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.

Intestinal serine protease inhibition increases FGF21 and improves metabolism in obese mice.

Trypsin is the major serine protease responsible for intestinal protein digestion. An inhibitor, camostat (CS), reduced weight gain, hyperglycemia, and dyslipidemia in obese rats; however, the mechanisms for these are largely unknown. We reasoned that CS creates an apparent dietary protein restriction, which is known to increase hepatic fibroblast growth factor 21 (FGF21). Therefore, metabolic responses to CS and a gut-restricted CS metabolite, FOY-251, were measured in mice. Food intake, body weight, blood glucose, branched-chain amino acids (LC/MS), hormone levels (ELISA), liver pathology (histology), and transcriptional changes (qRT-PCR) were measured in ob/ob, lean and diet-induced obese (DIO) C57BL/6 mice. In ob/ob mice, CS in chow (9-69 mg/kg) or FOY-251 (46 mg/kg) reduced food intake and body weight gain to a similar extent as pair-fed mice. CS decreased blood glucose, liver weight, and lipidosis and increased FGF21 gene transcription and plasma levels. In lean mice, CS increased liver FGF21 mRNA and plasma levels. Relative to pair feeding, FOY-251 also increased plasma FGF21 and induced liver FGF21 and integrated stress response (ISR) transcription. In DIO mice, FOY-251 (100 mg/kg po) did not alter peak glucose levels but reduced the AUC of the glucose excursion in response to an oral glucose challenge. FOY-251 increased plasma FGF21 levels. In addition to previously reported satiety-dependent (cholecystokinin-mediated) actions, intestinal trypsin inhibition engages non-satiety-related pathways in both leptin-deficient and DIO mice. This novel mechanism improves metabolism by a liver-integrated stress response and increased FGF21 expression levels in mice. NEW & NOTEWORTHY Trypsin inhibitors, including plant-based consumer products, have long been associated with metabolic improvements. Studies in the 1980s and 1990s suggested this was due to satiety hormones and caloric wasting by loss of protein and fatty acids in feces. This work suggests an entirely new mechanism based on the lower amounts of digested protein available in the gut. This apparent protein reduction may cause beneficial metabolic adaptation by the intestinal-liver axis to perceived nutrient stress.

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.

Active site conformational changes of prostasin provide a new mechanism of protease regulation by divalent cations.

Prostasin or human channel-activating protease 1 has been reported to play a critical role in the regulation of extracellular sodium ion transport via its activation of the epithelial cell sodium channel. Here, the structure of the extracellular portion of the membrane associated serine protease has been solved to high resolution in complex with a nonselective d-FFR chloromethyl ketone inhibitor, in an apo form, in a form where the apo crystal has been soaked with the covalent inhibitor camostat and in complex with the protein inhibitor aprotinin. It was also crystallized in the presence of the divalent cation Ca(+2). Comparison of the structures with each other and with other members of the trypsin-like serine protease family reveals unique structural features of prostasin and a large degree of conformational variation within specificity determining loops. Of particular interest is the S1 subsite loop which opens and closes in response to basic residues or divalent ions, directly binding Ca(+2) cations. This induced fit active site provides a new possible mode of regulation of trypsin-like proteases adapted in particular to extracellular regions with variable ionic concentrations such as the outer membrane layer of the epithelial cell.

Nafamostat mesilate is an extremely potent inhibitor of human tryptase.

Previously, nafamostat mesilate was found to be a potent inhibitor of human tryptase. In present study, we performed a kinetic study to determine its K(i) value for tryptase and compared it with that of gabexate mesilate. Nafamostat mesilate inhibited human tryptase in a competitive manner. The apparent K(i) value was estimated to be 95.3 pM, which was 1000 times lower than that of gabexate mesilate (95.1 nM). These results strongly indicated that nafamostat mesilate is an extremely potent inhibitor of tryptase and suggested that some of its beneficial effects in the treatment of clinical status may be due to tryptase inhibition.

Competitive inhibition of swine kidney copper amine oxidase by drugs: amiloride, clonidine, and gabexate mesylate.

Competitive inhibition of swine kidney copper amine oxidase by diuretic, antihypertensive, and anticoagulant drugs, amiloride, clonidine, and gabexate mesylate, respectively, is reported. The affinity of these compounds for swine kidney copper amine oxidase is similar to that observed for inhibitor binding to nitric oxide synthase and trypsin-like serine proteinases. This finding suggests that amiloride, clonidine, and gabexate mesylate should be administrated under careful control, since enzyme cross-inhibition may occur also in vivo.