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.

Gibbs Free Energy of Hydrolytic Water Molecule in Acyl-Enzyme Intermediates of a Serine Protease: A Potential Application for Computer-Aided Discovery of Mechanism-Based Reversible Covalent Inhibitors.

In order to predict the potencies of mechanism-based reversible covalent inhibitors, the relationships between calculated Gibbs free energy of hydrolytic water molecule in acyl-trypsin intermediates and experimentally measured catalytic rate constants (kcat) were investigated. After obtaining representative solution structures by molecular dynamics (MD) simulations, hydration thermodynamics analyses using WaterMap™ were conducted. Consequently, we found for the first time that when Gibbs free energy of the hydrolytic water molecule was lower, logarithms of kcat were also lower. The hydrolytic water molecule with favorable Gibbs free energy may hydrolyze acylated serine slowly. Gibbs free energy of hydrolytic water molecule might be a useful descriptor for computer-aided discovery of mechanism-based reversible covalent inhibitors of hydrolytic enzymes.

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.

Enhanced stability of oral insulin in targeted peptide ligand trimethyl chitosan nanoparticles against trypsin.

Oral insulin delivery is often limited by protease degradation. 2-(Dimethylamino)-2-oxoethyl 4-(4-guanidinobenzoyloxy)phenylacetate methanesulphonate (Camostat mesylate) is reported to have the ability to inhibit trypsin activity, which is the main protease responsible for protein degradation. This study attempted to form a novel nanoparticle by covalently conjugating 4-(2-(2-aminoethylamino)-2-oxoethyl)phenyl 4-guanidinobenzoyloxy (FOY-251), an active derivative of camostat mesylate, to the backbone of poly (γ-glutamic acid) (γ-PGA), in order to improve insulin stability against protease. Goblet cell targeting CSKSSDYQC (CSK) peptide was demonstrated to effectively improve the epithelial absorption of insulin. Therefore, the novel nanoparticle was prepared by mixing cationic peptide modified trimethyl chitosan (TMC-CSK) with anionic γPGA-FOY conjugate using multi-ion crosslinked method. Results showed that not only the γPGA-FOY conjugate but also the prepared novel nanoparticle could inhibit trypsin activity both in vitro environment and on the intestinal mucosal surface. This study would be beneficial for peptide modified nanoparticles in oral insulin delivery.

Combined derivatization and high-performance liquid chromatography with fluorescence and ultraviolet detection for simultaneous analysis of octreotide and gabexate mesylate metabolite in human pancreatic juice samples.

A simple and sensitive method based on the combination of derivatization and high-performance liquid chromatography with ultraviolet and fluorimetric detection was developed for the simultaneous determination of octreotide and gabexate mesylate metabolite in human pancreatic juice samples. Parameters of the derivatization procedure affecting extraction efficiency were optimized. The developed method was validated according to the International Conference on Harmonization guidelines. The calibration curves were linear over a range of 0.1-15 µg/mL for octreotide and 0.20-15 µg/mL for gabexate mesylate metabolite. Derivatized products of octreotide and gabexate mesylate metabolite were separated on a Luna C18 column (4.6 × 250 mm; 5 µm particle size) using a gradient with a run time of 36 min, without further purification. The limits of detection were 0.025 and 0.05, respectively, for octreotide and gabexate mesylate metabolite. This paper reports the validation of a quantitative high performance liquid chromatography-photodiode array-fluorescence (HPLC-PDA-FL) method for the simultaneous analysis of octreotide and gabexate mesylate metabolite in pancreatic juice by protein precipitation using zinc sulfate-methanol-acetonitrile containing the derivatizing reagent, 4-fluoro-7-nitro-[2,1,3]-benzoxadiazole (NBD-F). Derivatized products of octreotide and gabexate mesylate metabolite were separated on a Luna C18 column (4.6 × 250 mm; 5 µm particle size) using a gradient with a run time of 36 min, without further purification. The method was validated over the concentration ranges 0.1-15 and 0.2-15 µg/mL for octreotide and gabexate mesylate metabolite, respectively, in human pancreatic juice. Biphalin and methyl-p-hydroxybenzoate were used as the internal standards. This method was successfully utilized to support clinical studies in humans. The results from assay validations show that the method is selective, sensitive and robust. The limit of quantification of the method was 0.1 µg/mL for octreotide and 0.2 µg/mL for gabexate mesylate metabolite, and matrix matched standard curves showed a good linearity up to 15 µg/mL. In the entire analytical range the intra- and inter-day precision (RSD%) values were respectively ≤5.9% and ≤3.1% for octreotide and ≤2.0% and ≤3.9% for gabexate mesylate metabolite. For both analytes the intra- and inter-day accuracy (bias) values ranged respectively from -6.8 to -2.5% and from -4.6 to -5.7%. This method utilizes derivatization with NBD-F and provides adequate sensitivity for both drugs.

Comparison of ulinastatin, gabexate mesilate, and nafamostat mesilate in preservation solution for islet isolation.

For islet transplantation, maintaining organ viability after pancreas procurement is critically important for optimal graft function and survival. We recently reported that islet yield was significantly higher in the modified ET-Kyoto (MK) solution, which includes a trypsin inhibitor (ulinastatin), compared with the UW solution, and that the advantages of MK solution are trypsin inhibition and less collagenase inhibition. In this study, we compared ulinastatin with other trypsin inhibitors, gabexate mesilate, and nafamostat mesilate, in preservation solution for islet isolation. Ulinastatin was easily dissolved in ET-Kyoto solution, while ET-Kyoto with gabexate mesilate and nafamostat mesilate became cloudy immediately after addition. Although there were no significant differences in islet yield among the three groups, viability was significantly higher for the MK group than for the GK group or the NK group. The stimulation index was significantly higher for the MK group than for the GK group. In summary, there are no other trypsin inhibitors that are more effective than ulinastatin. Based on these data, we now use ET-Kyoto solution with ulinastatin for clinical islet transplantation.

Oral delivery system for two-pulse colonic release of protein drugs and protease inhibitor/absorption enhancer compounds.

It is well known that the intestinal stability and absorption of protein drugs are improved when enzyme inhibitors/permeation enhancers are coadministered. Recently, it was hypothesized that an increased effectiveness of these adjuvants might be achieved by timing their release prior to that of the protein, so that a more favorable environment would be established in advance. Therefore, an oral system was proposed for two-pulse colonic release of insulin and the protease inhibitor camostat mesilate/absorption enhancer sodium glycocholate. The device consisted of a drug-containing core, an inner swellable/erodible low-viscosity hydroxypropyl methylcellulose (HPMC) coating, an intermediate adjuvant layer, and an additional outer HPMC coating. HPMC coats and camostat mesilate/sodium glycocholate films with differing thicknesses were applied to immediate-release tablet cores by aqueous spray coating. The obtained units were characterized for weight, thickness, breaking force, and release performance. All systems showed satisfactory technological properties and the pursued pulsatile delivery behavior, with programmable delay phases preceding inhibitor/enhancer release and elapsing between inhibitor/enhancer and protein release, respectively. Indeed, both lag times linearly correlated with the relevant HPMC coating level. The system was thus proven suitable for yielding two-pulse release profiles, in which lag phases could be modulated to provide convenient concentration patterns for proteins and adjuvants.

Stability of gabexate mesilate products: Influence of the addition of mannitol.

Gabexate mesilate is a non-peptide protease inhibitor, developed in Japan, which is used in the treatment of acute pancreatitis and disseminated intravascular coagulation. This compound is readily hydrolyzed as it has ester bonds in its structure. It is now out of patent in Japan and there are many generic versions on the market. The crystal structure and the hydrolysate content of the branded product and nine generic versions were evaluated by X-ray diffractometry, thermal analysis and HPLC. The results showed that generic products containing mannitol as an additive had a higher content of hydrolysate as an impurity than the branded product or generic products formulated without mannitol, suggesting that the crystal structure might be altered and stability impaired in mannitol-containing drug products.

Properties of poly(lactic-co-glycolic acid) nanospheres containing protease inhibitors: camostat mesilate and nafamostat mesilate.

Poly(lactic-co-glycolic acid) (PLGA) nanospheres containing protease inhibitors, camostat mesilate (CM) and nafamostat mesilate (NM), were prepared by the emulsion solvent diffusion methods in water or in oil, and the w/o/w emulsion solvent evaporation method. The average diameter of PLGA nanospheres prepared in the water system were about 150-300 nm, whereas those prepared in the oil system were 500-600 nm. Among the three methods, these drugs were the most efficiently encapsulated up to 60-70% in PLGA nanospheres in the oil system. Other factors that may influence drug encapsulation efficiency and in vitro release such as drug load, molecular weight of polymer were also investigated. Both the CM- and NM-loaded nanospheres prepared in the water system immediately released about 85% of the drug upon dispersed in the release medium while the drug initial burst of nanospheres prepared by the emulsion solvent diffusion in oil method reduced to 30% and 60% for CM and NM, respectively. Poly(aspartic acid) (PAA), a complexing agent for cationic water soluble drugs, showed little effect on the encapsulation efficiency and release behavior for CM and NM. The DSC study and AFM pictures of nanospheres demonstrated that temperature-dependent drug release behavior was ascribable to the glass transition temperature of the polymer, which also affected the morphology of nanospheres upon dispersed in the release medium and influenced the drug release consequently.

Distribution of protease inhibitors in lipid emulsions: gabexate mesilate and camostat mesilate.

Gabexate mesilate (GM) and camostat mesilate (CM) are protease inhibitors used for the treatment of pancreatitis, and have been reported to show anticancer effects in vivo. Lipid emulsions (20% fractionated soybean oil) were investigated in terms of physicochemical interaction between the drugs and lipid emulsions as a possible drug carrier. The result showed that the drugs did not distribute in the oil phase but were adsorbed at the phospholipid interface of oil droplets. With increasing concentration of the drugs, the adsorption amount at the interface rose steeply to around 2.2x10(-11) mol/cm2 for GM and 1.2x10(-11) mol/cm2 for CM, respectively, followed by further adsorption deviated from the Langmuir adsorption manner after the inflection. To interpret this two-stage adsorption of the drugs, surface potential and fluorescence changes were examined in addition to thermodynamics for their interaction with the interfacial lipid layer. The primary adsorption was exothermic and was due to electrostatic interaction and van der Waals interaction between drug molecules and phospholipid molecules. Both acidic and neutral phospholipids in the lipid were involved in the adsorption of GM, while acidic phospholipids were mainly involved in the adsorption of CM. On the other hand, the secondary adsorption was endothermic and was entropy-driven most probably due to hydrophobic interaction for GM and CM in common, including peripheral penetration of drug molecules into the interfacial lipid layer.

Preparation and characterization of starch/cyclodextrin bioadhesive microspheres as platform for nasal administration of Gabexate Mesylate (Foy) in allergic rhinitis treatment.

Bioadhesive and biodegradable microspheres were obtained by chemical cross-linking with epichlorohydrin of an alkaline solution of a mixture of starch and alpha-, beta-, or gamma-cyclodextrin (CyD). Microspheres were characterized by scanning electron microscopy, swelling degree, and water retention. The percentage of the effective CyD in microspheres was estimated by measuring the amount of iodine and typical organic compounds (TOCs) retained in the hydrophobic cavity of CyD. Gabexate Mesylate (trade name Foy); GM), an antiallergic drug, was included in microspheres by soaking in an aqueous solution containing the drug, followed by solvent evaporation or lyophilization. UV, IR, and DSC data indicated that despite the fact that GM is a hydrophilic drug, its hydrophobic moiety close to the benzene ring is able to penetrate the CyD cavity and to form stable inclusion complexes. Values of the association equilibrium constant for GM binding to CyD, obtained by UV differential spectroscopy, indicated that the affinity of the drug for alpha- and gamma-CyD is higher than that for beta-CyD. In vitro, GM was gradually released during 1h. Even if the release rate of the drug is relatively fast, the microspheres might actually provide the best platform since the material adheres to the nasal mucosa which was proved by adhesion tests. The GM integrity was checked by comparing its anti-trypsin activity before and after release.

Therapeutic effect of camostat mesilate on Duchenne muscular dystrophy in mdx mice.

Duchenne muscular dystrophy is known to be caused by a defective gene of dystrophin, a 427-kDa cytoskeletal protein, but the effective therapeutic drug is presently unavailable. We previously reported that a trypsin-like protease designated as dystrypsin is markedly activated in the muscle microsomal fraction immediately before onset of the clinical signs in mdx mice, a dystrophin-deficient hereditary animal model for human Duchenne muscular dystrophy. In order to examine the possible participation of dystrypsin in the occurrence of the disease, we investigated the therapeutic effects of dystrypsin inhibitors on the occurrence and progress of muscular dystrophy. Here, we show that camostat mesilate, a low-molecular-weight inhibitor of trypsin-like proteases, including dystrypsin, is a candidate drug for Duchenne muscular dystrophy.

Gabexate mesilate, a synthetic protease inhibitor, inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production by inhibiting activation of both nuclear factor-kappaB and activator protein-1 in human monocytes.

Gabexate mesilate, a synthetic protease inhibitor, was shown to be effective in treating patients with sepsis-associated disseminated intravascular coagulation in which tumor necrosis factor-alpha (TNF-alpha) plays a critical role. We demonstrated that gabexate mesilate reduced lipopolysaccharide (LPS)-induced tissue injury by inhibiting TNF-alpha production in rats. In the present study, we analyzed the mechanism(s) by which gabexate mesilate inhibits LPS-induced TNF-alpha production in human monocytes in vitro. Gabexate mesilate inhibited the production of TNF-alpha in monocytes stimulated with LPS. Gabexate mesilate inhibited both the binding of nuclear factor-kappaB (NF-kappaB) to target sites and the degradation of inhibitory kappaBalpha. Gabexate mesilate also inhibited both the binding of activator protein-1 (AP-1) to target sites and the activation of mitogen-activated protein kinase pathways. These observations strongly suggest that gabexate mesilate inhibited LPS-induced TNF-alpha production in human monocytes by inhibiting activation of both NF-kappaB and AP-1. Inhibition of TNF-alpha production by gabexate mesilate might explain at least partly its therapeutic effects in animals given LPS and those in patients with sepsis.

Selective inhibition of human mast cell tryptase by gabexate mesylate, an antiproteinase drug.

Gabexate mesylate is a non-antigenic synthetic inhibitor of trypsin-like serine proteinases that is therapeutically used in the treatment of pancreatitis and disseminated intravascular coagulation and as a regional anticoagulant for hemodialysis. Considering the structural similarity between gabexate mesylate and arginine-based inhibitors of trypsin-like serine proteinases, the effect of gabexate mesylate on human and bovine mast cell tryptase action was investigated. Values of the inhibition constant (K(i)) for gabexate mesylate binding to human and bovine tryptase were 3.4 x 10(-9) M and 1.8 x 10(-7) M (at pH 7.4 and 37.0 degrees ), respectively. Furthermore, gabexate mesylate inhibited the fibrinogenolytic activity of human tryptase. On the basis of the available x-ray crystal structure of human tryptase, the possible binding mode of gabexate mesylate to human and bovine tryptase was analyzed. Human tryptase inhibition by gabexate mesylate may account for the reported prevention of inflammation, erosion, and ulceration of skin and mucosae.

Cross-enzyme inhibition by gabexate mesylate: formulation and reactivity study.

Gabexate mesylate (GM; commercialized under the brand name FOY) is a nonantigenic synthetic inhibitor of plasmatic and pancreatic serine proteinases that is used therapeutically in the treatment of pancreatitis and disseminated intravascular coagulation and as a regional anticoagulant for hemodialysis. The inhibitory effect of GM on nitric oxide synthase as well as serine proteinases and swine kidney copper amine oxidase, all acting on cationic substrates, has been investigated. On the basis of the available X-ray crystal structures of the enzymes considered, the possible binding mode(s) of GM has(have) been analyzed. The enzyme cross-inhibition by GM suggests that the use of this drug should be under careful control. With the aim to improve the scarce plasma stability of GM, the positively charged drug has been complexed to the surface of preformed anionic liposomes. The liposome-complexed GM half-life increases about five-fold, indicating the protective effect of liposomes on GM degradation. Moreover, the GM complexation with liposomes does not alter its inhibitory activity on NOS-I and porcine pancreatic trypsin.