Exoproduction and Biochemical Characterization of a Novel Serine Protease from Ornithinibacillus caprae L9T with Hide-Dehairing Activity.

This study is the first report on production and characterization of the enzyme from an Ornithinibacillus species. A 4.2-fold increase in the extracellular protease (called L9T) production from Ornithinibacillus caprae L9T was achieved through the one-factor-at-a-time approach and response surface methodological optimization. L9T protease exhibited a unique protein band with a mass of 25.9 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This novel protease was active over a range of pH (4-13), temperatures (30-80°C) and salt concentrations (0-220 g/l), with the maximal activity observed at pH 7, 70°C and 20 g/l NaCl. Proteolytic activity was upgraded in the presence of Ag+, Ca2+ and Sr2+, but was totally suppressed by 5 mM phenylmethylsulfonyl fluoride, which suggests that this enzyme belongs to the serine protease family. L9T protease was resistant to certain common organic solvents and surfactants; particularly, 5 mM Tween 20 and Tween 80 improved the activity by 63 and 15%, respectively. More importantly, L9T protease was found to be effective in dehairing of goatskins, cowhides and rabbit-skins without damaging the collagen fibers. These properties confirm the feasibility of L9T protease in industrial applications, especially in leather processing.

The Role of RASs /RVs in the Current Management of HCV.

The approval of combination therapies with direct-acting antiviral (DAA) regimens has led to significant progress in the field of hepatitis C virus (HCV) treatment. Although most patients treated with these agents achieve a virological cure, resistance to DAAs is a major issue. The rapid emergence of resistance-associated substitutions (RASs), in particular in the context of incomplete drug pressure, has an impact on sustained virological response (SVR) rates. Several RASs in NS3, NS5A and NS5B have been linked with reduced susceptibility to DAAs. RAS vary based on HCV characteristics and the different drug classes. DAA-resistant HCV variant haplotypes (RVs) are dominant in cases of virological failure. Viruses with resistance to NS3-4A protease inhibitors are only detected in the peripheral blood in a time frame ranging from weeks to months following completion of treatment, whereas NS5A inhibitor-resistant viruses may persist for years. Novel agents have been developed that demonstrate promising results in DAA-experienced patients. The recent approval of broad-spectrum drug combinations with a high genetic barrier to resistance and antiviral potency may overcome the problem of resistance.

A therapeutic combination of two small molecule toxin inhibitors provides broad preclinical efficacy against viper snakebite.

Snakebite is a medical emergency causing high mortality and morbidity in rural tropical communities that typically experience delayed access to unaffordable therapeutics. Viperid snakes are responsible for the majority of envenomings, but extensive interspecific variation in venom composition dictates that different antivenom treatments are used in different parts of the world, resulting in clinical and financial snakebite management challenges. Here, we show that a number of repurposed Phase 2-approved small molecules are capable of broadly neutralizing distinct viper venom bioactivities in vitro by inhibiting different enzymatic toxin families. Furthermore, using murine in vivo models of envenoming, we demonstrate that a single dose of a rationally-selected dual inhibitor combination consisting of marimastat and varespladib prevents murine lethality caused by venom from the most medically-important vipers of Africa, South Asia and Central America. Our findings support the translation of combinations of repurposed small molecule-based toxin inhibitors as broad-spectrum therapeutics for snakebite.

Sustainable production, biochemical and molecular characterization of thermo-and-solvent stable alkaline serine keratinase from novel Bacillus pumilus AR57 for promising poultry solid waste management.

The increasing amount of recalcitrant keratinous wastes generated from the poultry industry poses a serious threat to the environment. Keratinase have gained much attention to convert these wastes into valuable products. Ever since primitive feathers first appeared on dinosaurs, microorganisms have evolved to degrade this most recalcitrant keratin. In this study, we identified a promising keratinolytic bacterial strain for bioconversion of poultry solid wastes. A true keratinolytic bacterium was isolated from the slaughterhouse soil and was identified and designated as Bacillus pumilus AR57 by 16S rRNA sequencing. For enhanced keratinase production and rapid keratin degradation, the media components and substrate concentration were optimized through shake flask culture. White chicken feather (1% w/v) was found to be the good substrate concentration for high keratinase production when supplemented with simple medium ingredients. The biochemical characterization reveals astounding results which makes the B. pumilus AR57 keratinase as a novel and unique protease. Optimum activity of the crude enzyme was exhibited at pH 9 and 45 °C. The crude extracellular keratinase was characterized as thermo-and-solvent (DMSO) stable serine keratinase. Bacillus pumilus AR57 showed complete degradation (100%) of white chicken feather (1% w/v) within 18 h when incubated in modified minimal medium supplemented with DMSO (1% v/v) at 150 rpm at 37 °C. Keratinase from modified minimal medium supplemented with DMSO exhibits a half-life of 4 days. Whereas, keratinase from the modified minimal medium fortified with white chicken feather (1% w/v) was stable for 3 h only. Feather meal produced by B. pumilus AR57 was found to be rich in essential amino acids. Hence, we proposed B. pumilus AR57 as a potential candidate for the future application in eco-friendly bioconversion of poultry waste and the keratinase could play a pivotal role in the detergent industry. While feather meal may serve as an alternative to produce animal feed and biofertilizers.

Coumarin as a structural component of substrates and probes for serine and cysteine proteases.

Coumarins represent well-established structures to introduce fluorescence into tool compounds for biochemical investigations. They are valued for their small size, chemical stability and accessibility as well as their tunable photochemical properties. As components of fluorophore/quencher pairs or FRET donor/acceptor pairs, coumarins have frequently been applied in substrate mapping approaches for serine and cysteine proteases. This review also focuses on the incorporation of coumarins into the side chain of amino acids and the exploitation of the resulting fluorescent amino acids for the positional profiling of protease substrates. The protease-inhibiting properties of certain coumarin derivatives and the utilization of coumarin moieties to assemble activity-based probes for serine and cysteine proteases are discussed as well.

A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa.

Serine proteases play pivotal roles in normal physiology and a spectrum of patho-physiological processes. Accordingly, there is considerable interest in the discovery and design of potent serine protease inhibitors for therapeutic applications. This led to concerted efforts to discover versatile and robust molecular scaffolds for inhibitor design. This investigation is a bioprospecting study that aims to isolate and identify protease inhibitors from the cnidarian Actinia tenebrosa. The study isolated two Kunitz-type protease inhibitors with very similar sequences but quite divergent inhibitory potencies when assayed against bovine trypsin, chymostrypsin, and a selection of human sequence-related peptidases. Homology modeling and molecular dynamics simulations of these inhibitors in complex with their targets were carried out and, collectively, these methodologies enabled the definition of a versatile scaffold for inhibitor design. Thermal denaturation studies showed that the inhibitors were remarkably robust. To gain a fine-grained map of the residues responsible for this stability, we conducted in silico alanine scanning and quantified individual residue contributions to the inhibitor's stability. Sequences of these inhibitors were then used to search for Kunitz homologs in an A. tenebrosa transcriptome library, resulting in the discovery of a further 14 related sequences. Consensus analysis of these variants identified a rich molecular diversity of Kunitz domains and expanded the palette of potential residue substitutions for rational inhibitor design using this domain.

Citrus bioflavonoid, hesperetin, as inhibitor of two thrombin-like snake venom serine proteases isolated from Crotalus simus.

Around 5.5 million people suffer from snakebites per year, with about 400,000 cases with some type of sequelae, such as amputation, and 20,000 to 125,000 cases with the fatal end. Usually, the victim outcome depends on correct, agile and many times in situ intervention based on the proper identification of the snake venom type and its potential effects, among other factors. Therefore, knowledge on the snake venom composition and a research on inhibitors of snake venom target components might ameliorate envenoming dangerous outcome. Herein, two thrombin-like serine proteases from the Crotalus simus snake venom - SVSP1 and SVSP2 - were isolated in two chromatographic steps, using gel filtration and then RP-HPLC. They showed molecular masses of around 31.3 and 24.6 kDa, respectively, and mostly ß-sheet secondary structure features. The SVSP1 and SVSP2 were sequenced using tandem mass spectrometry (Q-TOF). Using the known serine protease structure (PDB entry: 4e7n), which was evaluated as homologous to the two target proteins, in silico docking results showed that hesperetin is its excellent inhibitor. Using in vitro tests with the commercial hesperetin, kinetic parameters were obtained for SVSPs against the synthetic substrate BApNA. Obtained results pointed that hesperetin might act as an uncompetitive (SVSP1) or mixed (SVSP2) inhibitor. Also, the fluorescence quenching upon inhibition was observed, as well as, red shift in maximums of around 20 nm, which indicate that the tryptophan residues in the target enzymes suffered conformational changes caused by hesperetin binding. Thus, a naturally occurring flavone that can easily be extracted from oranges might serve as low-cost inhibitor of the investigated snake venom proteases.

Serine protease inhibitors to treat inflammation: a patent review (2011-2016).

INTRODUCTION: Inflammation is a physiological part of the complex biological response of tissues to counteract various harmful signals. This process involves diverse actors such as immune cells, blood vessels, and nerves as sources of mediators for inflammation control. Among them serine proteases are key elements in both physiological and pathological inflammation. AREAS COVERED: Serine protease inhibitors to treat inflammatory diseases are being actively investigated by various industrial and academic institutions. The present review covers patent literature on serine protease inhibitors for the therapy of inflammatory diseases patented between 2011 and 2016. EXPERT OPINION: Serine proteases regulating inflammation are versatile enzymes, usually involved in proinflammatory cytokine production and activation of immune cells. Their dysregulation during inflammation can have devastating consequences, promoting various diseases including skin and lung inflammation, neuroinflammation, and inflammatory arthritis. Several serine proteases were selected for their contribution to inflammatory diseases and significant efforts that are spread to develop inhibitors. Strategies developed for inhibitor identification consist on either peptide-based inhibitor derived from endogenous protein inhibitors or small-organic molecules. It is also worth noting that among the recent patents on serine protease inhibitors related to inflammation a significant number are related to retinal vascular dysfunction and skin diseases.

The Alpha-Tocopherol Form of Vitamin E Boosts Elastase Activity of Human PMNs and Their Ability to Kill Streptococcus pneumoniae.

Despite the availability of vaccines, Streptococcus pneumoniae remains a leading cause of life-threatening infections, such as pneumonia, bacteremia and meningitis. Polymorphonuclear leukocytes (PMNs) are a key determinant of disease course, because optimal host defense requires an initial robust pulmonary PMN response to control bacterial numbers followed by modulation of this response later in infection. The elderly, who manifest a general decline in immune function and higher basal levels of inflammation, are at increased risk of developing pneumococcal pneumonia. Using an aged mouse infection model, we previously showed that oral supplementation with the alpha-tocopherol form of vitamin E (α-Toc) decreases pulmonary inflammation, in part by modulating neutrophil migration across lung epithelium into alveolar spaces, and reverses the age-associated decline in resistance to pneumococcal pneumonia. The objective of this study was to test the effect of α-Toc on the ability of neutrophils isolated from young (22-35 years) or elderly (65-69 years) individuals to migrate across epithelial cell monolayers in response to S. pneumoniae and to kill complement-opsonized pneumococci. We found that basal levels of pneumococcal-induced transepithelial migration by PMNs from young or elderly donors were indistinguishable, suggesting that the age-associated exacerbation of pulmonary inflammation is not due to intrinsic properties of PMNs of elderly individuals but rather may reflect the inflammatory milieu of the aged lung. Consistent with its anti-inflammatory activity, α-Toc treatment diminished PMN migration regardless of donor age. Unexpectedly, unlike previous studies showing poor killing of antibody-opsonized bacteria, we found that PMNs of elderly donors were more efficient at killing complement-opsonized bacteria ex vivo than their younger counterparts. We also found that the heightened antimicrobial activity in PMNs from older donors correlated with increased activity of neutrophil elastase, a serine protease that is required to kill pneumococci. Notably, incubation with α-Toc increased PMN elastase activity from young donors and boosted their ability to kill complement-opsonized pneumococci. These findings demonstrate that α-Toc is a potent modulator of PMN responses and is a potential nutritional intervention to combat pneumococcal infection.

Airway proteases: an emerging drug target for influenza and other respiratory virus infections.

To enter into airway epithelial cells, influenza, parainfluenza- and coronaviruses rely on host cell proteases for activation of the viral protein involved in membrane fusion. One protease, transmembrane protease serine 2 (TMPRSS2) was recently proven to be crucial for hemagglutinin cleavage of some human influenza viruses. Since the catalytic sites of the diverse serine proteases linked to influenza, parainfluenza- and coronavirus activation are structurally similar, active site inhibitors of these airway proteases could have broad therapeutic applicability against multiple respiratory viruses. Alternatively, superior selectivity could be achieved with allosteric inhibitors of TMPRSS2 or another critical protease. Though still in its infancy, airway protease inhibition represents an attractive host-cell targeting approach to combat respiratory viruses such as influenza.

Current therapy for chronic hepatitis C: The role of direct-acting antivirals.

One of the most exciting developments in antiviral research has been the discovery of the direct-acting antivirals (DAAs) that effectively cure chronic hepatitis C virus (HCV) infections. Based on more than 100 clinical trials and real-world studies, we provide a comprehensive overview of FDA-approved therapies and newly discovered anti-HCV agents with a special focus on drug efficacy, mechanisms of action, and safety. We show that HCV drug development has advanced in multiple aspects: (i) interferon-based regimens were replaced by interferon-free regimens; (ii) genotype-specific drugs evolved to drugs for all HCV genotypes; (iii) therapies based upon multiple pills per day were simplified to a single pill per day; (iv) drug potency increased from moderate (∼60%) to high (>90%) levels of sustained virologic responses; (v) treatment durations were shortened from 48 to 12 or 8 weeks; and (vi) therapies could be administered orally regardless of prior treatment history and cirrhotic status. However, despite these remarkable achievements made in HCV drug discovery, challenges remain in the management of difficult-to-treat patients.

Purification and biochemical characterization of two detergent-stable serine alkaline proteases from Streptomyces sp. strain AH4.

Streptomyces sp. strain AH4 exhibited a high ability to produce two extracellular proteases when cultured on a yeast malt-extract (ISP2)-casein-based medium. Pure proteins were obtained after heat treatment (30 min at 70 °C) and ammonium sulphate fractionation (30-60 %), followed by size exclusion HPLC column. Matrix assisted laser desorption ionization-time of flight mass spectrometry analysis revealed that the purified enzymes (named SAPS-P1 and SAPS-P2) were monomers with molecular masses of 36,417.13 and 21,099.10 Da, respectively. Their identified N-terminal amino acid displayed high homologies with those of Streptomyces proteases. While SAPS-P1 was optimally active at pH 12.0 and 70 °C, SAPS-P2 showed optimum activity at pH 10.0 and 60 °C. Both enzymes were completely stable within a wide range of temperature (45-75 °C) and pH (8.0-11.5). They were noted to be completely inhibited by phenylmethanesulfonyl fluoride and diisopropyl fluorophosphates, which confirmed their belonging to the serine proteases family. Compared to SAPS-P2, SAPS-P1 showed high thermostability and excellent stability towards bleaching, denaturing, and oxidizing agents. Both enzymes displayed marked stability and compatibility with a wide range of commercial laundry detergents and significant catalytic efficiencies compared to Subtilisin Carlsberg and Protease SG-XIV. Overall, the results indicated that SAPS-P1 and SAPS-P2 can be considered as potential promising candidates for future application as bioadditives in detergent formulations.

TMPRSS13 deficiency impairs stratum corneum formation and epidermal barrier acquisition.

Membrane-anchored serine proteases serve as important regulators of multiple developmental and homoeostatic processes in mammals. TMPRSS13 (transmembrane protease, serine 13; also known as mosaic serine protease large-form, MSPL) is a membrane-anchored serine protease with unknown biological functions. In the present study, we used mice with the Tmprss13 gene disrupted by a ß-galactosidase-neomycin fusion gene insertion to study the expression and function of the membrane-anchored serine protease. High levels of Tmprss13 expression were found in the epithelia of the oral cavity, upper digestive tract and skin. Compatible with this expression pattern, Tmprss13-deficient mice displayed abnormal skin development, leading to a compromised barrier function, as measured by the transepidermal fluid loss rate of newborn mice. The present study provides the first biological function for the transmembrane serine protease TMPRSS13.

Cathelicidin, kallikrein 5, and serine protease activity is inhibited during treatment of rosacea with azelaic acid 15% gel.

BACKGROUND: Excess cathelicidin and kallikrein 5 (KLK5) have been hypothesized to play a role in the pathophysiology of rosacea. OBJECTIVE: We sought to evaluate the effects of azelaic acid (AzA) on these elements of the innate immune system. METHODS: Gene expression and protease activity were measured in laboratory models and patients with rosacea during a 16-week multicenter, prospective, open-label study of 15% AzA gel. RESULTS: AzA directly inhibited KLK5 in cultured keratinocytes and gene expression of KLK5, Toll-like receptor-2, and cathelicidin in mouse skin. Patients with rosacea showed reduction in cathelicidin and KLK5 messenger RNA after treatment with AzA gel. Subjects without rosacea had lower serine protease activity (SPA) than patients with rosacea. Distinct subsets of patients with rosacea who had high and low baseline SPA were identified, and patients with high baseline exhibited a statistically significant reduction of SPA with 15% AzA gel treatment. LIMITATIONS: Study size was insufficient to predict clinical efficacy based on the innate immune response to AzA. CONCLUSIONS: These results show that cathelicidin and KLK5 decrease in association with AZA exposure. Our observations suggest a new mechanism of action for AzA and that SPA may be a useful biomarker for disease activity.

Proteome-wide reactivity profiling identifies diverse carbamate chemotypes tuned for serine hydrolase inhibition.

Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Inhibitors of serine hydrolases are used to treat many diseases, including obesity, diabetes, cognitive dementia, and bacterial and viral infections. Nonetheless, the majority of the 200+ serine hydrolases in mammals still lack selective inhibitors for their functional characterization. We and others have shown that activated carbamates, through covalent reaction with the conserved serine nucleophile of serine hydrolases, can serve as useful inhibitors for members of this enzyme family. The extent to which carbamates, however, cross-react with other protein classes remains mostly unexplored. Here, we address this problem by investigating the proteome-wide reactivity of a diverse set of activated carbamates in vitro and in vivo, using a combination of competitive and click chemistry (CC)-activity-based protein profiling (ABPP). We identify multiple classes of carbamates, including O-aryl, O-hexafluoroisopropyl (HFIP), and O-N-hydroxysuccinimidyl (NHS) carbamates that react selectively with serine hydrolases across entire mouse tissue proteomes in vivo. We exploit the proteome-wide specificity of HFIP carbamates to create in situ imaging probes for the endocannabinoid hydrolases monoacylglycerol lipase (MAGL) and α-ß hydrolase-6 (ABHD6). These findings, taken together, designate the carbamate as a privileged reactive group for serine hydrolases that can accommodate diverse structural modifications to produce inhibitors that display exceptional potency and selectivity across the mammalian proteome.

Inhibitory effect of isoniazid and orlistat combination on mycobacterial ES-31 serine protease in vitro and on the growth of M.tb bacilli in axenic culture.

BACKGROUND: Isoniazid and orlistat were reported to have inhibitory effect on mycobacterial ES-31 serine protease in vitro and bacterial cell growth in axenic culture. AIM: To study the cumulative effect and understand drug - drug interaction, if any, when isoniazid and orlistat used in combination. MATERIAL AND METHODS: Inhibition of mycobacterial ES-31 serine protease by different combinations of orlistat and isoniazid together and individually were studied using azocasein assay. Inhibition of secretion of excretory secretory ES-31 antigen in Sautan culture medium was studied under axenic condition and growth of M. tuberculosis H37Ra bacilli by CFU count on LJ-medium. RESULTS: Orlistat and isoniazid both showed inhibitory activity of ES-31 serine protease in in vitro as well as in vivo. Individually, isoniazid showed 90% inhibition at 200 ng/ml while orlistat at 250 ng/ml showed 65% inhibition of mycobacterial ES-31 serine protease in vitro. A combination of orlistat (250 ng/ml) and isoniazid (200 ng/ml) showed 86% inhibition in vitro while 73% inhibition was observed by orlistat (25 ng/ml) and isoniazid (200 ng/ml) on bacterial growth in axenic culture. CONCLUSION: Significant inhibition by orlistat suggests that it could be tried in patients with intolerance to isoniazid or in those already developed isoniazid resistance. It may also be explored in the suspected TB patients as initial medication in place of antibiotics for clinical relief.

Functions of rhomboid family protease RHBDL2 and thrombomodulin in wound healing.

The expression of thrombomodulin (TM), a membrane glycoprotein, is upregulated in neoepidermis during cutaneous wound healing. Rhomboid-like-2 (RHBDL2), an intramembrane serine protease, specifically cleaves TM at the transmembrane domain and causes the release of soluble TM (sTM). However, the physiological functions of TM and RHBDL2 in wound healing remain unclear. We demonstrated that both TM and RHBDL2 are upregulated in HaCaT cells stimulated by scratch wounds; furthermore, increased sTM was found in culture medium. Conversely, inhibition of RHBDL2 by 3,4-dichloroisocoumarin (DCI) or short hairpin RNA significantly inhibited wound-induced TM ectodomain shedding and wound healing. Both conditioned media from multiple-scratch-wounded HaCaT and recombinant sTM accelerated wound healing in HaCaT cells; such effects were abrogated by anti-TM antibodies. The RNA released from injured cells is involved in the induction of TM and RHBDL2. RHBDL2 and sTM were upregulated in ex vivo tissue culture of the injured skin. Furthermore, DCI inhibited sTM production and wound healing; this was reversed by recombinant sTM in mice. Thus, RHBDL2 and TM have important roles in wound healing via the release of sTM from keratinocytes; this may function as an autocrine/paracrine signal promoting wound healing.

HCV-NS3/4A protease inhibitory iridoid glucosides and dimeric foliamenthoic acid derivatives from Anarrhinum orientale.

Four new compounds were isolated from the methanol extract of the aerial parts of Anarrhinum orientale: 6'-O-cinnamoylmussaenosidic acid (1), 6'-O-cinnamoyl-8-O-(6'''-O-cinnamoylglucopyranosyl)mussaenosidic acid (2), (2E,6E)-8-{[(2E,6E)-8-hydroxy-2,6-dimethylocta-2,6-dienoyl]oxy}-2,6-dimethylocta-2,6-dienoic acid (3), and (2E,6E)-8-{[(2E,6E)-8-acetoxy-2,6-dimethylocta-2,6-dienoyl]oxy}-2,6-dimethylocta-2,6-dienoic acid (4). The known 8-O-cinnamoylmussaenosidic acid (5) was also identified. All five compounds were tested for inhibition of the hepatitis C virus (HCV) protease. Compounds 1 and 5 exhibited moderate activity, while 2 and 3 showed weak effects. No inhibitory activity on the human serine protease was observed for any of these compounds, which may infer the selectivity toward the viral protease. A computational docking study of the isolated compounds against HCV protease was used to formulate a hypothetical mechanism for the inhibitory activity of the active compounds on the enzymes tested.

[Recent advances in Saccharomyces boulardii research]. / Progrès récents dans la recherche sur Saccharomyces boulardii.

This review summarizes the probiotic mechanisms of action of Saccharomyces boulardii (S. boulardii) against inflammatory and non-inflammatory diarrheal conditions. S. boulardii is distributed in lyophilized form in many countries and used for the prevention of diarrhea in children and adults, including Clostridium difficile (C. difficile) associated infection. The main mechanisms of action of S. boulardii include inhibition of activities of bacterial pathogenic products, trophic effects on the intestinal mucosa, as well as modification of host signaling pathways involved in inflammatory and non-inflammatory intestinal diseases. S. boulardii inhibits production of pro-inflammatory cytokines by inhibiting main regulators of inflammation, including nuclear factor κB (NF-κB), and mitogen-activated protein kinases (MAP kinases), ERK1/2 and p38, but stimulates production of anti-inflammatory molecules such as peroxisome proliferator-activated receptor-gamma (PPAR-γ). Moreover, S. boulardii suppresses bacterial infection by inhibiting adhesion and/or overgrowth of bacteria, produces a serine protease that cleaves C. difficile toxin A, and stimulates antibody production against this toxin. Furthermore, S. boulardii may interfere with pathogenesis of Inflammatory Bowel Disease (IBD) by acting on T cells and acts in diarrheal conditions by improving the fecal biostructure in patients with diarrhea. These diverse mechanisms exerted by S. boulardii provide molecular clues for its effectiveness in diarrheal diseases and intestinal inflammatory conditions with an inflammatory component.