The FDA-approved CDK4/6 inhibitor Abemaciclib is a potent agonist of the β-arrestin-biased atypical chemokine receptor ACKR3
Atypical chemokine receptor 3 (ACKR3/CXCR7) is an intrinsically {beta}-arrestin-biased family-A GPCR that controls chemokine availability by scavenging CXCL12 and CXCL11, and consequently plays a key role in cancer, chronic inflammation, autoimmunity, cardiovascular disease, and CNS repair. Despite its pathophysiological significance, the chemical space available to interrogate and therapeutically modulate this receptor remains limited. Here, by using a drug-repurposing strategy to screen 2,342 FDA-approved compounds in a NanoBRET-based fluorescent chemokine-binding competition assay, we identified the clinically used CDK4/6 inhibitor abemaciclib (Verzenios(R)) as a nanomolar ACKR3 ligand. Abemaciclib inhibited CXCL12 binding to ACKR3 with comparable affinity to the ACKR3-reference agonist VUF15485, whereas the related clinically used CDK4/6 inhibitors palbociclib, ribociclib, dalpiciclib and trilaciclib were inactive on this receptor. Binding and functional profiling across classical and atypical chemokine receptors revealed selectivity for ACKR3, including lack of detectable activity at the CXCL12 receptors CXCR4 and ACKR5. Abemaciclib acted as a potent ACKR3 agonist, recruiting both {beta}-arrestin-1 and 2 and promoting rapid receptor internalization with potencies and efficacies, like the reference ACKR3 agonist VUF15485. Major CYP3A4-derived abemaciclib metabolites retained ACKR3 binding and agonist activity, albeit with reduced potency. Methionine 13C-HSQC NMR confirmed direct receptor engagement and revealed a small-molecule-like ACKR3 conformational signature distinct from the CXCL12-bound state. Cryo-EM of the abemaciclib-ACKR3-CID24 complex at 3.2 [A] uncovered a unique, inverted U-shaped ligand binding pose in the orthosteric pocket and activation-associated rearrangements of TM6, TM7 and helix 8. Functionally, abemaciclib blocked ACKR3-mediated uptake of CXCL11 and CXCL12 in vitro. Moreover, oral administration of abemaciclib in mice resulted in a time-dependent increase in plasma CXCL12 levels, indicative of in vivo ACKR3 target engagement. These findings establish this FDA-approved kinase inhibitor as a potent ACKR3 modulator and reveal an unexpected pharmacological link of this CDK4/6 inhibitor to atypical chemokine receptor biology.