Synthesis and Application of the First Radioligand Targeting the Allosteric Binding Pocket of Chemokine Receptor CXCR3



Strategies for the identification of allosteric modulators of chemokine receptors largely rely on various cell-based functional assays. Radioligand binding assays are typically not available for allosteric binding sites. We synthesized, purified, and applied the first tritium-labeled allosteric modulator of the human chemokine receptor CXCR3 (RAMX3, [3H]N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-[(1-methylpiperidin-4-yl)methyl]acetamide). RAMX3 is chemically derived from 8-azaquinazolinone-type allosteric modulators and binds to the CXCR3 receptor with a Kd value of 1.08 nM (specific activity: 80.4 Ci mmol−1). Radioligand displacement assays showed potent negative cooperativity between RAMX3 and chemokine CXCL11, providing a basis for the use of RAMX3 to investigate other potential allosteric modulators. Additionally, the synthesis and characterization of a number of other full and truncated 8-azaquinazoline analogues were used to validate the binding properties of RAMX3. We demonstrate that RAMX3 can be efficiently used to facilitate the discovery and characterization of small molecules as allosteric modulators of the CXCR3 receptor.