Huang Luping, Keyser Brian M, Tagmose Tina M, Hansen J Bondo, Taylor James T, Zhuang Hean, Zhang Min, Ragsdale David S, Li Ming
Department of Pharmacology SL-83, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA.
J Pharmacol Exp Ther. 2004 Apr;309(1):193-9. doi: 10.1124/jpet.103.060814. Epub 2004 Jan 12.
Mibefradil is a Ca2+ channel antagonist that inhibits both T-type and high-voltage-activated Ca2+ channels. We previously showed that block of high-voltage-activated channels by mibefradil occurs through the production of an active metabolite by intracellular hydrolysis. In the present study, we modified the structure of mibefradil to develop a nonhydrolyzable analog, (1S, 2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride (NNC 55-0396), that exerts a selective inhibitory effect on T-type channels. The acute IC(50) of NNC 55-0396 to block recombinant alpha(1)G T-type channels in human embryonic kidney 293 cells was approximately 7 microM, whereas 100 microM NNC 55-0396 had no detectable effect on high-voltage-activated channels in INS-1 cells. NNC 55-0396 did not affect the voltage-dependent activation of T-type Ca2+ currents but changed the slope of the steady-state inactivation curve. Block of T-type Ca2+ current was partially relieved by membrane hyperpolarization and enhanced at a high-stimulus frequency. Washing NNC 55-0396 out of the recording chamber did not reverse the T-type Ca2+ current activity, suggesting that the compound dissolves in or passes through the plasma membrane to exert its effect; however, intracellular perfusion of the compound did not block T-type Ca2+ currents, arguing against a cytoplasmic route of action. After incubating cells from an insulin-secreting cell line (INS-1) with NNC 55-0396 for 20 min, mass spectrometry did not detect the mibefradil metabolite that causes L-type Ca2+ channel inhibition. We conclude that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of L-type Ca2+ channels, thus rendering it more selective to T-type Ca2+ channels.
米贝拉地尔是一种钙通道拮抗剂，可抑制T型和高电压激活的钙通道。我们之前表明，米贝拉地尔对高电压激活通道的阻断是通过细胞内水解产生一种活性代谢物来实现的。在本研究中，我们对米贝拉地尔的结构进行了修饰，以开发一种不可水解的类似物，即（1S，2S）-2-（2-（N-[(3-苯并咪唑-2-基)丙基]-N-甲基氨基）乙基）-6-氟-1,2,3,4-四氢-1-异丙基-2-萘基环丙烷羧酸二盐酸盐（NNC 55-0396），它对T型通道具有选择性抑制作用。NNC 55-0396阻断人胚肾293细胞中重组α(1)G T型通道的急性半数抑制浓度（IC(50)）约为7微摩尔，而100微摩尔的NNC 55-0396对INS-1细胞中的高电压激活通道没有可检测到的影响。NNC 55-0396不影响T型钙电流的电压依赖性激活，但改变了稳态失活曲线的斜率。T型钙电流的阻断可通过膜超极化部分缓解，并在高刺激频率下增强。将NNC 55-0396从记录室中冲洗出去并没有使T型钙电流活性恢复，这表明该化合物溶解在质膜中或穿过质膜发挥其作用；然而，向细胞内灌注该化合物并没有阻断T型钙电流，这排除了其通过细胞质途径发挥作用的可能性。在用NNC 55-0396孵育胰岛素分泌细胞系（INS-1）的细胞20分钟后，质谱分析未检测到导致L型钙通道抑制的米贝拉地尔代谢物。我们得出结论，NNC 55-0396由于其结构修饰，不会产生导致L型钙通道抑制的代谢物，因此使其对T型钙通道更具选择性。
