Cryo-EM structures of human TRPC5 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 the first 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. The xanthine class of modulators includes 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. Targeting this pocket may be a promising strategy for TRPC1/4/5 drug discovery. Here we report the first structure of a small molecule-bound TRPC1/4/5 channelhuman TRPC5 in complex with the xanthine Pico145to 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 Zn 2+ ions. These studies lay the foundations for the structure-based design of new generations of TRPC1/4/5 modulators.

Transient receptor potential canonical 5 (TRPC5) mediates inflammatory mechanical pain

“…When sufficiently elevated, our data suggest that LPC can activate TRPC5 to induce spontaneous pain. CryoEM structures of mouse and human TRPC5 homomers have identified conserved phospholipid binding sites at the interface between individual monomer subunits (31,40,41). Since AC1903, HC-070, and ML204, decreased LPC-induced TRPC5 activity in our heterologous expression system, these compounds may be effective therapies for LPCrelated spontaneous pain in vivo.…”

“…Thresholds for mechanically-induced action potential firing were determined using a custom-built, feedback-controlled mechanical stimulator that applied a continuous force ramp from 0 to 100 mN. Firing frequencies were recorded during static force applications(2,5,10,20,40, 100, 150, and 200 mN for 10 s; 1 min inter-force interval). All data was recorded and analyzed in LabChart software.…”

Xanthine-based photoaffinity probes allow assessment of ligand engagement by TRPC5 channels

“…PAL of TRPC5 was successful within the same concentration range as the effects on TRPC5-mediated calcium influx, providing further evidence that xanthines modulate TRPC5 channels through direct interaction with TRPC5 protein, consistent with their behaviour in excised outside-out patch recordings 23,28 and with our cryo-EM and site-directed mutagenesis studies. 29 To the best of our knowledge, only one previous example of PAL on a TRPC channel had been reported: Kiyonaka et al used a pyrazole derivative (Pyr-PP) incorporating a diazirine photocrosslinker and a ketone handle (for oxime ligation with a biotin hydroxylamine probe) to suggest that the TRPC3 channel inhibitor Pyr3 directly interacts with TRPC3 protein. 39 Competition PAL experiments with Pico145 allowed the indirect, quantitative assessment of the relative affinities of Pico145 and photoaffinity probes.…”

“…Therefore, the different concentrations of EA used as activator in inhibition assays with TRPC4:C4, TRPC4-C1 and TRPC5-C1 may exaggerate differences in IC 50 values between compounds. Docking of compounds 4-7 to the Pico145/lipid binding site of TRPC5:C5 29 revealed potential binding modes. Similar to the best predictions for the binding mode of Pico145, 29 these simulations suggest that the xanthine cores of all four compounds 4-7 make p-stacking interactions with F576, while their 3-hydroxypropyl groups make hydrogen bonding interactions with Q573 and W577 (Fig.…”

“…We recently reported cryo-EM structures of human TRPC5:C5 in complex with Pico145, revealing that Pico145 binds to a conserved lipid binding site of TRPC5, where it displaces a bound phospholipid. 29 A subsequent report of a cryo-EM structure of TRPC5:C5 in complex with HC-070 30 suggested that HC-070 binds to the same site as Pico145, with a near-identical binding pose. These studies highlight targeting of the conserved lipid binding site of TRPC1/4/5 channels as a promising strategy for TRPC1/4/5 drug discovery.…”