Development and Validation of an Enzymatic Assay for TMPRSS4: Evaluation of Molecular Inhibitors

Abstract

TMPRSS4, a member of the transmembrane serine protease (TTSP) family, has earned significant attention due to its pronounced overexpression in various carcinoma types, involvement in disease processes, and viral entry.  However, despite its pivotal role in disease biology, little is known about its structural characteristics and potential drug targets. Accurate measurement of TMPRSS4 activity is crucial for understanding its role in these disease processes and for developing potential therapeutic interventions. In this context, we have expressed and purified TMPRSS4 protein and report the development of an enzymatic assay for the quantitative measurement of TMPRSS4 activity.  The assay is based on a synthetic fluorogenic peptide substrate that mimics the natural cleavage site of TMPRSS4/serine proteases substrates. The Assay development workflow includes enzyme preparation, optimization of assay materials, reagents, and conditions.  The assay is further validated using a known serine protease inhibitor, Aprotinin. Our results demonstrate that the developed enzymatic assay exhibits a wide dynamic range and successfully applied the assay to assess molecular inhibitors predicted by our computational studies. In conclusion, our study presents a robust enzymatic assay for TMPRSS4 activity that provides researchers and clinicians with a valuable tool for studying this protease's function and potential clinical applications.