Picomolar, selective, and subtype-specific small-molecule inhibition of TRPC1/4/5 channels

Abstract

The concentration of free cytosolic Ca2+ and the voltage across the plasma membrane are major determinants of cell function. Ca2+-permeable non-selective cationic channels are known to regulate these parameters, but understanding of these channels remains inadequate. Here we focus on transient receptor potential canonical 4 and 5 proteins (TRPC4 and TRPC5), which assemble as homomers or heteromerize with TRPC1 to form Ca2+-permeable non-selective cationic channels in many mammalian cell types. Multiple roles have been suggested, including in epilepsy, innate fear, pain, and cardiac remodeling, but limitations in tools to probe these channels have restricted progress. A key question is whether we can overcome these limitations and develop tools that are high-quality, reliable, easy to use, and readily accessible for all investigators. Here, through chemical synthesis and studies of native and overexpressed channels by Ca2+ and patch-clamp assays, we describe compound 31, a remarkable small-molecule inhibitor of TRPC1/4/5 channels. Its potency ranged from 9 to 1300 pm, depending on the TRPC1/4/5 subtype and activation mechanism. Other channel types investigated were unaffected, including TRPC3, TRPC6, TRPV1, TRPV4, TRPA1, TRPM2, TRPM8, and store-operated Ca2+ entry mediated by Orai1. These findings suggest identification of an important experimental tool compound, which has much higher potency for inhibiting TRPC1/4/5 channels than previously reported agents, impressive specificity, and graded subtype selectivity within the TRPC1/4/5 channel family. The compound should greatly facilitate future studies of these ion channels. We suggest naming this TRPC1/4/5-inhibitory compound Pico145.

Potent, selective, and subunit‐dependent activation of TRPC5 channels by a xanthine derivative

“…In these experiments, AM237 did not activate TRPC1:C5 currents (Figure c–e) but inhibited currents induced by EA in a concentration‐dependent manner, with full inhibition of both inward and outward currents observed at 3‐ to 100‐nM AM237 (Figure f–h). The characteristic ladle‐shape of the current–voltage plots was consistent with activation of heteromeric TRPC1:C5 channels (Figure d,g; Ludlow et al, ; Rubaiy, Ludlow, Henrot, et al, ). These data suggest that AM237 is a potent inhibitor of heteromeric TRPC1:5 channels.…”

“…This effect of AM237 on the EA‐mediated responses of TRPC5:C5 channels was confirmed by patch‐clamp electrophysiology experiments, in which application of AM237 partially suppressed inward and outward currents induced by EA in outside‐out membrane patches (Figure c–e). The characteristic chair‐shape of the current–voltage plots was consistent with activation of homomeric TRPC5:C5 channels (Figure d; Ludlow et al, ; Rubaiy, Ludlow, Henrot, et al, ). Incubation with AM237 did not lead to reduced HEK T‐REx cell viability (Figure S8).…”

“…AM237 was synthesized by modification of published procedures (Chenard & Gallaschun, ; Rubaiy, Ludlow, Henrot, et al, ) and purified to homogeneity according to 1 H NMR, 13 C NMR, 19 F NMR, and HPLC analysis (see Figures S1–S5). Pico145 was prepared according to previously reported procedures (Rubaiy, Ludlow, Henrot, et al, ). EA was obtained from PhytoLab (Vestenbergsgreuth, Germany).…”

“…Other promising TRPC1/4/5 activators include the natural product http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=10287 (Rubaiy et al, ), the marketed drug http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2326 (which has limited potency and selectivity but has been used for in vivo studies of TRPC5 channels; Richter, Schaefer, & Hill, ; Zhou et al, ; Zhu et al, ), and the synthetic benzothiadiazine derivative http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=10289, which activates TRPC5:C5 (EC 50 ~1 μM), TRPC1:C5, and TRPC4:C5 channels, but not TRPC4:C4 or TRPC1:C4 channels (Beckmann et al, ). The most promising TRPC1/4/5 inhibitors are two closely related xanthines, http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=10291 (also called HC‐608) and http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=10286 (Figure ; Just et al, ; Rubaiy, Ludlow, Bon, & Beech, ; Rubaiy, Ludlow, Henrot, et al, ), which were originally described in a patent (Chenard & Gallaschun, ). These compounds exhibit (sub)nanomolar potency against TRPC1/4/5 channels, while at 1–2 μM, no significant effects on hundreds of other targets were found (Just et al, ).…”

“…The compounds are orally bioavailable and suitable for in vivo studies (Cheung et al, ; Just et al, ). Pico145 also distinguishes between different TRPC1/4/5 tetramers, with the highest potency against heteromeric channels (Rubaiy, Ludlow, Henrot, et al, ).…”