A FluorescentProbe Enables the Discovery of ImprovedAntagonists Targeting the Intracellular Allosteric Site of the ChemokineReceptor 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 (<b>4</b>) targeting the CC chemokine receptor CCR7 at an intracellular allosteric site. We demonstrate its experimental power by applying <b>4</b> to identify two improved intracellular CCR7 antagonists, SLW131 (<b>10</b>) and SLW132 (<b>21m</b>), developed by converting two weakly active antagonists into single- or double-digit nanomolar ligands with minimal modifications. The thiadiazoledioxide <b>10</b> was derived from the CCR7 antagonist Cmp2105 by removing a methyl group from the benzamide moiety, while the squaramide <b>21m</b> was obtained from the CXCR1/CXCR2 antagonist and clinical candidate navarixin by replacing the ethyl substituent by a <i>tert</i>-butyl group to engage a lipophilic subpocket. We show that <b>10</b> and <b>21m</b> 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.