Human TRPC5 structures reveal interaction of a xanthine-based TRPC1/4/5 inhibitor with a conserved lipid binding site

Abstract

TRPC1/4/5 channels are non-specific cation channels implicated in a wide variety of diseases, and TRPC1/4/5 inhibitors have recently entered clinical trials. However, fundamental and translational studies require a better understanding of TRPC1/4/5 channel regulation by endogenous and exogenous factors. Although several potent and selective TRPC1/4/5 modulators have been reported, the paucity of mechanistic insights into their modes-of-action remains a barrier to the development of new chemical probes and drug candidates. Xanthine-based modulators include the most potent and selective TRPC1/4/5 inhibitors described to date, as well as TRPC5 activators. Our previous studies suggest that xanthines interact with a, so far, elusive pocket of TRPC1/4/5 channels that is essential to channel gating. Here we report the structure of a small-molecule-bound TRPC1/4/5 channel—human TRPC5 in complex with the xanthine Pico145—to 3.0 Å. We found that Pico145 binds to a conserved lipid binding site of TRPC5, where it displaces a bound phospholipid. Our findings explain the mode-of-action of xanthine-based TRPC1/4/5 modulators, and suggest a structural basis for TRPC1/4/5 modulation by endogenous factors such as (phospho)lipids and Zn2+ ions. These studies lay the foundations for the structure-based design of new generations of TRPC1/4/5 modulators.

Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

“…It is reported that Pico145, an HC-070 analogue, is more potent against hTRPC4/5 than HC-070 ( Just et al, 2018 ), possibly due to the substitution of chloride with trifluoromethoxy group on the chlorophenoxy ring of HC-070 ( Figure 6—figure supplement 2G–H ). Indeed, recent structure study showed that Pico145 binds to hTRPC5 channel using a similar pose as HC-070, except the chlorophenoxy ring with a trifluoromethoxy group on it inserts into a cavity surrounded by L521, Y524, F576, and V610 of hTRPC5 ( Figure 1—figure supplement 7J ; Wright et al, 2020 ). Another HC-070 analogue, AM237, is a TRPC5 activator with EC 50 around 15–20 nM ( Minard et al, 2019 ), and AM237 activates homomeric hTRPC5 channel instead of the heterotetrameric TRPC1: C5 channel ( Minard et al, 2019 ), suggesting that AM237 also binds at IBP-B which is at the interface between adjacent subunits.…”

“…Recently, two distinct inhibitor-binding sites were identified in the hTRPC6 channel ( Bai et al, 2020 ; Tang et al, 2018 ). During the editorial process of this work, inhibitor binding sites were also identified in TRPC4 ( Vinayagam et al, 2020 ) and TRPC5 ( Wright et al, 2020 ). Together with the sites identified in this study, we classify them into three groups, namely inhibitor-binding pocket (IBP) A-C ( Figure 6 , Figure 6—figure supplement 1 ).…”

“…Compared with Pico145, AM237 has an additional chloride atom on the phenoxy ring ( Figure 6—figure supplement 2I ), which makes the phenoxy group larger and this chloride atom might be in clash with L521 of hTRPC5. Therefore, AM237 might activate hTRPC5 by pushing L521 on S5 using its chloride-phenoxy group ( Wright et al, 2020 ). Consistent with our results of HC-070-binding site mutants, mutations in IBP-B, including Q573T, F576A, and W577A, decrease the potency of AM237 on hTRPC5 ( Wright et al, 2020 ).…”

“…Therefore, AM237 might activate hTRPC5 by pushing L521 on S5 using its chloride-phenoxy group ( Wright et al, 2020 ). Consistent with our results of HC-070-binding site mutants, mutations in IBP-B, including Q573T, F576A, and W577A, decrease the potency of AM237 on hTRPC5 ( Wright et al, 2020 ). Recently, it is reported that an hTRPC6 agonist AM-0883 binds at IBP-B as well ( Figure 6—figure supplement 1B ; Bai et al, 2020 ).…”

Canonical transient receptor potential channels and their modulators: biology, pharmacology and therapeutic potentials

“…Pico145 (C31, HC-608) is a xanthine derivative that potently inhibits TRPC1/4/5 channels with IC 50 of 1.3 nM (TRPC5:C5), 0.349 nM (TRPC4:C4), 0.199 nM (TRPC5:C1, heteromers), and 0.033 nM (TRPC4:C1, heteromers) (Rubaiy et al 2017b ). Pico145 can directly, reversibly, and competitively inhibits the AM237-mediated TRPC5:C5 channel activation (Rubaiy et al 2017a ; Minard et al 2019 ; Wright et al 2020 ). The potency of Pico145 depends on the concentration of the (−)-englerin A (Rubaiy et al 2017b ).…”