Pharmacological characterization and radiolabeling of VUF15485, a high-affinity small-molecule agonist for the atypical chemokine receptor ACKR3.

Atypical chemokine receptor 3 (ACKR3 is considered to be an interesting drug target. In this study we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new small-molecule agonist. VUF15485 binds with nanomolar affinity (pIC50 = 8.3) to human ACKR3, as measured in [125I]CXCL12 competition experiments. Moreover, in a BRET-based β-arrestin2 recruitment assay VUF15485 acts as an ACKR3 agonist with high potency (pEC50 = 7.6) and shows a similar extent of receptor activation compared to CXCL12 when using a newly developed, FRET-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485 was tested against a chemokine receptor panel (agonist and antagonist mode) and proves to be selective for ACKR3. VUF15485 was subsequently labeled with tritium at one of its methoxy groups affording [3H]VUF15485. The small-molecule agonist radioligand binds saturably and with high affinity to human ACKR3 (Kd = 8.2 nM). [3H]VUF15485 shows rapid binding kinetics and consequently a short residence time (RT < 2 min) for its binding to ACKR3. Displacement of [3H]VUF15485 binding to membranes of HEK293T cells, transiently expressing ACKR3, with a number of CXCR3, CXCR4 or ACKR3 small-molecule ligands confirmed the ACKR3 profile of the [3H]VUF15485 binding site. Interestingly, the chemokine ligands CXCL11 and CXCL12 are not able to displace the binding of [3H]VUF15485 to ACKR3. The radiolabeled VUF15485 was subsequently used to evaluate its binding pocket. Site-directed mutagenesis and docking studies using a recently solved cryo-EM structure propose VUF15485 to bind in the major and the minor binding pocket of ACKR3. Significance Statement The atypical chemokine receptor ACKR3 is considered an interesting drug target in relation to e.g. cancer. The study reports on new chemical biology tools for ACKR3, i.e. a new agonist that can also be radiolabelled and a new ACKR3 conformational sensor, that both can be used to directly study the interaction of ACKR3 ligands with the GPCR.