Exploring cathepsin G inhibition by vinyl-based ionic liquids: Insights from lab-on-valve integrated microsequential analysis and molecular docking approaches

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.