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Parasites & Vectors Apr 2017Aspartyl protease inhibitor (API) was thought to protect intestinal parasitic nematodes from their hostile proteolytic environment. Studies on Ostertagia ostertagi,...
BACKGROUND
Aspartyl protease inhibitor (API) was thought to protect intestinal parasitic nematodes from their hostile proteolytic environment. Studies on Ostertagia ostertagi, Ascaris suum and Brugia malayi indicated that aspins might play roles in nematode infection. In a recent study, proteins differentially expressed between free-living third-stage larvae (L3) and activated L3 (xL3) of Haemonchus contortus were identified by 2D-DIGE. API was found downregulated in xL3 when compared with L3. However, there was no report about the functions of H. contortus API in the parasite-host interaction. In this study, the gene encoding API from H. contortus was cloned, expressed, and part of its biological characteristics were studied.
RESULTS
A DNA fragment of 681 bp was amplified by RT-PCR. Ninety one percent of the amino acid sequence was similar with that for aspin from O. ostertagi. The recombinant API protein was fusion-expressed with a molecular weight of 48 × 10. Results of Western blot showed that the recombinant API could be recognized by serum from goat infected with H. contortus. It was found that API was localized exclusively in the subcutaneous tissue and epithelial cells of the gastrointestinal tract in adult H. contortus. qRT-PCR suggested that the API gene was differentially transcribed in different life-cycle stages, with the lowest level in female adults and the highest in free-living L3 larvae. Enzyme inhibition assay indicated that the recombinant API can inhibit the activity of pepsin significantly, and the optimal reaction pH and temperature were 4.0 and 37-50 °C respectively. In vitro study showed that the recombinant API could induce goat PBMCs to express IFN-γ, IL-4 and IL-10.
CONCLUSIONS
A new aspartyl protease inhibitor was cloned from H. contortus and its characteristics were studied for the first time. The results indicate that API may regulate the immune response of the host and play roles in the infection.
Topics: Animals; Aspartic Acid Proteases; Cloning, Molecular; Cytokines; Female; Goats; Haemonchiasis; Haemonchus; Host-Parasite Interactions; Leukocytes, Mononuclear; Life Cycle Stages; Male; Pepsin A; Protease Inhibitors; Real-Time Polymerase Chain Reaction; Recombinant Proteins
PubMed: 28420411
DOI: 10.1186/s13071-017-2137-1 -
Archives of Razi Institute Apr 2022Nowadays dengue virus infection (DENV) is one of the major health complications in the world. Although DENV is an old and common disease, unfortunately, until now, there...
Nowadays dengue virus infection (DENV) is one of the major health complications in the world. Although DENV is an old and common disease, unfortunately, until now, there are no specific relevant treatments available for it. This study, therefore, aimed to design, as well as synthesize selective peptide inhibitors, and investigate their activity by NS2B/NS3 protease inhibition assay. The design of the peptide ligands was based on studying the interactions with the dengue NS2B/NS3 protease using the computational docking technique in the MOE and AutoDock (version 4.2) software. To this end, the researchers designed 26 linear pentapeptides based on previous studies. It was revealed that two linear pentapeptides (i.e., GKRRK and KRRRK) are the best potential inhibitors. Furthermore, based on the findings of the two independent docking programs, the peptide GKRRK was synthesized by solid-phase peptide synthesis and its structure was confirmed. The protease inhibitor study was conducted for these two peptides to examine their activity against the dengue virus using a protin in as a control. It was found that the designed potential peptides possess interesting inhibition against the NS2B/NS3 protease. Additionally, the findings showed that the peptide GKRRK had the highest percentage of inhibition (71.11%) at 100 µM with the IC of 48.87 µM; therefore, this linear peptide could serve as a good inhibitor for the DENV.
Topics: Dengue; Dengue Virus; Molecular Docking Simulation; Peptide Hydrolases; Peptides; Protease Inhibitors
PubMed: 36284983
DOI: 10.22092/ARI.2022.357124.1980 -
Preventive Medicine Sep 1993There is now compelling evidence that certain protease inhibitors have strong anticarcinogenic activity. A particular protease inhibitor derived from soybeans, the... (Review)
Review
BACKGROUND
There is now compelling evidence that certain protease inhibitors have strong anticarcinogenic activity. A particular protease inhibitor derived from soybeans, the Bowman-Birk inhibitor, appears to be highly promising as a cancer chemopreventive agent. In this article, studies performed to characterize an extract of soybeans containing the Bowman-Birk inhibitor, termed Bowman-Birk inhibitor concentrate, are described.
METHODS
A number of different methods have been utilized to evaluate the efficacy and safety of Bowman-Birk inhibitor concentrate as a cancer chemopreventive agent in cells and animals. Bowman-Birk inhibitor concentrate has been studied extensively in animal carcinogenesis model systems and in vitro transformation systems.
RESULTS
Bowman-Birk inhibitor has been shown to prevent animal carcinogenesis in the absence of adverse side effects in several different systems. The dose proposed for human trials is approximately two orders of magnitude below doses shown to have no adverse effects in animal studies. At high doses, Bowman-Birk inhibitor concentrate has been shown to have a life-lengthening effect in mice. The intermediate marker endpoints known to be affected by Bowman-Birk inhibitor/Bowman-Birk inhibitor concentrate in in vitro and in vivo models systems include levels of oncogene expression and proteolytic activity. These intermediate marker endpoints, as well as histopathology, have been proposed for use in projected human trials which will be performed to evaluate Bowman-Birk inhibitor concentrate as an anticarcinogenic agent.
CONCLUSIONS
Bowman-Birk inhibitor concentrate is now ready for human trials and is likely to emerge as a powerful chemopreventive agent for human cancer.
Topics: Animals; Anticarcinogenic Agents; Cell Transformation, Neoplastic; Clinical Trials as Topic; Drug Screening Assays, Antitumor; Humans; Neoplasms; Neoplasms, Experimental; Protease Inhibitors
PubMed: 8234219
DOI: 10.1006/pmed.1993.1073 -
The Journal of Biological Chemistry Dec 1988A low molecular weight serine protease inhibitor, named trypstatin, was purified from rat peritoneal mast cells. It is a single polypeptide with 61 amino acid residues...
A low molecular weight serine protease inhibitor, named trypstatin, was purified from rat peritoneal mast cells. It is a single polypeptide with 61 amino acid residues and an Mr of 6610. Trypstatin markedly inhibits blood coagulation factor Xa (Ki = 1.2 x 10(-10) M) and tryptase (Ki = 3.6 x 10(-10) M) from rat mast cells, which have activities that convert prothrombin to thrombin. It also inhibits porcine pancreatic trypsin (Ki = 1.4 x 10(-8) M) and chymase (Ki = 2.4 x 10(-8) M) from rat mast cells, but not papain, alpha-thrombin, or porcine pancreatic elastase. Trypstatin forms a complex in a molar ratio of 1:1 with trypsin and one subunit of tryptase. The complete amino acid sequence of this inhibitor was determined and compared with those of Kunitz-type inhibitors. Trypstatin has a high degree of sequence homology with human and bovine inter-alpha-trypsin inhibitors, A4(751) Alzheimer's disease amyloid protein precursor, and basic pancreatic trypsin inhibitor. However, unlike other known Kunitz-type protease inhibitors, it inhibits factor Xa most strongly.
Topics: Amino Acid Sequence; Animals; Male; Mast Cells; Molecular Sequence Data; Protease Inhibitors; Proteins; Rats; Rats, Inbred Strains; Sequence Homology, Nucleic Acid; Serine Proteinase Inhibitors; Trypsin Inhibitor, Kunitz Soybean; Trypsin Inhibitors
PubMed: 3263966
DOI: No ID Found -
Bioorganic Chemistry Jul 2022The emerging COVID-19 pandemic generated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has severely threatened human health. The main protease (M) of...
The emerging COVID-19 pandemic generated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has severely threatened human health. The main protease (M) of SARS-CoV-2 is promising target for antiviral drugs, which plays a vital role for viral duplication. Development of the inhibitor against M is an ideal strategy to combat COVID-19. In this work, twenty-three hydroxamates 1a-i and thiosemicarbazones 2a-n were identified by FRET screening to be the potent inhibitors of M, which exhibited more than 94% (except 1c) and more than 69% inhibition, and an IC value in the range of 0.12-31.51 and 2.43-34.22 μM, respectively. 1a and 2b were found to be the most effective inhibitors in the hydroxamates and thiosemicarbazones, with an IC of 0.12 and 2.43 μM, respectively. Enzyme kinetics, jump dilution and thermal shift assays revealed that 2b is a competitive inhibitor of M, while 1a is a time-dependently inhibitor; 2b reversibly but 1a irreversibly bound to the target; the binding of 2b increased but 1a decreased stability of the target, and DTT assays indicate that 1a is the promiscuous cysteine protease inhibitor. Cytotoxicity assays showed that 1a has low, but 2b has certain cytotoxicity on the mouse fibroblast cells (L929). Docking studies revealed that the benzyloxycarbonyl carbon of 1a formed thioester with Cys145, while the phenolic hydroxyl oxygen of 2b formed H-bonds with Cys145 and Asn142. This work provided two promising scaffolds for the development of M inhibitors to combat COVID-19.
Topics: Animals; Antiviral Agents; Coronavirus 3C Proteases; Humans; Mice; Molecular Docking Simulation; Pandemics; Protease Inhibitors; SARS-CoV-2; Thiosemicarbazones; COVID-19 Drug Treatment
PubMed: 35462235
DOI: 10.1016/j.bioorg.2022.105799 -
Anesthesiology Jan 2009In cardiac surgery, the contact of blood with the artificial surfaces of the cardiopulmonary bypass results in activation of coagulation, fibrinolysis, and platelets,... (Comparative Study)
Comparative Study
BACKGROUND
In cardiac surgery, the contact of blood with the artificial surfaces of the cardiopulmonary bypass results in activation of coagulation, fibrinolysis, and platelets, which is recognized as reason for increased bleeding tendency. Antifibrinolytics like tranexamic acid or the broad-spectrum protease inhibitor aprotinin attenuate this response. The marketing of aprotinin has been suspended after a recent clinical trial suggested increased risks associated with aprotinin. Moreover, aprotinin is a protein of animal origin and has antigenic properties. As a result, alternative antifibrinolytic compounds are desirable.
METHODS
This in vitro study compared the antifibrinolytic efficacy of the synthetic small molecule CU-2010 with aprotinin and tranexamic acid. Antifibrinolytic activity in plasma and whole blood of ten healthy volunteers was examined with a turbidometric method and with tissue factor-activated thromboelastometry (ROTEM; Pentapharm, Munich, Germany). In addition, anticoagulant effects were assessed through measurement of plasma and whole blood clotting times and thrombin generation.
RESULTS
With its high affinity for plasmin (Ki, 2 nM), CU-2010 inhibited fibrinolysis comparable to aprotinin (Ki, 4 nM) and was ten times more potent than tranexamic acid. CU-2010 also inhibited plasma kallikrein (Ki < 1 nM) and factors Xa (Ki, 45 nM) and XIa (Ki, 18nM), which was reflected in prolongation of coagulation times and an attenuation of thrombin generation.
CONCLUSION
These findings suggest that CU-2010 has similar antifibrinolytic potency compared to aprotinin, is more potent than tranexamic acid, and possesses some anticoagulant effects.
Topics: Animals; Anticoagulants; Antifibrinolytic Agents; Aprotinin; Blood Coagulation; Cattle; Dose-Response Relationship, Drug; Humans; Protease Inhibitors; Serine Proteinase Inhibitors
PubMed: 19104179
DOI: 10.1097/ALN.0b013e318191408c -
European Journal of Medicinal Chemistry Oct 2021This paper presents the design and study of a first-in-class cyclic peptide inhibitor against the SARS-CoV-2 main protease (M). The cyclic peptide inhibitor is designed...
This paper presents the design and study of a first-in-class cyclic peptide inhibitor against the SARS-CoV-2 main protease (M). The cyclic peptide inhibitor is designed to mimic the conformation of a substrate at a C-terminal autolytic cleavage site of M. The cyclic peptide contains a [4-(2-aminoethyl)phenyl]-acetic acid (AEPA) linker that is designed to enforce a conformation that mimics a peptide substrate of M. In vitro evaluation of the cyclic peptide inhibitor reveals that the inhibitor exhibits modest activity against M and does not appear to be cleaved by the enzyme. Conformational searching predicts that the cyclic peptide inhibitor is fairly rigid, adopting a favorable conformation for binding to the active site of M. Computational docking to the SARS-CoV-2 M suggests that the cyclic peptide inhibitor can bind the active site of M in the predicted manner. Molecular dynamics simulations provide further insights into how the cyclic peptide inhibitor may bind the active site of M. Although the activity of the cyclic peptide inhibitor is modest, its design and study lays the groundwork for the development of additional cyclic peptide inhibitors against M with improved activities.
Topics: Coronavirus 3C Proteases; Drug Design; HEK293 Cells; Humans; Molecular Docking Simulation; Molecular Dynamics Simulation; Peptides, Cyclic; Protease Inhibitors; Protein Conformation
PubMed: 34023738
DOI: 10.1016/j.ejmech.2021.113530 -
International Journal of Molecular... Aug 2023Biodiversity within the animal kingdom is associated with extensive molecular diversity. The expansion of genomic, transcriptomic and proteomic data sets for...
Biodiversity within the animal kingdom is associated with extensive molecular diversity. The expansion of genomic, transcriptomic and proteomic data sets for invertebrate groups and species with unique biological traits necessitates reliable in silico tools for the accurate identification and annotation of molecules and molecular groups. However, conventional tools are inadequate for lesser-known organismal groups, such as eukaryotic pathogens (parasites), so that improved approaches are urgently needed. Here, we established a combined sequence- and structure-based workflow system to harness well-curated publicly available data sets and resources to identify, classify and annotate proteases and protease inhibitors of a highly pathogenic parasitic roundworm (nematode) of global relevance, called (barber's pole worm). This workflow performed markedly better than conventional, sequence-based classification and annotation alone and allowed the first genome-wide characterisation of protease and protease inhibitor genes and gene products in this worm. In total, we identified 790 genes encoding 860 proteases and protease inhibitors representing 83 gene families. The proteins inferred included 280 metallo-, 145 cysteine, 142 serine, 121 aspartic and 81 "mixed" proteases as well as 91 protease inhibitors, all of which had marked physicochemical diversity and inferred involvements in >400 biological processes or pathways. A detailed investigation revealed a remarkable expansion of some protease or inhibitor gene families, which are likely linked to parasitism (e.g., host-parasite interactions, immunomodulation and blood-feeding) and exhibit stage- or sex-specific transcription profiles. This investigation provides a solid foundation for detailed explorations of the structures and functions of proteases and protease inhibitors of and related nematodes, and it could assist in the discovery of new drug or vaccine targets against infections or diseases.
Topics: Animals; Male; Female; Haemonchus; Parasites; Host-Parasite Interactions; Peptide Hydrolases; Proteomics; Protease Inhibitors; Nematoda; Endopeptidases; Informatics
PubMed: 37569696
DOI: 10.3390/ijms241512320 -
Molecules (Basel, Switzerland) Feb 2023It was found that silkworm serine protease inhibitors BmSPI38 and BmSPI39 were very different from typical TIL-type protease inhibitors in sequence, structure, and...
It was found that silkworm serine protease inhibitors BmSPI38 and BmSPI39 were very different from typical TIL-type protease inhibitors in sequence, structure, and activity. BmSPI38 and BmSPI39 with unique structure and activity may be good models for studying the relationship between the structure and function of small-molecule TIL-type protease inhibitors. In this study, site-directed saturation mutagenesis at the P1 position was conducted to investigate the effect of P1 sites on the inhibitory activity and specificity of BmSPI38 and BmSPI39. In-gel activity staining and protease inhibition experiments confirmed that BmSPI38 and BmSPI39 could strongly inhibit elastase activity. Almost all mutant proteins of BmSPI38 and BmSPI39 retained the inhibitory activities against subtilisin and elastase, but the replacement of P1 residues greatly affected their intrinsic inhibitory activities. Overall, the substitution of Gly54 in BmSPI38 and Ala56 in BmSPI39 with Gln, Ser, or Thr was able to significantly enhance their inhibitory activities against subtilisin and elastase. However, replacing P1 residues in BmSPI38 and BmSPI39 with Ile, Trp, Pro, or Val could seriously weaken their inhibitory activity against subtilisin and elastase. The replacement of P1 residues with Arg or Lys not only reduced the intrinsic activities of BmSPI38 and BmSPI39, but also resulted in the acquisition of stronger trypsin inhibitory activities and weaker chymotrypsin inhibitory activities. The activity staining results showed that BmSPI38(G54K), BmSPI39(A56R), and BmSPI39(A56K) had extremely high acid-base and thermal stability. In conclusion, this study not only confirmed that BmSPI38 and BmSPI39 had strong elastase inhibitory activity, but also confirmed that P1 residue replacement could change their activity and inhibitory specificity. This not only provides a new perspective and idea for the exploitation and utilization of BmSPI38 and BmSPI39 in biomedicine and pest control, but also provides a basis or reference for the activity and specificity modification of TIL-type protease inhibitors.
Topics: Animals; Protease Inhibitors; Bombyx; Amino Acid Substitution; Amino Acid Sequence; Serine Proteinase Inhibitors; Subtilisins; Pancreatic Elastase
PubMed: 36903318
DOI: 10.3390/molecules28052073 -
Bioorganic & Medicinal Chemistry Letters Jul 2018A series of tripeptidyl transition state inhibitors with new P1 and warhead moieties were synthesized and evaluated in a GI-1 norovirus replicon system and against GII-4...
A series of tripeptidyl transition state inhibitors with new P1 and warhead moieties were synthesized and evaluated in a GI-1 norovirus replicon system and against GII-4 and GI-1 norovirus proteases. Compound 19, containing a 6-membered ring at the P1 position and a reactive aldehyde warhead exhibited sub-micromolar replicon inhibition. Retaining the same peptidyl scaffold, several reactive warheads were tested for protease inhibition and norovirus replicon inhibition. Of the six that were synthesized and tested, compounds 42, 43, and 45 potently inhibited the protease in biochemical assay and GI-1 norovirus replicon in the nanomolar range.
Topics: Animals; Antiviral Agents; Cell Line; Cell Survival; Chlorocebus aethiops; Dose-Response Relationship, Drug; Humans; Microbial Sensitivity Tests; Molecular Structure; Norovirus; Peptide Hydrolases; Peptidomimetics; Protease Inhibitors; Structure-Activity Relationship; Vero Cells; Virus Replication
PubMed: 29779977
DOI: 10.1016/j.bmcl.2018.05.012