TMPRSS13 promotes the cell entry of swine acute diarrhea syndrome coronavirus.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) has caused severe intestinal diseases in pigs. It originates from bat coronaviruses HKU2 and has a potential risk of cross-species transmission, raising concerns about its zoonotic potential. Viral entry-related host factors are critical determinants of susceptibility to cells, tissues, or species, and remain to be elucidated for SADS-CoV. Type II transmembrane serine proteases (TTSPs) family is involved in many coronavirus infections and has trypsin-like catalytic activity. Here we examine all 18 members of the TTSPs family through CRISPR-based activation of endogenous protein expression in cells, and find that, in addition to TMPRSS2 and TMPRSS4, TMPRSS13 significantly facilitates SADS-CoV infection. This is confirmed by ectopic expression of TMPRSS13, and specific to trypsin-dependent SADS-CoV. Infection with pseudovirus bearing SADS-CoV spike protein indicates that TMPRSS13 acts at the entry step and is sensitive to serine protease inhibitor Camostat. Moreover, both human and pig TMPRSS13 are able to enhance the cell-cell membrane fusion and cleavage of spike protein. Overall, we demonstrate that TMPRSS13 is another host serine protease promoting the membrane-fusion entry of SADS-CoV, which may expand its host tropism by using diverse TTSPs.

Enteral administration of the protease inhibitor gabexate mesilate preserves vascular function in experimental trauma/hemorrhagic shock.

Preserving vascular function is crucial for preventing multiorgan failure and death in ischemic and low-pressure states such as trauma/hemorrhagic shock (T/HS). It has recently been reported that inhibiting circulating proteases released from the bowel to the circulation during T/HS may preserve vascular function and improve outcomes following T/HS. This study aimed to evaluate the role of the serine protease inhibitor gabexate mesilate (GM) in preserving vascular function during T/HS when given enterally. We studied the vascular reactivity of mesenteric arteries from male Wistar rats treated with enteral GM (10 mg/kg) (GM-treated, n = 6) or control (Shock-control, n = 6) following (T/HS) using pressure myography. Concentration-response curves of endothelial-dependent and endothelial-independent agonists (e.g., acetylcholine, sodium nitroprusside) ranging from 10-10 to 10-5 M were performed. In a second set of experiments, ex-vivo arteries from healthy rats were perfused with plasma from shocked animals from both groups and vascular performance was similarly measured. Arteries from the GM-treated group demonstrated a preserved concentration-response curve to the α1 adrenergic agonist phenylephrine compared to arteries from Shock-control animals (- logEC50: - 5.73 ± 0.25 vs. - 6.48 ± 0.2, Shock-control vs. GM-treated, p = 0.04). When perfused with plasma from GM-treated rats, healthy arteries exhibited an even greater constriction and sensitivity to phenylephrine (- logEC50: - 6.62 ± 0.21 vs. - 7.13 ± 0.21, Shock-control vs. GM-treated, p = 0.02). Enteral GM also preserved the endothelium-dependent vascular response to agonists following T/HS and limited syndecan-1 shedding as a marker of glycocalyx compromise (41.84 ± 9 vs. 17.63 ± 3.97 ng/mL, Shock-control vs. GM-treated, p = 0.02). Syndecan-1 cleavage was correlated with plasma trypsin-like activity (r2 = 0.9611). Enteral gabexate mesilate was able to maintain vascular function in experimental T/HS, which was reflected by improved hemodynamics (mean arterial pressure 50.39 ± 7.91 vs. 64.95 ± 3.43 mmHg, Shock-control vs. GM treated, p = 0.0001). Enteral serine protease inhibition may be a potential therapeutic intervention in the treatment of T/HS.

Inhibition of Listeria Monocytogenes HtrA Protease with Camostat, Gabexate and Nafamostat Mesylates and the Binding Mode of the Inhibitors.

In many bacteria, the High Temperature requirement A (HtrA) protein functions as a chaperone and protease. HtrA is an important factor in stress tolerance and plays a significant role in the virulence of several pathogenic bacteria. Camostat, gabexate and nafamostat mesylates are serine protease inhibitors and have recently shown a great impact in the inhibition studies of SARS-CoV2. In this study, the inhibition of Listeria monocytogenes HtrA (LmHtrA) protease activity was analysed using these three inhibitors. The cleavage assay, using human fibrinogen and casein as substrates, revealed that the three inhibitors effectively inhibit the protease activity of LmHtrA. The agar plate assay and spectrophotometric analysis concluded that the inhibition of nafamostat (IC50 value of 6.6 ± 0.4 µM) is more effective compared to the other two inhibitors. Previous studies revealed that at the active site of the protease, these inhibitors are hydrolysed and one of the hydrolysates is covalently bound to the active site serine. To understand the mode of binding of these inhibitors at the active site of LmHtrA, docking of the inhibitors followed by molecular dynamics simulations were carried out. Analysis of the LmHtrA-inhibitor complex structures revealed that the covalently bound inhibitor is unable to occupy the S1 pocket of the LmHtrA which is in contrast to the previously determined camostat and nafamostat complex structures. This observation provides the first glimpse of the substrate specificity of LmHtrA which is not known. The obtained results also suggest that the development of novel inhibitors of LmHtrA and its homologs with active site architecture similar to LmHtrA can be pursued with suitable modification of these inhibitors. To date, only a very few studies have been carried out on identifying the inhibitors of HtrA proteolytic activity.

Mechanisms of NMDA receptor inhibition by nafamostat, gabexate and furamidine.

N-methyl-D-aspartate (NMDA) receptors are affected by many pharmaceuticals. In this work, we studied the action of the serine protease inhibitors nafamostat, gabexate and camostat, and an antiprotozoal compound, furamidine, on native NMDA receptors in rat hippocampal pyramidal neurons. Nafamostat, furamidine and gabexate inhibited these receptors with IC50 values of 0.20 ± 0.04, 0.64 ± 0.13 and 16 ± 3 µM, respectively, whereas camostat was ineffective. Nafamostat and furamidine showed voltage-dependent inhibition, while gabexate showed practically voltage-independent inhibition. Nafamostat and furamidine demonstrated tail currents, implying a 'foot-in-the-door' mechanism of action; gabexate did not demonstrate any signs of 'foot-in-the-door' or trapping channel block. Gabexate action was also not competitive, suggesting allosteric inhibition of NMDA receptors. Furamidine and nafamostat are structurally similar to the previously studied diminazene and all three demonstrated a 'foot-in-the-door' mechanism. They have a rather rigid, elongated structures and cannot fold into more compact forms. By contrast, the gabexate molecule can fold, but its folded structure differs drastically from that of typical NMDA receptor blockers, in agreement with its voltage-independent inhibition. These findings provide a better understanding of the structural determinants of NMDA receptor antagonism, while also supporting the potential clinical repurposing of these drugs as neuroprotectors for glaucoma and other neurodegenerative diseases.

Computational drug re-purposing targeting the spike glycoprotein of SARS-CoV-2 as an effective strategy to neutralize COVID-19.

COVID-19 has intensified into a global pandemic with over a million deaths worldwide. Experimental research analyses have been implemented and executed with the sole rationale to counteract SARS-CoV-2, which has initiated potent therapeutic strategy development in coherence with computational biology validation focusing on the characterized viral drug targets signified by proteomic and genomic data. Spike glycoprotein is one of such potential drug target that promotes viral attachment to the host cellular membrane by binding to its receptor ACE-2 via its Receptor-Binding Domain (RBD). Multiple Sequence alignment and relative phylogenetic analysis revealed significant sequential disparities of SARS-CoV-2 as compared to previously encountered SARS-CoV and MERS-CoV strains. We implemented a drug re-purposing approach wherein the inhibitory efficacy of a cluster of thirty known drug candidates comprising of antivirals, antibiotics and phytochemicals (selection contingent on their present developmental status in underway clinical trials) was elucidated by subjecting them to molecular docking analyses against the spike protein RBD model (developed using homology modelling and validated using SAVES server 5.0) and the composite trimeric structures of spike glycoprotein of SARS-CoV-2. Our results indicated that Camostat, Favipiravir, Tenofovir, Raltegravir and Stavudine showed significant interactions with spike RBD of SARS-CoV-2. Proficient bioavailability coupled with no predicted in silico toxicity rendered them as prospective alternatives for designing and development of novel combinatorial therapy formulations for improving existing treatment regimes to combat COVID-19.

Molecular Insights into Human Transmembrane Protease Serine-2 (TMPS2) Inhibitors against SARS-CoV2: Homology Modelling, Molecular Dynamics, and Docking Studies.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), which caused novel corona virus disease-2019 (COVID-19) pandemic, necessitated a global demand for studies related to genes and enzymes of SARS-CoV2. SARS-CoV2 infection depends on the host cell Angiotensin-Converting Enzyme-2 (ACE2) and Transmembrane Serine Protease-2 (TMPRSS2), where the virus uses ACE2 for entry and TMPRSS2 for S protein priming. The TMPRSS2 gene encodes a Transmembrane Protease Serine-2 protein (TMPS2) that belongs to the serine protease family. There is no crystal structure available for TMPS2, therefore, a homology model was required to establish a putative 3D structure for the enzyme. A homology model was constructed using SWISS-MODEL and evaluations were performed through Ramachandran plots, Verify 3D and Protein Statistical Analysis (ProSA). Molecular dynamics simulations were employed to investigate the stability of the constructed model. Docking of TMPS2 inhibitors, camostat, nafamostat, gabexate, and sivelestat, using Molecular Operating Environment (MOE) software, into the constructed model was performed and the protein-ligand complexes were subjected to MD simulations and computational binding affinity calculations. These in silico studies determined the tertiary structure of TMPS2 amino acid sequence and predicted how ligands bind to the model, which is important for drug development for the prevention and treatment of COVID-19.

The Anticoagulant Nafamostat Potently Inhibits SARS-CoV-2 S Protein-Mediated Fusion in a Cell Fusion Assay System and Viral Infection In Vitro in a Cell-Type-Dependent Manner.

Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 mM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat's safety, make it a likely candidate drug to treat COVID-19.

Comprehensive analysis of drugs to treat SARS­CoV­2 infection: Mechanistic insights into current COVID­19 therapies (Review).

The major impact produced by the severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) focused many researchers attention to find treatments that can suppress transmission or ameliorate the disease. Despite the very fast and large flow of scientific data on possible treatment solutions, none have yet demonstrated unequivocal clinical utility against coronavirus disease 2019 (COVID­19). This work represents an exhaustive and critical review of all available data on potential treatments for COVID­19, highlighting their mechanistic characteristics and the strategy development rationale. Drug repurposing, also known as drug repositioning, and target based methods are the most used strategies to advance therapeutic solutions into clinical practice. Current in silico, in vitro and in vivo evidence regarding proposed treatments are summarized providing strong support for future research efforts.

Virtual Screening of Natural Products against Type II Transmembrane Serine Protease (TMPRSS2), the Priming Agent of Coronavirus 2 (SARS-CoV-2).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused about 2 million infections and is responsible for more than 100,000 deaths worldwide. To date, there is no specific drug registered to combat the disease it causes, named coronavirus disease 2019 (COVID-19). In the current study, we used an in silico approach to screen natural compounds to find potent inhibitors of the host enzyme transmembrane protease serine 2 (TMPRSS2). This enzyme facilitates viral particle entry into host cells, and its inhibition blocks virus fusion with angiotensin-converting enzyme 2 (ACE2). This, in turn, restricts SARS-CoV-2 pathogenesis. A three-dimensional structure of TMPRSS2 was built using SWISS-MODEL and validated by RAMPAGE. The natural compounds library Natural Product Activity and Species Source (NPASS), containing 30,927 compounds, was screened against the target protein. Two techniques were used in the Molecular Operating Environment (MOE) for this purpose, i.e., a ligand-based pharmacophore approach and a molecular docking-based screening. In total, 2140 compounds with pharmacophoric features were retained using the first approach. Using the second approach, 85 compounds with molecular docking comparable to or greater than that of the standard inhibitor (camostat mesylate) were identified. The top 12 compounds with the most favorable structural features were studied for physicochemical and ADMET (absorption, distribution, metabolism, excretion, toxicity) properties. The low-molecular-weight compound NPC306344 showed significant interaction with the active site residues of TMPRSS2, with a binding energy score of -14.69. Further in vitro and in vivo validation is needed to study and develop an anti-COVID-19 drug based on the structures of the most promising compounds identified in this study.

Protease Inhibitors: Candidate Drugs to Inhibit Severe Acute Respiratory Syndrome Coronavirus 2 Replication.

The number of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly increased, although the WHO declared a pandemic. However, drugs that function against SARS-CoV-2 have not been established. SARS-CoV-2 has been suggested to bind angiotensin-converting enzyme 2, the receptor of the SARS coronavirus. SARS coronavirus and coronavirus 229E, the cause of the common cold, replicate through cell-surface and endosomal pathways using a protease, the type II transmembrane protease. To examine the effects of protease inhibitors on the replication of coronavirus 229E, we pretreated primary cultures of human nasal epithelial (HNE) cells with camostat or nafamostat, each of which has been used for the treatment of pancreatitis and/or disseminated intravascular coagulation. HNE cells were then infected with coronavirus 229E, and viral titers in the airway surface liquid of the cells were examined. Pretreatment with camostat (0.1-10 µg/mL) or nafamostat (0.01-1 µg/mL) reduced the titers of coronavirus 229E. Furthermore, a significant amount of type II transmembrane protease protein was detected in the airway surface liquid of HNE cells. Additionally, interferons have been reported to have antiviral effects against SARS coronavirus. The additive effects of interferons on the inhibitory effects of other candidate drugs to treat SARS-CoV-2 infection, such as lopinavir, ritonavir and favipiravir, have also been studied. These findings suggest that protease inhibitors of this type may inhibit coronavirus 229E replication in human airway epithelial cells at clinical concentrations. Protease inhibitors, interferons or the combination of these drugs may become candidate drugs to inhibit the replication of SARS-CoV-2.

TMPRSS2 and COVID-19: Serendipity or Opportunity for Intervention?

TMPRSS2 is both the most frequently altered gene in primary prostate cancer and a critical factor enabling cellular infection by coronaviruses, including SARS-CoV-2. The modulation of its expression by sex steroids could contribute to the male predominance of severe infections, and given that TMPRSS2 has no known indispensable functions, and inhibitors are available, it is an appealing target for prevention or treatment of respiratory viral infections.

Learning from our immunological history: What can SARS-CoV teach us about SARS-CoV-2?

Many strategies are being deployed to rapidly uncover targetable mechanisms of infection for SARS-CoV-2, and Hoffman et al exploit our understanding and immunological experience with SARS-CoV in our global race to understand, mitigate, and eventually prevent COVID-19.

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor.

The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.

Interactions between bradykinin and plasmin in the endothelial Ca2+ response.

Plasmin is a fibrinolytic factor and a serine protease that activates protease-activated receptors (PARs) to produce endothelium-derived relaxing factors such as nitric oxide and prostacyclin. Nitric oxide and prostacyclin production is regulated, at least in part, by the intracellular Ca2+ concentration in various blood vessel types. Bradykinin and plasmin stimulate vascular endothelial cells and work simultaneously in pathophysiological conditions such as thrombosis and inflammation. Here, we explored the interactions between bradykinin and plasmin in the endothelial Ca2+ response using the fluorescent indicator, Fura-2/AM, in primary cultures of porcine aortic endothelial cells (PAECs). Plasmin (0.15-15 µg/ml) and bradykinin (0.1-10 nM) increased intracellular Ca2+ concentrations in PAECs in a dose-dependent manner, and the plasmin-induced endothelial Ca2+ response occurred only once. Bradykinin (0.1-10 nM) inhibited the plasmin-induced endothelial Ca2+ response in a dose-dependent manner, however, plasmin did not affect the bradykinin-induced endothelial Ca2+ response. Pretreatment with gabexate mesilate (GM, 100 µM), a serine protease inhibitor, that blocks plasmin's proteolytic activity, fully suppressed the plasmin-induced Ca2+ response. After washout of GM and the first plasmin, the second administration of plasmin caused Ca2+ increases. However, when the first plasmin-induced Ca2+ response was blocked by pretreatment with bradykinin, the second plasmin (15 µg/ml) application did not cause any Ca2+ response, even 30 min after the washout of the first plasmin and bradykinin. Our data suggested that bradykinin regulated the plasmin-induced endothelial Ca2+ response by inhibiting the pathway downstream of the PARs' N-terminus cleavage.

Protection Provided by a Gabexate Mesylate Thermo-Sensitive In Situ Gel for Rats with Grade III Pancreatic Trauma.

BACKGROUND/AIMS: This study investigated the protection provided by gabexate mesylate thermo-sensitive in-situ gel (GMTI) against grade III pancreatic trauma in rats. METHODS: A grade III pancreatic trauma model with main pancreatic duct dividing was established, and the pancreas anatomical diagram, ascites, and serum biochemical indices, including amylase, lipase, C-reactive protein (CRP), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α), were examined. The pancreas was sliced and stained with hematoxylin eosin and subjected to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. RESULTS: Ascites, serum amylase, lipase, CRP, IL-6, and TNF-α levels were significantly increased in the pancreas trauma (PT) groups with prolonged trauma time and were significantly decreased after GMTI treatment. The morphological structure of the pancreas was loose, the acinus was significantly damaged, the nuclei were irregular and hyperchromatic, and there was inflammatory cell invasion in the PT group compared to the control. After GMTI treatment, the morphological structure of the pancreas was restored, and the damaged acinus and inflammatory cell invasion were decreased compared to the PT group. Moreover, the cell apoptosis index was significantly increased in the PT group and restored to the same levels as the control group after GMTI treatment. CONCLUSIONS: GMTI, a novel formulation and drug delivery method, exhibited specific effective protection against PT with acute pancreatitis therapy and has potential value as a minimally invasive adjuvant therapy for PT with acute pancreatitis.

Combination therapy with renin-angiotensin-aldosterone system inhibitor telmisartan and serine protease inhibitor camostat mesilate provides further renoprotection in a rat chronic kidney disease model.

We previously reported that camostat mesilate (CM) had renoprotective and antihypertensive effects in rat CKD models. In this study, we examined if CM has a distinct renoprotective effect from telmisartan (TE), a renin-angiotensin-aldosterone system (RAS) inhibitor, on the progression of CKD. We evaluated the effect of CM (400 mg/kg/day) and/or TE (10 mg/kg/day) on renal function, oxidative stress, renal fibrosis, and RAS components in the adenine-induced rat CKD model following 5-weeks treatment period. The combination therapy with CM and TE significantly decreased the adenine-induced increase in serum creatinine levels compared with each monotherapy, although all treatment groups showed similar reduction in blood pressure. Similarly, adenine-induced elevation in oxidative stress markers and renal fibrosis markers were significantly reduced by the combination therapy relative to each monotherapy. Furthermore, the effect of the combination therapy on plasma renin activity (PRA) and plasma aldosterone concentration (PAC) was similar to that of TE monotherapy, and CM had no effect on both PRA and PAC, suggesting that CM has a distinct pharmacological property from RAS inhibition. Our findings indicate that CM could be a candidate drug for an add-on therapy for CKD patients who had been treated with RAS inhibitors.

The feeding responses evoked by endogenous cholecystokinin are regulated by different gastrointestinal sites.

The current study tested the hypothesis that cholecystokinin (CCK) A receptor (CCKAR) in areas supplied by the celiac artery (CA), stomach and upper duodenum, and the cranial mesenteric artery (CMA), small and parts of the large intestine, is necessary for reduction of meal size, prolongation of the intermeal interval (time between first and second meal) and increased satiety ratio (intermeal interval/meal size or amount of food consumed during any given unit of time) by the non-nutrient stimulator of endogenous CCK release camostat. Consistent with our previous findings camostat reduced meal size, prolonged the intermeal interval and increased the satiety ratio. Here, we report that blocking CCKAR in the area supplied by the celiac artery attenuated reduction of meal size by camostat more so than the cranial mesenteric artery route. Blocking CCKAR in the area supplied by the cranial mesenteric artery attenuated prolongation of the intermeal interval length and increased satiety ratio by camostat more so than the celiac artery route. Blocking CCKAR in the areas supplied by the femoral artery (control) failed to alter the feeding responses evoked by camostat. These results support the hypothesis that CCKAR in the area supplied by the CA is necessary for reduction of meal size by camostat whereas CCKAR in the area supplied by the CMA is necessary for prolongation of the intermeal interval and increased satiety ratio by this substance. Our results demonstrate that meal size and intermeal interval length by camostat are regulated through different gastrointestinal sites.

A novel thermosensitive in-situ gel of gabexate mesilate for treatment of traumatic pancreatitis: An experimental study.

Gabexate mesilate (GM) is a trypsin inhibitor, and mainly used for treatment of various acute pancreatitis, including traumatic pancreatitis (TP), edematous pancreatitis, and acute necrotizing pancreatitis. However, due to the characteristics of pharmacokinetics, the clinical application of GM still needs frequently intravenous administration to keep the blood drug concentration, which is difficult to manage. Specially, when the blood supply of pancreas is directly damaged, intravenous administration is difficult to exert the optimum therapy effect. To address it, a novel thermosensitive in-situ gel of gabexate mesilate (GMTI) was developed, and the optimum formulation of GMTI containing 20.6% (w/w) P-407 and 5.79% (w/w) P188 with different concentrations of GM was used as a gelling solvent. The effective drug concentration on trypsin inhibition was examined after treatment with different concentrations of GMTI in vitro, and GM served as a positive control. The security of GMTI was evaluated by hematoxylin-eosin (HE) staining, and its curative effect on grade II pancreas injury was also evaluated by testing amylase (AMS), C-reactive protein (CRP) and trypsinogen activation peptide (TAP), and pathological analysis of the pancreas. The trypsin activity was slightly inhibited at 1.0 and 5.0 mg/mL in GM group and GMTI group, respectively (P<0.05 vs. P-407), and completely inhibited at 10.0 and 20.0 mg/mL (P<0.01 vs. P-407). After local injection of 10 mg/mL GMTI to rat leg muscular tissue, muscle fiber texture was normal, and there were no obvious red blood cells and infiltration of inflammatory cells. Furthermore, the expression of AMS, CRP and TAP was significantly increased in TP group as compared with control group (P<0.01), and significantly decreased in GM group as compared with TP group (P<0.01), and also slightly inhibited after 1.0 and 5.0 mg/mL GMTI treatment as compared with TP group (P<0.05), and significantly inhibited after 10.0 and 20.0 mg/mL GMTI treatment as compared with TP group (P<0.01). HE staining results demonstrated that pancreas cells were uniformly distributed in control group, and they were loosely arranged, partially dissolved, with deeply stained nuclei in TP group. Expectedly, after gradient GMTI treatment, pancreas cells were gradually restored to tight distribution, with slightly stained nuclei. This preliminary study indicated that GMTI could effectively inhibit pancreatic enzymes, and alleviate the severity of trauma-induced pancreatitis, and had a potential drug developing and clinic application value.

Divergent Inhibitor Susceptibility among Airway Lumen-Accessible Tryptic Proteases.

Tryptic serine proteases of bronchial epithelium regulate ion flux, barrier integrity, and allergic inflammation. Inhibition of some of these proteases is a strategy to improve mucociliary function in cystic fibrosis and asthmatic inflammation. Several inhibitors have been tested in pre-clinical animal models and humans. We hypothesized that these inhibitors inactivate a variety of airway protease targets, potentially with bystander effects. To establish relative potencies and modes of action, we compared inactivation of human prostasin, matriptase, airway trypsin-like protease (HAT), and ß-tryptase by nafamostat, camostat, bis(5-amidino-2-benzimidazolyl)methane (BABIM), aprotinin, and benzamidine. Nafamostat achieved complete, nearly stoichiometric and very slowly reversible inhibition of matriptase and tryptase, but inhibited prostasin less potently and was weakest versus HAT. The IC50 of nafamostat's leaving group, 6-amidino-2-naphthol, was >104-fold higher than that of nafamostat itself, consistent with suicide rather than product inhibition as mechanisms of prolonged inactivation. Stoichiometric release of 6-amidino-2-naphthol allowed highly sensitive fluorometric estimation of active-site concentration in preparations of matriptase and tryptase. Camostat inactivated all enzymes but was less potent overall and weakest towards matriptase, which, however was strongly inhibited by BABIM. Aprotinin exhibited nearly stoichiometric inhibition of prostasin and matriptase, but was much weaker towards HAT and was completely ineffective versus tryptase. Benzamidine was universally weak. Thus, each inhibitor profile was distinct. Nafamostat, camostat and aprotinin markedly reduced tryptic activity on the apical surface of cystic fibrosis airway epithelial monolayers, suggesting prostasin as the major source of such activity and supporting strategies targeting prostasin for inactivation.