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

Abstract

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.

In Silico Molecular Characterization of Human TMPRSS2 Protease Polymorphic Variants and Associated SARS-CoV-2 Susceptibility

“…The atomic coordinates of the human TMPRSS2 and SARS-CoV-2 S glycoprotein used in this study were retrieved from the RCSB Protein Data Bank (PDB) and the accession numbers were 7MEQ [31] and 6VSB [33], respectively. Both structures were subjected to manual inspection; ligands were removed, and all gaps and missing loops were built manually using Builder, as implemented in PyMOL.…”

“…The ectodomain is comprised of three subdomains: a low-density lipoprotein receptor type A (LDLR-A) domain, a class A scavenger receptor cysteine-rich (SRCR) domain and a trypsin-like serine peptidase. The crystal structure of the recombinant human TMPRSS2 was recently published [31]. The 1.95-Å structure shows a biologically active ectodomain, in which the LDLR-A domain is absent (Figure 2).…”

“…Molecular docking of TMPRSS2 with the spike glycoprotein. TMPRSS2 (PDB: 7MEQ) [31] bound in close proximity to cleavage sites 1 (R685/R686) (left) and 2 (R815/S816) (right) of the SARS-CoV-2 S glycoprotein (PDB: 6VSB) [33]. TMPRSS2 and the S protein were colored in magenta and green, respectively.…”

“…Interactions between the two molecules were strengthened by formation of several hydrogen bonds; site 1: between Q276 of TMPRSS2 and Q630/G655 of the S protein, between K392 and D643/T644, between D417 and N691/N692, between Q438 and D684, and between G462 and S686; site 2: between K300 of TMPRSS2 and K790 of the S protein, between K340 and K814/Y873, between K390 and D808, and between G464 and P809. The crystal structure of TMPRSS2 (PDB: 7MEQ) [31], comprised of two subdomains, a class A scavenger receptor cysteine-rich (SRCR) domain (red) and a trypsin-like serine peptidase (blue).…”

“…In this study, using the recently reported structural data of TMPRSS2 [31], we analyzed the molecular complex of TMPRSS2 and the S glycoprotein and expounded structurally in detail the intermolecular interactions of natural TMPRSS2 polymorphic variants. In addition, we investigated the intermolecular interactions between TMPRSS2 and its two potential inhibitors, namely camostat mesylate and nafamostat [32].…”