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. 2024 Jul 19;10(29):eadn2339. doi: 10.1126/sciadv.adn2339

Fig. 1. BTB09089 and BRD2813 induce species-selective GPR65-dependent cAMP production.

Fig. 1.

(A) Replicate plot from a high-throughput screen of 21,000 compounds showing relative cAMP production per compound for each of two technical replicates after 30-min stimulation of GPR65 KO HeLa cells reconstituted with hGPR65 at pH 7.1. (B) Chemical structure of BRD2813. (C) pH-dependent cAMP production in the presence of 0 to 50 μM BRD2813, showing positive allosteric modulation behavior. pH curves were normalized to pH 6.6 for maximum response and 7.5 for minimum cAMP response (mean ± SD, n = 2). (D and E) cAMP response after stimulation with 0 to 50 μM BTB09089 or BRD2813 in GPR65 KO HeLa cells reconstituted with (D) hGPR65 and (E) mGPR65 at pH 7.2 (mean ± SD, n = 2). (F) cAMP production in human PBMCs after 30-min stimulation with 0 to 50 μM BTB09089 or BRD2813 at pH 7.2 (mean ± SD, n = 2). (G and H) GPR65-dependent cAMP induction after 30-min stimulation with 0 to 50 μM BTB09089 or BRD2813 in (G) GPR65 WT and (H) GPR65 KO mouse splenocytes at pH 7.2 (mean ± SD, n = 3). The percent of maximum cAMP response was calculated relative to treatment with 100 μM forskolin and dimethyl sulfoxide. Data represent at least two independent experiments.