Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Jul;172(14):3495-509.
doi: 10.1111/bph.13140. Epub 2015 May 11.

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

Yingmin Zhu  1 , Yungang Lu  1   2 , Chunrong Qu  3 , Melissa Miller  4 , Jinbin Tian  1 , Dhananjay P Thakur  1 , Jinmei Zhu  3 , Zixin Deng  3 , Xianming Hu  2 , Meng Wu  4 , Owen B McManus  4 , Min Li  4 , Xuechuan Hong  3 , Michael X Zhu  1 , Huai-Rong Luo  5
Affiliations

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

Yingmin Zhu et al. Br J Pharmacol. 2015 Jul.

Abstract

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 Ca(2+) 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 Ca(2) (+) 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.

PubMed Disclaimer

Figures

Figure 1
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 receptors (μOR) (A, B) or 5-HT1A receptors (C) were seeded in wells of 96-well plates, loaded with Fluo-4 (A) or FMP (B, C) and fluorescence read in a microplate reader. M084 at different concentrations and buffer alone (0 μM) were added as indicated for 2.5 min 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, TRPC4β and M2 receptors. FMP II was used and stimulation was by CCh (1 μM). Because high concentrations of M084 caused a 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 colour 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 co-application of DAMGO (0.1 μM) and CCh (10 μM) to a cell that co-expressed TRPC4β and μ receptors. 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. 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 seven experiments with similar results.
Figure 2
Figure 2
M084 inhibited TRPC5 activity and exhibited minimal effects on TRPC3 and TRPC6. (A) M084 inhibited basal activity of TRPC5. Similar to Figure 1B, but the fluorescence membrane potential assay was performed using cells that stably co-expressed TRPC5 and μ receptors. The addition of M084 reduced the basal fluorescence in a concentration-dependent manner. (B) M084 inhibited TRPC5 currents. Similar to Figure 1E, but for a cell that co-expressed TRPC5 and μ receptors and voltage ramps were applied every 1 s. Currents were induced by the co-application 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 five experiments with similar results. (C and D) M084 weakly inhibited TRPC3 (C) and TRPC6 (D). Similar to Figure 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 receptors (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).
Figure 3
Figure 3
Structural analogues of M084 inhibited TRPC4 and TRPC5. (A) Concentration-dependence of the inhibitory effects of M084 and its structural analogues on DAMGO-evoked membrane depolarization in the stable HKE293 cell line that co-expressed TRPC4β and μ receptors. Fluorescence membrane potential assays were performed as in Figure 1B using the aminobenzimidazole compounds as indicated. DAMGO (0.1 μM)-evoked fluorescence increases (AUC) were normalized to that of 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 FMP II performed on cells that expressed TRPC4β and μ receptors (B), TRPC5 and μ receptors (C), TRPC1, TRPC4β and M2 receptors (D), TRPC3 only (E), TRPC6 and M5 receptors (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 and 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 in basal fluorescence for TRPC5-expressing cells (C). The compound did not inhibit CCh-evoked responses for TRPC3 (E), TRPC6 (F) and TRPC7 (G). (H) Compound 28 did not affect CCh-evoked Ca2+ responses in wild -type HEK293 cells. Untransfected cells were seeded in wells of a 96-well plate, loaded with Fluo-4 and fluorescence 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 colour code for compound 28 concentrations is used for all traces shown in (B–H).
Figure 4
Figure 4
M084 analogues inhibited agonist-evoked TRPC4 and TRPC5 currents. (A) Compound 28 inhibited TRPC4 currents. Similar to Figure 1E, but the voltage ramp was 200 ms and repeated every 1 s. Currents were elicited by the co-application of DAMGO (0.1 μM) and CCh (30 μM) in a cell that co-expressed TRPC4β and μ receptors. 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 and 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 five TRPC4-expressing and seven TRPC5-expressing cells. *P < 0.05, **P < 0.01 versus control (Cntl) by paired t-test. (E and F) % inhibition of agonist-evoked currents by compounds 9, 13 and 28 for TRPC4 and TRPC5 under the same protocol as shown in (A and B) at +100 mV (E) and −100 mV (F). Data (means ± SEM) for compound 28 were derived from (C and D). Data for compounds 9 and 13 were from separate experiments using cells that co-expressed μ receptors with either TRPC4β or TRPC5. Currents were elicited by co-stimulation with DAMGO (0.1 μM) and CCh (10 μM). Numbers of cells are indicated in parentheses. **P < 0.01, ***P < 0.001 by one sample t-tests comparing with 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 on the right. ***P < 0.001 versus Cntl by paired t-test. (H) Similar to (G), but the cells expressed TRPC1, TRPC4β and M2 receptors, and currents were evoked by CCh (10 μM). Summary data also represent n = 6 cells.
Figure 5
Figure 5
Pretreatment with compound 28 suppressed activation of TRPC4 and TRPC5 but not TRPC6 induced by agonist stimulation. (A–C) Currents evoked by DAMGO (0.1 μM) and CCh (30 μM) in cells that co-expressed TRPC4β and μ receptors without (A) or with (B) pretreatment with compound 28 (10 μM) for ∼30 s. 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 Figure 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 versus 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 in basal current at +100 mV upon application of compound 28 (E). The I–V curve for basal current before addition of compound 28 (grey 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 versus control by unpaired t-test. (G–I) Similar to (A–C), but the cells co-expressed TRPC6 and M5 receptors and the agonist was CCh (3 μM). For summary in (I), n = 6 for control, n = 6 for +28.
Figure 6
Figure 6
M084 and its analogues 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 the fluorescence read in a microplate reader for assessing 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: FFA (100 μM, A), menthol (200 μM, B), capsaicin (1 μM, C), 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 analogues, compounds 9, 13, 27 and 28. Agonists and their concentrations are the same as shown in (A–D). Integrated fluorescence changes (AUC) 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 versus 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 its washout (blue traces), recorded from dissociated mouse DRG 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 rundown in some cells. Number of cells tested are shown in parentheses.
Figure 7
Figure 7
M084 and analogues 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 nine 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 grey bar, are shown in upper panel). Traces from all nine 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 the 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 with DHPG alone.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alexander SP, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Spedding M, et al. The Concise Guide to PHARMACOLOGY 2013/14: G protein-coupled receptors. Br J Pharmacol. 2013a;170:1459–1581. - PMC - PubMed
    1. Alexander SP, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Catterall WA, et al. The Concise Guide to PHARMACOLOGY 2013/14: ion channels. Br J Pharmacol. 2013b;170:1607–1651. - PMC - PubMed
    1. Antolín AA, Mestres J. Distant polypharmacology among MLP chemical probes. ACS Chem Biol. 2015;10:395–400. - PubMed
    1. Calvo RR, Meegalla SK, Parks DJ, Parsons WH, Ballentine SK, Lubin ML, et al. Discovery of vinylcycloalkyl-substituted benzimidazole TRPM8 antagonists effective in the treatment of cold allodynia. Bioorg Med Chem Lett. 2012;22:1903–1907. - PubMed
    1. Eder P, Poteser M, Romanin C, Groschner K. Na+ entry and modulation of Na+/Ca2+ exchange as a key mechanism of TRPC signaling. Pflugers Arch. 2005;451:99–104. - PubMed

Publication types

MeSH terms

Associated data