Miller Melissa R., Shi Jie, Wu Meng, Engers Julie, Hopkins Corey R., Lindsley Craig W., Salovich James M., Zhu Yingmin, Tian Jin-Bin, Zhu Michael X., McManus Owen B., Li Min
Johns Hopkins Ion Channel Center, Baltimore, MD, 21205, USA
Vanderbilt Specialized Chemistry Center for Accelerated Probe Development (MLPCN), Nashville, TN, 37232, USA
ML204 was identified as a novel TRPC4 channel inhibitor following a high throughput fluorescent screen of the MLSMR library and SAR analysis of active compounds. ML204 inhibited calcium influx through TRPC4 channels activated by μ-opioid receptor stimulation with an IC50 value of 0.96 μM and exhibited 19-fold selectivity against TRPC6 channels in similar fluorescent assays. ML204 blocked TRPC4 channels in an electrophysiological assay with an IC value of 2.6 μM and was also active in fluorescent and electrophysiological assays in which TRPC4 channels were activated by different mechanisms, indicating direct block of TRPC4 channels. Selectivity for block of TRPC4 channels was examined in fluorescent and electrophysiological experiments against closely related TRPC channels and more distantly related TRPV, TRPA and TRPM channels, and against non-TRP ion channels. ML204 afforded good selectivity (19-fold) against TRPC6 channels and more modest selectivity against TRPC3 and TRPC5 (9-fold) channels. Little or no block of TRPV, TRPA, TRPM or voltage-gated ion channels was observed. ML204 exhibited properties useful for a variety of in vitro investigations.
在对MLSMR文库进行高通量荧光筛选并对活性化合物进行构效关系分析后，ML204被鉴定为一种新型的瞬时受体电位通道4（TRPC4）抑制剂。ML204可抑制由μ-阿片受体刺激激活的TRPC4通道介导的钙内流，其半数抑制浓度（IC50）值为0.96μM，并且在类似的荧光检测中对TRPC6通道表现出19倍的选择性。在电生理检测中，ML204以2.6μM的IC值阻断TRPC4通道，并且在TRPC4通道通过不同机制激活的荧光和电生理检测中也具有活性，表明其对TRPC4通道的直接阻断作用。在荧光和电生理实验中，针对密切相关的TRPC通道以及关系较远的瞬时受体电位香草酸亚型（TRPV）、瞬时受体电位锚蛋白1（TRPA）和瞬时受体电位M型（TRPM）通道，以及非TRP离子通道，检测了ML204对TRPC4通道阻断的选择性。ML204对TRPC6通道具有良好的选择性（19倍），对TRPC3和TRPC5通道的选择性适中（9倍）。未观察到对TRPV、TRPA、TRPM或电压门控离子通道的显著阻断或无阻断作用。ML204具有适用于各种体外研究的特性。
