NNC 55-0396 [(1S,2S)-2-(2-(N-[(3-Benzimidazol-2-yl)propyl]-N-
naphtyl cyclopropanecarboxylate dihydrochloride]: A New
Selective Inhibitor of T-Type Calcium Channels
Luping Huang, Brian M. Keyser, Tina M. Tagmose, J. Bondo Hansen, James T. Taylor,
Hean Zhuang, Min Zhang, David S. Ragsdale, and Ming Li
Department of Pharmacology, Tulane University Health Sciences Center, New Orleans, Louisiana (L.H., B.M.K., J.T.T., M.L.);
Department of Medicinal Chemistry, Novo Nordisk A/S, Måløv, Denmark (T.M.T., J.B.H.); Department of Anesthesiology and
Critical Care Medicine, Johns Hopkins University, Baltimore, Maryland (H.Z.); Department of Pharmacology, Medical College of
Virginia, Richmond, Virginia (M.Z.); and Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill
University, Montreal, Quebec, Canada (D.S.R.).
Received October 2, 2003; accepted December 5, 2003
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 mi-
befradil 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,
clopropanecarboxylate dihydrochloride (NNC 55-0396), that
exerts a selective inhibitory effect on T-type channels. The
acute IC50of NNC 55-0396 to block recombinant ?1G T-type
channels in human embryonic kidney 293 cells was ?7 ?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 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 cham-
ber 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 me-
tabolite that causes L-type Ca2?channel inhibition. We con-
clude 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
Voltage-gated Ca2?channels are transmembrane proteins
involved in the regulation of cellular excitability and intra-
cellular Ca2?signaling. Calcium channels have been divided
into various categories based on functional and pharmacolog-
ical 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 low-
voltage-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
This study was supported by American Heart Association Grant 0151047B
(to M.L.) and by Canadian Institutes of Health Research Grant MT13485 (to
Article, publication date, and citation information can be found at
ABBREVIATIONS: HVA, high-voltage-activated; HEK, human embryonic kidney; NNC 55-0395, (1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-
methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl valeroate dihydrochloride); 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; NNC 55-0397,
ride; PBS, phosphate-buffered saline; EI, electron impact; DMSO, dimethyl sulfoxide; G, conductance; MALDI-TOF, matrix-assisted laser
desorption ionization/time of flight instrument.
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics
JPET 309:193–199, 2004
Vol. 309, No. 1
Printed in U.S.A.
at ASPET Journals on November 6, 2015
no significant difference between the current in HEK 293/
?1G cells with (n ? 10) and without (n ? 6) NNC 55-0396
included in the pipette solution. For example, at the 10-min
timepoint, the p value is 0.12 with two-tailed unpaired Stu-
dent’s t test analysis. These data are inconsistent with the
idea that the site of action of NNC 55-0396 is on the cyto-
plasmic face of the T-type Ca2?channel. Thus, NNC 55-0396
appears to exhibit a relatively slow and wash-resistant bind-
ing to a site, perhaps within the transmembrane domains of
Mibefradil blocks both T-type Ca2?and HVA Ca2?chan-
nels. Although mibefradil appears to act directly on T-type
channels, we previously reported that its effect on HVA chan-
nels is not direct but instead involves cell permeation and
hydrolysis to an active metabolite that acts from the cyto-
plasmic side of the membrane (Wu et al., 2000). In the
present study, we developed a new compound, NNC 55-0396,
that has an inhibitory effect on T-type Ca2?current in HEK/
?1G cells but is not hydrolyzed to an active metabolite and
does not block HVA Ca2?currents in INS-1 cells. Thus, NNC
55-0396 is a selective inhibitor for T-type Ca2?channels. Our
data also suggest that the effect of NNC 55-0396 on T-type
channels is state-dependent; i.e., block is partially relieved by
membrane hyperpolarization and enhanced by high-fre-
quency channel activation.
Our previous findings (Wu et al., 2000) suggest that the
methoxyacetyl group of mibefradil is a critical determinant
for binding to HVA channels since its hydrolysis is the crit-
ical step in the binding of the compound to HVA channels. In
contrast, this moiety may play only a modest role in inter-
acting with the receptor domain of T-type Ca2?channels
since all three alternations—NNC 55-0395, NNC 55-0396,
and NNC 55-0397—retain the capability of blocking T-type
Ca2?current, although with varying potency. Our study
indicates that modification in the methoxyacetyl group of
mibefradil can improve its selectivity for T-type Ca2?chan-
nels over the HVA Ca2?channels without sacrificing the
potency of T-type Ca2?channel antagonism.
The primary objective of this study is to examine the struc-
ture-selectivity relationship of a small group of mibefradil
derivatives. Therefore, we chose HEK/?1G as a clean model
system. We do not know how effectively NNC 55-0396 will
block other T-type Ca2?channels, nor do we know its potency
on ?1G when other auxiliary subunits are present. However,
we speculate that NNC 55-0396 will block ?1H and ?1I, for it
has been shown that the IC50values of mibefradil blocking
?1G, ?1H, and ?1I are similar (Martin et al., 2000). In sum-
mary, our findings represent an important step toward de-
velopment of a specific T-type Ca2?inhibitor and thus have
potentially important scientific and therapeutic implications.
We thank Dr. B. Z. Peterson for constructive criticism on this
work. The mass spectrometric analyses were carried out in the
Louisiana State University Health Sciences Center New Orleans
Core Laboratories (New Orleans, LA).
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Address correspondence to: Dr. Ming Li, Department of Pharmacology
SL-83, 1430 Tulane Avenue, New Orleans, LA 70112. E-mail: email@example.com
Selective T-Type Ca2?Channel Blocker
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