Trypsin-like Serine Protease Research Articles

Camostat Does Not Inhibit the Proteolytic Activity of Neutrophil Serine Proteases.

Coronavirus disease 2019 (COVID-19) can lead to multi-organ failure influenced by comorbidities and age. Binding of the severe acute respiratory syndrome coronavirus 2 spike protein (SARS-CoV-2 S protein) to angiotensin-converting enzyme 2 (ACE2), along with proteolytic digestion of the S protein by furin and transmembrane protease serine subtype 2 (TMPRSS2), provokes internalization of SARS-CoV-2 into the host cell. Productive infection occurs through viral replication in the cytosol and cell-to-cell transmission. The catalytic activity of TMPRSS2 can be blocked by the trypsin-like serine protease inhibitor camostat, which impairs infection by SARS-CoV-2. At the site of infection, immune cells, such as neutrophils, infiltrate and become activated, releasing neutrophil serine proteases (NSPs), including cathepsin G (CatG), neutrophil elastase (NE), and proteinase 3 (PR3), which promote the mounting of a robust immune response. However, NSPs might be involved in infection and the severe outcome of COVID-19 since the uncontrolled proteolytic activity is responsible for many complications, including autoimmunity, chronic inflammatory disorders, cardiovascular diseases, and thrombosis. Here, we demonstrate that camostat does not inhibit the catalytic activity of CatG, NE, and PR3, indicating the need for additional selective serine protease inhibitors to reduce the risk of developing severe COVID-19.

Secondary Metabolites in Basil, Bio-Insecticide, Inhibition Effect, and In Silico Molecular Docking against Proteolytic Enzymes of the Red Palm Weevil (Rhynchophorus ferrugineus).

The purpose of this work was to determine the secondary metabolites generated by O. basilicum cell suspensions, as well as their insecticide and inhibitory activity against R. ferrugineus. The growth kinetics with inoculation Verticillium dahliae were determined and identified using LC-MS. Determination of total phenolic components (TFC), flavonoids (TF), and condensed tannins (TCT) were measured. Insecticidal activity of O. basilicum extract against R. ferrugineus (larva and adult) and proteolytic enzymes activity were assessed (in vitro and in vivo). The O. basilicum extract had an LC50 of 1238 µg/mL and an LD50 of 13.4 µg/larva. The LC50 of chicoric acid, ursolic acid, salvigenin, quercetin-3-O-rutinoside, rosmarinyl glucoside, and nepetoidin B demonstrated activity at an LC50 of 1132, 1167, 1189, 1214, 1275, and 1317 µg/mL, respectively. Chicoric acid, salvigenin, nepetoidin B, and rosmarinic acid demonstrated an LD50 activity of 10.23, 11.4, 11.9, and 12.4 µg/larva, respectively. The active extract of O. basilicum inhibited total protease, trypsin-like serine proteinases, elastase, cysteine, and metalloprotease activity with an IC50 (in vitro) of 119.4, 91, 102.4, 76.4, and 52.4 µg/mL, respectively. In silico studies of compounds were conducted, such as molecular docking and ADMET analysis. The study proposes using an efficient cell suspension technique to produce O. basilicum extract containing active secondary metabolites and accessible using as bio-insecticide.

A systematic approach for evaluating the role of surface-exposed loops in trypsin-like serine proteases applied to the 170 loop in coagulation factor VIIa

Proteases play a major role in many vital physiological processes. Trypsin-like serine proteases (TLPs), in particular, are paramount in proteolytic cascade systems such as blood coagulation and complement activation. The structural topology of TLPs is highly conserved, with the trypsin fold comprising two β-barrels connected by a number of variable surface-exposed loops that provide a surprising capacity for functional diversity and substrate specificity. To expand our understanding of the roles these loops play in substrate and co-factor interactions, we employ a systematic methodology akin to the natural truncations and insertions observed through evolution of TLPs. The approach explores a larger deletion space than classical random or directed mutagenesis. Using FVIIa as a model system, deletions of 1–7 amino acids through the surface exposed 170 loop, a vital allosteric regulator, was introduced. All variants were extensively evaluated by established functional assays and computational loop modelling with Rosetta. The approach revealed detailed structural and functional insights recapitulation and expanding on the main findings in relation to 170 loop functions elucidated over several decades using more cumbersome crystallization and single deletion/mutation methodologies. The larger deletion space was key in capturing the most active variant, which unexpectedly had a six-amino acid truncation. This variant would have remained undiscovered if only 2–3 deletions were considered, supporting the usefulness of the methodology in general protease engineering approaches. Our findings shed further light on the complex role that surface-exposed loops play in TLP function and supports the important role of loop length in the regulation and fine-tunning of enzymatic function throughout evolution.

Evolutionary history of type II transmembrane serine proteases involved in viral priming.

Type II transmembrane serine proteases (TTSPs) are a family of trypsin-like membrane-anchored serine proteases that play key roles in the regulation of some crucial processes in physiological conditions, including cardiac function, digestion, cellular iron homeostasis, epidermal differentiation, and immune responses. However, some of them, in particular TTSPs expressed in the human airways, were identified as host factors that promote the proteolytic activation and spread of respiratory viruses such as influenza virus, human metapneumovirus, and coronaviruses, including SARS-CoV-2. Given their involvement in viral priming, we hypothesized that members of the TTSP family may represent targets of positive selection, possibly as the result of virus-driven pressure. Thus, we investigated the evolutionary history of sixteen TTSP genes in mammals. Evolutionary analyses indicate that most of the TTSP genes that have a verified role in viral proteolytic activation present signals of pervasive positive selection, suggesting that viral infections represent a selective pressure driving the evolution of these proteases. We also evaluated genetic diversity in human populations and we identified targets of balancing selection in TMPRSS2 and TMPRSS4. This scenario may be the result of an ancestral and still ongoing host–pathogen arms race. Overall, our results provide evolutionary information about candidate functional sites and polymorphic positions in TTSP genes.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00439-022-02435-y.

Exploring the activity and inhibition of urokinase-type plasminogen activator (UPA)

Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that activates plasminogen into the active serine protease, plasmin. Studying the catalytic activity of uPA gives insight to plasmin’s involvement in anticoagulation, cell adhesion, and degradation of the extracellular matrix, among others. The uPA’s catalytic activity is influenced by the orientation of the catalytic triad and the substrate binding affinity at the specificity pocket. This enzyme consists of an Amino-Terminal Fragment (ATF), a protease domain, and a disordered linker which connects the ATF and the protease domain.

A Novel Fibrinolytic Protein From Pheretima vulgaris: Purification, Identification, Antithrombotic Evaluation, and Mechanisms Investigation.

Thrombotic diseases have been considered major causes of death around the world. Treatments with thrombolytic drugs, such as recombinant tissue-plasminogen activator, urokinase, and streptokinase, are reported to have a life-threatening bleeding tendency. On the contrary, lumbrokinase, identified from Lumbricus rubellus, is specific to fibrin and does not cause excessive bleeding. It possesses fibrinolytic activity and activation of plasminogen to dissolve fibrin. Hence, the purification of fibrinolytic protein monomer from earthworm and antithrombotic evaluation and investigation of mechanisms are needed. In this study, a novel fibrinolytic protein EPF3, with strong fibrinolytic activity, was purified from Pheretima vulgaris by ion exchange and size exclusion chromatography. SDS PAGE, bottom-up proteomics analysis, de novo sequencing, and circular dichroism (CD) analysis were carried out for identification and characterization of it. EPF3, with a molecular weight of 25136.24 Da, consisted of 241 amino acids and contained various forms of secondary structures, including α-helix (3.9%), β-sheet (42.8%), β-turn (21.2%), and random coil (32.1%). It was a trypsin-like serine protease and stable at pH 7.0 to 11.0 and below 40°C. EPF3 was confirmed to possess an antithrombotic effect by ex vivo clot lysis test and fibrinogen-thrombin time (Fib-TT) assay. The three-dimensional structure of EPF3 was predicted by SWISS-MODEL. Molecular docking analysis predicted that EPF3 could directly interact with antithrombotic target proteins (fibrin, fibrinogen, and plasminogen), which was further confirmed by further studies. The antithrombotic mechanism of EPF3 was clarified to be outstanding direct fibrinolysis, fibrinogenolytic activity, and certain activation of plasminogen. EPF3 possesses the potential to be developed into a promising antithrombotic agent.

In vitro interaction of potential antiviral TMPRSS2 inhibitors with human serum albumin and cytochrome P 450 isoenzymes

The interactions of four sulfonylated Phe(3-Am)-derived inhibitors (MI-432, MI-463, MI-482 and MI-1900) of type II transmembrane serine proteases (TTSP) such as transmembrane protease serine 2 (TMPRSS2) were examined with serum albumin and cytochrome P450 (CYP) isoenzymes. Complex formation with albumin was investigated using fluorescence spectroscopy. Furthermore, microsomal hepatic CYP1A2, 2C9, 2C19 and 3A4 activities in presence of these inhibitors were determined using fluorometric assays. The inhibitory effects of these compounds on human recombinant CYP3A4 enzyme were also examined. In addition, microsomal stability assays (60-min long) were performed using an UPLC-MS/MS method to determine depletion percentage values of each compound. The inhibitors showed no or only weak interactions with albumin, and did not inhibit CYP1A2, 2C9 and 2C19. However, the compounds tested proved to be potent inhibitors of CYP3A4 in both assays performed. Within one hour, 20%, 12%, 14% and 25% of inhibitors MI-432, MI-463, MI-482 and MI-1900, respectively, were degraded. As essential host cell factor for the replication of the pandemic SARS-CoV-2, the TTSP TMPRSS2 emerged as an important target in drug design. Our study provides further preclinical data on the characterization of this type of inhibitors for numerous trypsin-like serine proteases.

Phenotypic responses and potential genetic mechanism of lepidopteran insects under exposure to graphene oxide

Clarification of the interactions between engineered nanomaterials and multiple generations of insects is crucial to understanding the impact of nanotechnology on the environment and agriculture, particularly in toxicity management, pest management and genetic engineering. To date, there has been very limited information about nanoparticle-insect interactions at the genetic and proteomic levels. Here, we examined the phenotypic responses and potential mechanism of a lepidopteran insect Asian corn borer (ACB) to graphene oxide (GO). It was demonstrated that GO could significantly promote the growth of ACB. The transcriptomic and proteomic results consistently verified that GO might activate trypsin-like serine protease, glutathione S-transferase, heat shock protein and glycosyltransferase to further influence the development of ACB. RNA interference results indicated that the trypsin gene was one of the critical genes to accelerate the growth of ACB fed with GO diet. Moreover, physiological analysis showed potential alterations of the expression levels of genes and proteins, and more cholesterol (CE), triacylglycerides (TG) and lipids were accumulated in GO-exposed ACB. Our findings may help to reveal the phenotypic, physiological and genetic responses of insects under exposure to nanomaterials and to assess the environmental risks of other nanomaterials.

Spatio-Temporal Profiling of Metarhizium anisopliae-Responsive microRNAs Involved in Modulation of Plutella xylostella Immunity and Development.

Metarhizium anisopliae, a ubiquitous pathogenic fungus, regulates a wide array of the insect pest population. The fungus has been employed to control Plutella xylostella, an insecticide-resistant destructive lepidopteran pest, which causes substantial economic losses in crops worldwide. Integration of modern gene-silencing technologies in pest control strategies has become more crucial to counter pesticide-resistant insects. MicroRNAs (miRNA) play essential roles in the various biological process via post-transcriptional gene regulation. In the present study, RNA-seq analysis of control (CK36h, CK72h) and fungal-infected (T36h, T72h) midguts was performed to reveal underlying molecular mechanisms occurring in larval midgut at different time courses. We aimed at exploring M. anisopliae-responsive miRNAs and their target genes involved in development and immunity. After data filtration, a combined set of 170 miRNAs were identified from all libraries. Interestingly, miR-281, miR-263, miR-1, miR-6094 and miR-8 were listed among the most abundantly expressed conserved miRNAs. Furthermore, we experimentally studied the role of differentially expressed miR-11912-5p in regulating corresponding target trypsin-like serine proteinase (Px_TLSP). The luciferase assay (in vitro) revealed that miRNA-11912-5p significantly downregulated its target gene, suggesting it might play a crucial role in defense mechanism of P. xylostella against M.+ anisopliae infection. We used synthetic miRNA mimic/inhibitor (in vivo), to overexpress/silence miRNA, which showed harmful effects on larval duration, survival and adult fecundity. Additionally, fungal application in the presence of mimics revealed enhanced sensitivity of P. xylostella to infection. Our finding provides an insight into the relatively obscure molecular mechanisms involved in insect midgut during the fungal infection.

KLK6 mediates stemness and metabolism of gastric carcinoma cells via the PI3K/AKT/mTOR signaling pathway.

Gastric cancer is a common tumor of the digestive system, which can occur in any part of the stomach. Kallikrein 6 (KLK6) is a trypsin-like serine protease and has been found to be involved in extracellular matrix remodeling, tumor invasion and nervous system plasticity. Our previous study reported that KLK6 suppressed HGC-27 gastric cancer cell growth by inhibiting epithelial-mesenchymal transition; however, the mechanism of action underlying the effect of KLK6 still remains unclear. The aim of the present study was to investigate the effect and the underlying mechanism of KLK6 on stem cell-like properties and metabolism in gastric carcinoma cells. The HGC-27 cell line was transfected with KLK6 overexpression (OV-KLK6) and interference (short hairpin-KLK6) vectors, then the transfection efficiency was confirmed using western blot analysis and reverse transcription-quantitative PCR. The percentage of CD133+ and CD44+ cells was detected using flow cytometry, while the protein expression levels of the stem-associated genes, Nanog, Oct-4, SOX2 and Notch1, the metabolic markers, hexokinase (HK)1, HK2, GLUT1, and the proteins within the PI3K signaling pathway, phosphorylated (p)-PI3K, p-AKT and p-mTOR, were determined using western blot analysis. Biochemical kits were used to measure ATP production, lactic acid content and glucose uptake. A tumorigenicity assay was performed with nude mice to detect gastric tumor volume, and the protein expression level of Oct-4, Nanog, HK1, HK2 and GLUT1, and the mRNA expression level of KLK6 was also determined in gastric tumor tissues of mice. Compared with that in the control group, KLK6 protein and mRNA expression levels were significantly decreased in the four sh-RNA groups (P<0.05). Among them, sh-RNA-3 induced the lowest KLK6 expression and was used to silence KLK6 in subsequent experiments. Compared with that in the control and negative control groups, the percentage of CD133+ and CD44+ cells, the protein expression level of Oct-4, Nanog, HK1, HK2, GLUT1, p-PI3K, p-AKT and p-mTOR, and ATP content, lactic acid production, glucose uptake and gastric tumor volume were significantly decreased by sh-KLK6 (P<0.05), whereas KLK6 overexpression induced the opposite effect (P<0.05). In conclusion, KLK6 modulated stemness properties and cell metabolic profile in gastric carcinoma cells and the mechanism may be associated with the PI3K/AKT/mTOR signaling pathway.

First Crystal Structure of Bacterial Oligopeptidase B in an Intermediate State: The Roles of the Hinge Region Modification and Spermine.

Simple SummaryOligopeptidase B is a two-domain, trypsin-like peptidase from parasitic protozoa and bacteria which belongs to the least studied group of prolyloligopeptidases. In this study, we describe for the first time a crystal structure of bacterial oligopeptidase B and compare it with those of protozoan oligopeptidases B and related prolyloligopeptidases. The enzyme was crystallized in the presence of spermine and contained a modified sequence of the interdomain linker. Both factors were key for crystallization. The structure showed an uncommon intermediate conformation with a domain arrangement intermediate between open and closed conformations found in the crystals of ligand-free and inhibitor-bound prolyloligopeptidases, respectively. To evaluate the impact of the modification and spermine in the obtained conformation, small-angle X-ray scattering was applied, which showed that in solution wild-type enzymes adopt the open conformation and spermine causes a transition to the intermediate state, while the modification is associated with a partial transition. We suggest that spermine-dependent conformational transition replicates the behavior of the enzyme in bacterial cells and the intermediate state, which is rarely detected in vitro, and might be widely distributed in vivo, and so should be considered during computational studies, including those aimed wanting to develop the small molecule inhibitors targeting prolyloligopeptidases.Oligopeptidase B (OpB) is a two-domain, trypsin-like serine peptidase belonging to the S9 prolyloligopeptidase (POP) family. Two domains are linked by a hinge region that participates in the transition of the enzyme between two major states—closed and open—in which domains and residues of the catalytic triad are located close to each other and separated, respectively. In this study, we described, for the first time, a structure of OpB from bacteria obtained for an enzyme from Serratia proteomaculans with a modified hinge region (PSPmod). PSPmod was crystallized in a conformation characterized by a disruption of the catalytic triad together with a domain arrangement intermediate between open and closed states found in crystals of ligand-free and inhibitor-bound POP, respectively. Two additional derivatives of PSPmod were crystallized in the same conformation. Neither wild-type PSP nor its corresponding mutated variants were susceptible to crystallization, indicating that the hinge region modification was key in the crystallization process. The second key factor was suggested to be polyamine spermine since all crystals were grown in its presence. The influences of the hinge region modification and spermine on the conformational state of PSP in solution were evaluated by small-angle X-ray scattering. SAXS showed that, in solution, wild-type PSP adopted the open state, spermine caused the conformational transition to the intermediate state, and spermine-free PSPmod contained molecules in the open and intermediate conformations in dynamic equilibrium.

Antagonism of Protease Activated Receptor-2 by GB88 Reduces Inflammation Triggered by Protease Allergen Tyr-p3

The occurrence of allergic diseases induced by aeroallergens has increased in the past decades. Among inhalant allergens, mites remain the important causal agent of allergic diseases. Storage mites- Tyrophagus putrescentiae are found in stored products or domestic environments. Major allergen Tyr-p3 plays a significant role in triggering IgE-mediated hypersensitivity. However, its effects on pulmonary inflammation, internalization, and activation in human epithelium remain elusive. Protease-activated receptors (PARs) are activated upon cleavage by proteases. A549 cells were used as an epithelial model to examine the PAR activation by Tyr-p3 and therapeutic potential of PAR-2 antagonist (GB88) in allergic responses. Enzymatic properties and allergen localization of Tyr-p3 were performed. The release of inflammatory mediators, phosphorylation of mitogen-activated protein kinase (MAPK), and cell junction disruptions were evaluated after Tyr-p3 challenge. Enzymatic properties determined by substrate digestion and protease inhibitors indicated that Tyr-p3 processes a trypsin-like serine protease activity. The PAR-2 mRNA levels were significantly increased by nTyr-p3 but inhibited by protease inhibitors or GB88. Protease allergen of nTyr-p3 significantly increased the levels of pro-inflammatory cytokines (IL-6 and TNF-α), chemokine (IL-8), and IL-1β in epithelial cells. nTyr-p3 markedly increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and MAP kinase. When cells were pretreated with GB88 then added nTyr-p3, the phosphorylated ERK1/2 did not inhibit by GB88. GB88 increased ERK1/2 phosphorylation in human epithelium cells. GB88 is able to block PAR-2-mediated calcium signaling which inhibits the nTyr-p3-induced Ca2+ release. Among the pharmacologic inhibitors, the most effective inhibitor of the nTyr-p3 in the induction of IL-8 or IL-1β levels was GB88 followed by SBTI, MAPK/ERK, ERK, and p38 inhibitors. Levels of inflammatory mediators, including GM-CSF, VEGF, COX-2, TSLP, and IL-33 were reduced by treatment of GB88 or SBTI. Further, GB88 treatment down-regulated the nTyr-p3-induced PAR-2 expression in allergic patients with asthma or rhinitis. Tight junction and adherens junction were disrupted in epithelial cells by nTyr-p3 exposure; however, this effect was avoided by GB88. Immunostaining with frozen sections of the mite body showed the presence of Tyr-p3 throughout the intestinal digestive system, especially in the hindgut around the excretion site. In conclusion, our findings suggest that Tyr-p3 from domestic mites leads to disruption of the airway epithelial barrier after inhalation. Proteolytic activity of Tyr-p3 causes the PAR-2 mRNA expression, thus leading to the release of numerous inflammatory mediators. Antagonism of PAR2 activity suggests GB88 as the therapeutic potential for anti-inflammation medicine, especially in allergy development triggered by protease allergens.

Identification of 13 Guanidinobenzoyl- or Aminidinobenzoyl-Containing Drugs to Potentially Inhibit TMPRSS2 for COVID-19 Treatment.

Positively charged groups that mimic arginine or lysine in a natural substrate of trypsin are necessary for drugs to inhibit the trypsin-like serine protease TMPRSS2 that is involved in the viral entry and spread of coronaviruses, including SARS-CoV-2. Based on this assumption, we identified a set of 13 approved or clinically investigational drugs with positively charged guanidinobenzoyl and/or aminidinobenzoyl groups, including the experimentally verified TMPRSS2 inhibitors Camostat and Nafamostat. Molecular docking using the C-I-TASSER-predicted TMPRSS2 catalytic domain model suggested that the guanidinobenzoyl or aminidinobenzoyl group in all the drugs could form putative salt bridge interactions with the side-chain carboxyl group of Asp435 located in the S1 pocket of TMPRSS2. Molecular dynamics simulations further revealed the high stability of the putative salt bridge interactions over long-time (100 ns) simulations. The molecular mechanics/generalized Born surface area-binding free energy assessment and per-residue energy decomposition analysis also supported the strong binding interactions between TMPRSS2 and the proposed drugs. These results suggest that the proposed compounds, in addition to Camostat and Nafamostat, could be effective TMPRSS2 inhibitors for COVID-19 treatment by occupying the S1 pocket with the hallmark positively charged groups.

Hepatopancreas transcriptome analysis reveals the molecular responses to different dietary n-3 PUFA lipid sources in the swimming crab Portunus trituberculatus

n-3 LC-PUFA is vital to crustaceans for the growth, molting and development, while the precise molecular mechanism underlying the regulation of n-3 LC-PUFA is poorly investigated. Herein, an 8-week nutritional trial and transcriptome analysis were used to investigate the impacts of dietary n-3 PUFA lipid sources including fish oil (FO), krill oil (KO) and linseed oil (LO) on the transcriptome characteristics in hepatopancreas of swimming crab (Portunus trituberculatus). Crabs fed KO diet had significantly higher percent weight gain (PWG) and molting ratio (MR) than those of crabs fed other diets. The transcriptome analysis reveled that a total of 6567 unigenes were annotated in KEGG database, of which the expression of 1126 significantly altered in the KO vs FO group, while the expression of 921 unigenes changed in the LO vs FO group. The differentially expressed genes (DEGs) in KO vs FO comparison group involved in fat digestion and absorption, ABC transporters, adipocytokine signaling pathway, protein digestion and absorption, glycolysis/gluconeogenesis and HIF-1 signaling pathway. While dietary LO significantly altered the expression levels of genes involved in carbohydrate digestion and absorption, mineral absorption, protein processing in endoplasmic reticulum, glycosphingolipid biosynthesis and insect hormone biosynthesis. In addition, dietary KO significantly up-regulated the expression levels of cyp18a1 (participates in the process of ecdysteroids biosynthesis and further controls the molting of crustaceans), tryp (trypsin-like serine proteinases, resists the pathogen invasion), pla2 (phospholipase A2, regulates different inflammatory processes), ABC transporter (ATP-binding cassette transporter, transports amino acids, lipids and proteins into and out of the cell), cpt1 (carnitine palmitoyltransferase I, the major rate-limiting enzyme of mitochondrial fatty acid β-oxidation) and slc2 (solute carrier 2, mediates bidirectional glucose transport). Meanwhile, dietary LO could significantly down-regulated the expression level of cyp315a1 compared to dietary FO. To sum up, swimming crab fed with dietary krill oil could not only enhance the transport of glucose, lipids, fatty acids and other substrates, but also up-regulate the expression levels of genes related to molting, immunity and energy metabolism to promote molting and growth of swimming crab. The present study revealed the molecular responses of crabs to different dietary n-3 PUFA lipid sources, and further provided important clues with respect to elucidating the molecular mechanisms underlying the regulation of EPA and DHA on the molting and growth of swimming crab.

Structural and Computational Investigations of an Autoinhibitory Loop in Human Mesorypsin

There is evidence in the literature that many serine proteases, as typified by thrombin, possess an autoinhibitory loop (AIL) that controls substrate access to the active site. The presence of an AIL has not been documented in trypsins. Mesotrypsin is a human trypsin implicated in tumor cell invasiveness for pancreatic and other cancers. However, it is a challenging therapeutic target because of high structural similarity to other trypsin-like serine proteases, typically lower affinity for inhibitors compared to related serine proteases, and unique ability to cleave proteinaceous serine protease inhibitors. We have recently solved a high-resolution crystal structure of unbound mesotrypsin which revealed that residues 215-220 partially occluded the substrate binding site. Comparison of this new structure with previous inhibitor-bound crystal structures of mesotrypsin demonstrate the conformational transition of the AIL between closed and open states. These two conformational states were investigated with molecular dynamics simulations to discern the dynamical behavior of the AIL in different conformations. The AIL has increased local dynamics in the closed position, as well as a larger number of conformational clusters; this suggests there is a more well-defined open state ready to accept substrate, while there are a dynamic variety of closed states that preclude substrate binding to various extents. Additionally, we identified novel binding sites that are exposed by the conformational shift of the AIL to the closed state. We have conducted initial in silico screening for compounds predicted to bind preferentially to the AIL closed (but not open) structure and stabilize the closed conformation. These efforts have resulted in more than 40 compounds as initial hits. Exploiting the AIL as a novel allosteric binding site represents a new mechanism for targeting mesotrypsin and preventing its protumorigenic function.

Bio-Insecticide of Thymus vulgaris and Ocimum basilicum Extract from Cell Suspensions and Their Inhibitory Effect against Serine, Cysteine, and Metalloproteinases of the Red Palm Weevil (Rhynchophorus ferrugineus).

Simple SummaryMost thyme and basil species are characterized by a great chemical diversity. Uses have been developed through centuries in foods, as a source of essential oil, flavors, and antioxidants. The main aim of this study was to produce volatile metabolites using cell suspensions of Thymus vulgaris and Ocimum basilicum from the Al-Ahsa area in the sub-continental region. We surveyed the antifeedant activity of extracted volatile metabolites and the inhibition of extracts against total proteolytic enzyme activity from the red palm weevil Rhynchophorus ferrugineus (Oliver), (Coleoptera: Curculionidae). Thymus vulgaris was the most active extract, characterized not only by feeding inhibition but also by a growing deterrence on R. ferrugineus larvae. The O. basilicum extract also showed a lower range of biological activity; nevertheless, there was potent insecticidal activity. The high insecticidal activity of the T. vulgaris extract could be attributed to the high diversity of its volatile constituents. One of the merits of the current approach is that the outcomes are applicable and have the environmental goal of producing ecofriendly biopesticides.The current study was designed to investigate the insecticide role of volatile constituents produced from cell suspensions of T. vulgaris and O. basilicum against R. ferrugineus. Constituents were extracted from cell suspension after 40 days. Growth kinetics were measured with an inoculation of Verticillium dahliae and identified by GC-MS. Total volatile phenolic constituents were measured. Insecticidal activity against R. ferrugineus (adult) and proteolytic enzyme activity in larvae were assessed. GC-MS showed that the T. vulgaris extract has higher amounts of thymol, p-cymene, γ-terpinene, β-caryophyllene, and linalool in comparison to the O. basilicum extract, which is rich in estragole, β-terpineol, (E)-β-ocimene, 1,8-cineole, germacrene D, and eugenol. The T. vulgaris extract showed an LC50 of 1032 µg/mL, followed by O. basilicum with an LC50 of 1246 µg/mL. The IC50 values against the total proteases were 110.8 and 119.4 µg/mL for T. vulgaris and O. basilicum, respectively. The IC50 for the trypsin-like serine proteinase assessment was 81.6 and 91 µg/mL for T. vulgaris and O. basilicum, respectively. Cysteine, chymotrypsin, and metalloproteinase assessment showed an IC50 above 5000 µg/mL for both extracts. The study is proposed as a potential approach to use T. vulgaris and O. basilicum extract as a bio-insecticide against R. ferrugineus using an accessible and efficient cell suspension technique.

Screening of 5- and 6-Substituted Amiloride Libraries Identifies Dual-uPA/NHE1 Active and Single Target-Selective Inhibitors

The K+-sparing diuretic amiloride shows off-target anti-cancer effects in multiple rodent models. These effects arise from the inhibition of two distinct cancer targets: the trypsin-like serine protease urokinase-type plasminogen activator (uPA), a cell-surface mediator of matrix degradation and tumor cell invasiveness, and the sodium-hydrogen exchanger isoform-1 (NHE1), a central regulator of transmembrane pH that supports carcinogenic progression. In this study, we co-screened our library of 5- and 6-substituted amilorides against these two targets, aiming to identify single-target selective and dual-targeting inhibitors for use as complementary pharmacological probes. Closely related analogs substituted at the 6-position with pyrimidines were identified as dual-targeting (pyrimidine 24 uPA IC50 = 175 nM, NHE1 IC50 = 266 nM, uPA selectivity ratio = 1.5) and uPA-selective (methoxypyrimidine 26 uPA IC50 = 86 nM, NHE1 IC50 = 12,290 nM, uPA selectivity ratio = 143) inhibitors, while high NHE1 potency and selectivity was seen with 5-morpholino (29 NHE1 IC50 = 129 nM, uPA IC50 = 10,949 nM; NHE1 selectivity ratio = 85) and 5-(1,4-oxazepine) (30 NHE1 IC50 = 85 nM, uPA IC50 = 5715 nM; NHE1 selectivity ratio = 67) analogs. Together, these amilorides comprise a new toolkit of chemotype-matched, non-cytotoxic probes for dissecting the pharmacological effects of selective uPA and NHE1 inhibition versus dual-uPA/NHE1 inhibition.

Proteinase-Mediated Macrophage Signaling in Psoriatic Arthritis.

ObjectiveMultiple proteinases are present in the synovial fluid (SF) of an arthritic joint. We aimed to identify inflammatory cell populations present in psoriatic arthritis (PsA) SF compared to osteoarthritis (OA) and rheumatoid arthritis (RA), identify their proteinase-activated receptor 2 (PAR2) signaling function and characterize potentially active SF serine proteinases that may be PAR2 activators.MethodsFlow cytometry was used to characterize SF cells from PsA, RA, OA patients; PsA SF cells were further characterized by single cell 3’-RNA-sequencing. Active serine proteinases were identified through cleavage of fluorogenic trypsin- and chymotrypsin-like substrates, activity-based probe analysis and proteomics. Fluo-4 AM was used to monitor intracellular calcium cell signaling. Cytokine expression was evaluated using a multiplex Luminex panel.ResultsPsA SF cells were dominated by monocytes/macrophages, which consisted of three populations representing classical, non-classical and intermediate cells. The classical monocytes/macrophages were reduced in PsA compared to OA/RA, whilst the intermediate population was increased. PAR2 was elevated in OA vs. PsA/RA SF monocytes/macrophages, particularly in the intermediate population. PAR2 expression and signaling in primary PsA monocytes/macrophages significantly impacted the production of monocyte chemoattractant protein-1 (MCP-1). Trypsin-like serine proteinase activity was elevated in PsA and RA SF compared to OA, while chymotrypsin-like activity was elevated in RA compared to PsA. Tryptase-6 was identified as an active serine proteinase in SF that could trigger calcium signaling partially via PAR2.ConclusionPAR2 and its activating proteinases, including tryptase-6, can be important mediators of inflammation in PsA. Components within this proteinase-receptor axis may represent novel therapeutic targets.

Study on cocoonase, sericin, and degumming of silk cocoon: computational and experimental

BackgroundCocoonase is a proteolytic enzyme that helps in dissolving the silk cocoon shell and exit of silk moth. Chemicals like anhydrous Na2CO3, Marseille soap, soda, ethylene diamine and tartaric acid-based degumming of silk cocoon shell have been in practice. During this process, solubility of sericin protein increased resulting in the release of sericin from the fibroin protein of the silk. However, this process diminishes natural color and softness of the silk. Cocoonase enzyme digests the sericin protein of silk at the anterior portion of the cocoon without disturbing the silk fibroin. However, no thorough characterization of cocoonase and sericin protein as well as imaging analysis of chemical- and enzyme-treated silk sheets has been carried out so far. Therefore, present study aimed for detailed characterization of cocoonase and sericin proteins, phylogenetic analysis, secondary and tertiary structure prediction, and computational validation as well as their interaction with other proteins. Further, identification of tasar silkworm (Antheraea mylitta) pupa stage for cocoonase collection, its purification and effect on silk sheet degumming, scanning electron microscope (SEM)-based comparison of chemical- and enzyme-treated cocoon sheets, and its optical coherence tomography (OCT)-based imaging analysis have been investigated. Various computational tools like Molecular Evolutionary Genetics Analysis (MEGA) X and Figtree, Iterative Threading Assembly Refinement (I-TASSER), self-optimized predicted method with alignment (SOPMA), PROCHECK, University of California, San Francisco (UCSF) Chimera, and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) were used for characterization of cocoonase and sericin proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein purification using Sephadex G 25-column, degumming of cocoon sheet using cocoonase enzyme and chemical Na2CO3, and SEM and OCT analysis of degummed cocoon sheet were performed. ResultsPredicted normalized B-factors of cocoonase and sericin with respect to α and β regions showed that these regions are structurally more stable in cocoonase while less stable in sericin. Conserved domain analysis revealed that B. mori cocoonase contains a trypsin-like serine protease with active site range 45 to 180 query sequences while substrate binding site from 175 to 200 query sequences. SDS-PAGE analysis of cocoonase indicated its molecular weight of 25–26 kDa. Na2CO3 treatment showed more degumming effect (i.e., cocoon sheet weight loss) as compared to degumming with cocoonase. However, cocoonase-treated silk cocoon sheet holds the natural color of tasar silk, smoothness, and luster compared with the cocoon sheet treated with Na2CO3. SEM-based analysis showed the noticeable variation on the surface of silk fiber treated with cocoonase and Na2CO3. OCT analysis also exemplified the variations in the cross-sectional view of the cocoonase and Na2CO3-treated silk sheets. ConclusionsPresent study enlightens on the detailed characteristics of cocoonase and sericin proteins, comparative degumming activity, and image analysis of cocoonase enzyme and Na2CO3 chemical-treated silk sheets. Obtained findings illustrated about use of cocoonase enzyme in the degumming of silk cocoon at larger scale that will be a boon to the silk industry.

Exploration of Active Site-Directed Plasmin Inhibitors: Beyond Tranexamic Acid

Plasmin (Plm), a trypsin-like serine protease, is responsible for fibrinolysis pathway and pathologic events, such as angiogenesis, tumor invasion, and metastasis, and alters the expression of cytokines. A growing body of data indicates that a Plm inhibitor is a potential candidate as an anti-inflammatory and anti-cancer agent. A class of active site-directed plasmin inhibitors containing tranexamic acid residue has been designed. As evidenced by docking studies, the inhibitor binds to the active site not to the lysine binding site (LBS) in plasmin, thus preventing plasmin from digesting the substrate. Further optimization of the series, concerning both activity and selectivity, led to the second generation of inhibitors. This review focuses on the Plm inhibitory activity-structure relationship of Plm inhibitors with the goal of realizing their design and clinical application.