The calcium-selective TRPV5 channel activated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is involved in calcium homeostasis. Recently, cryoelectron microscopy (cryo-EM) provided molecular details of TRPV5 modulation by exogenous and endogenous molecules. However, the details of TRPV5 inhibition by the antifungal agent econazole (ECN) remain elusive due to the low resolution of the currently available structure. In this study, we employ cryo-EM to comprehensively examine how the ECN inhibits TRPV5. By combining our structural findings with site-directed mutagenesis, calcium measurements, electrophysiology, and molecular dynamics simulations, we determined that residues F472 and L475 on the S4 helix, along with residue W495 on the S5 helix, collectively constitute the ECN-binding site. Additionally, the structure of TRPV5 in the presence of ECN and PI(4,5)P2, which does not show the bound activator, reveals a potential inhibition mechanism in which ECN competes with PI(4,5)P2, preventing the latter from binding, and ultimately pore closure.
Copyright © 2023 Elsevier Ltd. All rights reserved.

The fungal disease of the nail, onychomycosis, which is also the most prevalent nail disturbance, demands effective topical treatment options considering the possible adverse effects of systemic antifungal therapy. The current work is focused on development of an adhesive and resistant, drug-delivering and permeation-enhancing polymeric film containing econazole nitrate (ECN) for topical antifungal treatment. The development of the lacquer formulation was guided by the Quality by Design approach to achieve the critical quality attributes needed to obtain the product of desired quality. Eudragit RSPO at 10% w/w was found to be the ideal adhesive polymer for the application and an optimal permeation-enhancing lacquer formulation was achieved by the optimization of other formulation excipients, such as plasticizer and the solvent system. Additionally, novel experimental enhancements introduced to the research included refined D50 drying time and drying rate tests for lacquer characterization as well as a multi-mechanism permeation-enhancing pre-treatment. Moreover, a practical implication was provided by a handwashing simulation designed to test the performance of the lacquer during actual use. In vitro drug release testing and ex vivo nail permeation testing demonstrated that the optimized nail lacquer performed better than control lacquer lacking the permeation enhancer by achieving a faster and sustained delivery of ECN. It can be concluded that this is a promising drug delivery system for topical antifungal treatment of onychomycotic nails, and the novel characterization techniques may be adapted for similar formulations in the future.