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The coronavirus disease-19 (COVID-19) outbreak that is caused by a highly contagious severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has become a zoonotic pandemic, with approximately 24.5 million positive cases and 8.3 lakhs deaths globally. The lack of effective drugs or vaccine provoked the research for drug candidates that can disrupt the spread and progression of the virus. The identification of drug molecules through experimental studies is time-consuming and expensive, so there is a need for developing alternative strategies like in silico approaches which can yield better outcomes in less time. Herein, we selected transmembrane protease serine 2 (TMPRSS2) enzyme, a potential pharmacological target against SARS-CoV-2, involved in the spread and pathogenesis of the virus. Since 3D structure is not available for this protein, the present study aims at homology modelling and validation of TMPRSS2 using Swiss-model server. Validation of the modelled TMPRSS2 using various online tools confirmed model accuracy, topology and stereochemical plausibility. The catalytic triad consisting of Serine-441, Histidine-296 and Aspartic acid-345 was identified as active binding site of TMPRSS2 using existing ligands. Molecular docking studies of various drugs and phytochemicals against the modelled TMPRSS2 were performed using camostat as a standard drug. The results revealed eight potential drug candidates, namely nafamostat, meloxicam, ganodermanontriol, columbin, myricetin, proanthocyanidin A2, jatrorrhizine and baicalein, which were further studied for ADME/T properties. In conclusion, the study unravelled eight high affinity binding compounds, which may serve as potent antagonists against TMPRSS2 to impact COVID-19 drug therapy., Highlights • 3D structure of TMPRSS2 was constructed using Homology modeling and was validated. • 50 K Ligand was utilized to reveal the active site of modelled TMPRSS2. • A total of twenty-five natural and marketed compounds were studied. • Nafamostatat, Meloxicam, Ganodermanontriol, Columbin, Myricetin, Proanthocyanidin A2, Jatrorrhizine and Baicalein shown good binding energies. • These potent TMPRSS2 inhibitors can prevent spread of SARS-CoV-2, aid in treatment of COVID-19.

Background: The pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). COVID-19 may take a severe course, resulting in sepsis, and antivirals are urgently needed to combat the disease. However, at present, only drugs developed against other diseases are available including the Malaria drug hydroxychloroquine but their therapeutic efficacy is unclear. Camostat mesylate, a clinically-proven protease inhibitor for treatment of pancreatitis, was shown to block SARS-CoV-2 entry into lung cells. Whether camostat mesylate dampens COVID-19 induced sepsis and the associated organ failure is unknown. Here we compare clinical courses of critically ill COVID-19 patients treated with either camostat mesylate or hydroxychloroquine. Methods: Eleven COVID-19 patients with organ failure were treated in intensive care unit (ICU) at our institution. Five patients (group 1) received hydroxychloroquine and six patients (group 2) camostat mesylate. Clinical disease status was assessed by Simplified Acute Physiology Score (SAPS) II and Sequential/ Sepsis-related Organ Failure Assessment (SOFA) score at day 1, 3 and 8. Viral load was determined from nasopharyngeal swabs. Findings: Patient age ranged from 45 to 76 years, all but one had histories of chronic disease and cardiovascular risk factors. Median symptomatic period was 2-10 days before being transferred to ICU. No differences in SAPS II scores or SOFA scores were observed upon ICU admission. In group 1, SOFA and SAPS II scores remained elevated (2 patients died) while in group 2 SOFA and SAPS II score decreased from 9 to 4 points (1 patient died). The decline in disease severity for group 2 was paralleled by a decline in inflammatory markers, which was not observed in group 1. The virus load in the nasopharynx was variable between patients, did not correlate with organ failure and no marked differences were observed between groups 1 and 2. Interpretation: The severity of COVID-19 sepsis markedly decreased upon camostat mesylate treatment within a period of 8 days and a similar effect was not observed upon hydroxychloroquine treatment. Camostat mesylate thus warrants further evaluation within randomized clinical studies. Funding Statement: Authors were employed at the University of Gottingen or at the Infection Biology Unit of the German Primate Center (Leibniz Institute for Primate Research). All expenses were covered by publicly funded institutions. Stefan Pohlmann received funding from the German Federal Ministry of Education and Research (BMBF), RAPID Fund (#01KI1723D). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Compassionate use was approved by our local ethical committee at the University of Gottingen.

Background: The trans-membrane protease serine 2 (TMPRSS2) is essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry and infection. Efficacy and safety of TMPRSS2 inhibitors in patients with coronavirus disease 2019 (Covid-19) have not been evaluated in randomized trials.Methods: We conducted an investigator-initiated, double-blind, randomized, placebo-controlled multicenter trial in patients hospitalized with confirmed SARS-CoV-2 infection from April 4, to December 31, 2020. Within 48 h of admission, participants were randomly assigned in a 2:1 ratio to receive the TMPRSS2 inhibitor camostat mesilate 200 mg three times daily for 5 days or placebo. The primary outcome was time to discharge or clinical improvement measured as ≥2 points improvement on a 7-point ordinal scale. Other outcomes included 30-day mortality, safety and change in oropharyngeal viral load.Findings: 137 patients were assigned to receive camostat mesilate and 68 to placebo. Median time to clinical improvement was 5 days (interquartile range [IQR], 3 to 7) in the camostat group and 5 days (IQR, 2 to 10) in the placebo group ( P = 0·31). The hazard ratio for 30-day mortality in the camostat compared with the placebo group was 0·82 (95% confidence interval [CI], 0·24 to 2·79; P = 0·75). The frequency of adverse events was similar in the two groups. Median change in viral load from baseline to day 5 in the camostat group was -0·22 log 10 copies/mL ( p Interpretation: Under this protocol, camostat mesilate treatment was not associated with increased adverse events during hospitalization for Covid-19 and did not affect time to clinical improvement, progression to ICU admission or mortality. ClinicalTrials.gov Identifier: NCT04321096. EudraCT Number: 2020-001200-42.

Graphical abstract, Coronavirus Disease 2019 (COVID-19) caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with a crude case fatality rate of about 0.5-10% depending on locality. A few clinically approved drugs, such as remdesivir, chloroquine, hydroxychloroquine, nafamostat, camostat, and ivermectin, exhibited anti-SARS-CoV-2 activity in vitro and/or in a small number of patients. However, their clinical use may be limited by anti-SARS-CoV-2 50% maximal effective concentrations (EC50) that exceeded their achievable peak serum concentrations (Cmax), side effects, and/or availability. To find more immediately available COVID-19 antivirals, we established a two-tier drug screening system that combines SARS-CoV-2 enzyme-linked immunosorbent assay and cell viability assay, and applied it to screen a library consisting 1528 FDA-approved drugs. Cetilistat (anti-pancreatic lipase), diiodohydroxyquinoline (anti-parasitic), abiraterone acetate (synthetic androstane steroid), and bexarotene (antineoplastic retinoid) exhibited potent in vitro anti-SARS-CoV-2 activity (EC50 1.13-2.01µM). Bexarotene demonstrated the highest Cmax:EC50 ratio (1.69) which was higher than those of chloroquine, hydroxychloroquine, and ivermectin. These results demonstrated the efficacy of the two-tier screening system and identified potential COVID-19 treatments which can achieve effective levels if given by inhalation or systemically depending on their pharmacokinetics.

We have shown that vagotomy (VGX) attenuates the reduction of meal size (MS) produced by cholecystokinin (CCK) -8 and -33 and that celiaco-mesenteric ganglionectomy (CMGX) attenuates the prolongation of the intermeal interval (IMI) produced by CCK-33. Here, we report the following novel data. First, by determining the distribution of CCK 1 receptor messenger RNA, which mediates reduction of MS and prolongation of IMI by CCK, in seven regions of the gastrointestinal tract in the adult rat we found that the duodenum contains the highest concentration of this receptor in the gut. Second, based on the previous finding we performed a unique surgical technique known as duodenal myotomy (MYO), which severs all the nerves of the gut wall in the duodenum including vagus, splanchnic and enteric nerves. Third, we determined MS and IMI in duodenal MYO rats in responses to endogenous CCK-58 released by the non-nutrient, trypsin inhibitor, camostat and CCK-8 to test the possibility that the duodenum is the site of action for reduction of MS and prolongation of IMI. We found that, similar to the previous work reported by using CCK-8 and MS, duodenal MYO also blocked reduction of MS by camostat. Forth, duodenal MYO blocked prolongation of IMI by camostat. As such, our current results suggest that the duodenum is the gut site that communicates both feeding signals of endogenous CCK, MS and IMI, with the brain through vagal and splanchnic afferents.

Camostat mesilate (or mesylate) releases endogenous cholecystokinin (CCK) or CCK-58, the only detectable endocrine form of CCK in the rat, and reduces cumulative food intake by activating CCK(1) receptor. However, the literature lacks meal pattern analysis and an appropriate dose-response curve for this peptide. Therefore, the current study determines meal size (MS), intermeal interval (IMI) and satiety ratio (SR) by orogastric gavage of camostat (0, 12.5, 25, 50, 100, 200, 300, 400, 800mg/kg) and compares them to those previously reported by a single dose of CCK-8 (1nmol/kg, i.p), the most utilized form of CCK. We found that camostat (200, 300, 400 and 800mg/kg) and CCK-8 reduced cumulative food intake and the size of the first meal, but only camostat prolonged IMI and increased SR. There was no change in the duration of the first two meals or in rated behaviors such as feeding, grooming, standing and resting in response to camostat and CCK-8, but there was more resting during the IMI in response to camostat. This study provides meal pattern analysis and an appropriate dose-response curve for camostat and CCK-8. Camostat reduces food intake by decreasing MS and prolonging IMI, whereas CCK-8 reduces food intake by reducing only meal size.

The pathogenesis of reflux esophagitis is not well understood and remains controversial. Distal gastrectomy serves as a model to assess the role of duodenal reflux with low gastric acidity in the development of reflux esophagitis. We investigated the clinical usefulness and antitrypsin activity after treatment with a trypsin inhibitor, camostat mesilate, against the reflux esophagitis after distal gastrectomy reconstructed with Billroth-I anastomosis.Twenty-eight patients with gastroesophageal reflux disease after distal gastrectomy were prescreened according to esophageal pH level and trypsin activity, and consequently 11 patients were enrolled in the present clinical study. Esophageal and duodenal washings were aspirated for the evaluation of the pretreatment trypsin activity. Then 100 mg of camostat mesilate was administered orally. At 30 and 120 minutes after the administration, duodenal washings were aspirated for the evaluation of posttreatment trypsin activity. Thereafter, 300 mg of camostat mesilate was administered orally 3 times daily for a 4-week period. On the 28th day of administration, the grade of reflux esophagitis (Los Angeles classification) was re-evaluated under endoscopy and the esophageal washings were aspirated for the evaluation of trypsin activity.The trypsin activities in the duodenum both at 30 and 120 minutes after oral ingestion of camostat mesilate were decreased significantly in comparison with those in the pretreatment period in each patient (P.001). In 6 of 7 patients with detectable trypsin activity in the esophagus, the activities after the 28th day of treatment were lower than those in the pretreatment period, and the symptoms were milder than those before treatment (P.05). Furthermore, endoscopic evaluation showed that 40% of patients were grade B, C, and D after treatment (28th day), whereas 70% of patients were grade B, C, and D before the treatment.Oral administration of trypsin inhibitor can improve reflux esophagitis after distal gastrectomy concomitant with decreased trypsin activity.

Camostat mesilate (CM), an oral protease inhibitor, has been used clinically for the treatment of chronic pancreatitis in Japan. However, the mechanism by which it operates has not been fully understood. Our aim was to evaluate the therapeutic efficacy of CM in the experimental pancreatic fibrosis model induced by dibutyltin dichloride (DBTC), and we also determined the effect of CM on isolated monocytes and panceatic stellate cells (PSCs). In vivo, chronic pancreatitis was induced in male Lewis rats by single administration of 7 mg/kg DBTC and a special diet containing 1 mg/g CM was fed to the DBTC+CM-treated group from day 7, while the DBTC-treated group rats were fed a standard diet. At days 0, 7, 14 and 28, the severity of pancreatitis and fibrosis was examined histologically and enzymologically in both groups. In vitro, monocytes were isolated from the spleen of a Lewis rat, and activated with lipopolysaccharide stimulation. Thereafter, the effect of CM on monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) production from monocytes was examined. Subsequently, cultured rat PSCs were exposed to CM and tested to see whether their proliferation, MCP-1 production and procollagen alpha1 messenger RNA expression was influenced by CM. In vivo, the oral administration of CM inhibited inflammation, cytokines expression and fibrosis in the pancreas. The in vitro study revealed that CM inhibited both MCP-1 and TNF-alpha production from monocytes, and proliferation and MCP-1 production from PSCs. However, procollagen alpha1 expression in PSCs was not influenced by CM. These results suggest that CM attenuated DBTC-induced rat pancreatic fibrosis via inhibition of monocytes and PSCs activity.

Cholecystokinin (CCK) is an important physiologic mediator that regulates satiety and gastric emptying. We demonstrated previously that soybean peptone acts directly on rat small intestinal mucosal cells to stimulate CCK release. In the present study, we examined the effects of beta-conglycinin, a major component of soy protein, and its peptone on food intake and gastric emptying after an intraduodenal infusion of beta-conglycinin peptone in relation to CCK release and interaction with the mucosal cell membrane. Intraduodenal infusion of beta-conglycinin peptone inhibited food intake in a dose-dependent manner, but that of whole soy peptone or camostat did not. The suppression of food intake by beta-conglycinin peptone was abolished by an intravenous injection of devazepide, a selective peripheral CCK receptor antagonist. The beta-conglycinin peptone infusion strongly suppressed gastric emptying with marked increases in portal CCK levels. We also observed that the beta-conglycinin peptone dose dependently and more potently stimulated CCK release from isolated dispersed mucosal cells of the rat jejunum than did beta-conglycinin itself. This stimulation corresponded to the binding activity of the peptide or protein to solubilized components of the rat jejunum membrane as evaluated by surface plasmon biosensor. These results indicate that beta-conglycinin peptone suppresses food intake, and this effect may be due to beta-conglycinin peptone in the lumen stimulating endogenous CCK release with direct acceptance to the intestinal cells.

The synthetic trypsin inhibitor camostat has been used for the treatment of acute and chronic pancreatitis in Japan based on the evidences obtained from a rat experimental model. However, rats differ from other rodents and from humans in terms of lacking a gallbladder and no response of pancreatic bicarbonate secretion to cholecystokinin (CCK). In the present study, we determined whether oral administration of camostat showed a trophic effect in mice as observed in rats and whether the trophic effect, if substantial, was mediated via the CCK-A receptor, using CCK-A receptor gene targeting mice. The chow containing 0.1% camostat was fed to 8-month-old mice. Three- and seven-day treatments with camostat did not affect pancreatic wet weight in CCK-A receptor (+/-) mice. After 14-day treatment, the ratio of pancreatic wet weight/body weight was significantly lower in CCK-A receptor (-/-) than (+/+) mice. The protein and chymotrypsin contents were lower and amylase content was higher in CCK-A receptor (-/-) mice, compared to (+/+) mice. No pathological findings were observed by histological examination. Camostat has a trophic effect on the pancreas in mice and this effect is mediated via the CCK-A receptor, but is less potent than in rats.

Feedback regulation of pancreatic enzyme secretion is well established in animals, and their pancreases are able to adapt to intraduodenal inhibition of pancreatic enzymes by proteinase inhibitors such as Camostate (FOY-305; Schwarz GmbH, Monheim, Germany). In this study, we addressed whether similar adaptive changes occur in the human pancreas after 4 weeks of 2 g/day Camostate application.Before, at the end of, and 2 weeks after 4-week Camostate treatment (four times 500 mg daily), pancreatic changes were analyzed with the use of a secretin-cerulein test, a test-meal stimulation, cholecystokinin plasma measurement, and standardized ultrasonographic investigations of the pancreas.Duodenal trypsin output after secretion stimulation was significantly increased (+44%; P0.01) and duodenal bicarbonate output decreased 22% (P0.05) after 4 weeks of Camostate application. The size of the pancreatic head (vertical) increased 8% (P0.05) at week 4 and decreased to pretreatment values 2 weeks after treatment (week 6). The other three diameters measured (head oblique, body, and tail) showed a similar pattern. Stimulated cholecystokinin plasma levels 15 minutes after application of a standard test meal increased 62% (P0.05).The human pancreas adapts to oral application of the proteinase inhibitor Camostate. These findings support the theory that feedback control of the exocrine pancreas operates in humans.

Leukocytes are thought to have a role in tissue injury induced by stress. Synthetic protease inhibitors can suppress the leukocyte-dependent tissue injury; however, the effect of camostat mesilate on microcirculatory disturbance has not been reported. Male Wistar rats were anesthetized, and a 30% full skin-thickness dorsal scald burn was inflicted. Rolling or sticking of leukocytes and deposits of Monastral blue B (MBB) in gastric venules was observed by using intravital microscopy. Oral administration of camostat mesilate 100 mg/kg was performed twice: 40 minutes before the thermal injury and 2.5 hours after injury. Microscopy was performed 5 hours after the thermal injury, and showed that camostat mesilate suppressed the percentage of MBB deposit areas, and the rolling or sticking of leukocytes in the gastric venules. Camostat mesilate appears to protect against gastric microcirculatory disturbance induced by thermal injury in rats.

The fourth component of complement (C4) in urine was measured in 19 patients with systemic lupus erythematosus (SLE). Urinary C4 was detectable in all SLE patients using an enzyme linked immunosorbent assay. In 11 of 13 patients whose urinary C4 was serially measured, a decrease of urinary C4 was found in parallel with a decrease of disease activity of SLE. In the remaining 2 patients, the amount of C4 increased preceding a flare-up of the disease. The measurement of urinary C4 may be useful in evaluating the disease activity of SLE in individual patients and sometimes in predicting the flare-up of the disease. The specific hemolytic activity of excreted C4 and Western blotting analysis showed that urinary C4 consisted mainly of degraded fragments of C4. In two cases, camostat, a serine protease inhibitor, rapidly decreased the urinary C4 excretion, suggesting that the complement activation occurred in the glomerulus in lupus nephritis.

1. 1. The role of endogenous CCK in the development of digestive enyme activities in small intestine and pancreas was investigated in suckling rats. Synthetic protease inhibitor (camostat 100 μg/g bwt) was orally administered twice daily for 5 days from 11 days of age. 2. 2. Pancreatic hypertrophy and hyperplasia, and alteration of pancreatic enzyme composition, especially decreases in amylase activity and increases in trypsin and chymotrypsin activities were produced by camostat treatment. These changes were completely suppressed by simultaneous administration of the potent CCK receptor antagonist L-364,718 (1 μg/g bwt). 3. 3. With camostat treatment, intestinal lactase activity decreased to 41%, while maltase and sucrase activities increased 3 and 2.5 times respectively. These changes in enzyme activities were not affected by the application of L-364,718. 4. 4. The mucosal disaccharidase and pancreatic enzyme activities could not be modified by chronic subcutaneous injection of camostat. The precocious induction of maltase and sucrase activities by camostat treatment was also observed in the adrenalectomized pups. 5. 5. These results indicate that pancreatic growth accompanied by alteration of digestive enzyme composition in the suckling rats is regulated by endogenous CCK, but the precocious induction of disaccharidase activities is not mediated by endogenous CCK released by camostat treatment.

To determine the effects of luminal protease inhibition on duodenal delivery and the intraluminal fate of pancreatic enzymes, six healthy subjects were intubated with an oro-ileal multilumen tube assembly. By using nonabsorbable markers, cumulative trypsin, chymotrypsin, lipase, and amylase activities were measured as delivered to duodenum, midjejunum, and distal ileum, with or without simultaneous duodenal perfusion of the protease inhibitor camostat at graded doses. Compared with saline, camostat (a) inhibited trypsin activity in the entire small intestinal lumen by up to 99%, and significantly reduced chymotrypsin activity by up to 89%; (b) significantly increased duodenal deliveries of lipase activity, amylase activity and volume; (c) did not influence plasma cholecystokinin concentrations; and (d) significantly increased jejunal and ileal deliveries of lipase but not amylase activity. Small intestinal transit and motility were not affected by camostat. In additional in vitro studies, camostat significantly reduced the spontaneous decline in lipase activity in fresh human duodenal juice incubated at 37°C. These findings demonstrate that duodenal deliveries of lipase and amylase activities increase when intraluminal protease activity is decreased; they suggest that this increase is not caused by slower proteolytic destruction of enzyme protein but by stimulation of pancreatic secretion. Thus, luminal protease-mediated feedback regulation of pancreatic secretion may be operative in humans. Because plasma cholecystokinin concentrations were not affected, these effects may in part be independent of cholecystokinin. The data further suggest that proteolytic digestion plays a major role in the rapid loss of luminal lipase activity on small intestinal transit.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disorder which remains refractory to therapies. Recently, type II transmembrane serine proteases have emerged as key actors in pathophysiology, by proteolysis of cell surface receptors and/or cell environment, thereby orchestrating cell crosstalks. Among them, deregulation of the matriptase contributes to various proliferative disorders. Hence, we sought to investigate the role of matriptase in pulmlonary fibrosis. Matriptase expression and activity were analyzed in human lung material from controls and IPF patients. Human fibroblasts were stimulated with recombinant matriptase and the activation of key signaling pathways involved in IPF was analyzed. Using the murine model of bleomycin-induced pulmonary fibrosis, mice were administrated for 14 days with 0 or 0.5mg of camostat mesylate, a matriptase inhibitor, and the markers of tissue fibrosis in lung homogenate, and inflammatory cell influx in the bronchoalveolar lavage fluid (BALF) of the animals were assessed. We show that matriptase is upregulated in the lung and BALF of IPF patients compared to controls. In fibroblasts, matriptase induced the activation of p42/44, Akt, and Smad2/3 pathways. Finally, camostat administration in bleomycin-treated mice led to a significant decrease in mortality (39.3% versus 60.4% in bleomycin-treated group). Fibronectin and collagen expression, and inflammatory cell influx, were not significantly different from saline-treated animals in the camostat group.Taken together, matriptase seems instrumental in pulmonary fibrosis, and camostat mesylat could be a promising target for thetreatment of IPF.

p-Carbethoxyphenyl episol-guanidinocaproate and p-(p'-guanidinobenzoyloxy)-phenyl derivatives were prepared, and their inhibitory effects on trypsin, plasmin, plasma kallikrein, thrombin, C1r- and C1 esterase were examined. Among the various inhibitors tested, p-nitrophenyl p'-guanidinobenzoate, N,N-dimethylamino p-(p'-guanidinobenzoyloxy)-benzoyl glycolate and N,N-dimethylamino p-(p'-guanidinobenzoyloxy)-benzilcarbonyloxy glycolate were the most effective inhibitors of trypsin, plasmin, plasma kallikrien and thrombin, and they strongly inhibited the esterolytic activities of C1r- and C1 esterase.

The effects of chronic oral treatment of rats with 400 mg/kg body weight camostat, a synthetic protease inhibitor, on weight gain and both pancreatic exocrine and endocrine secretion were studied and compared with pair-fed controls. Administration of camostat was found to result in a lower weight gain than in untreated rats fed ad libitum because of reduced food intake. The pancreas of treated rats showed hypertrophy and hyperplasia with significantly higher total contents of amylase, lipase, trypsinogen and chymotrypsinogen than that of pair-fed controls. The specific activity of lipase, trypsinogen and chymotrypsinogen remained unchanged but the specific activity of amylase was significantly lower than in pair-fed controls. Stimulation of pancreatic enzymes with CCK 8 in treated rats resulted in a greater output of proteases, no difference in secretion of lipase and a lower secretion of amylase than in pair-fed rats. Glucose-dependent insulin secretion was also significantly lower in camostat-treated rats than in controls. This effect could be reversed by gastric inhibitory polypeptide. After termination of treatment with camostat all enzymes were normalized within 14 d. Our results on the hypertrophied pancreas of rats suggest preferential synthesis and secretion of protease at the expense of amylase and diminished sensitivity of insulin to stimulation by glucose. All effects of camostat on the pancreas were reversible.