Xanthine-based photoaffinity probes allow assessment of ligand engagement by TRPC5 channels
Xanthine-based photoaffinity probes allow assessment of ligand engagement by TRPC5 channels
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DOI：
10.1039/d0cb00126k
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发表时间：
2020-09-18
影响因子：
4.1
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TRPC1/4/5 阳离子通道是治疗中枢神经系统 (CNS) 疾病、肾脏疾病、心血管和代谢疾病等的新兴药物靶点。已报道了各种小分子 TRPC1/4/5 调节剂，包括区分特定 TRPC1/4/5 四聚体的高效黄嘌呤衍生物。然而，缺乏在活细胞中分析 TRPC1/4/5 通道配体结合的工具。在这里，我们报告了一组有效的基于黄嘌呤的光亲和探针，其功能模仿黄嘌呤 Pico145 和 AM237。使用这些探针，我们开发了 TRPC5 通道的光亲和标记方案，为定量评估 TRPC5 与细胞中小分子的结合相互作用提供了第一种方法。该方法对于针对 TRPC1/4/5 通道的黄嘌呤/脂质结合位点的药物发现工作可能很重要。基于有效且选择性的 TRPC1/4/5 通道抑制剂 Pico145 的含二氮丙啶的光亲和探针，允许开发一种检测 TRPC5 蛋白和基于黄嘌呤的 TRPC5 通道调节剂之间细胞相互作用的检测方法。
TRPC1/4/5 cation channels are emerging drug targets for the treatment of, amongst others, central nervous system (CNS) disorders, kidney disease, and cardiovascular and metabolic disease. Various small-molecule TRPC1/4/5 modulators have been reported, including highly potent xanthine derivatives that distinguish between specific TRPC1/4/5 tetramers. However, tools to profile ligand engagement by TRPC1/4/5 channels in live cells are lacking. Here, we report a set of potent xanthine-based photoaffinity probes that functionally mimic the xanthines Pico145 and AM237. Using these probes, we have developed a photoaffinity labelling protocol for TRPC5 channels, providing the first method for the quantitative assessment of binding interactions of TRPC5 with small molecules in cells. This method could be important for drug discovery efforts targeting the xanthine/lipid binding site of TRPC1/4/5 channels. Diazirine-containing photoaffinity probes, based on the potent and selective TRPC1/4/5 channel inhibitor Pico145, allowed the development of an assay to probe cellular interactions between TRPC5 protein and xanthine-based TRPC5 channel modulators.