Development of Fluorescence and Mass Spectrometry Based Detection Assays for assessment of TMPRSS2 inhibition as Potential Treatment of COVID-19

The enzyme TMPRSS2 plays a critical role in entry of SARS-CoV-2 virus into a host cell. A fluorogenic tripeptide substrate was used to develop and validate biochemical screening assays for identification of TMPRSS2 inhibitors. In addition, HT-LC/MS/MS and Acoustic-Ejection MS (AEMS) assays were developed using a physiologically relevant, non-labeled peptide substrate. Validation of the LC/MS/MS assay was demonstrated by obtaining similar IC50 values to those obtained from the fluorogenic assay. Coherence of the AEMS assay was demonstrated by obtaining similar substrate Km values to those obtained from the LC/MS/MS assay. Herein we compare label and label-free detection methods with respect to analytical performance, cost, and routine usage in context of a screening campaign using two TMPRSS2 peptides as marker substrates.