Centre for Cardiovascular and Metabolic Research, Hull York Medical School, University of Hull, Hull HU6 7RX, UK.
Biochem Pharmacol. 2012 Apr 1;83(7):923-31. doi: 10.1016/j.bcp.2012.01.014. Epub 2012 Jan 20.
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used anti-inflammatory therapeutic agents, among which the fenamate analogues play important roles in regulating intracellular Ca²⁺ transient and ion channels. However, the effect of NSAIDs on TRPC4 and TRPC5 is still unknown. To understand the structure-activity of fenamate analogues on TRPC channels, we have synthesized a series of fenamate analogues and investigated their effects on TRPC4 and TRPC5 channels. Human TRPC4 and TRPC5 cDNAs in tetracycline-regulated vectors were transfected into HEK293 T-REx cells. The whole cell current and Ca²⁺ movement were recorded by patch clamp and calcium imaging, respectively. Flufenamic acid (FFA), mefenamic acid (MFA), niflumic acid (NFA) and diclofenac sodium (DFS) showed inhibition on TRPC4 and TRPC5 channels in a concentration-dependent manner. The potency was FFA>MFA>NFA>DFS. Modification of 2-phenylamino ring by substitution of the trifluoromethyl group in FFA with F, CH₃, OCH₃, OCH₂CH₃, COOH, and NO₂ led to the changes in their channel blocking activity. However, 2-(2'-methoxy-5'-methylphenyl)aminobenzoic acid stimulated TRPC4 and TRPC5 channels. Selective COX1-3 inhibitors (aspirin, celecoxib, acetaminophen, and indomethacin) had no effect on the channels. Longer perfusion (> 5 min) with FFA (100 μM) and MFA (100 μM) caused a potentiation of TRPC4 and TRPC5 currents after their initial blocking effects that appeared to be partially mediated by the mitochondrial Ca²⁺ release. Our results suggest that fenamate analogues are direct modulators of TRPC4 and TRPC5 channels. The substitution pattern and conformation of the 2-phenylamino ring could alter their blocking activity, which is important for understanding fenamate pharmacology and new drug development targeting the TRPC channels.
非甾体抗炎药（NSAIDs）是广泛使用的抗炎治疗药物，其中芬那酸类似物在调节细胞内 Ca²⁺瞬变和离子通道方面发挥重要作用。然而，NSAIDs 对 TRPC4 和 TRPC5 的影响尚不清楚。为了了解芬那酸类似物对 TRPC 通道的结构-活性，我们合成了一系列芬那酸类似物，并研究了它们对 TRPC4 和 TRPC5 通道的影响。四环素调控载体中的人 TRPC4 和 TRPC5 cDNA 转染到 HEK293 T-REx 细胞中。通过膜片钳和钙成像分别记录全细胞电流和 Ca²⁺运动。氟芬那酸（FFA）、甲芬那酸（MFA）、尼氟酸（NFA）和双氯芬酸钠（DFS）对 TRPC4 和 TRPC5 通道呈浓度依赖性抑制。效力为 FFA>MFA>NFA>DFS。FFA 中 2-苯氨基环的 2-苯基取代为三氟甲基的 F、CH₃、OCH₃、OCH₂CH₃、COOH 和 NO₂，导致其通道阻断活性发生变化。然而，2-（2'-甲氧基-5'-甲基苯基）氨基苯甲酸刺激 TRPC4 和 TRPC5 通道。选择性 COX1-3 抑制剂（阿司匹林、塞来昔布、对乙酰氨基酚和吲哚美辛）对通道没有影响。FFA（100 μM）和 MFA（100 μM）的长时间灌注（>5 分钟）在其初始阻断作用后引起 TRPC4 和 TRPC5 电流的增强，这似乎部分由线粒体 Ca²⁺释放介导。我们的结果表明，芬那酸类似物是 TRPC4 和 TRPC5 通道的直接调节剂。2-苯氨基环的取代模式和构象可以改变其阻断活性，这对于理解芬那酸药理学和靶向 TRPC 通道的新药开发很重要。
