Structural determinants of TRPV4 inhibition and identification of new antagonists with antiviral activity.

BACKGROUND AND PURPOSE: The transient receptor potential vanilloid 4 (TRPV4) cation channel participates in multiple physiological processes and is also at the core of different diseases, making this channel an interesting pharmacological target with therapeutic potential. However, little is known about the structural elements governing its inhibition. EXPERIMENTAL APPROACH: We have now combined in silico drug discovery and molecular dynamics simulation based on Xenopus tropicalis xTRPV4 structure with functional studies measuring cell Ca2+ influx mediated by human TRPV4 channel to characterize the binding site of known TRPV4 inhibitors and to identify novel small molecule channel modulators. KEY RESULTS: We have found that the inhibitor HC067047 binds to a pocket conformed by residues from S2-S3 linker (xTRPV4-D542), S4 (xTRPV4-M583 and Y587 and S5 (xTRPV4-D609 and F613). This pocket was also used for structure-based virtual screening in the search of novel channel modulators. Forty potential hits were selected based on the lower docking scores (from ~250,000 compounds) and their effect upon TRPV4 functionally tested. Three were further analysed for stability using molecular dynamics simulation and functionally tested on TRPV4 channels carrying mutations in the binding pocket. Compound NSC151066, shown to require residue xTRPV4-M583 for its inhibitory effect, presented an IC50 of 145 nM and demonstrated to be an effective antiviral against Zika virus with a potency similar to HC067047. CONCLUSION AND IMPLICATIONS: Together, we propose structural insights into the inhibition of TRPV4 and how this information can be used for the design of novel channel modulators. LINKED ARTICLES: This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc.

The mechanosensitive Piezo1 channel controls endosome trafficking for an efficient cytokinetic abscission.

Mechanical forces are exerted throughout cytokinesis, the final step of cell division. Yet, how forces are transduced and affect the signaling dynamics of cytokinetic proteins remains poorly characterized. We now show that the mechanosensitive Piezo1 channel is activated at the intercellular bridge (ICB) connecting daughter cells to regulate abscission. Inhibition of Piezo1 caused multinucleation both in vitro and in vivo. Piezo1 positioning at the ICB during cytokinesis depends on Pacsin3. Pharmacological and genetic inhibition of Piezo1 or Pacsin3 resulted in mislocation of Rab11-family-interacting protein 3 (Rab11-FIP3) endosomes, apoptosis-linked gene 2-interacting protein X (ALIX), and endosomal sorting complex required for transport III (ESCRT-III). Furthermore, we identified FIP3 as the link between Piezo1-generated Ca2+ signals and ALIX delivery to the ICB, where ALIX recruits the ESCRT-III component charged multivesicular body protein 4B, which promotes abscission. These results provide a different view of how mechanical forces participate in cytokinesis and identify Piezo1 as a key modulator of endosome trafficking.