Abstract：
A conserved intracellular allosteric binding site (IABS) was recently identified at several G protein-coupled receptors (GPCRs). This target site allows the binding of allosteric modulators and enables a new mode of GPCR inhibition. Herein, we report the development of a NanoBRET-based assay platform based on the fluorescent ligand LT221 (5), to detect intracellular binding to CCR6 and CXCR1, two chemokine receptors that have been pursued as promising drug targets in inflammation and immuno-oncology. Our assay platform enables cell-free as well as cellular NanoBRET-based binding studies in a nonisotopic and straightforward manner. By combining this screening platform with a previously reported CXCR2 assay, we investigated CXCR1/CXCR2/CCR6 selectivity profiles for both known and novel squaramide analogues derived from navarixin, a known intracellular CXCR1/CXCR2 antagonist and phase II clinical candidate for the treatment of pulmonary diseases. By means of these studies we identified compound 10, a previously reported tert-butyl analogue of navarixin, as a low nanomolar intracellular CCR6 antagonist. Further, our assay platform clearly indicated intracellular binding of the CCR6 antagonist PF-07054894, currently evaluated in phase I clinical trials for the treatment of ulcerative colitis, thereby providing profound evidence for the existence and the pharmacological relevance of a druggable IABS at CCR6.
摘要：
最近在几种G蛋白偶联受体（GPCRs）上发现了保守的细胞内变构结合位点（IABS）。该靶位点允许变构调节剂的结合并实现GPCR抑制的新模式。在这里，我们报告了基于荧光配体LT221的基于NanoBRET的测定平台的开发（5），用于检测细胞内与CCR6和CXCR1的结合，这两种趋化因子受体已被视为炎症和免疫肿瘤学中有希望的药物靶标。我们的分析平台能够以非同位素和直接的方式进行无细胞以及基于细胞的NanoBRET的结合研究。通过将此筛选平台与先前报道的CXCR2测定相结合，我们研究了CXCR1/CXCR2/CCR6的选择性谱已知和新的squaramide类似物衍生自纳瓦利辛，一种已知的细胞内CXCR1/CXCR2拮抗剂和治疗肺部疾病的II期临床候选药物。通过这些研究，我们确定了化合物10，一种先前报道的纳瓦利辛的叔丁基类似物，作为低纳摩尔细胞内CCR6拮抗剂。Further,我们的分析平台清楚地表明CCR6拮抗剂PF-07054894的细胞内结合，目前在治疗溃疡性结肠炎的I期临床试验中进行了评估，从而为CCR6处的可药用IABS的存在和药理学相关性提供了深刻的证据。
