Inhibition of the proteolytic processing of hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP) is an attractive approach for the drug discovery of novel anticancer therapeutics which prevent tumor progression and metastasis. Here, we utilized an improved and expanded version of positional scanning of substrate combinatorial libraries (PS-SCL) technique called HyCoSuL to optimize peptidomimetic inhibitors of the HGF/MSP activating serine proteases, HGFA, matriptase, and hepsin. These inhibitors have an electrophilic ketone serine trapping warhead and thus form a reversible covalent bond to the protease. We demonstrate that by varying the P2, P3, and P4 positions of the inhibitor with unnatural amino acids based on the protease substrate preferences learned from HyCoSuL, we can predictably modify the potency and selectivity of the inhibitor. We identified the tetrapeptide JH-1144 (8) as a single digit nM inhibitor of HGFA, matriptase and hepsin with excellent selectivity over Factor Xa and thrombin. These unnatural peptides have increased metabolic stability relative to natural peptides of similar structure. The tripeptide inhibitor PK-1-89 (2) has excellent pharmacokinetics in mice with good compound exposure out to 24 h. In addition, we obtained an X-ray structure of the inhibitor MM1132 (15) bound to matriptase revealing an interesting binding conformation useful for future inhibitor design.

The dysfunction of matriptase, a membrane-anchored protease, is highly related to the progression of skin and breast cancers. Epidermal growth factor (EGF)-induced matriptase activation and cancer invasion are known but with obscure mechanisms. Here, we demonstrate a vesicular-trafficking-mediated interplay between matriptase and EGF signaling in cancer promotion. We found that EGF induces matriptase to undergo endocytosis together with the EGF receptor, followed by acid-induced activation in endosomes. Activated matriptase is then secreted extracellularly on exosomes to catalyze hepatocyte growth factor precursor (pro-HGF) cleavage, resulting in autocrine HGF/c-Met signaling. Matriptase-induced HGF/c-Met signaling represents the second signal wave of EGF, which promotes cancer cell scattering, migration, and invasion. These findings demonstrate a role of vesicular trafficking in efficient activation and secretion of membrane matriptase and a reciprocal regulation of matriptase and EGF signaling in cancer promotion, providing insights into the physiological functions of vesicular trafficking and the molecular pathological mechanisms of skin and breast cancers.

Membrane expansion integrates multiple forces to mediate precise tube growth and network formation. Defects lead to deformations, as found in diseases such as polycystic kidney diseases, aortic aneurysms, stenosis, and tortuosity. We identified a mechanism of sensing and responding to the membrane-driven expansion of tracheal tubes. The apical membrane is anchored to the apical extracellular matrix (aECM) and causes expansion forces that elongate the tracheal tubes. The aECM provides a mechanical tension that balances the resulting expansion forces, with Dumpy being an elastic molecule that modulates the mechanical stress on the matrix during tracheal tube expansion. We show in Drosophila that the zona pellucida (ZP) domain protein Piopio interacts and cooperates with the ZP protein Dumpy at tracheal cells. To resist shear stresses which arise during tube expansion, Piopio undergoes ectodomain shedding by the Matriptase homolog Notopleural, which releases Piopio-Dumpy-mediated linkages between membranes and extracellular matrix. Failure of this process leads to deformations of the apical membrane, tears the apical matrix, and impairs tubular network function. We also show conserved ectodomain shedding of the human TGFβ type III receptor by Notopleural and the human Matriptase, providing novel findings for in-depth analysis of diseases caused by cell and tube shape changes.

Human influenza viruses cause acute respiratory symptoms that can lead to death. Due to the emergence of antiviral drug-resistant strains, there is an urgent requirement for novel antiviral agents and innovative therapeutic strategies. Using the peptidomimetic ketobenzothiazole protease inhibitor RQAR-Kbt (IN-1, aka N-0100) as a starting point, we report how substituting P2 and P4 positions with natural and unnatural amino acids can modulate the inhibition potency toward matriptase, a prototypical type II transmembrane serine protease (TTSP) that acts as a priming protease for influenza viruses. We also introduced modifications of the peptidomimetics N-terminal groups, leading to significant improvements (from μM to nM, 60 times more potent than IN-1) in their ability to inhibit the replication of influenza H1N1 virus in the Calu-3 cell line derived from human lungs. The selectivity towards other proteases has been evaluated and explained using molecular modeling with a crystal structure recently obtained by our group. By targeting host cell TTSPs as a therapeutic approach, it may be possible to overcome the high mutational rate of influenza viruses and consequently prevent potential drug resistance.

Cancerous B cells are almost indistinguishable from their non-malignant counterparts regarding their surface antigen expression. Accordingly, the challenge to be faced consists in elimination of the malignant B cell population while maintaining a functional adaptive immune system. Here, we present an IgM-specific antibody-drug conjugate masked by fusion of the epitope-bearing IgM constant domain. Antibody masking impaired interaction with soluble pentameric as well as cell surface-expressed IgM molecules rendering the antibody cytotoxically inactive. Binding capacity of the anti-IgM antibody drug conjugate was restored upon conditional protease-mediated demasking which consequently enabled target-dependent antibody internalization and subsequent induction of apoptosis in malignant B cells. This easily adaptable approach potentially provides a novel mechanism of clonal B cell lymphoma eradication to the arsenal available for non-Hodgkin\'s lymphoma treatment.

Prostasin and matriptase are extracellular membrane serine proteases with opposing effects in solid epithelial tumors. Matriptase is an oncoprotein that promotes tumor initiation and progression, and prostasin is a tumor suppressor that reduces tumor invasion and metastasis. Previous studies have shown that a subgroup of Burkitt lymphoma have high levels of ectopic matriptase expression but no prostasin. Reducing the matriptase level via small interfering RNAs in B lymphoma cells impeded tumor xenograft growth in mice. Here, we report a novel approach to matriptase regulation in B cancer cells by prostasin via exosomes to initiate a prostasin-matriptase protease activation cascade. The activation and shedding of matriptase were monitored by measuring its quantity and trypsin-like serine protease activity in conditioned media. Sustained activation of the protease cascade in the cells was achieved by the stable expression of prostasin. The B cancer cells with prostasin expression presented phenotypes consistent with its tumor suppressor role, such as reduced growth and increased apoptosis. Prostasin exosomes could be developed as an agent to initiate the prostasin-matriptase cascade for treating B lymphoma with further studies in animal models.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder that affects the processing of carbohydrates, proteins, and lipids. In T2DM, metabolic dysregulation occurs through various pathways caused by increased levels of many adipokines and inflammatory chemokines. Impaired insulin-glucose metabolism occurs in tissues. The proteolytic enzyme matriptase is thought to be closely related to glucose metabolism due to its glycolization sites.
OBJECTIVE: Our study aimed to evaluate the correlation between matriptase, a proteolytic enzyme, and metabolic parameters in individuals recently diagnosed with T2DM. We also sought to investigate the potential involvement of matriptase in the development of diabetes.
METHODS: We measured all participants\' metabolic laboratory parameters, including basic biochemical tests, hemograms, high-sensitivity C-reactive protein (hsCRP), and matriptase levels.
RESULTS: Our results showed a significant increase in circulating matriptase levels in individuals with T2DM compared to the control group. Furthermore, individuals with metabolic syndrome had significantly higher matriptase levels than those without in the T2DM and control groups. We also observed that T2DM patients had elevated levels of Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), hsCRP, and matriptase, which displayed a positive correlation.
CONCLUSIONS: Our study is the first to report elevated levels of matriptase in individuals with newly diagnosed T2DM and/or metabolic syndrome. Additionally, we found a significant positive correlation between matriptase levels and metabolic and inflammatory parameters, indicating a potential role for matriptase in the pathogenesis of T2DM and glucose metabolism. Further research on matriptase could lead to its recognition as a novel target for investigation.

Formation and maintenance of skin barrier function require tightly controlled membrane-associated proteolysis, in which the integral membrane Kunitz-type serine protease inhibitor, HAI-1, functions as the primary inhibitor of the membrane-associated serine proteases, matriptase and prostasin. Previously, HAI-1 loss in HaCaT human keratinocytes resulted in an expected increase in prostasin proteolysis but a paradoxical decrease in matriptase proteolysis. The paradoxical decrease in shed active matriptase is further investigated in this study with an unexpected discovery of novel functions of fibroblast growth factor-binding protein 1 (FGFBP1), which acts as an extracellular ligand that can rapidly elicit F-actin rearrangement and subsequently affect the morphology of human keratinocytes. This novel growth factor-like function is in stark contrast to the canonical activity of this protein through interactions with FGFs for its pathophysiological functions. This discovery began with the observation that HAI-1 KO HaCaT cells lose the characteristic cobblestone morphology of the parental cells and exhibit aberrant F-actin formation along with altered subcellular targeting of matriptase and HAI-2. The alterations in cell morphology and F-actin status caused by targeted HAI-1 deletion can be restored by treatment with conditioned medium from parental HaCaT cells, in which FGFBP1 was identified by tandem mass spectrometry. Recombinant FGFBP1 down to 1 ng/ml was able to revert the changes caused by HAI-1 loss. Our study reveals a novel function of FGFBP1 in the maintenance of keratinocyte morphology, which depends on HAI-1.

Type II transmembrane serine proteases represent pharmacological targets for blocking entry and spread of influenza or coronaviruses. In this study, the depletion rates of the 3-amidinophenylalanine (3-APhA)-derived matriptase/TMPRSS2 inhibitors MI-463, MI-472, MI-485 or MI-1900 were determined by LC-MS/MS measurements over a period of 300 min using suspensions of rat, dog and cynomolgus monkey primary hepatocytes. From these in vitro pharmacokinetic (PK) experiments, intrinsic clearance values (Clint) were evaluated, and in vivo pharmacokinetic parameters (hepatic clearance, hepatic extraction ratio and bioavailability) were predicted. It was found that rat hepatocytes were the most active in the metabolism of 3-APhA derivatives (Clint 31.9-37.8 mL/min/kg), whereas dog and monkey cells displayed somewhat lower clearance of these compounds (Clint 6.6-26.7 mL/min/kg). These data support elucidation of important PK properties of anti-TMPRSS2/anti-matriptase 3-APhAs using mammalian hepatocyte models and thus contribute to the optimization of lead compounds.

Since the proposition of the pro-invasive activity of proteolytic enzymes over 70 years ago, several roles for proteases in cancer progression have been established. About half of the 473 active human proteases are expressed in the prostate and many of the most well-characterized members of this enzyme family are regulated by androgens, hormones essential for development of prostate cancer. Most notably, several kallikrein-related peptidases, including KLK3 (prostate-specific antigen, PSA), the most well-known prostate cancer marker, and type II transmembrane serine proteases, such as TMPRSS2 and matriptase, have been extensively studied and found to promote prostate cancer progression. Recent findings also suggest a critical role for proteases in the development of advanced and aggressive castration-resistant prostate cancer (CRPC). Perhaps the most intriguing evidence for this role comes from studies showing that the protease-activated transmembrane proteins, Notch and CDCP1, are associated with the development of CRPC. Here, we review the roles of proteases in prostate cancer, with a special focus on their regulation by androgens.