NNC 55-0396 [(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]: A New Selective Inhibitor of T-Type Calcium Channels

Abstract

Mibefradil is a Ca 2ϩ channel antagonist that inhibits both Ttype and high-voltage-activated Ca 2ϩ 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 , that exerts a selective inhibitory effect on T-type channels. The acute IC 50 of NNC 55-0396 to block recombinant ␣ 1 G T-type channels in human embryonic kidney 293 cells was ϳ7 M, whereas 100 M 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 Ca 2ϩ currents but changed the slope of the steady-state inactivation curve. Block of T-type Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ channel inhibition. We conclude that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of Ltype Ca 2ϩ channels, thus rendering it more selective to T-type Ca 2ϩ channels.Voltage-gated Ca 2ϩ channels are transmembrane proteins involved in the regulation of cellular excitability and intracellular Ca 2ϩ signaling. Calcium channels have been divided into various categories based on functional and pharmacological criteria. High-voltage-activated (HVA) channels, which have been further subdivided into L-, N-, P/Q-, and R-types, require strong depolarizations for activation, whereas lowvoltage-activated or T-type channels activate over a much more negative voltage range and exhibit unique inactivation and deactivation kinetics (Armstrong and Matteson, 1985;Catterall, 1998;Perez-Reyes, 1998). The main structural component of the voltage-gated calcium channel is the ␣ 1

Role of T-type Ca2+ Channels in the Spontaneous Phasic Contraction of Pregnant Rat Uterine Smooth Muscle

“…However, in contrast, the recent investigation reported that mibefradil also blocked the L-type Ca 2＋ channel by active metabolite produced via intracellular hydrolysis. Therefore, non-hydrolyzable analogue of mibefradil, NNC 55-0396, was developed as a selective blocker of the T-type Ca 2＋ channel (Huang et al, 2004). We showed that cumulative addition of mibefradil and NNC 55-0396 produced concentration-dependent inhibition of frequency as well as amplitude of spontaneous Ca 2＋ transients and contractions, respectively.…”

Presynaptic T-Type Ca2+Channels Modulate Dendrodendritic Mitral–Mitral and Mitral–Periglomerular Connections in Mouse Olfactory Bulb

“…First, we applied NNC 55-0396, a nonhydrolyzable analog of mibefradil, which is more specific for T-type channels because it lacks the effects of the mibefradil metabolite on HVA Ca 2ϩ channels (Huang et al, 2004); 50 M NNC 55-0396 applied for Ͼ15 min inhibited LVA Ca 2ϩ influx (Fig. 4C 1 ), reduced the amplitude of the somatic APs (Fig.…”

T-Type Ca2+ Channel Inhibition Induces p53-Dependent Cell Growth Arrest and Apoptosis through Activation of p38-MAPK in Colon Cancer Cells

“…42). Notably, the reduction in cell proliferation in response to either mibefradil or TTL1177 was accompanied by a significant increase in activation of caspase-3/7 ( Fig.…”