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TMPRSS13, a member of the Type II Transmembrane Serine Proteases (TTSP) family, is involved in cancer progression and in cell entry of respiratory viruses. To date, no inhibitors have been specifically developed toward this protease. In this study, a chemical library of 65 ketobenzothiazole-based peptidomimetic molecules was screened against a proteolytically active form of recombinant TMPRSS13 to identify novel inhibitors. Following an initial round of screening, subsequent synthesis of additional derivatives supported by molecular modelling, uncovered important molecular determinants involved in TMPRSS13 inhibition. One inhibitor, N-0430, achieved low nanomolar affinity towards TMPRSS13 activity in a cellular context. Using a SARS-CoV-2 pseudovirus cell entry model, we further show the ability of N-0430 to block TMPRSS13-dependent entry of the pseudovirus. The identified peptidomimetic inhibitors and the molecular insights of their potency gained from this study will aid in the development of specific TMPRSS13 inhibitors.

The continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) urges better understanding of the functional motifs in the spike (S) protein and their tolerance to mutations. Here, we focused on the S2' motif, which, during virus entry, requires cleavage by a host cell protease to release the fusion peptide. Though belonging to an immunogenic region, the SARS-CoV-2 S2' motif (811-KPSKR-815) has shown hardly any variation, with its three basic (K/R) residues being >99.99% conserved thus far. By creating a series of mutant pseudoviruses bearing the spikes of Wuhan-Hu-1, its G614 mutant or the Delta and Omicron variants, we show that residue K 814 (preceding the scissile R 815 ) is dispensable for TMPRSS2 yet favored by the alternative TMPRSS13 protease. Activation by TMPRSS13 was drastically reduced when the SARS-CoV-2 S2' motif was swapped with that of the low pathogenic 229E coronavirus (685-RVAGR-689), and also, the reverse effect was seen. This swap had no impact on recognition by TMPRSS2. In the Middle East respiratory syndrome coronavirus (MERS-CoV) spike, introducing a dibasic scissile motif was easily accepted by TMPRSS13 but less so by TMPRSS2, confirming that TMPRSS13 favors a sequence rich in K/R residues. Pseudovirus entry experiments in Calu-3 cells confirmed that the S2' mutations have minor impact on TMPRSS2. Our findings are the first to demonstrate which S2' residues are important for SARS-CoV-2 spike activation by these two airway proteases, with TMPRSS2 being more tolerant to variation than TMPRSS13. This preemptive insight will help to estimate the impact of S2' motif changes as they appear in new SARS-CoV-2 variants. IMPORTANCE Since its introduction in humans, SARS-CoV-2 is evolving with frequent appearance of new variants. The surveillance would benefit from proactive characterization of the functional motifs in the spike (S) protein, the most variable viral factor. This is linked to immune evasion but also influences spike functioning. Remarkably, though located in a strongly immunogenic region, the S2' cleavage motif has, thus far, remained highly conserved. This suggests that its sequence is critical for spike activation by airway proteases. To investigate this, we assessed how pseudovirus entry is affected by changes in the S2' motif. We demonstrate that TMPRSS2 readily accepts variations in this motif, whereas the alternative TMPRSS13 protease is more fastidious. The Wuhan-Hu-1, G614, Delta and Omicron spikes showed no difference in this regard. Being the first in its kind, our study will help to assess the impact of S2' variations as soon as they are detected during variant surveillance.

TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, harbors four N-linked glycosylation sites in its extracellular domain. Two of the glycosylated residues are located in the scavenger receptor cysteine-rich (SRCR) protein domain, while the remaining two sites are in the catalytic serine protease (SP) domain. In this study, we examined the role of N-linked glycosylation in the proteolytic activity, autoactivation, and cellular localization of TMPRSS13. Individual and combinatory site-directed mutagenesis of the glycosylated asparagine residues indicated that glycosylation of the SP domain is critical for TMPRSS13 autoactivation and catalytic activity toward one of its protein substrates, the prostasin zymogen. Additionally, SP domain glycosylation-deficient TMPRSS13 displayed impaired trafficking of TMPRSS13 to the cell surface, which correlated with increased retention in the endoplasmic reticulum. Importantly, we showed that N-linked glycosylation was a critical determinant for subsequent phosphorylation of endogenous TMPRSS13. Taken together, we conclude that glycosylation plays an important role in regulating TMPRSS13 activation and activity, phosphorylation, and cell surface localization., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

TMPRSS13 is a member of the type II transmembrane serine protease (TTSP) family. Although various TTSPs have been characterized in detail biochemically and functionally, the basic properties of TMPRSS13 remain unclear. Here, we investigate the activation, inhibition, post-translational modification, and localization of TMPRSS13. We show that TMPRSS13 is a glycosylated, active protease and that its own proteolytic activity mediates zymogen cleavage. Full-length, active TMPRSS13 exhibits impaired cell-surface expression in the absence of the cognate Kunitz-type serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 or HAI-2. Concomitant presence of TMPRSS13 with either HAI-1 or -2 mediates phosphorylation of residues in the intracellular domain of the protease, and it coincides with efficient transport of the protease to the cell surface and its subsequent shedding. Cell-surface labeling experiments indicate that the dominant form of TMPRSS13 on the cell surface is phosphorylated, whereas intracellular TMPRSS13 is predominantly non-phosphorylated. These data provide novel insight into the cellular properties of TMPRSS13 and highlight phosphorylation of TMPRSS13 as a novel post-translational modification of this TTSP family member and potentially other members of this family of proteases., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)