Pharmacokinetics of Nafamostat,a Potent Serine Protease Inhibitor,by a Novel LC-MS/MS Analysis

Nafamostat, a synthetic serine protease inhibitor, has been used for the treatment of inflammatory diseases such as pancreatitis. Recently, an increasing number of studies have shown the promising antiviral effects of nafamostat for the treatment of coronavirus disease-19 (COVID-19). This study aimed to develop a novel liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis and to characterize the pharmacokinetics of nafamostat in rats. Nafamostat in the rat plasma was extracted by solid phase extraction, and ¹³C₆-nafamostat was used as an internal standard. The quantification limit of nafamostat in the rat plasma was 0.5 ng/mL. The LC-MS/MS method was fully validated and applied to characterize the pharmacokinetics of nafamostat in rats. Following intravenous injection (2 mg/kg), nafamostat in the plasma showed a multiexponential decline with an average elimination half-life (t_1/2) of 1.39 h. Following oral administration of nafamostat solutions (20 mg/kg) in 10% dimethyl sulfoxide (DMSO) and in 10% DMSO with 10% Tween 80, nafamostat was rapidly absorbed, and the average oral bioavailability was 0.95% and 1.59%, respectively. The LC-MS/MS method and the pharmacokinetic information of nafamostat could be helpful for the further preclinical and clinical studies of nafamostat.     

Identification of the Most Stable Sc2C80 Isomers: Structure,Electronic Property,and Molecular Spectra Investigations

A systematic density functional theory investigation has been carried out to explore the possible structures of Sc₂C₈₀ at the BMK/6‐31G(d) level. The results clearly show that Sc₂@C₈₀‐I_h, Sc₂@C₈₀‐D_5h, and Sc₂C₂@C₇₈‐C_2v can be identified as three isomers of Sc₂C₈₀ metallofullerene with the lowest energy. Frontier molecular orbital analysis indicates that the two Sc₂@C₈₀ isomers have a charge state as (Sc³⁺)₂@C₈₀^6?and the Sc₂C₂@C₇₈ has a charge state of (Sc³⁺)₂C₂^2?@C₇₈^4?. Moreover, the metal‐cage covalent interactions have been studied to reveal the dynamics of endohedral moiety. The vertical electron affinity, vertical ionization potential, infrared spectra and ¹³C nuclear magnetic resonance spectra have been also computed to further disclose the molecular structures and properties.     

Crystal Structures of the Serine Protease Domain of Murine Plasma Kallikrein

Plasma kallikrein(PK), a serine protease in the trypsin clan(SA), plays critical roles in many physiological and pathological pathways. Regulating the abnormal activity of PK has been successfully used in the clinical therapy of hereditary angioedema. In this study, the serine protease domain of murine plasma kallikrein(m PK) was expressed in the pichia pastoris system. The recombinant protein was a glycosylated active enzyme after purification by the cation exchange and size-exclusion chromatography, and was crystallized at the precipitant condition of 25% PEG 3350, 0.1 M Tris-HCl pH 8.5 and 0.1 M Na Cl. The crystal structure of m PK was determined at 2.6 ?. This is the first published crystal structure of m PK, showing some distinctive features at S2' and S3' pockets when compared to its human analogue(human plasma kallikrein, h PK). In addition, m PK show unique structural features in the non-conservative 67-72 and 76-81 loops when compared to other serine proteases. These results provide insights for the design of potent and selective PK inhibitors.     

Purification, Characterization, and Specificity Determination of a New Serine Protease Secreted by Penicillium waksmanii

The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 °C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl₂, KCl, and BaCl, and partially inhibited by CuCl₂, CoCl₂ and totally inhibited by AlCl₃ and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k _cat/K _m of 10,666 mM^?1?s^?1, followed by the peptide Abz-GLRSSKQ-EDDnp with a k _cat/K _m of 7,500 mM^?1?s^?1. Basic and acidic side chain-containing amino acids performed best at subsite S₁. Subsites S₂, S₃, S^′ ₂, and S^′ ₁, S^′ ₃ showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k _cat/K _m were observed for the subsites S₂, S₃, and S^′ _2. The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level.     

Calorimetric Investigation of Conversion to The Most Stable Subgel Phase of Phosphatidylethanolamine-water System

The conversion of either the gel or the liquid crystal phase to the most stable subgel phase in dimyristoylphosphatidylethanolamine (DMPE)-water system at a water content of 25 mass% was studied by differential scanning calorimetry and isothermal calorimetry. The calorimetric experiments were performed for two samples depending on whether the thermal treatment of cooling to -60°C was adopted or not. In DSC of varying heating rate, exothermic peaks due to the partial conversion were observed at either temperatures just below the gel-to-liquid crystal phase transition at 50°C or temperatures where the liquid crystal phase is present as a metastable state. The enthalpies of conversion for both the gel and the liquid crystal phase were measured directly by the isothermal calorimetries at 47 and 53°C, respectively, where the exothermic peaks were observed by DSC and were compared with the enthalpy difference between the gel and subgel phases and that between the liquid crystal and subgel phases. This revised version was published online in July 2006 with corrections to the Cover Date.     

Rapid Degradation of SARS-CoV-2 Spike S Protein by A Specific Serine Protease

The S protein of SARS-CoV-2 is a crucial structural and functional component for virus entry. Due to the constant mutation of the virus, there are very limited ways to prevent and control COVID-19. This experiment used a macroscopic SDS-PAGE method and proved that the S protein of wild-type SARS-CoV-2 virus, especially the S1 subunit, is very sensitive to alkaline serine protease with acidic pI (ASPNJ), NJ represents Neanthes japonica (Izuka) from which ASP is purified). ASPNJ cleaves proteins when the carbonyl group of the peptide bond is contributed by arginine or lysine. ASPNJ can degrade the S protein very quickly and effectively in vitro with relative selectivity. It can be inferred that the S, S1 and RBD of SARS-CoV-2 variants can also be easily degraded by ASPNJ. This rapid and strong degradation of the S protein by ASPNJ may become a potential new treatment strategy.     

Identification of Non-Macrocyclic Small Molecule Inhibitors against the NS3/4A Serine Protease of Hepatitis C Virus through in Silico Screening

Drug discovery and design for inhibition of the Hepatitis C Virus (HCV) NS3/4A serine protease is a major challenge. The broad, shallow, and generally featureless nature of the active site makes it a difficult target for "hit" selection especially using standard docking programs. There are several macrocyclic NS3/4A protease inhibitors that have been approved or are in clinical trials to treat chronic HCV (alone or as combination therapy), but most of the current therapies for HCV infection have untoward side effects, indicating a continuing medical need for the discovery of novel therapeutics with improved efficacy. In this study, we designed and implemented a two-tiered and progressive docking regime that successfully identified five non-macrocyclic small molecules that show inhibitory activity in the low micromolar range. Of these, four compounds show varying inhibition against HCV subgenotypes 1b, 1a, 2a, and 4d. The top inhibitor (3) has an IC(50) value of 15 μM against both subgenotypes 1b and 2a of the NS3/4A protease enzyme. Another inhibitor, 1, inhibits all four subgenotypes with moderate activity, showing highest activity for genotype 2a (24 μM). The five inhibitors presented in this study could be valuable candidates for future hit to lead optimization. Additionally, enzyme-inhibitor interaction models presented herein provide key information regarding structural differences between the active sites of the NS3/4A protease of the HCV subgenotype 1a and 1b that might explain the variable inhibitory activity between subgenotypes of the small molecule inhibitors identified here.     

Purification and Characterization of a New Serine Protease with Fibrinolytic Activity from the Marine Invertebrate, Urechis unicinctus

A non-hemorrhagic, chymotrypsin-like serine protease, UFEII, was purified from the marine echiuroid worm, Urechis unicinctus, after a combination of chromatography steps. UFEII was monomeric, with an apparent molecular weight of 26.7 kDa via SDS-PAGE. The isoelectric point of UFEII was 4.03, and the maximum activity of the enzyme was observed at 50 °C and pH?8.0. According to fibrin plate assays, UFEII could not only directly degrade fibrin and fibrinogen but also activate plasminogen. Further, UFEII preferentially hydrolyzed the fibrinogen γ-chain, followed by the Bβ-chains and Aα-chains. Moreover, ufeII, full length of the gene encoding UFEII, was obtained by RT-PCR, degenerated PCR, and nested PCR. The ufeII was determined to be a 906-bp cDNA containing an open reading frame of 795 bp encoding a putative protein of 264 amino acids with a predicted molecular weight of 27.03 kDa. Besides, UFEII exhibited no hemorrhagic effect. Overall, U. unicinctus may represent a potential source of new therapeutic agents in thrombolytic therapy.     

The SARS-CoV-2 Entry Inhibition Mechanisms of Serine Protease Inhibitors,OM-85, Heparin and Soluble HS Might Be Linked to HS Attachment Sites

This article discusses the importance of D-xylose for fighting viruses (especially SARS-CoV-2) that use core proteins as receptors at the cell surface, by providing additional supporting facts that these viruses probably bind at HS/CS attachment sites (i.e., the hydroxyl groups of Ser/Thr residues of the core proteins intended to receive the D-xylose molecules to initiate the HS/CS chains). Essentially, the additional supporting facts, are: some anterior studies on the binding sites of exogenous heparin and soluble HS on the core proteins, the inhibition of the viral entry by pre-incubation of cells with heparin, and additionally, corroborating studies about the mechanism leading to type 2 diabetes during viral infection. We then discuss the mechanism by which serine protease inhibitors inhibit SARS-CoV-2 entry. The biosynthesis of heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (DS), and heparin (Hep) is initiated not only by D-xylose derived from uridine diphosphate (UDP)-xylose, but also bioactive D-xylose molecules, even in situations where cells were previously treated with GAG inhibitors. This property of D-xylose shown by previous anterior studies helped in the explanation of the mechanism leading to type 2 diabetes during SARS-CoV-2 infection. This explanation is completed here by a preliminary estimation of xyloside GAGs (HS/CS/DS/Hep) in the body, and with other previous studies helping to corroborate the mechanism by which the D-xylose exhibits its antiglycaemic properties and the mechanism leading to type 2 diabetes during SARS-CoV-2 infection. This paper also discusses the confirmatory studies of regarding the correlation between D-xylose and COVID-19 severity.     

丙型肝炎病毒抑制剂的三维药效团和构效关系

通过ＣＡＴＡＬＹＳＴ软件包得到了两类ＨＣＶ ＮＳ３丝氨酸蛋白酶抑制剂的三维药效团模型。尽管这两类抑制剂具有完全不同的骨架结构,但得到的药效团却具有共同的特性。这当这两类抑制剂和受体发生相互作用时,可能采用了相似的结合模式。根据药效团模型,进行了三维构效关系的研究。结果表明,得到的药效团模型具有良好的预测能力（线性回归系数Ｒ＝０．８９）。     

Anhydrous Amorphous Calcium Oxalate Nanoparticles from Ionic Liquids: Stable Crystallization Intermediates in the Formation of Whewellite

The mechanisms by which amorphous intermediates transform into crystalline materials are not well understood. To test the viability and the limits of the classical crystallization, new model systems for crystallization are needed. With a view to elucidating the formation of an amorphous precursor and its subsequent crystallization, the crystallization of calcium oxalate, a biomineral widely occurring in plants, is investigated. Amorphous calcium oxalate (ACO) precipitated from an aqueous solution is described as a hydrated metastable phase, as often observed during low‐temperature inorganic synthesis and biomineralization. In the presence of water, ACO rapidly transforms into hydrated whewellite (monohydrate, CaC₂O₄ ? H₂O, COM). The problem of fast crystallization kinetics is circumvented by synthesizing anhydrous ACO from a pure ionic liquid (IL‐ACO) for the first time. IL‐ACO is stable in the absence of water at ambient temperature. It is obtained as well‐defined, non‐agglomerated particles with diameters of 15–20 nm. When exposed to water, it crystallizes to give (hydrated) COM through a dissolution/recrystallization mechanism.     

Neutral Serine Protease from Penicillium italicum. Purification,Biochemical Characterization,and Use for Antioxidative Peptide Preparation from Scorpaena notata Muscle

In the present study, purification and properties of an extracellular neutral serine protease from the fungus Penicillium italicum and its potential application as an antioxidant peptides producer are reported. The protease was purified to homogeneity using ammonium sulfate precipitation, Sephacryl S-200 gel filtration, diethylaminoethanol (DEAE)-Sepharose ion exchange chromatography, and TSK-HPLC gel filtration with a 10.2-fold increase in specific activity and 25.8 % recovery. The purified enzyme appeared as single protein band with a molecular mass of 24 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the proteolytic activity were pH 7.0 and 50 °C, respectively. The enzyme was stable in the pH range of 6.0–9.0. The protease was activated by divalent cations such as Ca²⁺ and Mg²⁺. Complete inhibition of the purified enzyme by phenylmethylsulfonyl fluoride confirmed that the protease was of serine-type. The purified enzyme revealed high stability and relatively broad specificity. Scorpaena notata muscle protein hydrolysates prepared using purified serine protease (protease from P. italicum (Prot-Pen)) showed good in vitro antioxidative activities. The antioxidant activities of Scorpaena muscle protein hydrolyzed by Prot-Pen (SMPH-PP) were evaluated using various antioxidant assays: 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, reducing power, ferrous chelating activity, and DNA nicking assay. SMPH-PP showed varying degrees of antioxidant activity and almost the same strongest protection against hydroxyl radical induced DNA breakage.