Discovery and pharmacological characterization of a novel potent inhibitor of diacylglycerol-sensitive TRPC cation channels

Abstract

Background and purpose: The cation channel transient receptor potential canonical (TRPC) 6 has been associated with several pathologies including focal segmental glomerulosclerosis, pulmonary hypertension and ischaemia reperfusion-induced lung oedema. We set out to discover novel inhibitors of TRPC6 channels and investigate the therapeutic potential of these agents.

Experimental approach: A library of potential TRPC channel inhibitors was designed and synthesized. Activity of the compounds was assessed by measuring intracellular Ca(2+) levels. The lead compound SAR7334 was further characterized by whole-cell patch-clamp techniques. The effects of SAR7334 on acute hypoxic pulmonary vasoconstriction (HPV) and systemic BP were investigated.

Key results: SAR7334 inhibited TRPC6, TRPC3 and TRPC7-mediated Ca(2+) influx into cells with IC50 s of 9.5, 282 and 226 nM, whereas TRPC4 and TRPC5-mediated Ca(2+) entry was not affected. Patch-clamp experiments confirmed that the compound blocked TRPC6 currents with an IC50 of 7.9 nM. Furthermore, SAR7334 suppressed TRPC6-dependent acute HPV in isolated perfused lungs from mice. Pharmacokinetic studies of SAR7334 demonstrated that the compound was suitable for chronic oral administration. In an initial short-term study, SAR7334 did not change mean arterial pressure in spontaneously hypertensive rats (SHR).

Conclusions and implications: Our results confirm the role of TRPC6 channels in hypoxic pulmonary vasoregulation and indicate that these channels are unlikely to play a major role in BP regulation in SHR. SAR7334 is a novel, highly potent and bioavailable inhibitor of TRPC6 channels that opens new opportunities for the investigation of TRPC channel function in vivo.

Figure 1

Discovery of SAR7334 using pharmacophore-guided design of focused aminoindanol libraries. Pharmacophoric information derived from a selection of known analogues of the cation channel inhibitor SKF96365 were translated into library designs and synthesized. Promising aminoindanol derivatives were further investigated with emphasis on stereochemical positioning of relevant substituents. (A) Mapping of SAR7334 to the TRPC6 pharmacophore model. The red sphere corresponds to a positive ionizable moiety, the light blue sphere indicates lipophilic features, while brown spheres mark ring aromatic features. The mapping of SAR7334 to the pharmacophore was generated with LigandScout (Inteligand Software-Entwicklungs und Consulting GmbH, Maria Enzersdorf, Austria). (B) Aminoindanol derivatives with cis or trans geometries were accessed from 2-bromo-1-indanones by nucleophilic substitution with amines, carbonyl reduction and subsequent O-alkylation/arylation. Trans geometries, in particular with aryloxy substituents (R5 = aryl) were realized by epoxide opening of indene oxide with amines and a Mitsunobu reaction with double inversion. (C) Structure of SAR7334.

Figure 2

Inhibition of Ca²⁺ influx via diacylglycerol-sensitive TRPC channels by SAR7334. Time-dependent changes of intracellular Ca²⁺ concentration in Fluo-4-loaded induced TRPC6- HEK-FITR cells (A), TRPC3-CHO cells (C) and TRPC7 HEK-FITR cells (E) were measured using FLIPR. TRPC-mediated Ca²⁺ influx following application of 30 μM OAG (+OAG) was dose-dependently reduced in cells pre-incubated with the indicated concentrations of SAR7334. Control traces (con) were recorded during application of extracellular solution. Representative traces are shown. The relative inhibition of TRPC6 (B), TRPC3 (D) and TRPC7 channels (F) was calculated from the AUC for each concentration SAR7334 and IC₅₀s were derived from the best fit of the data (solid lines) with a logistic model. Data are given as means ± SEM (n = 5–10).

Figure 3

SAR7334 potently inhibits TRPC6 whole-cell currents. TRPC6 currents were elicited in TRPC6-HEK-FITR cells by application of 50 μM OAG. (A) Current traces measured during voltage ramps before and after application of the TRPC6 channel activator OAG and after consecutive additions of SAR7334. (B) Time course and dose dependence of SAR7334-induced TRPC6 inhibition. Whole-cell currents were measured at −70 mV during voltage ramps and plotted versus time. For better comparison of responses, current amplitudes were normalized to the maximal current after addition of OAG and maximal currents were superimposed. The arrow indicates application of SAR7334. (C) TRPC6 currents were measured as in (B) and inhibition was determined in steady state after application of the respective concentration of SAR7334. The dose–response relationship was fitted to a logistic function. Data are given as means ± SEM (n = 3–12).

Figure 4

Inhibition of receptor-stimulated TRPC6 currents by of SAR7334. Whole-cell currents were measured in TRPC6-HEK-FITR cells. (A) The time course of TRPC6 currents at −70 mV during application of 200 nM trypsin and 100 nM SAR7334 is depicted. Trypsin and SAR7334 were added to the extracellular solution during the time indicated by the bars. (B) Current responses measured during voltage ramps at the time points indicated in A are shown.

Figure 5

Effect of SAR7334 on hypoxia-induced vasoconstriction in isolated perfused and ventilated lungs. (A) Acute hypoxic pulmonary vasoconstriction (HPV) was assessed as the increase in pulmonary artery pressure (ΔPAP) upon hypoxic ventilation in isolated perfused mouse lungs. Data were normalized to the reference pressure response in the absence of SAR7334 for each experiment. SAR7334 dose-dependently reduced the strength of HPV. SAR7334 had no significant effect on normoxic vascular tone (not shown). Data shown are means ± SEM (n = 5). ***P < 0.001, significantly different from control response. (B) Original recordings of PAP in the absence (left) or presence (right) of 1 μM SAR7334. Hypoxic challenge is indicated by the bars.

Figure 6

Pharmacokinetic profile of SAR7334. Plasma concentrations of SAR7334 were determined after single oral administration of 10 mg·kg⁻¹ of the compound to male Sprague Dawley rats. Data are given as means ± SEM (n = 3).

Figure 7

SAR7334 does not affect BP in conscious SHR. Mean arterial pressure (MAP) was measured telemetrically in SHR. The time course of average MAP in vehicle-treated (n = 8) and SAR7334-treated (n = 5) groups is illustrated (A). Statistical comparison of MAP in vehicle- and SAR7334-treated animals 24 h before compound application (pre) and after treatment (post) did not show a time-dependent or treatment-related effect or a significant interaction of both factors (repeated measures two-way anova) (B).