leupeptins and nafamostat

The unprecedented coronavirus SARS-CoV-2 outbreak at Wuhan, China, caused acute respiratory infection to humans. There is no precise vaccine/therapeutic agents available to combat the COVID-19 disease. Some repurposed drugs are saving the life of diseased, but the complete cure is relatively less. Several drug targets have been reported to inhibit the SARS-CoV-2 virus infection, in that TMPRSS2 (transmembrane protease serine 2) is one of the potential targets; inhibiting this protease stops the virus entry into the host human cell. Camostat mesylate, nafamostat, and leupeptin are the drugs, in which the first two drugs are being used for COVID-19 and leupeptin also tested. To consider these drugs as the repurposed drug for COVID-19, it is essential to understand their binding affinity and stability with TMPRSS2. In the present study, we performed the molecular docking and molecular dynamics (MD) simulation of these molecules with the TMPRSS2. The docking study reveals that leupeptin molecule strongly binds with TMPRSS2 protein than the other two drug molecules. The RMSD and RMSF values of MD simulation confirm that leupeptin and the amino acids of TMPRSS2 are very stable than the other two molecules. Furthermore, leupeptin forms interactions with the key amino acids of TMPRSS2 and the same have been maintained during the MD simulations. This structural and dynamical information is useful to evaluate these drugs to be used as repurposed drugs, however, the strong binding profile of leupeptin with TMPRSS2, suggests, it may be considered as a repurposed drug for COVID-19 disease after clinical trial.

Topics: Antiviral Agents; Benzamidines; COVID-19; COVID-19 Drug Treatment; Drug Repositioning; Esters; Guanidines; Humans; Leupeptins; Molecular Docking Simulation; Molecular Dynamics Simulation; Protein Binding; SARS-CoV-2; Serine Endopeptidases

Programmed cell death (PCD) is an important process in development and disease, as it allows the body to rid itself of unwanted or damaged cells. However, PCD pathways can also be activated in otherwise healthy cells. One such case occurs in sensory hair cells of the inner ear following exposure to ototoxic drugs, resulting in hearing loss and/or balance disorders. The intracellular pathways that determine if hair cells die or survive following this or other ototoxic challenges are incompletely understood. We use the larval zebrafish lateral line, an external hair cell-bearing sensory system, as a platform for profiling cell death pathways activated in response to ototoxic stimuli. In this report the importance of each pathway was assessed by screening a custom cell death inhibitor library for instances when pathway inhibition protected hair cells from the aminoglycosides neomycin or gentamicin, or the chemotherapy agent cisplatin. This screen revealed that each ototoxin likely activated a distinct subset of possible cell death pathways. For example, the proteasome inhibitor Z-LLF-CHO protected hair cells from either aminoglycoside or from cisplatin, while D-methionine, an antioxidant, protected hair cells from gentamicin or cisplatin but not from neomycin toxicity. The calpain inhibitor leupeptin primarily protected hair cells from neomycin, as did a Bax channel blocker. Neither caspase inhibition nor protein synthesis inhibition altered the progression of hair cell death. Taken together, these results suggest that ototoxin-treated hair cells die via multiple processes that form an interactive network of cell death signaling cascades.

Topics: Animals; Antioxidants; Benzamidines; Calpain; Caspase Inhibitors; Cell Death; Cells, Cultured; Cisplatin; Cross-Linking Reagents; Gentamicins; Guanidines; Hair Cells, Auditory, Inner; Lateral Line System; Leupeptins; Methionine; Neomycin; Oligopeptides; Protease Inhibitors; Protein Synthesis Inhibitors; Reactive Oxygen Species; Zebrafish

Transforming growth factor beta (TGFbeta) is a key remodelling factor in asthma. It is produced as a latent complex and the main limiting step in TGFbeta bioavailability is its activation. Mast cell tryptase has been shown to stimulate the release of functionally active TGFbeta from human airway smooth muscle (ASM) cells [P. Berger, P.O. Girodet, H. Begueret, O. Ousova, D.W. Perng, R. Marthan, A.F. Walls, J.M. Tunon de Lara, Tryptase-stimulated human airway smooth muscle cells induce cytokine synthesis and mast cell chemotaxis, FASEB J. 17 (2003) 2139-2141]. The aim of this study was to determine if tryptase could cause TGFbeta activation as well as expression in ASM cells via its receptor, proteinase-activated receptor 2 (PAR2). Tryptase caused TGFbeta activation without affecting levels of total TGFbeta. This effect was inhibited by the selective tryptase inhibitor FUT175 and leupeptin but not mimicked by the PAR2 activating peptide SLIGKV-NH(2). Furthermore, the ASM cells used in the study did not express PAR2. The results indicate that tryptase activates TGFbeta via a PAR2-independent proteolytic mechanism in human ASM cells and may help understanding the role of tryptase in asthma.

Topics: Benzamidines; Bronchi; Guanidines; Humans; Leupeptins; Myocytes, Smooth Muscle; Oligopeptides; Receptor, PAR-2; Transforming Growth Factor beta; Trypsin Inhibitors; Tryptases

The pruritogenic potency of tryptase and its involvement in anti-pruritic effect of intravenous nafamostat mesilate (NFM) were studied in mice. An intradermal injection of tryptase (0.05-1 ng/site) elicited scratching in ICR mice, while chymase was without effects at doses of 0.05-50 ng/site. The dose-response curve of tryptase action was bell-shaped and the effect peaked at 0.1 ng/site (approximately 0.7 fmol/site). NFM (10 mg/kg) inhibited scratching induced by tryptase but not by histamine and serotonin. NFM (1-10 mg/kg) produced the dose-dependent inhibition of scratching induced by intradermal compound 48/80 (10 microg/site). The inhibition by NFM (10 mg/kg) was abolished in mast cell-deficient (WBB6F1 W/W(V)) mice, but not in wild-type (WBB6F1 +/+) mice. NFM (10 mg/kg) suppressed tryptase activity in the mouse skin. Proteinase-activated receptor-2 (PAR-2) neutralizing antibody (0.1 and 1 microg/site) and the PAR-2 antagonist FSLLRY (10 and 100 microg/site) inhibited scratching induced by tryptase (0.1 ng/site) and compound 48/80 (10 microg/site). These results suggest that mast cell tryptase elicits itch through PAR-2 receptor and that NFM inhibits itch-associated responses mainly through the inhibition of mast cell tryptase.

Topics: Animals; Antibodies; Antipruritics; Benzamidines; Dose-Response Relationship, Drug; Extravasation of Diagnostic and Therapeutic Materials; Guanidines; Histamine; Histamine H1 Antagonists; Injections, Intradermal; Leupeptins; Male; Mast Cells; Mice; Mice, Inbred ICR; Mice, Mutant Strains; Oligopeptides; p-Methoxy-N-methylphenethylamine; Pruritus; Receptor, PAR-2; Serine Endopeptidases; Serotonin; Skin; Terfenadine; Time Factors; Tryptases

Inhibitory effects of nafamostat mesilate (nafamostat) on various enzymes were investigated, and they were compared with those of gabexate mesilate (gabexate), leupeptin, aprotinin and urinastatin in vitro. Nafamostat inhibited trypsin, plasmin, thrombin, pancreatic kallikrein, Clr and Cls more potently than gabexate and leupeptin. Gabexate and leupeptin did not inhibit pancreatic kallikrein and thrombin, respectively. Aprotinin inhibited trypsin, plasmin, pancreatic kallikrein and chymotrypsin. Urinastatin inhibited trypsin and chymotrypsin. Nafamostat inhibited the complement-mediated hemolysis in diluted serum more potently than gabexate and leupeptin, but aprotinin and urinastatin did not. Nafamostat, furthermore, inhibited the complement-mediated hemolysis in undiluted serum, but gabexate did not. Unlike aprotinin and urinastatin, nafamostat and gabexate inhibited alpha 2-macroglobulin bound trypsin as well as free trypsin to the same extent. The inhibitory effect of gabexate toward trypsin was reduced more markedly than that of nafamostat after incubation with plasma at 37 degrees C. These results show that nafamostat is more useful than other inhibitors such as gabexate, leupeptin, aprotinin and urinastatin.

Topics: alpha-Macroglobulins; Aprotinin; Benzamidines; Complement Inactivator Proteins; Gabexate; Glycoproteins; Guanidines; Hemolysis; In Vitro Techniques; Leupeptins; Protease Inhibitors; Protein Binding