Abstract 4343: Characterization and comparison of inhibitors of GPR68

Abstract Background: GPR68 is a proton-sensing GPCR reported to play a role promoting tumor aggressiveness and immune-evasion, as well as in inflammatory disease. Most data supporting these findings come from genetic studies, and disclosed small molecule compounds targeting GPR68 are scarce. Nevertheless, two distinct chemical entities have recently been reported as GPR68 inhibitors. They have been used to define in vitro mechanisms by which GPR68 may affect disease biology. One of these, FT011, has completed a Phase 2 trial for scleroderma and is described as a GPR68 antagonist, however no pharmacological characterization at GPR68 has been reported to date. A chemical probe, ogremorphin, identified through a chemical genetic screen, is described as a GPR68 antagonist with anti-cancer efficacy in vitro. Methods: Here we describe the identification of three series of novel, drug-like GPR68 antagonists from a screening campaign, which were further optimized via our proprietary generative AI design methods. We present the pharmacological characterization of these series and compare representative examples to FT011 and ogremorphin in primary pharmacology assays and benchmark their activity in fibroblast and glioblastoma (GBM) cell lines. Results: Three unique chemical series were identified as potent antagonists of GPR68. Detailed characterization using the TRUPATH system suggests that all three scaffolds act as negative allosteric modulators. In comparison, in our hands, FT011 does not inhibit GPR68 activation when evaluated with classical signaling technologies, nor in TRUPATH. Ogremorphin does weakly antagonise GPR68 as reported, with the potency of these effects dependent on the assay technology. All three of our designed GPR68 antagonist series potently inhibit the release of GPR68-stimulated cytokines in a GPR68-expressing cancer associated fibroblast line, as does ogremorphin to a lesser degree. In our hands, FT011 does not antagonise GPR68-mediated cytokine release. We confirm the reported inhibition on GBM lines by ogremorphin, but this effect is absent in our three unique GPR68 antagonist series. Conclusions: Here we describe multiple highly-potent chemical scaffolds targeting GPR68 as negative allosteric modulators with our data strongly supporting the characterization of our drug-like molecules as first-in-class antagonists of GPR68. In contrast, our profiling of FT011 does not support its purported activity at GPR68. Ogremorphin is confirmed as a weak antagonist of GPR68 which inhibits GBM cell proliferation but it is possible the effect of ogremorphin is due to activity against other targets, or polypharmacology not represented in our GPR68 antagonists. Our first-in-class compounds are currently in development for oncology indications. Citation Format: Piotr Stepien, Duncan Bellamy, Alice E. Berizzi, Alex Ceroni, Elena Fonfria, Major Gooyit, Mizuho Horioka, Katherine Lansu, Sam R. Lawrence, Manisha Naik, Maninder Panesar, Kostantinos Papachristos, Martin Redhead, Simon Richards, David A. Smith-Parkin, Jake Swadling, Diogo M. Tomaz, Bojan Vilagos, Andrew S. Walker, Michael Prime, Reid H. Olsen. Characterization and comparison of inhibitors of GPR68 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4343.