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The global health emergency imposed by COVID-19 necessitates the development of antiviral therapeutics that disable SARS-CoV-2 in a manner insensitive to emergent viral mutations. Transmembrane serine protease 2 (TMPRSS2) is an airway-expressed host factor that acts in concert with furin to proteolytically activate the SARS-CoV-2 Spike (S) protein to drive virus-host membrane fusion. Disabling TMPRSS2 would shut down a conserved entry pathway for coronaviruses. Unfortunately, current recombinant TMPRSS2 production methods afford mostly inactive zymogens which could lead to inaccurate assessment of inhibitor potencies. We have established a fully functional source of extracellular TMPRSS2 engineered for secretion from Sf9 insect cells as a zymogen that was activated on-demand at mg yields. The activated protein had >90% molar enzyme activity at apparent homogeneity. Its proteolytic activity was characterized through fluorogenic peptidase assays as well as gel-based S protein cleavage assays. Covalent protease inhibitors nafamostat and camostat diverged in potency with k_inact/K_i values of (0.27±0.02) and (0.0071±0.0006) s^-1nM^-1, respectively, despite forming a common phenylguanidino acyl-enzyme intermediate. Nafamostat blocked S protein cleavage at 1:1 stoichiometry with TMPRSS2. Competitive inhibitors benzamidine and sunflower trypsin inhibitor-1 had values of (240±10) and (1.6±0.3) µM, respectively. The availability of highly active enzyme now enables experimentally-informed design of inhibitors that potently and selectively disrupt TMPRSS2 activity, in support of effective therapeutics to complement vaccines in combating an evolving COVID-19 pandemic and potential future coronavirus outbreaks.

<b>Figure 1</b> Inhibition of recombinant TMPRSS2 protease activity. (A) TMPRSS2 zymogen activation (B) Engineered extracellular TMPRSS2 (dasTMPRSS2) amenable to on-demand self-activation. (C) TMPRSS2-mediated SARS-CoV-2 fusion step and formation of S1’ and S2’ products. (D) TMPRSS2 fluorogenic peptidase assays enabling ranking of active site inhibitors.

Figure 1 Inhibition of recombinant TMPRSS2 protease activity. (A) TMPRSS2 zymogen activation (B) Engineered extracellular TMPRSS2 (dasTMPRSS2) amenable to on-demand self-activation. (C) TMPRSS2-mediated SARS-CoV-2 fusion step and formation of S1’ and S2’ products. (D) TMPRSS2 fluorogenic peptidase assays enabling ranking of active site inhibitors.

Bryan Fraser

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