The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced owing to emerging variants of concern1,2. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against variants of concern3,4. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs) such as TMPRSS2; these proteases cleave the viral spike protein to expose the fusion peptide for cell entry, and thus have an essential role in the virus lifecycle5,6. Here we identify and characterize a small-molecule compound, N-0385, which exhibits low nanomolar potency and a selectivity index of higher than 106 in inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids7. In Calu-3 cells it inhibits the entry of the SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Notably, in the K18-human ACE2 transgenic mouse model of severe COVID-19, we found that N-0385 affords a high level of prophylactic and therapeutic benefit after multiple administrations or even after a single administration. Together, our findings show that TTSP-mediated proteolytic maturation of the spike protein is critical for SARS-CoV-2 infection in vivo, and suggest that N-0385 provides an effective early treatment option against COVID-19 and emerging SARS-CoV-2 variants of concern.

Objective. Increasing evidence implicates serine proteinases in pathologic tissue turnover. The aim of this study was to assess the role of the transmembrane serine proteinase matriptase in cartilage destruction in osteoarthritis (OA).Methods. Serine proteinase gene expression in femoral head cartilage obtained from either patients with hip OA or patients with fracture to the neck of the femur (NOF) was assessed using a low-density array. The effect of matriptase on collagen breakdown was determined in cartilage degradation models, while the effect on matrix metalloproteinase (MMP) expression was analyzed by real-time polymerase chain reaction. ProMMP processing was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Nterminal sequencing, while its ability to activate proteinase-activated receptor 2 (PAR-2) was determined using a synovial perfusion assay in mice.Results. Matriptase gene expression was significantly elevated in OA cartilage compared with NOF cartilage, and matriptase was immunolocalized to OA chondrocytes. We showed that matriptase activated proMMP-1 and processed proMMP-3 to its fully active form. Exogenous matriptase significantly enhanced cytokine-stimulated cartilage collagenolysis, while matriptase alone caused significant collagenolysis from OA cartilage, which was metalloproteinase-dependent. Matriptase also induced MMP-1, MMP-3, and MMP-13 gene expression. Synovial perfusion data confirmed that matriptase activates PAR-2, and we demonstrated that matriptase-dependent enhancement of collagenolysis from OA cartilage is blocked by PAR-2 inhibition.Conclusion. Elevated matriptase expression in OA and the ability of matriptase to activate selective proMMPs as well as induce collagenase expression make this serine proteinase a key initiator and inducer of cartilage destruction in OA. We propose that the indirect effects of matriptase are mediated by PAR-2, and a more detailed understanding of these mechanisms may highlight important new therapeutic targets for OA treatment.Metalloproteinases, especially matrix metalloproteinases (MMPs), are considered to be the most important class of proteinase in terms of cartilage degradation, because collectively they can degrade all components of this complex extracellular matrix (ECM) (1). Indeed, type II collagen is a major structural component of this ECM, and collagenolysis is an essentially irreversible step (2), making such proteolysis a major therapeutic target (3). Collagens are remarkably resistant to prote-

Type II transmembrane serine proteases (TTSPs) are a newly recognized family of S1 class proteolytic enzymes, with 20 distinct members known in mice and humans. TTSPs are divided into four subfamilies based on their modular structure [1]. The HAT ⁄ DESC sub-family is the largest and is comprised of HAT, DESC1-4 and HAT-like HATL3-5. It exhibits the simplest modular structure of the stem region, which consists of a single sea urchin sperm protein, an entero-peptidase and an agrin domain (SEA). The matriptase subfamily contains three highly homologous proteases: matriptase, matriptase-2 and matriptase-3. All matrip-tases have similar stem regions, with one SEA, two C1r ⁄ C1s, urchin embryonic growth factor, bone morphogenic protein-1 (CUB), and three (matriptase-2 and matriptase-3) or four (matriptase) low-density Type II transmembrane serine proteases are an emerging class of proteo-lytic enzymes involved in tissue homeostasis and a number of human disorders such as cancer. To better define the biochemical functions of a subset of these proteases, we compared the enzymatic properties of matriptase, matriptase-2, hepsin and DESC1 using a series of internally quenched fluorogenic peptide substrates containing o-aminobenzoyl and 3-nitro-tyro-sine. We based the sequence of the peptides on the P4 to P4¢ activation sequence of matriptase (RQAR-VVGG). Positions P4, P3, P2 and P1¢ were substituted with nonpolar (Ala, Leu), aromatic (Tyr), acid (Glu) and basic (Arg) amino acids, whereas P1 was fixed to Arg. Of the four type II trans-membrane serine proteases studied, matriptase-2 was the most promiscuous , and matriptase was the most discriminating, with a distinct specificity for Arg residues at P4, P3 and P2. DESC1 had a preference similar to that of matriptase, but with a propensity for small nonpolar amino acids (Ala) at P1¢. Hepsin shared similarities with matriptase and DESC1, but was markedly more permissive at P2. Matriptase-2 manifested broader specifici-ties, as well as substrate inhibition, for selective internally quenched fluorescent substrates. Lastly, we found that antithrombin III has robust inhibitory properties toward matriptase, matriptase-2, hepsin and DESC1, whereas plasminogen activator inhibitor-1 and a 2-antiplasmin inhibited matriptase-2, hepsin and DESC1, and to a much lesser extent, matriptase. In summary, our studies revealed that these enzymes have distinct substrate preferences. Abbreviations a 1-ACT, a 1-antichymotrypsin; AEBSF, 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride; AMC, 7-amino-4-methylcoumarin; a 1-AP, a 1-antiplasmin;; a 1-AT, a 1-antitrypsin; AT III, antithrombin III; IQF, internally quenched fluorescent; PAI-I, plasminogen activator inhibitor I; PAR-2, protease-activated receptor-2; proMSP-1, macrophage-stimulating protein 1 precursor; PS-SCL, positional scanning-synthetic combinatorial libraries; TTSP, type II transmembrane serine protease.

Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place.

Matriptase is a member of the type II transmembrane serine protease family. Several studies have reported deregulated matriptase expression in several types of epithelial cancers, suggesting that matriptase constitutes a potential target for cancer therapy. We report herein a new series of slow, tightbinding inhibitors of matriptase, which mimic the P1−P4 substrate recognition sequence of the enzyme. Preliminary structure−activity relationships indicate that this benzothiazole-containing RQAR-peptidomimetic is a very potent inhibitor and possesses a good selectivity for matriptase versus other serine proteases. A molecular model was generated to elucidate the key contacts between inhibitor 1 and matriptase.

Background & Aims Matriptase is a membrane-anchored serine protease encoded by Suppression of Tumorigenicity-14 (ST14) that is required for epithelial barrier homeostasis. However its functional role in inflammatory bowel disease (IBD) is unexplored. Methods Matriptase expression in control, Crohn's disease and ulcerative colitis tissue specimens was studied by qPCR and immunostaining. Matriptase function was investigated by subjecting St14 hypomorphic and control littermates to dextran sodium sulfate (DSS)-induced colitis and by siRNA silencing in cultured monolayers. Mice were analyzed for clinical, histological, molecular and cellular effects. Results Matriptase protein and ST14 mRNA levels are significantly down-regulated in inflamed colonic tissues from Crohn's disease and ulcerative colitis patients. Matriptase deficient St14 hypomorphic mice administered DSS for 7 days followed by water without DSS for 3 days develop a severe colitis with only 30% of the St14 hypomorphic mice surviving to day 14, compared with 100% of control littermates. Persistent colitis in surviving St14 hypomorphic mice was associated with sustained cytokine production, an inability to recover barrier integrity, and enhanced claudin-2 expression. Cytokines implicated in barrier disruption during IBD suppress matriptase expression in T84 epithelial monolayers and restoration of matriptase improves barrier integrity in the cytokine-perturbed monolayers. Conclusions These data demonstrate a critical role for matriptase in restoring barrier function to injured intestinal mucosa during colitis, which is suppressed by excessive activation of the immune system. Strategies to enhance matriptase-mediated barrier recovery could be important for intervening in the cycle of inflammation associated with IBD.

Background: Matriptase promotes intestinal epithelial barrier formation. Results: Matriptase and prostasin constitute a common proteolytic pathway where matriptase is indirectly activated by prostasin and functions on the cell surface to regulate barrier formation. Conclusion: A prostasin 3 matriptase protease cascade regulates barrier formation in simple intestinal epithelia. Significance: Cell surface serine proteases have complex cascade relationships that are context-and tissue-specific.

The type II transmembrane serine protease TMPRSS6 (also known as matriptase-2) controls iron homeostasis through its negative regulation of expression of hepcidin, a key hormone involved in iron metabolism. Upstream of the hepcidin-regulated signaling pathway, TMPRSS6 cleaves its target substrate hemojuvelin (HJV) at the plasma membrane, but the dynamics of the cell-surface expression of the protease have not been addressed. Here, we report that TMPRSS6 undergoes constitutive internalization in transfected HEK293 cells and in two human hepatic cell lines, HepG2 and primary hepatocytes, both of which express TMPRSS6 endogenously. Cell surface-labeled TMPRSS6 was internalized and was detected in clathrin-and AP-2-positive vesicles via a dynamin-dependent pathway. The endocytosed TMPRSS6 next transited in early endosomes and then to lysosomes. Internalization of TMPRSS6 is dependent on specific residues within its N-terminal cytoplasmic domain, as site-directed mutagenesis of these residues abrogated internalization and maintained the enzyme at the cell surface. Cells coexpressing these mutants and HJV produced significantly decreased levels of hepcidin compared with wild-type TMPRSS6 due to the sustained cleavage of HJV at the cell surface by TMPRSS6 mutants. Our results underscore for the first time the importance of TMPRSS6 trafficking at the plasma membrane in the regulation of hepcidin expression, an event that is essential for iron homeostasis.The serine protease superfamily of hydrolytic enzymes is composed of Ͼ200 members (MEROPS Database (1)), most of them being secreted soluble proteins. TMPRSS6 (matriptase-2), a member of a novel family of type II transmembrane serine proteases that function at the cell surface (2), was originally characterized as a protease expressed mostly in the liver (3) and capable of processing proteins such as type I collagen, fibronectin, and fibrinogen (4, 5). The protease is a mosaic protein composed of multiple domains, including a 52-residue N-terminal cytoplasmic domain (6), a transmembrane domain that anchors the enzyme within membranes, and an extracellular domain itself constituted of multiple regions, including the catalytic region. Until now, no function had been associated with the cytoplasmic tail of the enzyme. TMPRSS6 is synthesized as an inactive zymogen with the catalytic region being disulfidelinked to the main chain following activation. Moreover, the enzyme undergoes cell-surface shedding, releasing a soluble and active form in the extracellular milieu (7). It exhibits many of the enzymatic specificities of other members of the type II transmembrane serine protease family, preferring arginine residues in P4, P3, and P1 positions relative to the cleaved peptide bond (5,8).Although TMPRSS6 has been associated with breast and prostate cancer (9, 10), recent reports have demonstrated its direct involvement in iron homeostasis. Indeed, genetic analysis of kindred suffering from iron-refractory iron deficiency anemia identified sequence variants in the TMPRSS6 gene, ...