A Fluorescent Probe Enables the Discovery of Improved Antagonists Targeting the Intracellular Allosteric Site of the Chemokine Receptor CCR7

Intracellular ligands of G protein-coupled receptors (GPCRs) are gaining significant interest in drug discovery. Here, we report the development of the fluorescent ligand Mz437 (4) targeting the CC chemokine receptor CCR7 at an intracellular allosteric site. We demonstrate its experimental power by applying 4 to identify two improved intracellular CCR7 antagonists, SLW131 (10) and SLW132 (21m), developed by converting two weakly active antagonists into single- or double-digit nanomolar ligands with minimal modifications. The thiadiazoledioxide 10 was derived from the CCR7 antagonist Cmp2105 by removing a methyl group from the benzamide moiety, while the squaramide 21m was obtained from the CXCR1/CXCR2 antagonist and clinical candidate navarixin by replacing the ethyl substituent by a tert-butyl group to engage a lipophilic subpocket. We show that 10 and 21m qualify to probe CCR7 biology in recombinant and primary immune cells and expect our novel probes to facilitate the design of next-generation intracellular CCR7 ligands.