Discovery of a Highly Selective and Potent TRPC3 Inhibitor with High Metabolic Stability and Low Toxicity

Abstract

The overactivation of transient receptor potential canonical 3 (TRPC3) is associated with neurodegenerative diseases and hypertension. Pyrazole 3 (Pyr3) is reported as the most selective TRPC3 inhibitor, but it has two inherent structural limitations: (1) the labile ester moiety leads to its rapid hydrolysis to the inactive Pyr8 in vivo, and (2) the alkylating trichloroacrylic amide moiety is known to be toxic. To circumvent these limitations, we designed a series of conformationally restricted Pyr3 analogues and reported that compound 20 maintains high potency and selectivity for human TRPC3 over its closely related TRP channels. It has significantly improved metabolic stability compared with Pyr3 and has a good safety profile. Preliminary evaluation of 20 demonstrated its ability to rescue Aβ-induced neuron damage with similar potency to that of Pyr3 in vitro. Collectively, these results suggest that 20 represents a promising scaffold to potentially ameliorate the symptoms associated with TRPC3-mediated neurological and cardiovascular disorders.

Figure 1

Chemical structures of reported TRPC3 inhibitors.

Figure 2

Design of metabolically stable and low toxic selective TRPC3 inhibitors by addressing structural liabilities in Pyr3.

Scheme 1. Synthesis of Ester Compounds 2–8 with Different Chlorine Substitution Patterns

Reagents and conditions: (i) EtOH, 60 °C, 6 h; (ii) CuI, DMCDA, K₂CO₃, toluene, reflux, 12 h; (iii) NCS, DMF, 100 °C, 8 h.

Scheme 2. Synthesis of Amide Compounds 17–22

Reagents and conditions: (i) selectfluor, MeCN, 50 °C, 3 h; (ii) NBS, DMF, r.t., 8 h. (iii) 1 M KOH (aq.), EtOH, H₂O, rt, 8 h; (iv) SOCl₂, CH₂Cl₂, reflux for 4 h; (v) ethylamine, TEA, r.t., 12 h.

Figure 3

Inhibition of TRPC3-mediated currents by synthesized compounds. (a) Cartoon depicting the experimental conditions used to test the effect of the synthesized compounds on HEK293 cells overexpressing hTRPC3. TRPC3 currents were measured in the whole-cell configuration of the patch-clamp technique, and cells were challenged with agonists/antagonists through perfusion. (b) Percentage of hTRPC3 currents elicited by GSK₁₇₀ and inhibited by the synthesized compounds in transfected HEK293 cells. Bars represent mean ± SD; n is denoted above the x-axis. (c) Representative traces of hTRPC3 currents activated by 1 μM GSK₁₇₀ and inhibited at different concentrations of compound 20 (blue traces). Each concentration was tested on independent cells and normalized against its internal control (maximum amplitude) to avoid tachyphylaxis. (d) Dose–response profile of hTRPC3 currents elicited by 1 μM GSK₁₇₀ and inhibited at different concentrations of compound 20. Circles represent mean ± SD. (e) Representative traces of hTRPC3 currents activated by 1 μM GSK₁₇₀ and inhibited at different concentrations of Pyr3 (gray traces). Each concentration was tested on independent cells and normalized against its internal control (maximum amplitude) to avoid tachyphylaxis. (f) Dose–response profile of hTRPC3 currents elicited by 1 μM GSK₁₇₀ and inhibited at different concentrations of Pyr3. Circles represent mean ± SD. (g) Representative traces of hTRPC3 currents activated by 1 μM GSK₁₇₀ and inhibited at different concentrations of compound 20 (blue traces) in the presence of extracellular Ca²⁺ (2 mM). Each concentration was tested on independent cells and normalized against its internal control (maximum amplitude) to avoid tachyphylaxis. (h) Dose–response profile of hTRPC3 currents elicited by 1 μM GSK₁₇₀ and inhibited at different concentrations of compound 20 in the presence of extracellular Ca²⁺ (2 mM). Circles represent mean ± SD. (i) Representative traces of hTRPC3 currents activated by 1 μM GSK₁₇₀ and inhibited at different concentrations of Pyr3 (gray traces) in the presence of extracellular Ca²⁺ (2 mM). Each concentration was tested on independent cells and normalized against its internal control (maximum amplitude) to avoid tachyphylaxis. (j) Dose–response profile of hTRPC3 currents elicited by 1 μM GSK₁₇₀ and inhibited at different concentrations of Pyr3 in the presence of extracellular Ca²⁺ (2 mM). Circles represent mean ± SD. GSK₁₇₀: GSK1702934A.

Figure 4

Compound 20 is a selective inhibitor of the TRPC subfamily. (a) Representative whole-cell recordings of HEK293 cells overexpressing hTRPA1, rTRPV1, rTRPV4, hTRPC3, mmTRPC6, hTRPC7, and hTRPM8. Currents were evoked with high concentrations of the agonist (red) and challenged with 20 (10 μM) in the presence of the respective agonist (blue). (b) Percentage of peak current blocked by 20 (10 μM) in the presence of each agonist. AITC, Allyl isothiocyanate; Cap, capsaicin; GSK₁₀₁, GSK1016790A. Bars are mean ± SD. n is denoted above the x-axis.

Figure 5

LC/MS analyses of the purified 20–TRPC3 complexes (right) together with the prepared control (left) support the direct binding of 20 to TRPC3 proteins. Spectra of MS data collected from 2.5 min to avoid buffer salts to MS.

Figure 6

Compound 20 shows a good safety profile in mice. (a) Mouse survival curves. (b) Mean percent change in mouse body weight ± SD relative to body weight at the time of initiating drug treatment. All mice received a daily dose at 200 mg/kg body weight of compounds 20, 27, or Pyr3 (n = 5) for 5 consecutive days. Dashed lines indicate mouse weight relative to its respective baseline weight at day 0 and 15% weight loss.

Figure 7

Effects on neuron dendritic morphology. (a) Compound 20 and Pyr3 protect against Aβ-induced damage to dendrites. Mature hippocampal neurons (DIV = 14) were treated with CHO control medium, or naturally secreted Aβ-containing conditioned medium (7PA2) or in combination with compound 20 or Pyr3 at different concentrations, for 16 h, followed by immunocytochemistry using an antibody against MAP2 (green) and DAPI (blue). Scale bar: 100 μm. (b) Lack of toxicity of the inhibitor compounds in neuronal culture. Hippocampal neurons (DIV = 14) were treated with compound 20 or Pyr3 alone at different concentrations, followed by immunocytochemistry staining with MAP2 (green) and DAPI (blue). Scale bar: 100 μm. (c) Quantification of dendritic length. Bars are mean ± SEM. **p < 0.01 (one-way ANOVA). ****p < 0.0001 (one-way ANOVA). n = 50 dendrites.