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Biomolecules Jul 2022Protease inhibitors are widely studied since the unrestricted activity of proteases can cause extensive organ lesions. In particular, elastase activity is involved in...
Protease inhibitors are widely studied since the unrestricted activity of proteases can cause extensive organ lesions. In particular, elastase activity is involved in the pathophysiology of acute lung injury, for example during SARS-CoV-2 infection, while serine proteases and thrombin-like proteases are involved in the development and/or pathology of the nervous system. Natural protease inhibitors have the advantage to be reversible and with few side effects and thus are increasingly considered as new drugs. Kunitz-type protease inhibitors (KTPIs), reported in the venom of various organisms, such as wasps, spiders, scorpions, and snakes, have been studied for their potent anticoagulant activity and widespread protease inhibitor activity. Putative KTPI anticoagulants have been identified in transcriptomic resources obtained for two blister beetle species, and . The KTPIs of and were characterized by combined transcriptomic and bioinformatics methodologies. The full-length mRNA sequences were divided on the base of the sequence of the active sites of the putative proteins. In silico protein structure analyses of each group of translational products show the biochemical features of the active sites and the potential protease targets. Validation of these genes is the first step for considering these molecules as new drugs for use in medicine.
Topics: Animals; COVID-19; Coleoptera; Protease Inhibitors; SARS-CoV-2; Serine Proteases
PubMed: 35883544
DOI: 10.3390/biom12070988 -
MBio Aug 2022Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to represent a global health emergency as a highly transmissible, airborne virus. An important...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to represent a global health emergency as a highly transmissible, airborne virus. An important coronaviral drug target for treatment of COVID-19 is the conserved main protease (M). Nirmatrelvir is a potent M inhibitor and the antiviral component of Paxlovid. The significant viral sequencing effort during the ongoing COVID-19 pandemic represented a unique opportunity to assess potential nirmatrelvir escape mutations from emerging variants of SARS-CoV-2. To establish the baseline mutational landscape of M prior to the introduction of M inhibitors, M sequences and its cleavage junction regions were retrieved from ~4,892,000 high-quality SARS-CoV-2 genomes in the open-access Global Initiative on Sharing Avian Influenza Data (GISAID) database. Any mutations identified from comparison to the reference sequence (Wuhan-Hu-1) were catalogued and analyzed. Mutations at sites key to nirmatrelvir binding and protease functionality (e.g., dimerization sites) were still rare. Structural comparison of M also showed conservation of key nirmatrelvir contact residues across the extended family (α-, β-, and γ-coronaviruses). Additionally, we showed that over time, the SARS-CoV-2 M enzyme remained under purifying selection and was highly conserved relative to the spike protein. Now, with the emergency use authorization (EUA) of Paxlovid and its expected widespread use across the globe, it is essential to continue large-scale genomic surveillance of SARS-CoV-2 M evolution. This study establishes a robust analysis framework for monitoring emergent mutations in millions of virus isolates, with the goal of identifying potential resistance to present and future SARS-CoV-2 antivirals. The recent authorization of oral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antivirals, such as Paxlovid, has ushered in a new era of the COVID-19 pandemic. The emergence of new variants, as well as the selective pressure imposed by antiviral drugs themselves, raises concern for potential escape mutations in key drug binding motifs. To determine the potential emergence of antiviral resistance in globally circulating isolates and its implications for the clinical response to the COVID-19 pandemic, sequencing of SARS-CoV-2 viral isolates before, during, and after the introduction of new antiviral treatments is critical. The infrastructure built herein for active genetic surveillance of M evolution and emergent mutations will play an important role in assessing potential antiviral resistance as the pandemic progresses and M inhibitors are introduced. We anticipate our framework to be the starting point in a larger effort for global monitoring of the SARS-CoV-2 M mutational landscape.
Topics: Animals; Antiviral Agents; COVID-19; Coronavirus 3C Proteases; Cysteine Endopeptidases; Drug Combinations; Humans; Lactams; Leucine; Nitriles; Pandemics; Proline; Protease Inhibitors; Ritonavir; SARS-CoV-2; Viral Nonstructural Proteins
PubMed: 35862764
DOI: 10.1128/mbio.00869-22 -
International Journal of Molecular... Jun 2009Antimicrobial proteins (peptides) are known to play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians... (Review)
Review
Antimicrobial proteins (peptides) are known to play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals. They are also known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses. Recently, the rapid emergence of microbial pathogens that are resistant to currently available antibiotics has triggered considerable interest in the isolation and investigation of the mode of action of antimicrobial proteins (peptides). Plants produce a variety of proteins (peptides) that are involved in the defense against pathogens and invading organisms, including ribosome-inactivating proteins, lectins, protease inhibitors and antifungal peptides (proteins). Specially, the protease inhibitors can inhibit aspartic, serine and cysteine proteinases. Increased levels of trypsin and chymotrypsin inhibitors correlated with the plants resistance to the pathogen. Usually, the purification of antimicrobial proteins (peptides) with protease inhibitor activity was accomplished by salt-extraction, ultrafiltration and C(18) reverse phase chromatography, successfully. We discuss the relation between antimicrobial and anti-protease activity in this review. Protease inhibitors from plants potently inhibited the growth of a variety of pathogenic bacterial and fungal strains and are therefore excellent candidates for use as the lead compounds for the development of novel antimicrobial agents.
Topics: Anti-Infective Agents; Bacteria; Candida albicans; Fabaceae; Peptides; Plants; Protease Inhibitors; Solanum tuberosum
PubMed: 19582234
DOI: 10.3390/ijms10062860 -
BMC Oral Health Dec 2021Oral dryness is a common symptom that may interfere with swallowing, chewing, and taste. The most common reason for oral dryness is hyposalivation. Some individuals...
BACKGROUND
Oral dryness is a common symptom that may interfere with swallowing, chewing, and taste. The most common reason for oral dryness is hyposalivation. Some individuals experiencing oral dryness do not have hyposalivation, however, and the reverse is also true. Here, we focused on healthy individuals with a lower salivary flow rate and evaluated the relationship between the perception of oral dryness and salivary parameters to clarify the cause underlying the perception of oral dryness.
METHODS
A total of 59 participants were divided into 2 groups with a lower or higher salivary flow rate according to the median salivary flow rate. In participants with a lower salivary flow rate, we assessed salivary bacterial counts, protease activities, protein concentrations, oral parameters, and the subjective perception of oral dryness.
RESULTS
Protease activities and concentrations of protease inhibitors such as cystatin-D and cystatin-SA in the saliva of participants experiencing oral dryness were significantly higher and lower, respectively, than in those not experiencing oral dryness, even though no difference in the salivary flow rate was detected. Salivary cystatin-D and cystatin-SA concentrations correlated negatively with salivary protease activities.
CONCLUSIONS
The composition of salivary protease inhibitors and increased protease activities affect the subjective perception of oral dryness.
Topics: Anti-Infective Agents; Health Status; Humans; Protease Inhibitors; Saliva; Xerostomia
PubMed: 34930236
DOI: 10.1186/s12903-021-02024-x -
Proceedings of the National Academy of... Dec 2011Hepatitis C virus (HCV) infection is a global health burden with over 170 million people infected worldwide. In a significant portion of patients chronic hepatitis C...
Hepatitis C virus (HCV) infection is a global health burden with over 170 million people infected worldwide. In a significant portion of patients chronic hepatitis C infection leads to serious liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV NS3 protein is essential for viral polyprotein processing and RNA replication and hence viral replication. It is composed of an N-terminal serine protease domain and a C-terminal helicase/NTPase domain. For full activity, the protease requires the NS4A protein as a cofactor. HCV NS3/4A protease is a prime target for developing direct-acting antiviral agents. First-generation NS3/4A protease inhibitors have recently been introduced into clinical practice, markedly changing HCV treatment options. To date, crystal structures of HCV NS3/4A protease inhibitors have only been reported in complex with the protease domain alone. Here, we present a unique structure of an inhibitor bound to the full-length, bifunctional protease-helicase NS3/4A and show that parts of the P4 capping and P2 moieties of the inhibitor interact with both protease and helicase residues. The structure sheds light on inhibitor binding to the more physiologically relevant form of the enzyme and supports exploring inhibitor-helicase interactions in the design of the next generation of HCV NS3/4A protease inhibitors. In addition, small angle X-ray scattering confirmed the observed protease-helicase domain assembly in solution.
Topics: Carrier Proteins; Chromatography, Gel; Crystallization; Crystallography, X-Ray; Escherichia coli; Hepatitis C; Inhibitory Concentration 50; Intracellular Signaling Peptides and Proteins; Models, Molecular; Protease Inhibitors; Protein Conformation; Scattering, Small Angle; Viral Nonstructural Proteins
PubMed: 22160684
DOI: 10.1073/pnas.1110534108 -
Bioscience Reports Apr 2017Frog skin secretions contain complex peptidomes and peptidic protease inhibitors that are one of the biologically and structurally described groups of components. In the...
Frog skin secretions contain complex peptidomes and peptidic protease inhibitors that are one of the biologically and structurally described groups of components. In the present study, by use of molecular 'shotgun' cloning and LC MS/MS fractionation sequencing, a novel Bowman-Birk-type heptadecapeptide (AALKGCWTKSIPPKPCF-amide), named rypsin nhibitor (OSTI), with a canonical Cys-Cys disulfide bridge, was isolated and identified in piebald odorous frog () skin secretion. A synthetic replicate of OSTI-exhibited trypsin inhibitory activity with a value of 0.3 ± 0.04 nM and also a tryptase inhibitory effect with a of 2.5 ± 0.6 μM. This is the first time that this property has been reported for a peptide originating from amphibian sources. In addition, substituting lysine (K) with phenylalanine (F) at the presumed P1 position, completely abrogated the trypsin and tryptase inhibition, but produced a strong chymotrypsin inhibition with a of 1.0 ± 0.1 μM. Thus, the specificity of this peptidic protease inhibitor could be optimized through modifying the amino acid residue at the presumed P1 position and this novel native OSTI, along with its analogue, [Phe]-OSTI, have expanded the potential drug discovery and development pipeline directed towards alleviation of serine protease-mediated pathologies.
Topics: Amino Acid Sequence; Animals; Base Sequence; Chromatography, High Pressure Liquid; Cloning, Molecular; DNA, Complementary; Oligopeptides; Protease Inhibitors; Ranidae; Skin; Trypsin; Trypsin Inhibitors; Tryptases
PubMed: 28356487
DOI: 10.1042/BSR20160593 -
European Journal of Trauma and... Jun 2022Trauma and hemorrhagic shock (T/HS) is a major cause of morbidity and mortality. Existing treatment options are largely limited to source control and fluid and blood...
PURPOSE
Trauma and hemorrhagic shock (T/HS) is a major cause of morbidity and mortality. Existing treatment options are largely limited to source control and fluid and blood repletion. Previously, we have shown that enteral protease inhibition improves outcomes in experimental models of T/HS by protecting the gut from malperfusion and ischemia. However, enteral protease inhibition was achieved invasively, by laparotomy and direct injection of tranexamic acid (TXA) into the small intestine. In this study, we tested a minimally invasive method of enteral protease inhibitor infusion in experimental T/HS that can be readily adapted for clinical use.
METHODS
Wistar rats were exsanguinated to a mean arterial blood pressure (MABP) of 40 mmHg, with laparotomy to induce trauma. Hypovolemia was maintained for 120 min and was followed by reperfusion of shed blood. Animals were monitored for an additional 120 min. A modified orogastric multi-lumen tube was developed to enable rapid enteral infusion of a protease inhibitor solution while simultaneously mitigating risk of reflux aspiration into the airways. The catheter was used to deliver TXA (T/HS + TXA) or vehicle (T/HS) continuously into the proximal small intestine, starting 20 min into the ischemic period.
RESULTS
Rats treated with enteral protease inhibition (T/HS + TXA) displayed improved outcomes compared to control animals (T/HS), including significantly improved MABP (p = 0.022) and lactate (p = 0.044). Mass spectrometry-based analysis of the plasma peptidome after T/HS indicated mitigation of systemic proteolysis in T/HS + TXA.
CONCLUSION
Minimally invasive, continuous enteral protease inhibitor delivery improves outcomes in T/HS and is readily translatable to the clinical arena.
Topics: Animals; Disease Models, Animal; Humans; Intestine, Small; Ischemia; Protease Inhibitors; Rats; Rats, Wistar; Shock, Hemorrhagic; Tranexamic Acid
PubMed: 33483765
DOI: 10.1007/s00068-020-01591-y -
Seminars in Immunopathology Jan 2018Activation and regulation of the cascade systems of the blood (the complement system, the coagulation/contact activation/kallikrein system, and the fibrinolytic system)... (Review)
Review
Activation and regulation of the cascade systems of the blood (the complement system, the coagulation/contact activation/kallikrein system, and the fibrinolytic system) occurs via activation of zymogen molecules to specific active proteolytic enzymes. Despite the fact that the generated proteases are all present together in the blood, under physiological conditions, the activity of the generated proteases is controlled by endogenous protease inhibitors. Consequently, there is remarkable little crosstalk between the different systems in the fluid phase. This concept review article aims at identifying and describing conditions where the strict system-related control is circumvented. These include clinical settings where massive amounts of proteolytic enzymes are released from tissues, e.g., during pancreatitis or post-traumatic tissue damage, resulting in consumption of the natural substrates of the specific proteases and the available protease inhibitor. Another example of cascade system dysregulation is disseminated intravascular coagulation, with canonical activation of all cascade systems of the blood, also leading to specific substrate and protease inhibitor elimination. The present review explains basic concepts in protease biochemistry of importance to understand clinical conditions with extensive protease activation.
Topics: Animals; Complement Activation; Complement C3-C5 Convertases, Classical Pathway; Complement System Proteins; Disease Susceptibility; Enzyme Activation; Humans; Kinetics; Molecular Targeted Therapy; Peptide Hydrolases; Protease Inhibitors; Protein Binding; Proteolysis; Signal Transduction; Substrate Specificity
PubMed: 28900700
DOI: 10.1007/s00281-017-0646-9 -
The Biochemical Journal Mar 2004The proteins that inhibit peptidases are of great importance in medicine and biotechnology, but there has never been a comprehensive system of classification for them.... (Review)
Review
The proteins that inhibit peptidases are of great importance in medicine and biotechnology, but there has never been a comprehensive system of classification for them. Some of the terminology currently in use is potentially confusing. In the hope of facilitating the exchange, storage and retrieval of information about this important group of proteins, we now describe a system wherein the inhibitor units of the peptidase inhibitors are assigned to 48 families on the basis of similarities detectable at the level of amino acid sequence. Then, on the basis of three-dimensional structures, 31 of the families are assigned to 26 clans. A simple system of nomenclature is introduced for reference to each clan, family and inhibitor. We briefly discuss the specificities and mechanisms of the interactions of the inhibitors in the various families with their target enzymes. The system of families and clans of inhibitors described has been implemented in the MEROPS peptidase database (http://merops.sanger.ac.uk/), and this will provide a mechanism for updating it as new information becomes available.
Topics: Biological Evolution; Disulfides; Protease Inhibitors; Protein Conformation; Sequence Homology, Amino Acid; Terminology as Topic
PubMed: 14705960
DOI: 10.1042/BJ20031825 -
Clinical Microbiology and Infection :... Feb 2011Improved understanding of the hepatitis C virus (HCV) life cycle has led to the discovery of numerous potential targets for antiviral therapy. HCV polyprotein processing... (Review)
Review
Improved understanding of the hepatitis C virus (HCV) life cycle has led to the discovery of numerous potential targets for antiviral therapy. HCV polyprotein processing and replication have been identified as the most promising viral targets. However, viral entry and fusion, RNA translation, virus assembly and release and several host cell factors may provide alternative attractive targets for future anti-HCV therapies. Inhibitors of the HCV NS3/4A protease are currently the most advanced in clinical development. Monotherapy with protease inhibitors has shown high antiviral activity, but is associated with frequent selection of resistant HCV variants, often resulting in viral breakthrough. However, there is encouraging evidence from phase 2/3 trials indicating that the addition of a protease inhibitor (e.g. telaprevir and boceprevir) to pegylated interferon-α/ribavirin substantially improves sustained virological response rates in both treatment-naïve and treatment-experienced patients with HCV genotype 1. Nucleos(t)ide inhibitors of the HCV NS5B polymerase have shown variable antiviral activity against different HCV genotypes, but seem to have a higher genetic barrier to resistance than protease inhibitors. In addition, several allosteric binding sites have been identified for non-nucleoside inhibitors of the NS5B polymerase. However, the development of a substance with high antiviral activity and a high genetic barrier to resistance seems to be difficult. Among the different host cell-targeting compounds in early clinical development, cyclophilin inhibitors have shown the most promising results. Although advances have also been made in improving interferons, combinations of antiviral agents with different mechanisms of action may lead to the eventual possibility of interferon-free regimens.
Topics: Antiviral Agents; Clinical Trials as Topic; Drug Discovery; Drug Resistance, Viral; Drug Therapy, Combination; Hepacivirus; Hepatitis C; Humans; Interferon alpha-2; Interferon-alpha; Polyethylene Glycols; Protease Inhibitors; Recombinant Proteins; Ribavirin
PubMed: 21087349
DOI: 10.1111/j.1469-0691.2010.03430.x