Identification and optimization of 2-aminobenzimidazole derivatives as novel inhibitors of TRPC4 and TRPC5 channels

Article abstract of DOI:10.1111/bph.13140

Background and Purpose Transient receptor potential canonical (TRPC) channels play important roles in a broad array of physiological functions and are involved in various diseases. However, due to a lack of potent subtype-specific inhibitors the exact roles of TRPC channels in physiological and pathophysiological conditions have not been elucidated. Experimental Approach Using fluorescence membrane potential and Ca²⁺ assays and electrophysiological recordings, we characterized new 2-aminobenzimidazole-based small molecule inhibitors of TRPC4 and TRPC5 channels identified from cell-based fluorescence high-throughput screening. Key Results The original compound, M084, was a potent inhibitor of both TRPC4 and TRPC5, but was also a weak inhibitor of TRPC3. Structural modifications of the lead compound resulted in the identification of analogues with improved potency and selectivity for TRPC4 and TRPC5 channels. The aminobenzimidazole derivatives rapidly inhibited the TRPC4- and TRPC5-mediated currents when applied from the extracellular side and this inhibition was independent of the mode of activation of these channels. The compounds effectively blocked the plateau potential mediated by TRPC4-containing channels in mouse lateral septal neurons, but did not affect the activity of heterologously expressed TRPA1, TRPM8, TRPV1 or TRPV3 channels or that of the native voltage-gated Na⁺, K⁺ and Ca²⁺ channels in dissociated neurons. Conclusions and Implications The TRPC4/C5-selective inhibitors developed here represent novel and useful pharmaceutical tools for investigation of physiological and pathophysiological functions of TRPC4/C5 channels.

Full text of DOI:10.1111/bph.13140

Identification and optimization of 2-aminobenzimidazole derivatives as
novel inhibitors of TRPC4 and TRPC5 channels1
Yingmin Zhua,#, Yungang Lua,b,#,¶, Chunrong Quc, Melissa Millerd,¶, JinbinTiana,
Dhananjay P. Thakura, Jinmei Zhuc, Zixin Dengc, Xianming Huc, Meng Wud,¶, Owen B.
McManusd,¶, Min Lid,¶, Xuechuan Hongc,*, Michael X. Zhua,*, Huai-Rong Luoe,*,
a
Department of Integrative Biology and Pharmacology, The University of Texas Health
Science Center at Houston, Houston, TX, 77030, USA
b
The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510150,
China
c
State Key Laboratory of Virology, Key Laboratory of Combinatorial Biosynthesis and
Drug Discovery (Wuhan University), Ministry of Education, Wuhan University School
of Pharmaceutical Sciences, Wuhan 430071, China
d
Department of Neuroscience, High Throughput Biology Center and Johns Hopkins Ion
Channel Center, Johns Hopkins University School of Medicine, Baltimore, Maryland,
21205, USA
e
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming
Institute of Botany, the Chinese Academy of Sciences, Kunming, Yunnan, China
Running title: Novel 2-aminobenzimidazole-based TRPC4/C5 inhibitors
# Equal contribution authors
*Corresponding authors: Dr. Michael X. Zhu, Dr. Xuechuan Hong, and Dr. Huai-Rong
Luo
Department of Integrative Biology and Pharmacology
The University of Texas Health Science Center at Houston
6431 Fannin St, MSB 4.128
Houston, TX, 77030, USA
Phone: 713-500-7505
E-mail: michael.x.zhu@uth.tmc.edu; xhy78@whu.edu.cn; luohuairong@mail.kib.ac.cn
This article has been accepted for publication and undergone full peer review but has not been through the
copyediting, typesetting, pagination and proofreading process, which may lead to differences between this
version and the Version of Record. Please cite this article as doi: 10.1111/bph.13140
1
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¶Current affiliations: University of California at Los Angeles (YL), University of
California at Berkeley (MM), High-Throughput Screening Facility, University of Iowa
(MW), Essen Bioscience (OM), GlaxoSmithKline (ML)
Author contributions
OM, ML, X Hong, HRL, and MXZ designed the research; YZ, YL, CQ, MM, JT, DPT,
JZ, ZD, X Hu, MW performed experiments; YZ, YL, MM, JT, MW, OM, X Hong, MXZ,
HRL performed data analyses; YZ, YL, MW, OM, ML, X Hong, HRL, and MXZ wrote
the paper.
Abbreviations used:
ACPD, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid; 2APB, 2-
aminoethoxydiphenyl borate; [Ca2+]i, intracellular Ca2+ concentration; CCh, Carbachol;
cpd, compound; DHPG, (S)-3,5-dihydroxyphenylglycine (DHPG); DRG, dorsal root
ganglion; FBS, fetal bovine serum; FFA, flufenamic acid; FMP, FLIPR membrane
potential dye; FMP II, FLIPR membrane potential dye II; GPCR, G protein-coupled
receptor; 5-HT, 5-hydroxytryptamine; 5-HT1AR, 5-HT receptor type 1A; I-V, current-
voltage; M2R and M5R, M2 and M5 muscarinic receptor, respectively; MLPCN,
Molecular Libraries Probe Production Centers Network; MLSMR, Molecular Libraries
Small Molecule Repository; µOR, μ-opioid receptor; PLC, phospholipase C; SAR,
Structure and activity relationship; TRP, Transient Receptor Potential; TRPC, TRP
canonical
2
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Abstract:
Background and Purpose: Transient receptor potential canonical (TRPC) channels play
important roles in a broad array of physiological functions and are implicated in diseases.
However, the lack of potent subtype-specific inhibitors has limited delineation of the
roles of TRPC channels in physiological and pathophysiological conditions.
Experimental approach: Using fluorescence membrane potential and Ca2+ assays and
electrophysiological recording, we characterized new 2-aminobenzimidazole-based small
molecular inhibitors of TRPC4 and TRPC5 channels identified from cell-based
fluorescence high throughput screening.
Key Results: The original compound, M084, had IC50 values of 10.3 and 8.2 µM against
TRPC4 and TRPC5, respectively, but was also weakly inhibitory to TRPC3 (IC50 ~50
µM). Structure modifications of the lead compound resulted in the identification of
structural analogs with improved potency (IC50 < 5 µM) and selectivity for TRPC4 and
TRPC5 channels. The aminobenzimidazole derivatives exhibited fast action on inhibiting
TRPC4 and TRPC5 currents when applied from the extracellular side and the inhibition
was independent of the mode of activation of these channels. The compounds effectively
blocked the plateau potential mediated by TRPC4-containing channels in mouse lateral
septal neurons but did not affect the activity of heterlogously expressed TRPA1, TRPM8,
TRPV1, or TRPV3 channels as well as that of the native voltage-gated Na+, K+ and Ca2+
channels in dissociated neurons.
Conclusions & Implications: The TRPC4/C5-selective inhibitors developed here
represent novel and useful pharmaceutical tools for investigation of physiological and
pathophysiological functions of TRPC4/C5 channels.
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INTRODUCTION
The superfamily of Transient Receptor Potential (TRP) cation channels in mammals
consists of six subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin),
TRPA (ankyrin), TRPP (polycystin), and TRPML (mucolipin) (Montell et al., 2002).
TRPC channels are typically activated downstream from stimulation of phospholipase C
(PLC) (Plant and Schaefer, 2003; Trebak et al., 2007). However, which step(s) or
constituent(s) of the PLC pathway is the most critical for TRPC activation is not well
defined. The mammalian TRPC family has seven members, TRPC1-7, and based on
sequence similarities, they are separated into four groups: TRPC1, TRPC2,
TRPC3/C6/C7, and TRPC4/C5. TRPC2 is a pseudogene in humans. TRPC3/C6/C7 can
be directly activated by diacylglycerols (Hofmann et al., 1999). TRPC4/C5 appears to
respond to receptors that activate Gi/o signaling in addition to the Gq/11-PLC pathway
(Jeon et al., 2012; Jeon et al., 2008; Kim et al., 2014; Otsuguro et al., 2008). Because
PLC activation is commonly achieved by the stimulation of G protein-coupled receptors
(GPCRs) or receptor tyrosine kinases, the TRPC channels are also referred to as receptor-
operated channels (Plant and Schaefer, 2003).
Functionally, TRPC channels are non-selective cation channels that mediate Na+ and
Ca2+ entry into cells, leading to membrane depolarization and intracellular Ca2+
concentration ([Ca2+]i) elevation. The rise in Na+ level at the cytoplasmic side could also
be important for Na+-dependent transport (Eder et al., 2005). However, because of the
lack of pharmacological tools, the TRPC-mediated physiological functions are often
revealed by the studies of TRPC mutant animals or human patients who bear naturally
occurring mutations in TRPC genes. These studies have generated a long list of TRPC-
dependent functions, including neurotransmission, fear response, and neurodegeneration
in the nervous system (Munsch et al., 2003; Phelan et al., 2012; Riccio et al., 2009;
Riccio et al., 2014), excitation and contraction coupling and muscle tone of smooth
muscles (Tsvilovskyy et al., 2009; Welsh et al., 2002), as well as regulation of
endothelial permeability in the vasculature and filtration in the kidney (Tiruppathi et al.,
2002; Winn et al., 2005). Currently, the roles of TRPC channels in normal and
pathological conditions are under extensive investigation.
The scarcity of TRPC probes has severely hampered the characterization of these
channels in their assembly, function, and role in pathophysiology. Using a cell-based
high-throughput fluorescence assay to screen for TRPC4 probes from the Molecular
Libraries Small Molecule Repository (MLSMR) supported by the Molecular Libraries
Probe Production Centers Network (MLPCN), we have identified a small group of
compounds showing antagonist activity on TRPC4. We have previously reported that
ML204, a quinoline compound, potently and selectively inhibited TRPC4 with an IC50
value of 0.96 µM (Miller et al., 2011a). ML204 was later shown to inhibit visceral pain
in a dose-dependent manner (Westlund et al., 2014) and to protect kidney filter function
(Schaldecker et al., 2013). Here, we report characterization and optimization of an
(amino)benzimidazole-based compound, M084, from the same screen. Although not as
potent as ML204, the M084 series affords improved stability and kinetics of inhibiting
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TRPC4/C5 and provides an alternative structural scaffold for further development of
more potent TRPC4/C5-selective antagonists.
METHODS
Cell Lines and Cell Culture—HEK293 cells were grown in DMEM (high glucose)
supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml
penicillin, and 100 µg/ml streptomycin at 37 °C, 5% CO2. The stable cell line that
inducibly expresses human TRPA1 and the conditions for induction has been described
previously (Hu et al., 2009). Rat TRPV1 was transiently transfected into HEK293 cells
seeded in wells of 96-well plates and used for Ca2+ assay 20 hrs post-transfection as
described (Hu et al., 2004). Stable cell lines that express human TRPC3, or mouse
TRPC4, TRPC5, TRPC6, TRPC7, TRPV3, or TRPM8 were established as described
previously (Miller et al., 2011a) and maintained in the above medium supplemented with
G418 (400 µg/ml; Invitrogen). The stable cell lines over-expressing TRPC4 and TRPC5
were also co-expressed with μ-opioid receptor (μOR) or serotonin 5-HT1A receptor (5-
HT1AR). The TRPC6 cell line also stably expressed the M5 muscarinic receptor (M5R).
For all GPCRs, the receptor cDNA was placed in the pIREShyg2 vector (Clontech) and
the cell lines stably coexpressing TRPC and GPCR were maintained in the medium
containing 400 μg/ml G418 and 50 μg/ml hygromycin B (Calbiochem). For the stable
cell line co-expressing TRPC1 and TRPC4, human TRPC1 cDNA was placed in a
modified pIRESneo vector (Clontech) with the neomycin-resistant gene replaced by the
zeocin-resistant one and transfected to the stable cell line that expressed TRPC4 and M2
muscarinic receptor (M2R). The cells were maintained in 100 μg/ml zeocin (Invitrogen),
in addition to G418 and hygromycin B. The nomenclature for receptors and ion channels
conforms to BJP's Concise Guide to Pharmacology (Alexander et al., 2013a, 2013b).
Fluorescence Ca2+ and Membrane Potential Assays—HEK293 cells stably
expressing the desired channel and receptor types were seeded in wells of 96-well plates
pre-coated with polyornithine (20 µg/ml, molecular weight >30,000; Sigma) at 1 X 105
cells/well and grown for >16 hrs. Cells were loaded with either Fluo 4-AM to monitor
intracellular Ca2+ changes or the FLIPR membrane potential dye (FMP, Molecular
Devices) to monitor membrane potential changes by using the FlexStation microplate
reader (Molecular Devices) following previously described protocols (Hu et al., 2004;
Otsuguro et al., 2008). Either the original FMP or FLIPR membrane potential dye II
(FMP II) was used. Extracellular solution for all FlexStation assays contained (in mM)
140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, with pH adjusted to 7.4
by NaOH. Probenecid (2 mM) was included in all Ca2+ assays except for TRPV1. Assays
were run at the 32 °C, except for TRPV1 and TRPA1, which were carried out at the room
temperature (~22 °C).
Electrophysiological Recordings—HEK293 cells stably expressing desired TRPC
channels were seeded in 35-mm dishes one day before whole-cell recordings were
performed. Recording pipettes were pulled from standard wall borosilicate tubing with
filament (Sutter Instrument) to 2–4 MΩ when filled with a pipette solution containing (in
mM) 110 CsCl, 1 MgCl2, 6.46 CaCl2, 10 BAPTA, (with the estimated free [Ca2+] of ~400
nM), 10 HEPES, with the pH adjusted to 7.2 with CsOH, and placed in the bath solution
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of the same composition as the extracellular solution used for the Ca2+ assay. Isolated
cells were voltage-clamped in the whole-cell mode using an EPC9 (HEKA Instruments)
amplifier. Voltage commands were made from the PatchMaster program (version 2.60;
HEKA), and currents were recorded at 5 kHz. Voltage ramps of either 200 or 500 ms to -
100 mV after a brief (20-ms) step to +100 mV from holding potential of 0 mV were
applied every 1 or 2 s. Cells were continuously perfused with the bath solution through a
gravity-driven multi-outlet device with the desired outlet placed about 50 µm away from
the cell being recorded. Drugs were diluted in the extracellular solution to the desired
final concentrations and applied to the cell through perfusion.
All animal procedures were carried out in accordance with the Guide for the Care and
Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of
Health. The use of mice and animal protocols were approved by the Animal Welfare
Committee of The University of Texas Health Science Center at Houston. Preparation of
mouse brain slices and recordings of agonist-evoked plateau potential from lateral septal
neurons in brain slices by whole-cell current clamp methods were described previously
(Tian et al., 2014).
Isolation of mouse dorsal root ganglion (DRG) neurons and whole-cell recordings to test
the effects of the compound on voltage-gated Na+, K+, and Ca2+ channels in isolated
DRG neurons were essentially carried out as previously described(Miller et al., 2011a).
For Na+ channels, the pipette solution had (in mM): 140 CsCl, 5 NaCl, 4 MgCl2, 10
HEPES, 10 EGTA, 2 Na-ATP, 10 HEPES, pH 7.2; bath had: 35 NaCl, 110 choline-Cl,
1.2 MgCl2, 0.15 CaCl2, 0.2 CdCl2, 10 glucose, 10 HEPES, pH 7.4. For K+ channels, the
pipette solution had (in mM): 130 K-methanesulfonate, 7 KCl, 0.05 EGTA, 1 Na2ATP, 3
MgATP, 0.5 Na2GTP, 10 HEPES, pH 7.2; bath had: 140 mM choline-Cl, 5 KCl, 2 CoCl2,
1 MgCl2, 10 glucose, 10 HEPES, pH 7.4. For Ca2+ channels (in mM): pipette solution had
(in mM): 117 CsCl, 1.8 MgCl2, 9 EGTA, 14 Tris-creatine phosphate,4 MgATP, 0.3
TrisGTP, 9 HEPES, pH 7.2; bath had: 130 tetraethylammonium-Cl, 10 BaCl2, 1 MgCl2,
0.0004 tetrodotoxin, 10 glucose, 10 HEPES, pH 7.4. Voltage protocols are shown in the
figure.
Compounds and Combinatorial Chemical Library Synthesis— A series of novel
2-aminobenzimidazole derivatives were prepared as reported previously (Zhu et al.,
2013). Most of the compounds were synthesized from the commercially available 2-
chloro-1H benzo[d]imidazole and amines using methyl-1-butanol or MeOH as the
solvent under microwave irradiation. A small number of compounds were purchased
from the Sigma-Aldrich and ChemBridge. The structures and sources of all compounds
tested in the current study are listed in Table 1. 2-Amino-6-
(trifluoromethoxy)benzothiazole (Riluzole) was from Matrix Scientific. Capsaicin,
carbamoylcholine (carbachol or CCh), [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin
(DAMGO), flufenamic acid, 5-hydroxytryptamine (5-HT), menthol were from Sigma-
Aldrich. 2-Aminoethoxydiphenyl borate (2APB) was from Cayman Chemical Co.
RESULTS:
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M084 selectively inhibits TRPC4 and TRPC5 — Using a stable HEK293 cell line
co-expressing mouse TRPC4β and µ opioid receptor (µOR), we conducted high
throughput screening for compounds that affect TRPC4 channel function by measuring µ
agonist, DAMGO-evoked intracellular Ca2+ rise (Miller et al., 2011a; Miller et al.,
2011b). One of the primary hits (CID 284016) identified from the screening of a total of
305,000 compounds in the MLSMR library is n-butyl-1h-benzimidazol-2-amine (M084,
Fig. 1E inset). The compound was resynthesized and tested for activity on TRPC4β
stably co-expressed with µOR using the Ca2+ assay (Fig. 1A), from which the IC50 was
estimated to be 3.7 ± 0.5 µM (n = 10) when DAMGO was used at 0.1 µM. Using the
same cell line, we also performed a fluorescence membrane potential assay, which
represents a different assay from the primary screening. As shown in Fig. 1B, with the
cells loaded with the FLIPR membrane potential dye (FMP), DAMGO (0.1 µM) induced
a robust increase in fluorescence intensity in the TRPC4 + µOR cells, indicating
membrane depolarization. This response was specific for µOR-mediated TRPC4
activation as DAMGO failed to induce fluorescence increases in cells that expressed
either TRPC4β or µOR alone or wild type HEK293 cells (data not shown, but see (Miller
et al., 2011b)). Preincubation with M084 did not cause an appreciable change in the
fluorescence signal but attenuated the DAMGO-evoked increase in a concentration
dependent manner (IC50 = 10.3 ± 0.5 µM, n = 12, Table 2). Similarly, in cells that co-
expressed TRPC4 with serotonin (5-hydroxytryptamine, or 5-HT) receptor type 1A (5-
HT1AR), the 5-HT (1 µM) evoked fluorescence increase was also inhibited by
preincubation of M084 (Fig. 1C). In addition, M084 inhibited carbachol (CCh, 1 µM)
evoked membrane depolarization in cells that co-expressed TRPC1, TRPC4 and M2R in
a concentration-dependent manner (Fig. 1D), with an estimated IC50 of 8.3 ± 1.7 µM (n =
6), suggesting that the compound also acts on the TRPC1/C4 heteromeric channels. In
whole-cell voltage clamp recordings, co-application of DAMGO (0.1 µM) and CCh (10
µM), which acts at endogenous Gq/11-coupled muscarinic receptors to facilitate TRPC4
current triggered through stimulation of Gi/o signaling, to the TRPC4β/µOR co-
expressing cells elicited a double rectifying current with an “N-shaped” current-voltage
(I-V) relationship, typical for TRPC4/C5 currents (Fig. 1E). Application of M084 (8 µM)
immediately decreased the currents, which recovered only moderately and slowly upon
washout of M084 (Fig. 1E). These results confirmed that M084 is an inhibitor of the
TRPC4-containing channels.
TRPC5 is a close homolog of TRPC4 but it shows constitutive activity when expressed in
HEK293 cells (Yamada et al., 2000; Zeng et al., 2004). The application of M084 to cells
that stably co-expressed TRPC5 and µOR immediately led to decreases in fluorescence
intensity in a concentration dependent manner in the membrane potential assay (Fig. 2A),
indicative of blockade of TRPC5 basal activity in these cells. At the highest concentration
tested (22 µM), M084 also inhibited the DAMGO-induced fluorescence increase, but at
lower concentrations, the inhibitory effect was not obvious. In fact, lower concentrations
of M084 actually increased the DAMGO-evoked fluorescence increase (Fig. 2A, see 0.8,
2.5, and 7.4 µM M084), likely because the partial inhibition of the constitutive currents
produced a lower membrane potential preceding the DAMGO addition. Overall, these
data suggest that M084 also inhibits TRPC5. Supporting this conclusion, M084 (8 µM)
caused an immediate inhibition of DAMGO/CCh-evoked whole-cell current in cells that
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co-expressed TRPC5 and µOR (Fig. 2B). On the other hand, M084 caused weak
inhibition of TRPC3 and even more moderate inhibition of TRPC6, as shown by CCh-
induced membrane depolarization in cells that stably expressed human TRPC3 alone or
mouse TRPC6 together with the Gq/11-coupled M5 muscarinic receptor (M5R) (Fig. 2C,
2D). The M5R was introduced because the stable monoclonal TRPC6 cell line had a poor
response to CCh as compared to the wild type HEK293 cells (data not shown). These
data indicated that among the related TRPC channels, M084 is relatively selective to
TRPC4 and TRPC5. M084 exhibited a very good solubility in aqueous buffers and was
relatively quick with its inhibitory action on these channels. However, the potency of
M084 was low, with IC50 values of 10.3 ± 0.5 and 8.2 ± 0.7 µM for TRPC4 and TRPC5,
respectively, as determined by the FMP assay using DAMGO to stimulate Gi/o via the co-
expressed µOR (Table 2). In addition, M084 displayed a weak but clear inhibitory effect
on TRPC3, with an IC50 of ~50 µM (Table 2). Therefore, we searched and synthesized
structural analogs of M084 in order to identify more potent and selective inhibitors for
TRPC4 and/or TRPC5.
Structure and activity relationship (SAR) of M084 — We have tested a total of
28 structural analogs of M084, obtained either from commercial sources or through new
synthesis (Table 1), against the TRPC4β/µOR cells using the FMP assay. The results
revealed that the amino group linked to position 2 of the benzimidazole backbone was
absolutely required for activity. This could be either a primary (M084 and compound 27)
or secondary amine in the form of a ring structure either as a piperidine (compounds 9, 13,
16, and 28) or a pyrrolidine (17). Therefore, the 2-aminobenzimidazol skeleton forms the
basic scaffold required for inhibiting TRPC4. Generally, a carbonyl functional group and
π system were not well tolerated at the amino position of benzimidazole, as indicated by
for example, acylation (4 and 10), aromatic substitution (7, 18, 25, and 29), or addition of
alcohol or ether (3 and 22) or the substituted morpholine (15); neither was an additional
amine, such as the aliphatic heteroalkane chains on the amino position (23 and 24) and
the substituted piperazine (11 and 12), tolerated. Dialkylation of the amine (20, 21, and
25) also resulted in the loss of function on TRPC4. Pyrrolidine and methylpiperidine at
position 2 of the benzimidazole backbone appeared to work slightly better than the
original n-butylamine, suggesting that the n-butyl chain might loop around to interact
with the channel. This is supported by the inhibitory action of compound (cpd) 27, in
which the primary amine is sterically hindered. However, the piperidine has to be linked
with the benzimidazole backbone via the amino group (16) instead of an alkyl group (14),
further emphasizing the essential scaffold being the 2-aminobenzimidazole. The methyl
group of the methylpiperidine could be added at either the 4th (9 and 13) or the 2nd
position (28) of the piperidine. Modifications at position 1 of the benzimidazole
backbone tended to reduce the inhibitory effect on TRPC4 (e.g. cpds 8 and 19), while
substitution of the proton with Cl- at position 5 of the benzimidazole backbone was well
tolerated (compare cpds 9 and 13).
We have determined the IC50 values for cpds 9, 13, 16, 17, 27, and 28 on DAMGO-
induced depolarization in cells that co-expressed TRPC4β and µOR (Fig. 3A and Table
2). Cpds 9, 13, 17, and 28 showed improved potency over the original compound, with
about 50% decrease in IC50 values. Similar improvement was also found for cpds 9, 13,
and 28 against TRPC5 activated by DAMGO through the co-expressed µOR (Table 2).
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However, both cpd 9 and cpd 13 still showed weak inhibition on TRPC3 and slight
inhibition on TRPC6 at high concentrations (Table 2). On the other hand, cpds 16, 17, 27
and 28 did not inhibit TRPC3 or C6 (Table 2). Cpd 28 also inhibited CCh-evoked
membrane depolarization in cells that co-expressed TRPC1, TRPC4 and M2R (Fig. 3D),
with an estimated IC50 of 11.0 ± 1.7 µM (n = 6). Although the IC50 value did not improve
from M084, the analog cpd 28 did not induce the slow depolarization seen with the high
concentration of M084 on the TRPC1/C4 heterometic channel (compare 22.2 µM traces
between Fig. 1D and Fig. 3D). In addition, cpd 28 did not affect CCh-induced Ca2+
response in wild type HEK293 cells (Fig. 3H) and it did not inhibit CCh-evoked
membrane depolarization through TRPC7 (Fig. 3G). Example traces for cpd 28 effects on
TRPC3, C4, C1/C4, C5, C6, and C7, as measured by agonist-evoked membrane
depolarization using the FLIPR membrane potential dye II (FMP II) are shown in Fig.
3B-3G. Note that FMP II exhibited slower kinetics and gave smaller fold fluorescence
increases in response to TRPC channel-mediated membrane depolarization than the
original FMP dye; however, it also yielded less fluorescence decrease upon inhibition of
the constitutive activity of TRPC5 in response to application of the antagonist (compare
Fig. 2A and Fig. 3C).
In whole-cell recordings, acute application of cpd 28 (10 µM) led to inhibition of
TRPC4β currents activated by co-stimulation of Gi/o and Gq/11 pathways with DAMGO
(0.1 µM) plus CCh (30 µM) in cells that co-expressed TRPC4β and µOR (Fig. 4A). It
also inhibited TRPC5 currents activated by CCh (100 µM) via endogenous muscarinic
receptors in the TRPC5-expressing cells (Fig. 4B). Note the much smaller inward
currents at negative potentials for TRPC5 in the absence of Gi/o stimulation. For both
TRPC4 and TRPC5, the inhibition by cpd 28 was more pronounced at negative than at
positive potentials (Fig. 4C-4F). Quantification of the degree of inhibition by cpd 28 at -
100 mV and +100 mV revealed 83.4 ± 4.1 and 48.9 ± 8.6% reduction of TRPC4 and 86.8
± 3.5 and 62.7 ± 4.8% reduction of TRPC5 currents, respectively (Fig. 4E, 4F). Similar
inhibition of TRPC4 and TRPC5 currents were also observed with the acute application
of cpds 9 and 13 (Fig. 4E, 4F). Recently, riluzole was shown to activate TRPC5
independent of receptor/PLC activation (Richter et al., 2014a). Application of cpd 28 (10
µM) also caused instantaneous inhibition of riluzole-evoked TRPC5 current (Fig. 4G). In
addition, in the TRPC1/C4/M2R coexpressing cells, cpd 28 (10 µM) also strongly
depressed the current evoked by CCh.
Pre-exposure of the cells to cpd 28 for ~30 sec also strongly inhibited the activation of
TRPC4β by the co-stimulation of DAMGO and CCh by ~80 and 98% at +100 and -100
mV, respectively (Fig. 5A-5C). Similarly, pretreatment with cpd 28 also blocked the
CCh-evoked TRPC5 currents by ~85 and 90% at +100 and -100 mV, respectively (Fig.
5D-5F). Noticeably, bath application of cpd 28 also reduced the constitutive outward
current in TRPC5-expressing cells (Fig. 5E), consistent with the observation in
fluorescence membrane potential measurements (Fig. 3C). By contrast, pretreatment with
cpd 28 did not significantly alter the CCh-evoked TRPC6 currents in cells that co-
expressed TRPC6 and M5R (Fig. 5G-5I), confirming the lack of its effect on TRPC6.
Therefore, the results from the electrophysiological experiments corroborate the
conclusions from the fluorescence membrane potential assay that cpds 9, 13, and 28 are
TRPC4/C5 blockers and cpd 28 has an improved selectivity over M084 on the
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TRPC4/C5 subgroup of TRPC channels. The fast onset of action of these drugs makes
them ideally suited in electrophysiological experiments to isolate currents mediated by
native TRPC4/C5 channels.
M084 and its analogs have minimal effect on related channels — Using stable
cell lines that expressed TRPA1, TRPM8, TRPV1, and TRPV3 channels, we examined
the effects of M084 and a selected number of its analogs, cpds 9, 13, 27 and 28, on these
distantly related TRP channels. The cells were loaded with Fluo-4 and then stimulated
with the respective agonist for each channel, with changes in [Ca2+]i monitored by a
fluorescence plate reader. As exemplified in Fig. 6A-6D for cpd 28 and summarized in
Fig. 6E for all five compounds, M084 and cpds 9, 13, and 28 showed neither agonistic
nor antagonistic effect on the tested TRP channels. Cpd 27 was ineffective on TRPV1
and TRPV3, but moderately inhibited TRPA1 and TRPM8. The inhibition on TRPM8
was not very surprising as a number of benzimidazole-containing compounds have been
shown to be potent antagonists of TRPM8 (Parks et al., 2011; Calvo et al., 2012).
Furthermore, using freshly isolated mouse DRG neurons, we recorded native voltage-
gated Na+, K+, and Ca2+ currents and found that M084 had no effect on these channels
(Fig. 6F). These data further confirm the selectivity of the M084 compound series on
TRPC, especially TRPC4/C5 channels.
M084 and its analogs inhibit native TRPC4-like activity in lateral septal
neurons — We and others have shown that TRPC4-containing channels mediate a
plateau potential response when stimulated by the agonist of group I metabotropic
glutamate receptors, (S)-3,5-dihydroxyphenylglycine (DHPG) or (1S,3R)-1-
aminocyclopentane-1,3-dicarboxylic acid (ACPD), in rodent lateral septal neurons
(Phelan et al., 2012; Tian et al., 2014). This response was greatly facilitated by the
injection of a positive current while DHPG (30 µM) was applied (Fig. 7A). While co-
application of the previously reported TRPC4/C5 blocker, ML204 (30 µM), significantly
suppressed the DHPG-induced plateau depolarization (Fig. 7B, 7G), the inhibition was
incomplete, probably because of the slow mechanism of action and the relatively poor
aqueous stability of the compound. On the other hand, when coapplied without
preincubation, M084 (100 µM) and its analogs, cpds 9, 13 and 28 (all at 30 µM),
inhibited the DHPG-induced plateau depolarization by ~80% (Fig. 7C-7G),
demonstrating the effectiveness of these blockers in acute suppression of native TRPC4-
containing channels, in agreement with their actions on heterologously expressed
channels.
DISCUSSION & CONCLUSIONS
Small molecular probes for TRPCs are of critical value for analyzing physiological and
pathophysiological functions of these channels. Because of the lack of specific inhibitors,
testing of native TRPC channel functions has been limited to the use of nonspecific
blockers, such as SKF96365 and 2-aminoethoxydiphenyl borate (2APB) and occasionally
flufenamic acid (Hu et al., 2004; Inoue et al., 2001; Merritt et al., 1990). However, these
compounds have low potency on TRPC channels and are either equally effective or better
at against other non-TRPC targets (Merritt et al., 1990; Singh et al., 2010). Therefore,
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several groups have made efforts to identify novel small molecular probes for TRPC
channels (Kiyonaka et al., 2009; Majeed et al., 2011; Miehe et al., 2012; Miller et al.,
2011a; Schleifer et al., 2012; Urban et al., 2012; Washburn et al., 2013; Richter et al.,
2014a, 2014b). Previously, we have reported the characterization of ML204 as a
selective inhibitor of TRPC4/C5 (Miller et al., 2011a). Others have found ML204 to be
useful in demonstrating the functions of native TRPC4 and/or TRPC5 channels in
visceral pain (Westlund et al., 2014), disruption of kidney filtration barrier (Schaldecker
et al., 2013), and neuronal excitability regulation (Zhang et al., 2013; Kolaj et al., 2014).
The M084 series reported here represents a different hit from the same screen that
identified ML204, in which a cell-based assay was used to monitor DAMGO-evoked
[Ca2+]i rise via mouse TRPC4β by activation of the co-expressed Gi/o-coupled µOR
(Miller et al., 2011a; Miller et al., 2011b). The primary hit, M084, contains a 2-
aminobenzimidazole scaffold. Through SAR studies, we have shown the amine at the 2nd
position of benzimidazole being essential for the inhibitory action on TRPC4, indicating
that indeed the basic structural backbone for this series is 2-aminobenzimidazole. This
differs from the series of benzimidazole-containing antagonists for TRPM8, in which an
amino group is typically not present at such position (Parks et al., 2011; Calvo et al.,
2012). However, just the 2-aminobenzimidazole backbone itself (cpd 2) exhibited no
activity on TRPC4. The addition of a four-carbon alkyl radical (n-butyl) or the joining of
the amine by four or five carbons to form pyrrolidine or piperidine, respectively, was
necessary to confer the inhibition on TRPC4. This suggests that a ring-shaped structure
around the amine may be important for binding to and/or the inhibitory action on the
TRPC channel. It is possible that the n-butyl chain of M084 also loops around to form a
pseudo “ring-shaped” structure when binding to the channel. This explains the lower
apparent affinity of M084, based on the IC50 value, than its analogs with the pyrrolidine
or piperidine substitution. Yet, we found that methylpiperidine worked better than
piperdine in this position. Although addition of the methyl group at either the 2nd or the
4th position of the piperdine worked equally well on TRPC4 and C5, the methyl group at
the 4th position seems to confer some activity, albeit weak, on TRPC3 and TRPC6. Thus,
without the methyl at the 4th position, cpds 16, 17, and 28 did not show appreciable
inhibition on TRPC3 and TRPC6 at 22 µM. Also interesting is that although the
substitution of the n-butylamine with a sterically hindered primary amine (cpd 27) was
allowed, other substitutions, e. g. aromatic structures, acylation, dialkylation, and
introduction of alcohol, ether or additional amine, all resulted in the loss of inhibitory
action on TRPC4. Therefore, a heterocyclic aliphatic amine attached to the 2nd position of
a benzimidazol backbone forms the basic structure for binding to and/or inhibiting
TRPC4/C5 channels. The requirement for a heterocyclic aliphatic amine is similar to the
SAR profile of ML204 (Miller et al., 2011a), suggesting that the two compound series
may share a similar mechanism of action. Therefore, even though the original hits
ML204 and M084 did not look alike, the inhibitory action on TRPC channels probably
involves pyrrolidine- or piperidine-like structures with a methyl group allowed at certain
positions. The quinoline (ML204) and benzimidazol (M084) backbones are probably not
essential for binding to the channels and the inhibitory action, but may influence the
compound stability and its kinetics for interaction with the channels.
11
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The M084 series was found to inhibit homomeric TRPC4 and TRPC5 as well as
heteromeric TRPC1/C4 channels in both the fluorescence membrane potential assay and
whole-cell voltage clamp recordings. The inhibition was not dependent on the receptor
type employed to trigger channel activation. Importantly, not only the activation of
TRPC4 or TRPC5 by the Gi/o-coupled µOR or 5-HT1AR, but also that of TRPC5 by the
endogenous Gq/11-coupled muscarinic receptors, as well as its direct agonist, riluzole, was
inhibited by M084 and its analogs. Furthermore, the constitutive activity of TRPC5 was
reduced by these compounds in both the fluorescence membrane potential and
electrophysiological assays. These, together with the finding that only some, but not all of
these inhibitors weakly blocked Gq/11-mediated activation of TRPC3 and TRPC6, suggest
a direct inhibitory effect of the aminobenzimidazole compounds on TRPC4/C5 channels.
We found M084 and it analogs to be relatively selective to TRPC, especially TRPC4/C5,
channels. At the highest concentration tested (22 µM), these compounds did not affect the
functions of TRPA1, TRPM8, TRPV1, and TRPV3 heterologously expressed in HEK293
cells in Ca2+ influx assays. In whole-cell recordings of mouse DRG neurons, M084 did
not significantly alter the current density of voltage-gated Na+, K+, or Ca2+ channels.
Importantly, unlike ML204 (Miller et al., 2011a), cpd 28 did not affect intracellular Ca2+
release induced through activation of endogenous muscarinic receptors. Among the
analogs analyzed here, cpd 28 yielded low IC50 values against TRPC4 and TRPC5 and
showed no inhibitory effect on TRPC3/C6/C7, indicating that it is an excellent TRPC4
and TRPC5 selective inhibitor. In electrophysiological recordings, all
aminobenzimidazole compounds produced immediate inhibition of TRPC4 and TRPC5
currents following bath application, indicating a direct and fast action by the compounds.
This feature is particularly important in electrophysiological studies to determine the
contribution of native TRPC4/C5 channels. Indeed, recordings of DHPG-induced plateau
potential in mouse lateral septal neurons demonstrated the effectiveness of M084 and its
analogs, cpds 9, 13 and 28, on inhibiting native TRPC4-containing channels. Under the
conditions used for these experiments, the M084 series exhibited better inhibitory effect
than ML204, suggesting improved stability and/or action kinetics. Curiously, however,
we have found that cpds 16 and 28 blocked butyrylcholinesterase in a cell-free assay at
similar to or slightly better potency than their inhibition of TRPC4 or TRPC5 functions
(Zhu et al., 2013). A recent study also showed that ML204 inhibited
acetylcholinestearase in the cell-free assay at similar potency as its inhibition of TRPC4
(Antolín and Mestres, 2015). Although the cholinesterases are not known to regulate
TRPC channels, cautions should be taken when using these compounds to study native
TRPC4/C5 function in the presence of cholinesterase activities. Since cpd 27 exhibited
no effect on cholinesterases (Zhu et al., 2013), it may be used as an alternative or an
additional control to confirm the involvement of TRPC4/C5 channels. However, other
off-target effects of cpd 27, e.g. TRPA1 and TRPM8, should also be carefully evaluated
before a conclusion is reached. Further improvements of the aminobenzimidazoles may
yield compounds with higher potency and better selectivity against TRPC4 and/or
TRPC5.
Pharmacological tools are essential for revealing the function of TRP channels. Currently
available compounds tend to broadly affect voltage-gated channels, intracellular Ca2+
release channels, chloride channels, and/or multiple types of TRP channels from several
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subfamilies (Hofmann et al., 1999; Inoue et al., 2001; Merritt et al., 1990). Therefore,
identification of isoform specific probes for TRP channels is important for advancing the
studies and understanding on these channels. Because TRPC4/C5 channels are implicated
in diseases (Jung et al., 2011; Schaldecker et al., 2013; von Spiczak et al., 2010), the
pharmacological tools may also have therapeutic potential. The specific TRPC4/C5
inhibitors reported here should be excellent tools for physiological and pathological
studies defining the functional significance of TRPC4/C5-containing channels and may
facilitate the development of therapeutics targeting TRPC channels.
Acknowledgements
We thank Dr. Corey Hopkins for initial evaluation of the lead compounds and
suggestions on structural analogs. This work was supported by grants from National
Institutes of Health (NS056942, NS092377, and DK081654 to MXZ, U54 MH084691 to
ML), National Natural Science Foundation of China (81373254 and 21390402 to X.
Hong), Natural Science Foundation of Yunnan Province (2012FB181 and 2014BC011 to
HRL), postdoctoral fellowship from the Third Affiliated Hospital of Guangzhou Medical
University (to YL), predoctoral fellowship from American Heart Association-Southwest
Affiliate (to DPT), Fundamental Research Funds for the Central Universities (to CQ), and
Innovation Seed Fund of Wuhan University School of Medicine (to X Hong).
Conflicts of Interest
None.
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FIGURE LEGENDS
Figure 1 | M084 inhibited agonist-evoked TRPC4 activity. A-C, Pretreatment with
M084 inhibited TRPC4-mediated Ca2+ influx (A) and membrane depolarization (B, C) in
a concentration-dependent manner. HEK293 cells stably co-expressing TRPC4β and µ
opioid receptor (µOR, A, B) or 5-hydroxytryptamine (5-HT) 1A receptor (5-HT1AR, C)
were seeded in wells of 96-well plates, loaded with Flou-4 (A) or FLIPR membrane
potential dye (FMP, B, C) and read in a microplate reader. M084 at different
concentrations and buffer alone (0 µM) were added as indicated for 2.5 minutes before
DAMGO (0.1 µM, A, B) or 5-HT (1 µM, C) was introduced. Increases in fluorescence
intensity indicate intracellular Ca2+ elevation (A) or membrane depolarization (B, C). D,
Similar to B and C, but cells stably co-expressed TRPC1, TRPC4 and M2R. FLIPR
membrane potential dye II (FMPII) was used and stimulation was by carbachol (CCh, 1
µM). Because high concentrations of M084 caused slow fluorescence increase in these
cells, the fluorescence changes were normalized to the fluorescence intensity
immediately preceding CCh addition (F180) instead of that in the beginning of the
experiment (F0) as in all other examples. The same color code for M084 concentrations is
used for all traces shown in A-D. E, M084 inhibited TRPC4 currents. Representative
traces showing currents at +100 and -100 mV evoked by coapplication of DAMGO (0.1
µM) and CCh (10 µM) to a cell that co-expressed TRPC4β and µOR. M084 (8 µM) was
added as indicated. Currents were elicited by 500-ms voltage ramps from +100 to -100
mV from the holding potential of 0 mV applied every 2 s. Dashed line indicates zero
current. Current-voltage (I-V) relationships obtained from the voltage ramps at the time
points indicated are shown below the time courses. Inset shows the structure of M084.
Representative of 7 experiments with similar results.
Figure 2 | M084 inhibited TRPC5 activity and exhibited minimal effects on TRPC3
and TRPC6. A, M084 inhibited basal activity of TRPC5. Similar to Fig. 1B, but the
fluorescence membrane potential assay was performed using cells that stably co-
expressed TRPC5 and µOR. The addition of M084 reduced the basal fluorescence in a
concentration-dependent manner. B, M084 inhibited TRPC5 currents. Similar to Fig. 1E,
but for a cell that co-expressed TRPC5 and µOR and voltage ramps were applied every 1
s. Currents were induced by the coapplication of DAMGO (0.1 µM) and CCh (10 µM).
Addition of M084 (8 µM) in the presence of DAMGO and CCh immediately suppressed
the currents, which partially recovered upon washout of M084. I-V relationships obtained
from the voltage ramps at the time points indicated are shown below the time courses.
Representative of 5 experiments with similar results. C & D, M084 weakly inhibited
TRPC3 (C) and TRPC6 (D). Similar to Fig. 1B, but the fluorescence membrane potential
assay was performed using cells that stably expressed human TRPC3 (C) or co-expressed
mouse TRPC6 and M5 muscarinic receptor (M5R, D). The addition of M084 caused little
fluorescence change and CCh-evoked membrane depolarization was only weakly
inhibited by M084. The concentrations of CCh used were 100 µM (C) and 0.3 µM (D).
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Figure 3 | Structural analogs of M084 inhibited TRPC4 and TRPC5. A,
Concentration dependence of M084 and its structural analogs for inhibition of DAMGO-
evoked membrane depolarization in the stable HKE293 cell line that co-expressed
TRPC4β and µOR. Fluorescence membrane potential assays were performed as in Fig.
1B using the aminobenzimidazole compounds as indicated. DAMGO (0.1 µM) evoked
fluorescence increases (area under the curve) were normalized to that from the control
pretreated with the buffer alone. Data are means ± SEM for n = 12 measurements for all
compounds. Data points were fitted with the Hill equation. B-G, Representative traces of
the fluorescence membrane potential assays using FMPII performed on cells that
expressed TRPC4β and µOR (B), TRPC5 and µOR (C), TRPC1, TRPC4 and M2R (D),
TRPC3 only (E), TRPC6 and M5R (F), or TRPC7 only (G). Compound 28 was applied
as indicated and the respective receptor agonist was added 2.5 min later. Receptor agonist
concentrations used were: DAMGO, 0.1 µM (B & C), CCh, 0.3 µM (F), 1 µM (D) and
100 µM (E, G). Note the concentration-dependent suppression of DAMGO-evoked
depolarization for TRPC4β (B), TRPC5 (C), and TRPC1/C4 (D) by compound 28, as
well as the decrease of basal fluorescence for TRPC5-expressing cells (C). The
compound did not inhibit CCh-evoked response for TRPC3 (E), TRPC6 (F), and TRPC7
(G). H, Compound 28 did not affect CCh-evoked Ca2+ response in wild type HEK293
cells. Untransfected cells were seeded in wells of a 96-well plate, loaded with Fluo-4 and
read in a microplate reader. Compound 28 or buffer alone (0 µM) was applied as
indicated. The addition of CCh (100 µM) immediately increased fluorescence, indicating
a rise in [Ca2+]i, which was unaffected by the pretreatment with the compound. The same
color code for compound 28 concentrations is used for all traces shown in B-H.
Figure 4 | M084 analogs inhibited agonist-evoked TRPC4 and TRPC5 currents. A,
Compound 28 inhibited TRPC4 currents. Similar to Fig. 1E, but the voltage ramp was
200 ms and repeated every 1 s. Currents were elicited by the coapplication of DAMGO
(0.1 µM) and CCh (30 µM) in a cell that co-expressed TRPC4β and µOR. Compound 28
(10 µM) was applied as indicated after the currents had developed and this led to
immediate decreases of currents at both positive and negative potentials. I-V relationships
obtained from the voltage ramps at the time points indicated are shown to the right. B,
Similar to A, but for a cell that expressed only TRPC5. The currents were elicited by 100
µM CCh. Compound 28 decreased the CCh-evoked currents. C & D, Current amplitudes
immediately before (Control, Cntl) and at the end of the application of compound 28
(+28) for TRPC4 and TRPC5 at +100 mV (C) and -100 mV (D). Data are means ± SEM
for 5 TRPC4-expressing and 7 TRPC5-expressing cells. * P < 0.05, ** P < 0.01 vs Cntl
by paired t test. E & F, % inhibition of agonist-evoked currents by compounds 9, 13, and
28 for TRPC4 and TRPC5 under the same protocol as shown in A & B at +100 mV (E)
and -100 mV (F). Data (means ± SEM) for compound 28 were derived from C & D. Data
for compounds 9 and 13 were from separate experiments using cells that co-expressed
µOR with either TRPC4β or TRPC5. Currents were elicited by costimulation with
DAMGO (0.1 µM) and CCh (10 µM). Numbers of cells are indicated in parentheses. **
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P < 0.01, *** P < 0.001 by one sample t tests comparing to 100% (no inhibition). G,
Compound 28 inhibited riluzole-induced TRPC5 currents. Similar to B, but riluzole (50
µM) was applied to elicit TRPC5 currents. Summary data (means ± SEM, n = 6) for
current amplitudes immediately before (Cntl) and at the end of compound 28 (10 µM)
application (+28) are shown at right. *** P < 0.001 vs Cntl by paired t test. H, Similar to
G, but the cells expressed TRPC1, TRPC4 and M2R, and currents were evoked by CCh
(10 µM). Summary data also represent n = 6 cells.
Figure 5 | Pretreatment with compound 28 suppressed activation of TRPC4 and
TRPC5 but not TRPC6 by agonist stimulation. A-C, Currents evoked by DAMGO
(0.1 µM) and CCh (30 µM) in cells that co-expressed TRPC4 and µOR without (A) or
with (B) a pretreatment by compound 28 (10 µM) for ~30 sec. Compound 28 was also
present throughout the exposure to the agonists. Shown are time courses of currents at
+100 and -100 mV (left) and I-V relationships obtained by the voltage ramp protocol
(same as Fig. 4A) before (black trace) and during (red trace) agonist stimulation (right).
Summary data (means ± SEM) for agonist-induced peak current amplitudes (absolute
values) at +100 and -100 mV are shown in C. n = 7 for control, n = 5 for +28. ** P <
0.01. *** P < 0.001 vs control by unpaired t test. D-F, Similar to A-C, but the cells
expressed TRPC5 and the agonist was CCh (100 µM). Note the decrease of basal current
at +100 mV upon application of compound 28 (E). The I-V curve for basal current before
addition of compound 28 (gray trace) is indicated by the solid arrow, while that for
current in the presence of compound 28 but before CCh (black trace) is indicated by the
open arrowhead. For summary in F, n = 7 for control, n = 6 for +28. * P < 0.05. *** P <
0.001 vs control by unpaired t test. G-I, Similar to A-C, but the cells co-expressed
TRPC6 and M5R and the agonist was CCh (3 µM). For summary in I, n = 6 for control, n
= 6 for +28.
Figure 6 | M084 and analogs did not inhibit other channels. A-D, Compound 28 had
no effect on TRPA1, TRPM8, TRPV1, and TRPV3. HEK293 cells stably expressing
human TRPA1 (A), mouse TRPM8 (B), mouse TRPV3 (D) or transiently expressing rat
TRPV1 (C) were seeded in wells of 96-well plates, loaded with Fluo-4, and read in a
microplate reader for changes in [Ca2+]i. Compound 28 (7.4 and 22.2 µM) or buffer alone
(0 µM) was added as indicated for 2.5 min before the application of the corresponding
agonist: flufenamic acid (FFA, 100 µM, A), menthol (200 µM, B), capsaicin (1 µM, C),
2-aminoethoxydiphenyl borate (2APB, 200 µM, D). E, Summary (means ± SEM) for
agonist-induced Fluo-4 fluorescence changes in cells that expressed TRPA1, TRPM8,
TRPV1, and TRPV3 in the presence of 22.2 µM M084 and its analogs, compounds 9, 13,
27, and 28. Agonists and their concentrations are the same as shown in A-D. Integrated
fluorescence changes (area under the curve) were normalized to that in the absence of the
2-aminobenzimidazole drug (control). n = 6 measurements for each. Only compound 27
showed moderate inhibition of TRPA1 and TRPM8. * P < 0.05 vs corresponding control.
F, Representative current traces of voltage-gated Na+, K+, and Ca2+ channels (INa, IK, ICa)
before (black traces) and during (red traces) the application of M084 (30 µM) and after
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its washout (blue traces), recorded from dissociated mouse dorsal root ganglion neurons.
Voltage protocols are shown above the traces. Current traces are overlaid for comparison,
with the one during M084 application placed in the front. Histogram shows means ±
SEM of current densities, at step voltages that yielded the maximal currents, in the
presence of M084 normalized to the average values before M084 and after washout to
correct for run-down in some cells. Numbers of cells tested are shown in parentheses.
Figure 7 | M084 and analogs inhibited TRPC4-mediated plateau potentials in lateral
septal neurons. A, Plateau potentials evoked by pressure ejection of DHPG (30 µM) and
concomitant current injection in lateral septal neurons. The lateral septal neuron in mouse
brain slice was held at -80 mV under whole-cell current clamp mode. A series of 9
current injections (20 ms, 0.2-1 nA, with a 0.1-nA increment and 1.3 s intervals) were
applied immediately before initiation of DHPG ejection (5-20 psi, 30 ms) (current
protocol and time of DHPG application, indicated by the gray bar, shown in upper panel).
Traces from all 9 sweeps are overlaid, with the one that yielded the maximal
depolarization response shown in black (lower panel). B-F, Similar to A, but DHPG was
co-ejected with ML204 (B), M084 (C), compound 9 (D), 13 (E), or 28 (F). G, Summary
of maximal depolarization response, as determined by area under the trace from the
sweep with the longest depolarization period. Data are means ± SEM for the numbers of
neurons indicated in parentheses. All drugs were used at 30 µM except for M084, which
was used at 100 µM. * P < 0.05, ** P < 0.01, compared to DHPG alone.
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22
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23
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24
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Table 1. SAR evaluation of benzimidazole derivatives on TRPC4
Benzimidazole backbone
Compound
Position 2
-NH2
Position 1
Position 5
Source
Resynthesized
SA: 171778
Inhibition @ 22 µM
M084
2
-H
-H
-H
-H
93%
No
3
4
-H
-H
-H
-H
SA: 572721
No
SA: L200263
13%
5
6
-H
-H
SA: L202517
SA: S441503
24%
No
-NH2
-H
-H
7
-H
SA: S62597
No
8
9
-H
SA: T320684
SA: T320722
21%
92%
-H
-H
-Cl
10
-Cl
SA: T135674
No
11
12
13
14
15
-H
-H
-H
-H
-H
-Cl
-Cl
-H
-H
-H
SA: T320625
SA: T320633
SA: T320676
CB: 4003377
CB: 4033874
No
No
94%
No
No

16
17
-H
-H
-H
-H
CB: 4034369
CB: 4034623
87%
95%
18
-H
-H
New Synthesis
No
19
20
21
22
23
-CH3
-CH3
-H
-H
-H
-H
-H
-H
New Synthesis
New Synthesis
New Synthesis
New Synthesis
New Synthesis
No
No
No
No
No
-H
-H
24
25
-H
-H
-H
-H
New Synthesis
New Synthesis
No
No
26
27
-H
-H
-H
-H
New Synthesis
New Synthesis
31%
80%
28
-H
-H
-H
-H
New Synthesis
New Synthesis
97%
No
29
SA: Sigma Aldrich, CB: ChemBridge
Functional assay performed by fluorescence membrane potential measurements of DMAGO-evoked
response in cells co-expressing TRPC4β and µOR.

Table 2. IC50 values (µM) of 2-aminobenzimidazole compounds on
TRPC3/C4/C5/C6
TRPC4β+µOR
(vs DAMGO)
TRPC5+µOR
(vs DAMGO)
TRPC6+M5R
(vs CCh)
TRPC3
(vs CCh)
10.3 ± 0.5 (n = 12)
4.1 ± 0.6 (n = 12)
5.2 ± 0.7 (n = 12)
11.0 ± 1.0 (n = 12)
5.5 ± 0.5 (n = 12)
10.2 ± 1.4 (n = 12)
4.3 ± 0.4 (n = 12)
8.2 ± 0.7 (n = 12)
3.1 ± 0.5 (n = 12)
6.6 ± 0.9 (n = 12)
11.1 ± 1.1 (n = 6)
8.0 ± 1.0 (n = 6)
12.4 ± 1.3 (n = 6)
3.5 ± 0.3 (n = 6)
59.6 ± 16.3 (n = 6)
57.1 ± 9.5 (n = 6)
63.8 ± 17.1 (n = 6)
>100 (n = 6)
48.6 ± 9.5 (n = 6)
30.4 ± 5.3 (n = 6)
19.3 ± 0.6 (n = 6)
>100 (n = 6)
M084
9
13
16
>100 (n = 6)
>100 (n = 6)
17
>100 (n = 6)
>100 (n = 6)
27
>100 (n = 6)
>100 (n = 6)
28
Fluorescence membrane potential measurements on stable TRPC cell lines were performed as in
Fig. 3 using either FMP or FMP II. Agonist evoked fluorescence increases (area under the curve)
were normalized to that from the control pretreated with the buffer alone. For TRPC5, the
remaining fluorescence intensity in the highest concentration of the antagonist for each set of the
experiments was assumed to represent no activity and used for baseline subtraction for
calculating the area under the curve. Data points were fitted with the Hill equation to determine
IC50 values, expressed as means ± SEM for the numbers of measurements indicted in parentheses.