At least 120 non-olfactory G-protein-coupled receptors in the human genome are ‘orphans’ for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs.
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This work was supported by National Institutes of Health (NIH) grants U01104974 (B.L.R., B.K.S. and W.K.K.), R01 DA017204 (B.L.R. and W.K.K.) and the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH PDSP) (X.-P.H., H.Z., M.S.F., W.K.K., T.J.M., A.J. and B.L.R.), the Michael Hooker Chair for Protein Therapeutics and Translational Proteomics to B.L.R.; Genentech Foundation Predoctoral Fellowship (J.K.); NIH grants GM59957 and GM71896 (B.K.S.) and the Structural Genomics Consortium (B.K.S.); grant P01 HL114471 (R.B.P. and D.A.D.); NICHD grant U54 HD079124 (M.S.M., K.A.S., V.N.); NIH grant U19MH082441 (B.L.R., J.J. and X.C.). We thank Mark Pausch (Merck & Co.) for providing us Gs- and Gq-yeast strains for yeast screening assays.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Validation and confirmation of GPCR activation assays.
a–o, Yeast (a–k) and HEK293T cell (l–o) GPCR activation assays. a–d, Concentration-dependent growth of GPR43-expressing Gs yeast (a), GPR43-expressing Gq yeast (b), GPR41-expressing Gs yeast (c), and GPR41-expressing Gq yeast (d) in response to various short-chain fatty acids (SCFAs). e–h, Concentration-dependent growth of GPR39-expressing Gs yeast (GPR39s) in response to zinc ions (e), chromium ions (f), cadmium ions (g) and iron ions (h). i–k, Concentration-dependent cAMP responses of GPR39-expressing HEK293T cells to ZnCl2 (i), ZnSO4 (j) or CdSO4 (k) as measured by luciferase cAMP reporter assay. l, N-unsubstituted benzodiazepines (lorazepam, clonazepam, desmethyldiazepam and norfludiazepam; 10 μM) stimulated cAMP production in a GPR68- and pH-dependent manner. Data are mean ± s.e.m. (n = 3–66 measurements). m–p, Concentration–response curves of N-unsubstituted benzodiazepines lorazepam (m), desmethyldiazepam (n), clonazepam (o) and norfludiazepam (p) at pH 6.50 or 7.40 in GPR68-transfected HEK293T cells (structures in Supplementary Table 1). Normalized results represent mean ± s.e.m. (n = 3) and curves were analysed in GraphPad Prism using the built-in 4 parameter logistic function.
Extended Data Figure 2 Lorazepam and ogerin have minimal GPR4 or GPR65 activity.
a–d, Effect of lorazepam (a, b) or ogerin (c, d) on GPR4 (a, c) or GPR65 (b, d); data represent normalized mean ± s.e.m. (n = 3). Sequencing alignment proton-sensing receptor and docking poses for ogerin and its analogues. e, GPR68 snake plot showing extracellular loops and transmembrane domains (upper portion); important residues are highlighted. Glu160, Arg189 and His269 were mutated in this study. f, Sequence alignment of GPR4, GPR65 and GPR68 to CXCR4 (PDB code 3ODU) (PROMALS-3D) was manually refined to reduce gaps and to position conserved residues. TM, transmembrane regions; IL, intracellular loop; EL, extracellular loop. Conserved residues highlighted in blue by degree of conservation while red boxes indicate residues important for receptor function. Red stars indicate residues mutated in this study. g, Sampling different regions for lorazepam binding modes in GPR68. Yellow and grey surfaces contour the binding site of 1T1t and CVX15 in CXCR4 crystal structures (PDB codes 3ODU and 3OE0, respectively), while green and red surfaces sample the entire binding pocket. The magenta surface represents the canonical orthosteric biogenic amine site. h, ZINC32547799 in its predicted orientation and interactions with GPR68. i, Optimization of ogerin (magenta, thin lines) to C2 (brown, structure in Fig. 3a) by insertion of a single methylene is predicted to improve packing in the aryl pocket of the ogerin site. Adding a second methylene, thus creating a propyl linker in C3 (yellow, structure in Fig. 3a), is predicted to disrupt the packing and thus to reduce the allosteric effect.
Extended Data Figure 3 Heat map of off-target activities of lead compounds at potential CNS drug targets.
Radioligand binding assays were carried out by the National Institute of Mental Health Psychoactive Drug Screening Program (NIMH PDSP) as described previously56,57 (online protocols available at http://pdsp.med.unc.edu/pdspw/binding.php). Values represent mean binding affinities (pKi, n = 2–4). Affinities lower than a pKi of 5, or less than 50% inhibition at 10 μM, are shown as a minimum of 5 on the pKi scale. The hERG inhibition activity was tested in a hERG functional assay as previously published59. AMPA, aminomethylphosphonic acid receptor; BZP, benzodiazepine receptor; DAT, dopamine transporter; DOR, delta (δ) opioid receptor; KA, kainate acid receptor; KOR, kappa (κ) opioid receptor; MOR, mu (μ) opioid receptor; NAT, noradrenaline transporter; NMDA, N-methyl-D-aspartate receptor; ND, not determined; PBR, peripheral benzodiazepine binding site; SERT, serotonin transporter; hERG, human ether-a-go-go-related gene (potassium channel Kv11.1).
Extended Data Figure 4 Confirmation of modelling results via mutagenesis.
a, b, Protons showed agonist activity at GPR68 wild-type and mutant receptors in cAMP production (a) and calcium release (b); parameters are in Supplementary Table 4. c, Relative GPR68 wild-type and mutant receptor expression levels determined by anti-Flag immunoblotting (n = 3). d, Proton-mediated cAMP production in untransfected cells (n = 16). e, Calcium release by lorazepam and selected ZINC compounds (10 μM at pH 8.0, n = 6–22 measurements). f–j, Effect of ogerin and ZINC32547799 (10 μM) on proton-mediated cAMP production (f and g, n = 4), calcium release (h and i, n = 3), and phosphatidylinositol hydrolysis (j, n = 3) at GPR68 wild-type or mutant-transfected HEK293T cells. k, Effect of ogerin and ZINC32547799 on phosphatidylinositol hydrolysis at pH 8.4 at GPR68-transfected GPR68 HEK293T cells (n = 3). Normalized results represent mean ± s.e.m. and curves were analysed using a four-parameter logistic function.
Extended Data Figure 5 Control experiments for signalling and pharmacology.
a, Basal cAMP production of GPR68 wild-type and mutant receptors (mean ± s.e.m., n = 24–46 measurements). b, pH-dependent activity of ogerin at GPR68 wild type (mean ± s.e.m., n = 3). c, Ogerin concentration–responses at GPR68 wild-type and mutant receptors at pH 9.0 (c, mean ± s.e.m., n = 3), under which cAMP reporter assay was not affected (d–f). d–g, Proton modulated isoproterenol-mediated Gs-activation via β2-adrenergic receptors in untransfected (d, f) and GPR68-transfected (e, g) cells. Normalized results (basal at pH 9.5 for d and e; or corresponding buffer control for f and g) represent mean ± s.e.m. (n = 6). h, i, Inverse agonist and antagonist activity (Ki of 220 nM) of ogerin at A2A (cAMP production, h) and weak antagonist activity (Ki of 736 nM) at 5-HT2B receptors (calcium mobilization, i). 5′-N-ethylcarboxamidoadenosine (NECA) and 2-chloro-N6-cyclopentyladenosine (CCPA) served as agonist controls, while CGS15943 is an inverse agonist control for A2A receptors. Normalized results represent mean ± s.e.m. (n = 3). Curves were analysed in GraphPad Prism with the built-in four-parameter logistic function. j, k, Lead compounds (10 μM) showed no agonist (j) or antagonist (k) activity at CXCR4 receptors (cAMP production) with CXCL12 as an agonist control (1 or 3 μM) or AMD 3100 (10 μM) as an antagonist control. Results represent mean ± s.d. (n = 2).
Extended Data Figure 6 Primary screening and comparison of allosteric parameters of 13 ogerin analogues at GPR68.
The 13 ogerin analogues (structures in Supplementary Table 9) identified from docking a virtual library of more than 600 ogerin derivatives were synthesized (Supplementary Information). a–e, Production of cAMP was measured in transiently transfected HEK293T cells at 10 μM and five different pH conditions, pH 8.4 (a); pH 7.9 (b); pH 7.4 (c); pH 7.0 (d); and pH 6.5 (e), to reveal any pH-dependent potentiation activity. Normalized results represent mean ± s.e.m. (n = 8–16 measurements). f, Graphic comparison of the allosteric parameters logα and logβ. Proton concentration–responses were carried out in the absence and presence of increasing concentrations of ogerin and its analogues, results were analysed using a standard allosteric operational model to obtain allosteric parameters. Values represent mean ± s.e.m. (n ≥ 3; see details in Supplementary Table 8).
Extended Data Figure 7 Characterization of potent GPR68 PAMs.
a–c, Concentration–response curves of H+ in the absence and presence of increasing concentrations of CGH2466 (a, a′), tracazolate (b, b′) and SLV320 (c, c′) and in the absence (left column, a, b, c) and presence (right column, a′, b′, c′) of phosphodiesterase inhibitor (Ro 20-1724, 30 μM) at GPR68-expressing cells. Normalized results (mean ± s.e.m., n = 8 for CGH2466; n = 5 for tracazolate; n = 5 for SLV320 for left column and n = 3 for right column) were analysed using a four-parameter logistic function and the standard allosteric operational model (not shown). Allosteric parameters in absence of Ro 20-1724 are summarized in Supplementary Table 8. For each pair of fittings, the proton potency value (negative logarithm of the half-maximum effective concentration (pEC50)) from the agonist concentration–response curve (right) in the absence of testing compound was used as the pKA for the allosteric operational model (left). d, Schematic showing the shared pharmacology among GABAA, adenosine GPCRs and GPR68 ligands. Molecules along each edge of the triangle have been shown to have activity at both targets, whereas tracazolate, in the middle, shows activity at all three.
Extended Data Figure 8 GPR68 mouse biology.
a, Ogerin (Og) and lorazepam (Lo) activate PKA and p42/p44 MAP kinase in HEK293 cells stably expressing haemagglutinin (HA)-tagged GPR68 but not HApcDNA; vehicle (Ve). b, c, GPR68 knockout (n = 7) mice exhibited no differences in contextual memory retrieval (b) or cued memory retrieval (c) as compared to wild-type mice (n = 8). d–f, At 10 mg kg−1, the ogerin isomer ZINC32547799 had no effect on learning (d) or contextual and cue memory (e, f) in wild-type mice (vehicle, n = 6; drug, n = 7). g–i, At 30 mg kg−1, ZINC32547799 enhanced wild-type learning (g, drug × time interaction, F(3.39) = 3.58, P = 0.022; drug alone F(1.39) = 1.19, P = 0.295; Bonferroni post-hoc test revealed a significant effect (P < 0.05) at the third unconditioned/conditioned stimulus training point, two-way ANOVA), but had no effect at contextual and cue memory (h, i) (vehicle, n = 7; drug n = 8). Male mice at age of 6–8 weeks were used in the test. Normalized contextual memory retrieval (d) and cued memory retrieval (f) are presented in Fig. 4c, d.
Extended Data Figure 9 Screening of ZINC compounds predicted to be active at GPR65 based on BTB09089 docking poses.
a–d, Primary screening with ZINC compounds (30 μM) for agonist activity at GPR65 when receptors were kept inactive at pH 8.40 (a); at control HEK293T cells for nonspecific activity (b); at GPR65 when receptors were activated at pH7.40 for modulator or antagonist activity (c); at GPR65 when receptors were activated by BTB09089 (30 μM) at pH 8.40 for modulator or antagonist activity (d). Normalized results represent mean ± s.e.m. from a minimum of three assays (each in minimum of triplicate and a total of ≥16 measurements). The red dashed line in b–d indicates the 20% inhibition line (an arbitrary cut-off line).
Extended Data Figure 10 Characterization of GPR65 allosteric modulators at wild-type and mutant receptors.
a, BTB09089 showed weak agonist activity, but failed to potentiate proton activity at GPR65 (n = 8). b, Selected compounds from Extended Data Fig. 9b, c were tested for GPR65 specific inhibition (n = 16–56 measurements). Several compounds (such as ZINC41613384, ZINC9468042 and ZINC62678696) showed GPR65-specific inhibition. c, Selected compounds from Extended Data Fig. 9b, d were tested for antagonist activity against BTB09089-activated signal at GPR65 (n = 16–64 measurements). ZINC62678696 showed GPR65 specific inhibition when it was activated by either proton or BTB09089. d, ZINC62678696 inhibited GPR65 activity. e–g, Proton concentration–responses (e), BTB09089 concentration–responses (f), and ZINC13684400 concentration–responses (g) at GPR65 mutant receptors. Normalized results represent mean ± s.e.m. (n ≥ 3) and curves were analysed in GraphPad Prism with a standard four-parameter logistic function. Corresponding curves of proton at GPR65 wild-type receptors (from Extended Data Fig. 4a) and BTB09089 and ZINC13684400 (from Fig. 5e) are also included (dashed lines) for comparison. Pharmacological parameters are listed in Supplementary Table 13.
Extended Data Figure 11 In vivo behavioural profiling of GPR68-knockout mice.
a, b, No effects of GPR68 deletion on distance travelled in an open field. Data represent mean ± s.e.m. for each group for a one-hour test session. c, d, No difference on latency to fall from an accelerating rotarod. Data represent mean ± s.e.m. for each group. e–h, Decreased startle responses in GPR68 knockout mice after presentation of acoustic stimuli (e, f). Data represent mean ± s.e.m. for each group. No effects of genotype were found for levels of prepulse inhibition (g, h). Data represent mean ± s.e.m. for each group (*P < 0.05). i–l, No difference at acquisition and reversal learning in the Morris water maze. Data represent mean ± s.e.m. of four trials per day. Subject numbers were 9 wild-type and 7 knockout male mice, and 12 wild-type and 11 knockout female mice.
This file contains Supplementary Methods ‘Chemistry procedures for ogerin and its analogues’ and additional references. (PDF 537 kb)
This file contains Supplementary Tables 1-13 and additional references. (PDF 2596 kb)
This zipped file contains 4 PDB files as follows: for modeled complex of GPR65 with BTB09089; for modeled complex of GPR65 with both BTB09089 and ZINC62678696; for modeled complex of GPR68 with lorazepam; and for modeled complex of GPR68 with ogerin. (ZIP 230 kb)
Supplementary Figure 1
This file contains Supplementary Figure 1 ‘Yeast-based screening to identify potential ligands for GPR68’, which is a heat map showing growth responses of yeast expressing 24 different GPCRs (horizontal axis) to 446 compounds in the NCC library (vertical axis). Blue color is 0% (or less than 0%) above background; white is 100% above background, red color indicates 200% (or more) above background growth stimulation, i.e., agonist activity. Data were clustered using GENE-E software (http://www.broadinstitute.org/cancer/software/GENE-E/) using complete linkage algorithm. GPR68 is the right-most column in the heat map and details are shown in Figure 1a. (ZIP 172 kb)
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Huang, XP., Karpiak, J., Kroeze, W. et al. Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65. Nature 527, 477–483 (2015). https://doi.org/10.1038/nature15699
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