G protein-coupled receptors (GPCRs): advances in structures, mechanisms, and drug discovery

Abstract

G protein-coupled receptors (GPCRs), the largest family of human membrane proteins and an important class of drug targets, play a role in maintaining numerous physiological processes. Agonist or antagonist, orthosteric effects or allosteric effects, and biased signaling or balanced signaling, characterize the complexity of GPCR dynamic features. In this study, we first review the structural advancements, activation mechanisms, and functional diversity of GPCRs. We then focus on GPCR drug discovery by revealing the detailed drug-target interactions and the underlying mechanisms of orthosteric drugs approved by the US Food and Drug Administration in the past five years. Particularly, an up-to-date analysis is performed on available GPCR structures complexed with synthetic small-molecule allosteric modulators to elucidate key receptor-ligand interactions and allosteric mechanisms. Finally, we highlight how the widespread GPCR-druggable allosteric sites can guide structure- or mechanism-based drug design and propose prospects of designing bitopic ligands for the future therapeutic potential of targeting this receptor family.

Fig. 1

Phylogenetic tree of GPCRs indicating GPCR structures that have been solved in complex with modulators. Nodes represent GPCRs named according to their UniProt gene name and are organized according to the GPCR database. GPCR structures bound to modulators are highlighted by color

Fig. 2

Timeline of major advancements in GPCR structure study using X-ray crystallography and cryo-EM

Fig. 3

a Schematic representation of GPCR activation process. Upon agonist (red circle) binding, the receptor proceeds into a pre-activation state coupling with the G protein heterotrimer, where the exchange of GDP and GTP in G protein α subunit leads to G protein dissociation and mediate G protein signaling pathway. The phosphorylation of the receptor C-terminal tail by GRK binding promotes arrestin recruitment and signaling. When the antagonists (blue circle) bind, the receptor stabilizes in an inactive state. b Crosstalk of downstream pathway of Gs, Gq, Gi and arrestin

Fig. 4

a Categories of Representative human diseases caused by GPCR dysfunctions. b Classification of the effects of mutations on GPCR dysfunctions

Fig. 5

a A bridged general view of fentanyl and oliceridine inducing distinct pharmacological profiles. b 2D structure of fentanyl and oliceridine shown for clarity. c Superimposed views of μOR–fentanyl (gray cartoon, gray sticks; PDB: 8EF5) and μOR–oliceridine (light green cartoon, light green sticks; PDB: 8EFB) complex structure, together with the comparison of ligand binding modes and arrestin coupling interfaces, are presented. 2D structures of two designed biased modulators are also presented

Fig. 6

a Detailed binding modes of S1PR5 in complex with siponimod. Labels of the residues engaged in polar contacts with siponimod are colored in blue, with hydrogen bonds presented by orange dashes. The residues of the hydrophobic pocket that stabilizes ligand binding are marked with green labels, while residues that are critical for signal transduction are labeled in red. b Superimposed views of S1PR1 (orange cartoon, PDB: 7T6B), S1PR2 (light green cartoon, PDB: 7C4S), S1PR3 (light purple cartoon, PDB: 7YXA), and S1PR5 (yellow cartoon, PDB: 7TD4) GPCR structures, where TM1 and TM7 of S1PR5 are highlighted for clarity. c Superimposed views of active S1PR1-siponimod complex (cyan cartoon, cyan stick, PDB: 7TD4) and inactive S1PR1 structure (gray cartoon, gray stick, PDB: 3V2Y) to illustrate the “toggle switch” activation mechanism. d 2D structure of siponimod is shown for clarity

Fig. 7

a Detailed binding mode of lemborexant in complex with OX2R (receptor: light orange, ligand: cyan, PDB: 7XRR), where steric hindrance of T135^3.33 only allows one orientation of the ligand. b Detailed binding mode of lemborexant in complex with OX1R (receptor: light pink, ligand: yellow, PDB: 6TOT), where small side chain of A127^3.33 accounts for two orientations of the ligand. c Abridged general view of employing MD simulation to predict the conformation of the ligand before receptor binding, to improve K_on values. d 2D structure of lemborexant is shown for clarity

Fig. 8

a Detailed binding mode of 5-HT_1F in complex with lasmiditan. Hydrogen bonds are presented by orange dashes, while halogen bonds are presented by green dashes. b Superimposed views of 5-HT_1A (light green cartoon, PDB: 7E2X), 5-HT_1B (light orange cartoon, PDB: 5V54), 5-HT_1D (light gray cartoon, PDB: 7E32), 5-HT_1E (light pink cartoon, PDB: 7E33), and 5-HT_1F (light purple cartoon, PDB: 7EXD). The TM4-ECL2-TM5 region of the 5-HT_1F receptor is highlighted for clarity. c The structure alignment comparison of αN helices of G protein coupling with their corresponding 5-HT receptors. αN helix of G_i protein coupled with 5-HT_1F is highlighted for clarity. d 2D structure of lasmiditan

Fig. 9

a Detailed binding mode of GnRH1 in complex with elagolix (receptor: light gray, ligand: light pink, PDB: 7BR3), where N-termini of GnRH1R is highlighted in a light purple to present its co-occupation with elagolix in the orthosteric pocket. b Overview of the special signal transduction mechanism in GnRH1R. c 2D structure of elagolix for clarity

Fig. 10

a Detailed binding mode of A₂AR in complex with compound 4d (receptor: light gray, ligand: orange, PDB: 3UZA). b SAR study of A₂AR antagonist. c Comparison of the orthosteric binding site of A₂AR–Adenosine complex (light blue, PDB: 2YDO), A₂AR–ZM241385 complex (light yellow, PDB: 4EIY), A₂AR–Compound 4e complex (light green, PDB: 3UZC), the difference in cavity occupation is highlighted by red circles and arrows

Fig. 11

a Detailed binding mode of D₂R in complex with compound 1 (12) (receptor: light gray, ligand: salmon, the receptor is modeled from PDB: 3PBL). TM5 of the receptor is colored in pink and ECL2 is colored in blue for clarity. b SAR study of β-arrestin biased agonists of D₂R

Fig. 12

11 allosteric binding sites reported across GPCRs mapped onto representative class A GPCR CB1R. Gray pockets represent binding pockets within 7TMD, and white pockets represent binding pockets outside 7TMD. For each pocket, the number of unique ligands is indicated using boldface type, and the number of GPCRs containing the pocket is provided in parentheses. The boundary of the lipid bilayer is indicated by gray dashes

Fig. 13

Three extracellular allosteric binding sites in GPCRs and the corresponding small-molecule allosteric modulators. Stick models of small-molecule ligands are mapped to representative members of outside 7TMD (I and II) (GLP-1R, PDB: 6VCB), outside 7TMD (II and III) (GPR101, PDB: 8W8S), and within 7TMD (M4R, PDB: 7V68) GPCRs. The position of an orthosteric ligand of M4R (shown in gray and sphere-and-stick representation) is mapped onto the overview of allosteric modulators for comparison. For each pocket, the number of unique modulators is indicated in boldface type, and the number of GPCRs containing the pocket is indicated in parentheses

Fig. 14

Two-dimensional (2D) chemical structures of synthetic allosteric ligands targeting the GPCR extracellular vestibule

Fig. 15

a Schematic representation of PAM LSN3160440 and orthosteric GLP-1 bound to GLP-1R (PDB: 6VCB). GLP-1 is indicated in pink. b Detailed binding modes of GLP-1R bound to LSN3160440; π–π stacking is indicated in gray dashes. c 2D structure of small-molecule allosteric ligand LSN3160440 presented for clarity. d Superposition of orthosteric small-molecule agonists Boc5 (displayed with purple sticks), TT-OAD2 (displayed with salmon sticks), LY3502970 (displayed with yellow sticks), and CHU-128 (displayed with blue sticks) to LSN3160440–GLP-1–GLP-1R structure reveals a partial overlap in the TM1-TM2 cleft. The conserved residue Tyr145^1.40 is highlighted

Fig. 16

a Schematic representation of allosteric agonist AA-14 bound to GPR101 (PDB: 6VCB). b Detailed binding modes of GPR101 bound to AA-14. c 2D structure of small-molecule allosteric ligand AA-14 presented for clarity

Fig. 17

a Superposition of PAM LY2119620 bound to M2 receptor (pink cartoon, pink sticks; PDB: 4MQT) and M4 receptor (yellow cartoon, yellow sticks; PDB: 7V68). b Detailed binding modes of M2 receptor bound to LY2119620. c Detailed binding modes of M4 receptor bound to LY2119620. Hydrogen bonds are presented as orange dashes and π–π stacking is presented as gray dashes. d Superimposed views of highlighted residue Trp422^7.36 on M2 receptor–iperoxo–LY2119620 (pink cartoon, pink sticks; PDB: 6U1N), M2 receptor–LY2119620 (purple cartoon, purple sticks; PDB: 4MQT), and M2 receptor–iperoxo (blue cartoon, blue sticks; PDB: 4MQS) structures. The orthosteric agonist iperoxo is presented in orange

Fig. 18

a Schematic representation of PAM cinacalcet bound to CaSR (PDB: 7M3F). b Detailed binding modes of CaSR bound to cinacalcet in extended conformations. c Detailed binding modes of CaSR bound to cinacalcet in bent conformations. Hydrogen bonds are presented as orange dashes and π–π stackings are presented as gray dashes

Fig. 19

a Schematic representation of NAM NPS-2143 bound to CaSR (PDB: 7M3E). b Detailed binding modes of CaSR bound to NPS-2143. Hydrogen bond is presented as orange dashes and π–π stacking is presented as gray dashes. c 2D structure of small-molecule allosteric ligand NPS-2143 provided for clarity. d Superimposed views of highlighted residues on CaSR–Cinacalcet–Ca²⁺–Trp (blue cartoon, blue sticks; PDB: 7M3F), CaSR–Ca²⁺–Trp (pink cartoon, pink sticks; PDB: 7DD6), and CaSR–NPS-2143–Ca²⁺–Trp (purple cartoon, purple sticks; PDB: 7M3E) structures. e Workflow of discovery of novel CaSR PAMs utilizing structural information

Fig. 20

a Five allosteric binding sites in the transmembrane domain outside 7TMD of GPCRs and the corresponding small-molecule allosteric modulators. Stick models of small-molecule ligands are mapped to representative members of outside 7TMD (I–III) (P2Y₁, PDB: 4XNV), outside 7TMD (II–IV) (CB1R, PDB: 6KQI), outside 7TMD (III and V) (C5aR1, PDB: 6C1R), outside 7TMD (V and VI) (CXCR3, PDB: 8HNN), and outside 7TMD (I, VI, and VII) (A1R, PDB: 7LD3) GPCRs. For each pocket, the number of unique modulators is indicated in boldface type, and the number of GPCRs containing the pocket is provided in parentheses. b 2D chemical structures of synthetic small-molecule allosteric ligands targeting the transmembrane domain outside 7TMD of GPCRs. c Extracellular view of the five allosteric sites

Fig. 21

a Schematic representation of the allosteric antagonist BPTU bound to P2Y₁ receptor (PDB code 4XNV). b Detailed binding modes of P2Y₁ receptor bound to BPTU. Hydrogen bonds are presented as orange dashes. c Superimposed views of the highlighted residues on 2MeSADP − P2Y₁R − G₁₁ (pink cartoon, pink sticks; PDB: 7XXH) and P2Y₁R − BPTU structures (blue cartoon, blue sticks; PDB: 4XNV)

Fig. 22

a Superposition of the cocrystal structures of NAM ORG27569 (yellow, PDB: 6KQI) and PAM ZCZ011 (pink, PDB: 7FEE) with orthosteric ligand-bound CB1R. b Detailed binding modes of CB1R binding to ORG27569. c Detailed binding modes of CB1R binding to ZCZ011. Hydrogen bond is presented as orange dashes, and π–π stacking is presented as gray dashes. d Superimposed views of highlighted residues on CB1R–CP55940–ORG27569 (pink cartoon, pink sticks; PDB: 6KQI) and CB1R–AMG315–G_i structures (blue cartoon, blue sticks; PDB: 8GHV). e Superimposed views of highlighted residues on CB1R–AM6538 (pink cartoon, pink sticks; PDB: 5TGZ) and CB1R-CP55940–ZCZ011 structures (blue cartoon, blue sticks; PDB: 7FEE)

Fig. 23

a Superposition of the cocrystal structures of PAM LY3154207 bound to dopamine D1 receptor in upright (pink, PDB: 7CKZ) and boat (yellow, PDB: 7LJC and 7X2F) conformations. b Detailed binding modes of dopamine D1 receptor binding to LY3154207 in upright conformations. c Detailed binding modes of dopamine D1 receptor binding to LY3154207 in boat conformations. Hydrogen bonds are presented as orange dashes; π–π stacking and π–cation stacking interactions are presented as gray dashes

Fig. 24

a Schematic representation of the allosteric antagonist avacopan bound to C5a receptor 1 (PDB: 6C1R). b Detailed binding modes of C5a receptor 1 bound to avacopan. Hydrogen bond is presented as orange dashes. c Superimposed views of highlighted residue Trp213^5.49 on C5aR1–PMX53–avacopan (pink cartoon, pink sticks; PDB: 6C1R) and C5aR1–C5a–G_o (blue cartoon, blue sticks; PDB: 8IA2) structures

Fig. 25

a Schematic representation of the allosteric antagonist SCH546738 bound to CXCR3 (PDB code 8HNN). b Detailed binding modes of CXCR3 binding to SCH546738. c 2D structure of small-molecule allosteric ligand SCH546738 provided for clarity

Fig. 26

a Schematic representation of PAM MIPS521 bound to A1R (PDB code 7LD3). b Detailed binding modes of A1R binding to MIPS521. Hydrogen bonds are presented as orange dashes. c 2D structure of small-molecule allosteric ligand MIPS521 provided for clarity

Fig. 27

Allosteric binding sites in the transmembrane domain within 7TMD of GPCRs and corresponding small-molecule allosteric modulators. Stick models of small-molecule ligands are mapped to representative member CRF1R, PDB: 4K5Y. The number of unique modulators is indicated in boldface type, and the number of GPCRs containing the pocket is provided in parentheses

Fig. 28

2D chemical structures of synthetic small-molecule allosteric ligands targeting the transmembrane domain within 7TMD of GPCRs

Fig. 29

a Schematic representation of the NAM fenobam bound to mGlu5 receptor (PDB: 5CGC). b Detailed binding modes of mGlu5 receptor binding to compound 14; polar interactions are shown in orange dashes. c SAR optimization of mGlu5 receptor NAMs. d Superimposed views of the highlighted residues on mGlu5–mavoglurant (purple cartoon, purple sticks; PDB: 4OO9), mGlu5–HTL14242 (orange cartoon, orange sticks; PDB: 5CGD), mGlu5–fenobam (blue cartoon, blue sticks; PDB: 6FFH), and mGlu5–CDPPB (pink cartoon, pink sticks; PDB: 8TAO) structures

Fig. 30

a Schematic representation of the allosteric agonist PCO371 bound to PTH1R (PDB: 8GW8). b Detailed binding modes of PTH1R binding to PCO371. Hydrogen bonds are presented as orange dashes. c Superposition of the PTH1R bound to PCO371 (displayed in pink) and PTH (displayed in blue) reveals conformational changes upon PCO371 binding

Fig. 31

Two intracellular allosteric binding sites in GPCRs and the corresponding small-molecule allosteric modulators. Stick models of small-molecule ligands are mapped to representative members of outside 7TMD (V−VII) (GCGR, PDB: 5EE7) and inside 7TMD (CCR2, PDB: 5T1A) GPCRs. For each pocket, the number of unique modulators is indicated in boldface type, and the number of GPCRs containing the pocket is provided in parentheses

Fig. 32

2D structures of synthetic allosteric ligands targeting the GPCR intracellular surface

Fig. 33

a Schematic representation of PAM BHFF bound to GABA_B receptor (PDB: 7C7Q). b Detailed binding modes of GABA_B receptor binding to BHFF. Hydrogen bonds are presented by orange dashes. c Superimposed views of the N-terminal α-helix on compound 2–GLP-1R–G_s (blue cartoon, blue sticks; PDB: 7DUR) and GLP-1–GLP-1R–G_s (pink cartoon, pink sticks; PDB: 6×18) structures. d Schematic representation of ago-PAM compound 2 bound to GLP-1R (PDB: 7EVM). e Detailed binding modes of GLP-1R bound to compound 2. f Superimposed views of highlighted residues on compound 2–GLP-1R–G_s (blue cartoon, blue sticks; PDB: 7DUR), GLP-1–GLP-1R–G_s (pink cartoon, pink sticks; PDB: 6×18), and compound 2–GLP-1–GLP-1R–G_s (purple cartoon, purple sticks; PDB: 7DUQ) structures

Fig. 34

a Schematic representation of the allosteric antagonist MK-0893 bound to GCGR (PDB: 5EE7). b Detailed binding modes of GCGR binding to MK-0893. Hydrogen bonds are presented as orange dashes. c 2D structure of small-molecule allosteric ligand MK-0893 shown for clarity

Fig. 35

a Schematic representation of the allosteric antagonist vercirnon bound to CCR9 (PDB: 5LWE). b Detailed binding modes of CCR9 binding to vercirnon. Hydrogen bonds are presented as orange dashes. c 2D structure of small-molecule allosteric ligand vercirnon shown for clarity