TY - JOUR
T1 - Pharmacological Characterization and Radiolabeling of VUF15485, a High-Affinity Small-Molecule Agonist for the Atypical Chemokine Receptor ACKR3
AU - Zarca, Aurelien M.
AU - Adlere, Ilze
AU - Viciano, Cristina P.
AU - Arimont-Segura, Marta
AU - Meyrath, Max
AU - Simon, Icaro A.
AU - Bebelman, Jan Paul
AU - Laan, Dennis
AU - Custers, Hans G.J.
AU - Janssen, Elwin
AU - Versteegh, Kobus L.
AU - Buzink, Maurice C.M.L.
AU - Nesheva, Desislava N.
AU - Bosma, Reggie
AU - de Esch, Iwan J.P.
AU - Vischer, Henry F.
AU - Wijtmans, Maikel
AU - Szpakowska, Martyna
AU - Chevigné, Andy
AU - Hoffmann, Carsten
AU - de Graaf, Chris
AU - Zarzycka, Barbara A.
AU - Windhorst, Albert D.
AU - Smit, Martine J.
AU - Leurs, Rob
N1 - This research was funded by European Union’s Horizon 2020 Marie Skłodowska-Curie Actions (MSCA) Program under Grant Agreement 641833 (ONCOgenic Receptor Network of Excellence and Training, ONCORNET) to A.M.Z., I.A., C.P.V., M.A.-S., C.H., H.F.V., M.W., C.d.G., R.L., I.J.P.dE., M.J.S., ONCORNET2.0 “ONCOgenic Receptor Network of Excellence and Training” (MSCA-ITN-2020-ETN) to I.J.P.dE., M.J.S., R.L., C.d.G., M.J.S., C.H., M.W., H.F.V., A.C., the Luxembourg Institute of Health (LIH) to M.M., M.S., and A.C., Luxembourg National Research Fund (INTER/FNRS grants 20/15084569 and AFR HOPE-IOID, PRIDE-14254520 “I2TRON” and PRIDE-16749720 “NextImmune2”), F.R.S.-FNRS-Télévie (Grants 7.8504.20, 7.4502.21, and 7.8508.22) to M.M., M.S., and A.C.
Publisher Copyright:
Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.
PY - 2024/3/14
Y1 - 2024/3/14
N2 - 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 β-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 bioluminescence resonance energy transfer-based β-arrestin2 recruitment assay VUF15485 acts as a potent ACKR3 agonist (pEC50 = 7.6) and shows a similar extent of receptor activation compared with CXCL12 when using a newly developed, fluorescence resonance energy transfer-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485, tested against a (atypical) chemokine receptor panel (agonist and antagonist mode), proves to be selective for ACKR3. VUF15485 labeled with tritium at one of its methoxy groups ([3H]VUF15485), binds ACKR3 saturably and with high affinity (Kd = 8.2 nM). Additionally, [3H]VUF15485 shows rapid binding kinetics and consequently a short residence time (<2 minutes) for binding to ACKR3. The selectivity of [3H]VUF15485 for ACKR3, was confirmed by binding studies, whereupon CXCR3, CXCR4, and ACKR3 small-molecule ligands were competed for binding against the radiolabeled agonist. 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 that VUF15485 binds in the major and the minor binding pocket of ACKR3. SIGNIFICANCE STATEMENT: The atypical chemokine receptor atypical chemokine receptor 3 (ACKR3) is considered an interesting drug target in relation to cancer and multiple sclerosis. The study reports on new chemical biology tools for ACKR3, i.e., a new agonist that can also be radiolabeled and a new ACKR3 conformational sensor, that both can be used to directly study the interaction of ACKR3 ligands with the G protein-coupled receptor.
AB - 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 β-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 bioluminescence resonance energy transfer-based β-arrestin2 recruitment assay VUF15485 acts as a potent ACKR3 agonist (pEC50 = 7.6) and shows a similar extent of receptor activation compared with CXCL12 when using a newly developed, fluorescence resonance energy transfer-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485, tested against a (atypical) chemokine receptor panel (agonist and antagonist mode), proves to be selective for ACKR3. VUF15485 labeled with tritium at one of its methoxy groups ([3H]VUF15485), binds ACKR3 saturably and with high affinity (Kd = 8.2 nM). Additionally, [3H]VUF15485 shows rapid binding kinetics and consequently a short residence time (<2 minutes) for binding to ACKR3. The selectivity of [3H]VUF15485 for ACKR3, was confirmed by binding studies, whereupon CXCR3, CXCR4, and ACKR3 small-molecule ligands were competed for binding against the radiolabeled agonist. 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 that VUF15485 binds in the major and the minor binding pocket of ACKR3. SIGNIFICANCE STATEMENT: The atypical chemokine receptor atypical chemokine receptor 3 (ACKR3) is considered an interesting drug target in relation to cancer and multiple sclerosis. The study reports on new chemical biology tools for ACKR3, i.e., a new agonist that can also be radiolabeled and a new ACKR3 conformational sensor, that both can be used to directly study the interaction of ACKR3 ligands with the G protein-coupled receptor.
UR - http://www.scopus.com/inward/record.url?scp=85187961735&partnerID=8YFLogxK
U2 - 10.1124/molpharm.123.000835
DO - 10.1124/molpharm.123.000835
M3 - Article
C2 - 38346795
AN - SCOPUS:85187961735
SN - 0026-895X
VL - 105
SP - 301
EP - 312
JO - Molecular Pharmacology
JF - Molecular Pharmacology
IS - 4
ER -