Pharmacological characterization and radiolabeling ofVUF15485, a high-affinity small-molecule agonist for theatypical chemokine receptor ACKR3

Zarca, A.; Adlere, I.; Viciano, C. P.; Arimont-Segura, M.; Meyrath, M.; Simon, I. A.; Bebelman, J. P. M.; Laan, D.; Custers, H. G. J.; Janssen, E. C.; Versteegh, K. L.; Bosma, R.; De Esch, I. J. P.; Vischer, H. F.; Wijtmans, M.; Szpakowska, M.; Chevigne, A.; Hoffmann, C.; de Graaf, C.; Zarzycka, B. A.; Windhorst, A. D.; Smit, M. J.; Leurs, R.

doi:10.1101/2023.07.12.548622

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bioRxiv · 10.1101/2023.07.12.548622

Pharmacological characterization and radiolabeling ofVUF15485, a high-affinity small-molecule agonist for theatypical chemokine receptor ACKR3

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Abstract

Atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7, is considered to be an interesting drug target. In this study we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new ACKR3 small-molecule agonist, that will serve as an important new tool to study this {beta}-arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC50 = 8.3) to human ACKR3, as measured in [125I]CXCL12 competition binding experiments. Moreover, in a BRET-based {beta}-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 (atypical) 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.

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Zarca, A., Adlere, I., Viciano, C. P., Arimont-Segura, M., Meyrath, M., Simon, I. A., Bebelman, J. P. M., Laan, D., Custers, H. G. J., Janssen, E. C., Versteegh, K. L., Bosma, R., De Esch, I. J. P., Vischer, H. F., Wijtmans, M., Szpakowska, M., Chevigne, A., Hoffmann, C., de Graaf, C., Zarzycka, B. A., Windhorst, A. D., Smit, M. J., Leurs, R.. 2023-07-12. Pharmacological characterization and radiolabeling ofVUF15485, a high-affinity small-molecule agonist for theatypical chemokine receptor ACKR3. https://doi.org/10.1101/2023.07.12.548622

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