Palmer R Kyle, Atwal Karnail, Bakaj Ivona, Carlucci-Derbyshire Stacy, Buber M Tulu, Cerne Rok, Cortés Rosa Y, Devantier Heather R, Jorgensen Vincent, Pawlyk Aaron, Lee S Paul, Sprous Dennis G, Zhang Zheng, Bryant Robert
Redpoint Bio Inc., Ewing, New Jersey 08628, USA.
Assay Drug Dev Technol. 2010 Dec;8(6):703-13. doi: 10.1089/adt.2010.0334.
Transient receptor potential melastatin-5 (TRPM5) is a calcium-gated monovalent cation channel expressed in highly specialized cells of the taste bud and gastrointestinal tract, as well as in pancreatic β-cells. Well established as a critical signaling protein for G protein-coupled receptor-mediated taste pathways, TRPM5 also has recently been implicated as a regulator of incretin and insulin secretion. To date, no inhibitors of practical use have been described that could facilitate investigation of TRPM5 functions in taste or secretion of metabolic hormones. Using recombinant TRPM5-expressing cells in a fluorescence imaging plate reader-based membrane potential assay, we identified triphenylphosphine oxide (TPPO) as a selective and potent inhibitor of TRPM5. TPPO inhibited both human (IC₅₀ = 12 μM) and murine TRPM5 (IC₅₀ = 30 μM) heterologously expressed in HEK293 cells, but had no effect (up to 100 μM) on the membrane potential responses of TRPA1, TRPV1, or TRPM4b. TPPO also inhibited a calcium-gated TRPM5-dependent conductance in taste cells isolated from the tongues of transgenic TRPM5(+/)⁻ mice. In contrast, TPP had no effect on TRPM5 responses, indicating a strict requirement of the oxygen atom for activity. Sixteen additional TPPO derivatives also inhibited TRPM5 but none more potently than TPPO. Structure-activity relationship of tested compounds was used for molecular modeling-based analysis to clarify the positive and negative structural contributions to the potency of TPPO and its derivatives. TPPO is the most potent TRPM5 inhibitor described to date and is the first demonstrated to exhibit selectivity over other channels.
瞬时受体电位褪黑素5（TRPM5）是一种钙门控单价阳离子通道，表达于味蕾和胃肠道的高度特化细胞以及胰腺β细胞中。TRPM5作为G蛋白偶联受体介导的味觉通路的关键信号蛋白已被充分证实，最近还被认为是肠促胰岛素和胰岛素分泌的调节因子。迄今为止，尚未描述可促进对TRPM5在味觉或代谢激素分泌中的功能进行研究的实用抑制剂。在基于荧光成像酶标仪的膜电位测定中，我们使用表达重组TRPM5的细胞，鉴定出三苯基氧化膦（TPPO）是TRPM5的一种选择性强效抑制剂。TPPO抑制了在HEK293细胞中异源表达的人TRPM5（IC₅₀ = 12 μM）和小鼠TRPM5（IC₅₀ = 30 μM），但对TRPA1、TRPV1或TRPM4b的膜电位反应没有影响（高达100 μM）。TPPO还抑制了从转基因TRPM5(+/)⁻小鼠舌头分离的味觉细胞中钙门控的TRPM5依赖性电导。相比之下，TPP对TRPM5反应没有影响，表明活性严格需要氧原子。另外16种TPPO衍生物也抑制TRPM5，但没有一种比TPPO更有效。利用测试化合物的构效关系进行基于分子建模的分析，以阐明对TPPO及其衍生物效力的正性和负性结构贡献。TPPO是迄今为止描述的最有效的TRPM5抑制剂，并且是第一个被证明对其他通道具有选择性的抑制剂。
