. Glucagon-like peptide-1 receptor (GLP-1R), as a member of the secretin-like class B family of GPCR, is activated by the endogenous GLP-1 peptides such as GLP-1 (1−37), GLP-1 (7−37), GLP-1 (1−36) amide, and GLP-1 (7−36) L



Perspective

Figure 18. (A) Overview of the cocrystal structure of an allosteric mavoglurant antagonist-bound mGlu5 receptor (PDB code 4OO9). The mGlu5 receptor is light pink, and the mavoglurant is shown in stick representation. (B) Key residues in mGlu5 receptor for mavoglurant binding. Hydrogen bonds are yellow dotted lines.

neurotransmitter glutamate.122 The activation of mGlu receptors plays a key role in the regulation of the strength of synaptic transmission.123 The mGlu receptors include eight members that can be divided into three subgroups based on sequence homology, pharmacological properties, and Gprotein-coupling profile, namely, group I (mGlu1 and mGlu5), group II (mGlu2 and mGlu3), and group III (mGlu4, mGlu6, mGlu7, and mGlu8).124 Among them, mGlu5 receptor is largely expressed in the frontal cortex, hippocampus, striatum, and amygdala. These areas are associated with motivation, emotion, and cognition. The mGlu5 receptor represents an important therapeutic target, and the NAMs of the mGlu5 receptor that decrease mGlu5 receptor activation are instrumental for the treatment of fragile X syndrome (FXS), anxiety, and depression.125−128 Mavoglurant (AFQ056) (Figure 3) is a NAM of mGlu5 receptor that was previously under phase III clinical trials for the treatment of FXS.129 It has now been discontinued given the negative results in the clinical trials.130 Recently, a cocrystal structure of binary mGlu5−mavoglurant complex has been resolved.68 In the structure, the mavoglurant is located deep within the 7TM helical bundle that is wrapped by residues from helices II, III, and V−VII (Figure 18A). The carbamate carbonyl of the mavoglurant in the allosteric binding site (Figure 18B) forms a hydrogen bond with the side chain of Asn7475.47, and the hydroxyl group of the ligand forms hydrogen bonds with the side chains of Ser8057.35 and Ser8097.39 as well as the backbone carbonyl of Ser8057.35. For hydrophobic interactions, the bicyclic ring of the ligand is located in the main hydrophobic pocket created by residues Ile6513.36, Pro6553.40, Leu7445.44, Trp7856.50, Phe7886.53, Met8027.32, and Val8067.36. The 3-methylphenyl ring fills a hydrophobic pocket created by residues Ile6252.46, Pro6553.40, Tyr6593.44, and Ala8107.40. The alkyne linker establishes contacts with residues Pro6553.40, Tyr6593.44, Ser8097.39, and Val8067.36. The methoxyl terminal portion of the carbamate creates hydrophobic contacts with residues Ile651 3.36 , Pro7435.43, and Leu7445.44. HTL14242 (Figure 3) is another NAM of mGlu5 receptor that was identified by means of fragment- and structure-based drug discovery.69 Unlike mavoglurant, which contains a central acetylene that is potentially susceptible to metabolic activation

group forms hydrophobic interactions with Ser3556.44 , Met3976.38, and Leu4016.42 and protrudes into the lipid bilayer. In summary, the binding mode of PF-06372222 in the GLP-1R is similar to that of MK-0893 in the GCGR. Similarly, the tetrazole group of NNC0640 in the allosteric NNC0640 binding site (Figure 17D) forms hydrogen bonds with the side chains of Ser3526.41 and Asn4067.61. The amide group of the ligand forms hydrogen bonds with the side chains of Ser3526.41 and Lys3516.40, and the urea amine moiety of the ligand engages in a hydrogen bond with the side chain of Ser3556.44. For hydrophobic interactions, the methylsulfonephenyl group of the ligand is located in a hydrophobic pocket defined by residues Phe3245.54, Ile3285.58, Phe3476.36, Leu3546.43 and the alkyl chain of Lys3516.40. The benzamidetetrazole group creates hydrophobic contacts with the alkyl chains of Arg3486.37 and Lys3516.40, Leu4017.56, and Val4057.60. The phenylcyclohexyl group forms hydrophobic interactions with Ser3556.44, Leu3596.48, Phe3906.31, Met3976.38, and Leu4016.42 and protrudes into the lipid bilayer. In summary, the binding modes of two allosteric antagonists PF-06372222 and NNC0640 in the GLP-1R are extremely similar. Comparative MD simulations of complex GLP-1R with NAMs PF-0637222 and NNC0634 and a positive allosteric modulator (PAM) 6,7-dichloro-3-methanesulfonyl-2-tertbutylaminoquinoxaline were performed to further understand the allosteric activation of GLP-1R.67 The model of agonist PAM-mediated receptor activation was proposed based on the simulations of antagonist NAM-bound and agonist PAM-bound GLP-1R. In the NAM-bound GLP-1R, the binding of NAMs restrains the movement of helix VI away from helix VII via the ionic lock interactions between helices II, III, VI, and VII, which prevent G-protein coupling. On the contrary, in the agonist PAM-bound GLP-1R, the binding of PAM induces a conformational rearrangement of helices VI and VII, thereby resulting in the disruption of the ionic lock interactions and facilitating efficient G-protein coupling.

5. CLASS C ALLOSTERIC GPCR−SMALL-MOLECULE MODULATOR INTERACTIONS 5.1. mGlu 5−Mavoglurant and mGlu 5 −HTL14242 Structures. The metabotropic glutamate (mGlu) receptors, as a family of class C GPCRs, are activated by the endogenous M



Perspective

Figure 19. (A) Overview of the cocrystal structure of an allosteric HTL-14242-bound mGlu5 receptor (PDB code 5CGD). The mGlu5 receptor is light pink, and the HTL-14242 is shown in stick representation. (B) Structural comparisons of mGlu5−HTL-14242 complex structure (light pink) versus mGlu5−mavoglurant complex structure (lime). (C) Key residues in mGlu5 receptor for HTL-14242 binding. Hydrogen bonds are yellow dotted lines. The water molecule involved in water-mediated hydrogen bonds is represented by a red sphere.

Figure 20. (A) Overview of the cocrystal structure of an allosteric FITM-bound mGlu1 receptor (PDB code 4OR2). The mGlu1 receptor is light pink, and the FITM is shown in stick representation. (B) Key residues in mGlu1 receptor for FITM binding. Hydrogen bonds are yellow dotted lines.

FITM (Figure 3) is a small-molecule NAM of the mGlu1 receptor that was identified via virtual screening and subsequent optimization of the hit compounds.134 It shows excellent subtype selectivity for the mGlu1 receptor against the mGlu5 receptor. The determination of a cocrystal structure of FITM bound to the mGlu1 7TM domain (residues 581−860) clearly shows that FITM binds inside the 7TM helical bundle adjacent to the extracellular side (Figure 20A).70 The allosteric FITM binding site of the mGlu1 receptor partially overlaps with the orthosteric binding site for the class A GPCRs, but it is distinct from its own orthosteric glutamate binding site at the Nterminal extracellular domain. The allosteric FITM binding site is formed by residues from the ECL2, helices II, III, and V−VII. The pyrimidineamine group of the FITM in the allosteric FITM binding site (Figure 20B) forms a hydrogen bond with the side chain of Thr8157.38. For hydrophobic interactions, the p-fluorophenyl group of the ligand is situated deep into a pocket defined by residues Ile7645.51, Thr7946.44, Ile7976.47, Trp7986.48, and Phe8016.51. The central thiazole linker of the ligand creates hydrophobic interactions with residues Val6643.32, Pro7565.43, and Phe8016.51. The 5-N-isopropylpyrimidine group of the ligand establishes hydrophobic interactions with residues Leu6482.60, Gln6603.28, Val6643.32, Thr748ECL2, Tyr8056.55, Thr8157.38 and the alkyl chain of Arg6613.29. Structural comparison between the NAM-bound mGlu1 receptor and other inactive states of class A GPCRs

via glutathione conjugation occurring at the acetylene, HTL14242 is a novel, non-acetylene small molecule that does not pose any known bioactivation risk. The determination of a cocrystal structure of HTL14242-bound mGlu5 receptor shows that HTL14242 occupies a pocket lined by residues from helices II, III, and V−VII (Figure 19A).69 In fact, the allosteric binding site of HTL-14242 in the mGlu5 receptor is greatly similar to that of mavoglurant in the mGlu5 receptor (Figure 19B). The pyridine nitrogen of the HTL-14242 in the allosteric HTL-14242 binding site (Figure 19C) engages a hydrogen bond with the side chain of Ser8097.39, and the 5-cyano of the ligand is involved in a water-mediated hydrogen bond with the backbone carbonyl oxygen of Val7405.40. For hydrophobic interactions, the pyridyl ring of the ligand fits into a hydrophobic pocket defined by residues Ile6252.46, Tyr6593.44, Ser8097.39, and Ala8107.40. The pyrimidine linker is situated in a hydrophobic pocket created by residues Ser6543.39, Pro6553.40, and Val8067.36. The phenyl ring creates hydrophobic contacts with residues Trp7856.50 and Phe7886.53. The cyano substituent establishes contacts with residues Leu7445.44, Phe7886.53, and Met8027.32. 5.2. mGlu1−FITM Structure. The mGlu1 receptor, which is another member of group I of the mGlu receptors, represents a potential therapeutic target for the treatment of schizophrenia and other disorders such as substance abuse and ataxia.131−133 N



Perspective

Figure 21. (A) Overview of the cocrystal structure of an allosteric vismodegib-bound SMO (PDB code 5L7I). The N-terminal large cysteine-rich domain (CRD), the small linker domain (LD), and the central 7TM domain are lime, orange, and light pink, respectively. Vismodegib is shown in stick representation. (B) Key residues in SMO for vismodegib binding. Hydrogen bonds are yellow dotted lines. (C) Structural superimposition of two SMO structures in the presence (light pink) and absence (PDB code 5L7D, cyan) of vismodegib.

Figure 22. Multiplicity of allosteric sites across all GPCR subfamilies. The representative crystal structure of β2AR−Cmp-15PA complex is selected as a template, and the other crystal structures of GPCR−small-molecule allosteric modulator complexes are superimposed to that of β2AR−Cmp15PA complex. The β2AR is gray, and all small-molecule allosteric modulators are shown in stick representation.

reveals that the conformation of the intracellular side of the mGlu1 receptor responsible for G-protein coupling is similar to those of the inactive states of class A GPCRs. In the mGlu1 receptor, the formation of a salt bridge between the side chain of Lys6783.46 at the intracellular end of helix III and the side chain of Glu7836.33 at the intracellular end of helix VI inactivates the receptor by restraining the activation-related outward movement of helix VI.

The HH signaling pathway plays a critical role in a wide range of cancers and has been considered a therapeutic target for cancer therapy.136−139 The full-length SMO contains multiple domains, including an extracellular region composed of a large N-terminal cysteinerich domain (CRD), followed by a small linker domain (LD), a central 7TM domain, and a C-terminal intracellular domain.140 The orthosteric binding site of SMO is located at the CRD, where the endogenous ligands (such as lipoprotein WNT) are bound. Vismodegib (Figure 4) is a NAM of SMO that was approved by the United States Food and Drug Administration (FDA) in 2012 to treat metastatic or locally advanced basal cell carcinoma (BCC).141 Recently, the cocrystal structure of vismodegib-bound SMO has been solved. The binary complex

6. CLASS F ALLOSTERIC GPCR−SMALL-MOLECULE MODULATOR INTERACTIONS 6.1. SMO−Vismodegib Structure. SMO is a member of class F GPCRs that is responsible for transduction of developmental signals of the Hedgehog (Hh) pathway.135 O



Perspective

Figure 23. Enlarged view of allosteric sites located at the intracellular side of the receptors CCR2, CCR9, and β2AR (A), at the extracellular side of the receptors M2, CCR5, and SMO (B), and at the central 7TM helical bundle of receptors mGlu5, mGlu1, and CRF1R (C).

Figure 24. Enlarged view of allosteric sites located at the interface of receptor−lipid bilayer in the receptors GPR40, P2Y1, PAR2, GLP-1R, and GCGP.

sodium ion to the Asp2.50 residue allosterically stabilizes class A GPCRs in their inactive states.142 Here, we superimpose the currently solved receptor−ligand complexes to the crystal structure of β2AR−Cmp-15PA complex to highlight the different locations of small-molecule allosteric sites across four GPCR subfamilies. The latter, as a representative class A GPCR, is selected as a template. As shown in Figure 22, the allosteric sites are located not only at the 7TM helical bundle but also at the interface of receptor−lipid bilayer. Prior to crystallographic studies, previous combinations of long-scale accelerated MD simulations and subsequent fragment-based mapping algorithm predicted the location of allosteric sites for the β1AR,143 β2AR,143 M2,144 and A2A adenosine receptor.145 Dramatically, the predicted allosteric sites on the β2AR and M2 are confirmed by the currently solved β2AR/M2−allosteric modulator complexes, thereby suggesting the feasibility of using computational methods to identify the putative allosteric sites on the GPCRs if the crystal structures of allosteric GPCR− modulator complexes are unavailable. In the 7TM helical bundle, the allosteric sites can be classified into three subgroups as follows: at the extracellular side of the receptors (M2, CCR5, and SMO), at the central 7TM helical bundle (mGlu5, mGlu1, and CRF1R), and at the intracellular side of the receptors (CCR2, CCR9, and β2AR). Remarkably, the two allosteric sites of homologous CCR2 and CCR9 at the intracellular side (Figure 23A) are highly overlapping, whereas the allosteric site of β2AR is partially overlapped with that of CCR2 and CCR9. However, the allosteric sites at the extracellular side (Figure 23B) and at the central 7TM (Figure 23C) are nonoverlapping with each other. In addition, the allosteric sites at the interface of receptor−lipid bilayer (GPR40, P2Y1, PAR2, GLP-1R, and GCGP) are located at the different sides of the receptors (Figure 24). In addition to the existence of partial overlap

structure clearly shows that vismodegib is bound to the 7TM helical bundle adjacent to the extracellular side (Figure 21A).71 The vismodegib binding site is defined by residues from the LD, ECL2, ECL3, and helices I, II, and V−VII. The pyridine nitrogen and the amide amine of vismodegib in the allosteric vismodegib binding site (Figure 21B) are involved in a hydrogen bond with the side chain of Tyr394ECL2 and Asp384ECL2, respectively. For hydrophobic interactions, the pyridinechlorophenyl moiety of the ligand is deeply buried, forming numerous hydrophobic interactions with residues Met2301.32, Trp2812.58, Val386ECL2, Ser387ECL2, Ile389ECL2, Phe391ECL2, Tyr394ECL2, Arg4005.43, His4706.51, Glu518ECL3, Leu5227.42, and Met5257.45. The methylsulfonephenyl moiety of the ligand creates hydrophobic contacts with residues Leu221LD, Tyr394ECL2, Gln4776.58, Trp4806.61, Phe4846.65, and Pro513ECL3. Further structural superimposition of SMO in the presence and absence of vismodegib shows that vismodegib binding to the 7TM domain significantly induces conformational changes in the extracellular domains and the ECL3 loop (Figure 21C), thereby ultimately leading to the conformational rearrangement of the sterol-binding site in the CRD and subsequently preventing cholesterol binding to the sterolbinding site.

7. ALLOSTERIC SITES ON GPCRS To date, the cocrystal structures of GPCR−small-molecule modulator complexes have been available across all human GPCR classes A, B, C, and F. In addition to the small-molecule allosteric sites on the GPCRs, the allosteric sites for the sodium ion and amilorides at the center of the 7TM helical bundle of class A GPCRs are revealed by high-resolution crystallographic structures.142 The comparison of inactive-state and active-state crystal structures of class A GPCRs shows that binding of P



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between the allosteric sites of GPR40 and PAR2, the other allosteric sites are nonoverlapping. In particular, in the GPR40, two distinct allosteric sites outside the 7TM helical bundle are observed, thereby revealing multiple allosteric-binding sites in a single receptor. Collectively, a comparison of allosteric sites across four GPCR subfamilies suggests the structural diversity of allosteric sites for GPCRs, which is beneficial for the design of allosteric modulators with high subtype selectivity.

Jian Zhang: 0000-0002-6558-791X Notes

The authors declare no competing financial interest. Biographies Shaoyong Lu received his undergraduate Applied Chemistry degree in Hangzhou Normal University, China, in 2007, after which, he obtained the Ph.D. degree in Chemistry in Zhejiang University, China, in 2012. Then he continued postdoctoral research at Shanghai Jiao Tong University, School of Medicine, China, in Prof. Jian Zhang’s group and now is an Associate Professor. During 2014−2015, he studied as a Visiting Scholar in Prof. Ruth Nussinov’s group at the National Cancer InstituteFrederick. His main interests include allosteric protein− ligand interactions and the development of allosteric methods and applications.

8. CONCLUSIONS AND PERSPECTIVES GPCRs, which constitute the largest class of membraneassociated receptors in the human genome, are most frequently targeted by therapeutic drugs. GPCRs possess a conserved orthosteric site bound by endogenous ligands among receptor subtypes, and such structure poses a key challenge in designing selective GPCR drugs between receptor subtypes. Such drug discovery challenge can be addressed by tailoring allosteric GPCR modulators for nonconserved allosteric sites. Allosteric GPCR modulators directed at allosteric sites that are topographically distinct from orthosteric sites create a promising opportunity to modulate receptor function through many methods, thereby providing the potential for differential selectivity and improved safety against traditional orthosteric ligands. The recent great progress in GPCR crystallography has solved 14 distinct GPCRs in complex with their small-molecule allosteric modulators across all human GPCR classes A, B, C, and F such as CCR5, CCR2, CCR9, M2, GPR40, P2Y1, β2AR, PAR2, CRF1R, GCGP, GLP-1R, mGlu5, mGlu1, and SMO. These receptor−modulator structural complexes demonstrate that allosteric sites are located within or outside the 7TM helical bundle and are nonoverlapping with one another except the two homologous receptors CCR2 and CCR9. This finding supports the notion of the structural diversity of allosteric sites on GPCRs. The structures of GPCRs that have been solved in complex with small-molecule allosteric modulators pave the way for virtual screening or structure-based drug design to identify novel allosteric leads and to improve the binding affinity of existing allosteric modulators. The computational identification of allosteric sites on GPCRs can be developed to predict the location of potential allosteric sites in a receptor of interest to produce more structures of GPCRs that solve complex with allosteric modulators in the future.146−150 In silico screening of large compound libraries directed at the predicted allosteric sites can then be conducted to find new allosteric chemotypes151−155 with the potential to confer novel paradigms of GPCR biology in even well-established receptors. For example, a combination of empirical and homology models assisted the structure-based screening and identified allosteric modulators for the orphan receptors, GPR68 and GPR65, as validated by functional assays.156 Computational enhanced sampling simulations coupled with iterative molecule docking and experimental testing identified allosteric modulators for the M2 receptor.157 The recently solved crystal structures of allosteric GPCR− small-molecule modulator complexes, together with the continued campaigns against new allosteric sites on GPCRs, indicate progress in the field of GPCR allosteric drug discovery.

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Jian Zhang received a B.M. degree in Pharmacology in 2002 from Peking University and a Ph.D. in 2007 from Shanghai Institute of Materia Medica, Chinese Academy of Sciences. After receiving his Ph.D., he moved to University of Michigan to carry out postdoctoral research in Prof. Shaomeng Wang’s group. In 2009, he joined the Shanghai Jiao Tong University, School of Medicine, where he is a fulltime researcher and doctoral supervisor. Now, he is also the director of Medicinal Bioinformatics Center. His fields of research include first-inclass drug discovery and chemical biology that mainly pertain to the repertoire of allostery. His website provides further details: http://mdl. shsmu.edu.cn/.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (973 Program) (2015CB910403); National Natural Science Foundation of China (21778037, 81603023, 81322046, 91753117, 81473137); Shanghai Health and Family Planning Commission (20154Y0058).

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ABBREVIATIONS USED ADP, adenosine 5′-diphosphate; β2AR, β2 adrenergic receptor; CCL, CC chemokine ligand; CCR2, CC chemokine receptor 2; CCR5, CC chemokine receptor 5; CCR9, CC chemokine receptor 9; CRD, cysteine-rich domain; CRF, corticotropinreleasing factor; CRF1R, corticotropin-releasing factor 1 receptor; ECL, extracellular loop; FDA, Food and Drug Administration; FFA, free fatty acid; FXS, fragile X syndrome; GCGR, glucagon receptor; GLP-1R, glucagon-like peptide-1 receptor; GPCR, G-protein-coupled receptor; Hh, Hedgehog; HIV-1, human immunodeficiency virus type 1; ICL, intracellular loop; LD, linker domain; M2R, muscarinic 2 acetylcholine receptor; MD, molecular dynamics; mGlu5, metabotropic glutamate 5; NAM, negative allosteric modulator; PAM, positive allosteric modulator; PAR2, protease-activated receptor 2; PPI, protein−protein interaction; SMO, smoothened receptor; 7TM, seven-transmembrane

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Small Molecule Allosteric Modulators of G-Protein-Coupled Receptors

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