Structure, activity and inhibition of human TMPRSS2, a protease implicated in SARS-CoV-2 activation

• Published in: bioRxiv
• Main Authors: Fraser, Bryan J, Beldar, Serap, Seitova, Almagul, Hutchinson, Ashley, Mannar, Dhiraj, Li, Yanjun, Kwon, Daniel, Tan, Ruiyan, Wilson, Ryan P, Leopold, Karoline, Subramaniam, Sriram, Halabelian, Levon, Arrowsmith, Cheryl H, Benard, Francois
• Format: Paper
• Language: English; Japanese
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 24.06.2021
• Subjects: Coronaviruses; Crystal structure; Crystallization; Drug development; Enzymatic activity; Enzymes; Fusion protein; Membrane fusion; Membrane proteins; Proteinase inhibitors; Serine; Severe acute respiratory syndrome coronavirus 2
• DOI: 10.1101/2021.06.23.449282

Transmembrane protease, serine 2 (TMPRSS2) has been identified as key host cell factor for viral entry and pathogenesis of SARS-coronavirus-2 (SARS-CoV-2). Specifically, TMPRSS2 proteolytically processes the SARS-CoV-2 Spike (S) Protein, enabling virus-host membrane fusion and infection of the lungs. We present here an efficient recombinant production strategy for enzymatically active TMPRSS2 ectodomain enabling enzymatic characterization, and the 1.95 A X-ray crystal structure. To stabilize the enzyme for co-crystallization, we pre-treated TMPRSS2 with the synthetic protease inhibitor nafamosat to form a stable but slowly reversible (15 hour half-life) phenylguanidino acyl-enzyme complex. Our study provides a structural basis for the potent but non-specific inhibition by nafamostat and identifies distinguishing features of the TMPRSS2 substrate binding pocket that will guide future generations of inhibitors to improve selectivity. TMPRSS2 cleaved recombinant SARS-CoV-2 S protein ectodomain at the canonical S1/S2 cleavage site and at least two additional minor sites previously uncharacterized. We established enzymatic activity and inhibition assays that enabled ranking of clinical protease inhibitors with half-maximal inhibitory concentrations ranging from 1.7 nM to 120 uM and determination of inhibitor mechanisms of action. These results provide a body of data and reagents to support future drug development efforts to selectively inhibit TMPRSS2 and other type 2 transmembrane serine proteases involved in viral glycoprotein processing, in order to combat current and future viral threats. Competing Interest Statement The authors have declared no competing interest.