Biological characterization of ligands targeting the human CC chemokine receptor 8 (CCR8) reveals the biased signaling properties of small molecule agonists.

The human CC chemokine receptor 8 (CCR8) is a promising drug target for cancer immunotherapy and autoimmune disease. Besides human and viral chemokines, previous studies revealed diverse classes of CCR8-targeting small molecules. We characterized a selection of these CCR8 ligands (hCCL1, vCCL1, ZK756326, AZ6; CCR8 agonists and a naphthalene-sulfonamide-based CCR8 antagonist), in in vitro cell-based assays (hCCL1 binding, calcium mobilization, cellular impedance, cell migration, β-arrestin 1/2 recruitment), and used pharmacological tools to determine G protein-dependent and -independent signaling pathways elicited by these ligands. Our data reveal differences in CCR8-mediated signaling induced by chemokines versus small molecules, which was most pronounced in cell migration studies. Human CCL1 most efficiently induced cell migration whereby Gβγ signaling was indispensable. In contrast, Gβγ signaling did not contribute to cell migration induced by other CCR8 ligands (vCCL1, ZK756326, AZ6). Although all tested CCR8 agonists were full agonists for calcium mobilization, a significant contribution for Gβγ signaling herein was only apparent for human and viral CCL1. Despite both Gα- and Gα-signaling regulate intracellular Ca-release, cellular impedance experiments showed that CCR8 agonists predominantly induce Gα-dependent signaling. Finally, small molecule agonists displayed higher efficacy in β-arrestin 1 recruitment, which occurred independently of Gα signaling. Also in this latter assay, only hCCL1-induced activity was dependent on Gβγ-signaling. Our study provides insight into CCR8 signaling and function and demonstrates differential CCR8 activation by different classes of ligands. This reflects the ability of CCR8 small molecules to evoke different subsets of the receptor's signaling repertoire, which categorizes them as biased agonists.