Department of Medicinal Chemistry (A.M.Z., M.A.-S., I.A.S., J.P.B., H.G.J.C., K.L.V., M.C.M.L.B., D.N.N., R.B., I.J.P.dE., H.F.V., M.W., C.dG., B.A.Z., M.J.S., R.L.) and Department of Chemistry & Pharmaceutical Sciences (E.J.), Amsterdam Institute for Molecular Life Sciences, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HV Amsterdam, Netherlands; Griffin Discoveries BV, Amsterdam, Netherlands (I.A., I.J.P.dE., R.L.); Bio-Imaging-Center/Rudolf-Virchow-Zentrum, Institut für Pharmakologie, Versbacher Strasse 9, 97078 Würzburg, Germany (C.P.V., C.H.); Institute for Molecular Cell Biology, CMB - Center for Molecular Biomedicine, University Hospital Jena, Friedrich Schiller University Jena, Jena, Germany (C.P.V., C.H.); Immuno-Pharmacology and Interactomics, Department of Infection and Immunity, Luxembourg Institute of Health, 29, rue Henri Koch, L-4354, Esch-sur-Alzette, Luxembourg (M.M., M.S., A.C.); and Department of Radiology and Nuclear Medicine, VU University Medical Center Amsterdam, Netherlands (D.L., A.D.W.).
Department of Medicinal Chemistry (A.M.Z., M.A.-S., I.A.S., J.P.B., H.G.J.C., K.L.V., M.C.M.L.B., D.N.N., R.B., I.J.P.dE., H.F.V., M.W., C.dG., B.A.Z., M.J.S., R.L.) and Department of Chemistry & Pharmaceutical Sciences (E.J.), Amsterdam Institute for Molecular Life Sciences, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HV Amsterdam, Netherlands; Griffin Discoveries BV, Amsterdam, Netherlands (I.A., I.J.P.dE., R.L.); Bio-Imaging-Center/Rudolf-Virchow-Zentrum, Institut für Pharmakologie, Versbacher Strasse 9, 97078 Würzburg, Germany (C.P.V., C.H.); Institute for Molecular Cell Biology, CMB - Center for Molecular Biomedicine, University Hospital Jena, Friedrich Schiller University Jena, Jena, Germany (C.P.V., C.H.); Immuno-Pharmacology and Interactomics, Department of Infection and Immunity, Luxembourg Institute of Health, 29, rue Henri Koch, L-4354, Esch-sur-Alzette, Luxembourg (M.M., M.S., A.C.); and Department of Radiology and Nuclear Medicine, VU University Medical Center Amsterdam, Netherlands (D.L., A.D.W.)
Mol Pharmacol. 2024 Mar 14;105(4):301-312. doi: 10.1124/molpharm.123.000835.
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 -arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC = 8.3) to human ACKR3, as measured in [I]CXCL12 competition binding experiments. Moreover, in a bioluminescence resonance energy transfer-based -arrestin2 recruitment assay VUF15485 acts as a potent ACKR3 agonist (pEC = 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 ([H]VUF15485), binds ACKR3 saturably and with high affinity ( = 8.2 nM). Additionally, [H]VUF15485 shows rapid binding kinetics and consequently a short residence time (<2 minutes) for binding to ACKR3. The selectivity of [H]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 [H]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.
非典型趋化因子受体 3（ACKR3），以前称为 CXCR7，被认为是一个有趣的药物靶点。在这项研究中，我们报告了 VUF15485 的合成、药理学特征和放射性标记，这是一种新的 ACKR3 小分子激动剂，将作为研究这种 -arrestin 偏向趋化因子受体的重要新工具。VUF15485 以纳摩尔亲和力（pIC = 8.3）与人 ACKR3 结合，如[I]CXCL12 竞争结合实验所示。此外，在基于生物发光共振能量转移的-arrestin2 募集测定中，VUF15485 作为一种有效的 ACKR3 激动剂（pEC = 7.6）起作用，并且当使用新开发的基于荧光共振能量转移的 ACKR3 构象传感器时，与 CXCL12 相比，其受体激活程度相似。此外，针对（非典型）趋化因子受体组（激动剂和拮抗剂模式）测试的 ACKR3 激动剂 VUF15485 被证明对 ACKR3 具有选择性。用氚标记其一个甲氧基（[H]VUF15485）的 VUF15485 以高亲和力（ = 8.2 nM）饱和结合 ACKR3。此外，[H]VUF15485 显示出快速结合动力学，因此与 ACKR3 的结合停留时间较短（<2 分钟）。通过结合研究证实了[H]VUF15485 对 ACKR3 的选择性，其中趋化因子配体 CXCL11 和 CXCL12 不能置换放射性标记激动剂与 ACKR3 的结合。有趣的是，趋化因子配体 CXCL11 和 CXCL12 不能置换放射性标记激动剂与 ACKR3 的结合。随后使用放射性标记的 VUF15485 来评估其结合口袋。使用最近解决的冷冻电镜结构进行的定点突变和对接研究表明，VUF15485 结合在 ACKR3 的主要和次要结合口袋中。意义陈述：非典型趋化因子受体 3（ACKR3）被认为是与癌症和多发性硬化症相关的一个有趣的药物靶点。该研究报告了 ACKR3 的新的化学生物学工具，即一种新的激动剂，也可以进行放射性标记，以及一种新的 ACKR3 构象传感器，两者都可用于直接研究 ACKR3 配体与 G 蛋白偶联受体的相互作用。
