Exploring cathepsin G inhibition by vinyl-based ionic liquids: Insights from lab-on-valve integrated microsequential...

Cathepsin G is a serine protease involved in inflammatory and tissue remodeling processes associated with chronic wounds. In this study, an automatic flow-based analytical method based on a Lab-on-Valve integrated microsequential analysis (μSIA-LOV) system was developed for assessing Cathepsin G activity and the inhibitory effects of vinyl-based ionic liquids. The enzymatic reaction was based on the hydrolysis of N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, with product formation monitored spectrophotometrically at 405 nm. The developed system enabled accurate, reproducible, environmentally sustainable analyses, and efficient determination of IC₅₀ values, while reducing reagent consumption, analysis time, and waste generation. Its miniaturized configuration allows rapid and reliable screening of enzyme inhibitors under controlled conditions, allowed 6 assays per hour. Molecular docking and molecular dynamics simulations revealed that ionic liquids containing polar or electron-withdrawing groups exhibited stronger inhibitory effects due to favorable electrostatic and π-π interactions within the Cathepsin G active site, providing molecular-level insight into the structure-activity relationships governing ionic liquids - Cathepsin G interactions. These findings demonstrate how ionic liquid structure modulates Cathepsin G inhibition and highlight the potential of the μSIA-LOV platform as a green and efficient tool for enzyme inhibition studies, with potential applications in the development of targeted therapeutic strategies for chronic wound management.