Vercirnon Sodium

A conserved intracellular allosteric binding site (IABS) was recently identified at several G protein‐coupled receptors (GPCRs). This target site allows the binding of allosteric modulators and enables a new mode of GPCR inhibition. Herein, we report the development of a NanoBRET‐based assay platform based on the fluorescent ligand LT221 (5), to detect intracellular binding to CCR6 and CXCR1, two chemokine receptors that have been pursued as promising drug targets in inflammation and immuno‐oncology. Our assay platform enables cell‐free as well as cellular NanoBRET‐based binding studies in a nonisotopic and straightforward manner. By combining this screening platform with a previously reported CXCR2 assay, we investigated CXCR1/CXCR2/CCR6 selectivity profiles for both known and novel squaramide analogues derived from navarixin, a known intracellular CXCR1/CXCR2 antagonist and phase II clinical candidate for the treatment of pulmonary diseases. By means of these studies we identified compound 10, a previously reported tert‐butyl analogue of navarixin, as a low nanomolar intracellular CCR6 antagonist. Further, our assay platform clearly indicated intracellular binding of the CCR6 antagonist PF‐07054894, currently evaluated in phase I clinical trials for the treatment of ulcerative colitis, thereby providing profound evidence for the existence and the pharmacological relevance of a druggable IABS at CCR6.

A conserved intracellular allosteric binding site (IABS) has recently been identified at several G protein‐coupled receptors (GPCRs). Starting from vercirnon, an intracellular C−C chemokine receptor type 9 (CCR9) antagonist and previous phase III clinical candidate for the treatment of Crohn's disease, we developed a chemical biology toolbox targeting the IABS of CCR9. We first synthesized a fluorescent ligand enabling equilibrium and kinetic binding studies via NanoBRET as well as fluorescence microscopy. Applying this molecular tool in a membrane‐based setup and in living cells, we discovered a 4‐aminopyrimidine analogue as a new intracellular CCR9 antagonist with improved affinity. To chemically induce CCR9 degradation, we then developed the first PROTAC targeting the IABS of GPCRs. In a proof‐of‐principle study, we succeeded in showing that our CCR9‐PROTAC is able to reduce CCR9 levels, thereby offering an unprecedented approach to modulate GPCR activity.