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Methods in Molecular Biology (Clifton,... 2022Activity-based protein profiling (ABPP) is a powerful tool in biological chemistry to monitor protein activity using chemical probes that bind covalently and...
Activity-based protein profiling (ABPP) is a powerful tool in biological chemistry to monitor protein activity using chemical probes that bind covalently and irreversible to active site of enzymes such as proteases. To date, there are three different ways to experimentally use ABPP: comparative, competitive, and convolution ABPP. Here we use and describe the convolution ABPP approach, a method used to detect changes in protease inhibitor abundance in different proteomes. We have applied this method to monitor the activity of Lolium perenne apoplastic cysteine proteases during the interaction with the fungal endophyte Epichloë festucae. We describe the method to isolate apoplastic fluids from infected and uninfected L. perenne ryegrass leaves and the protocol to perform a convolution ABPP experiment. Furthermore, we report how to quantify and analyze fluorescent gels obtained from the ABPP labeling.
Topics: Cysteine Proteases; Enzyme Inhibitors; Lolium; Protease Inhibitors; Proteome; Symbiosis
PubMed: 35583775
DOI: 10.1007/978-1-0716-2079-3_8 -
Plant Physiology and Biochemistry : PPB Sep 2023Kunitz-like protease inhibitors (KTIs) have been identified to play critical roles in insect defense, but evidence for their involvement in drought stress is sparse. The...
Heterologous expression of the Glycine soja Kunitz-type protease inhibitor GsKTI improves resistance to drought stress and Helicoverpa armigera in transgenic Arabidopsis lines.
Kunitz-like protease inhibitors (KTIs) have been identified to play critical roles in insect defense, but evidence for their involvement in drought stress is sparse. The aim of this study was to identify and functionally characterize a Kunitz-like protease inhibitor, GsKTI, from the wild soybean (Glycine soja) variety ED059. Expression patterns suggest that drought stress and insect herbivory may induce GsKTI transcript levels. Transgenic Arabidopsis lines overexpressing GsKTI have been shown to exhibit enhanced drought tolerance by regulating the ABA signaling pathway and increasing xylem cell number. Transgenic Arabidopsis leaves overexpressing GsKTI interfered with insect digestion and thus had a negative effect on the growth of Helicoverpa armigera. It is concluded that GsKTI increases resistance to drought stress and insect attack in transgenic Arabidopsis lines.
Topics: Animals; Arabidopsis; Glycine max; Protease Inhibitors; Droughts; Plant Proteins; Fabaceae; Moths; Glycine; Plants, Genetically Modified; Stress, Physiological; Gene Expression Regulation, Plant
PubMed: 37536218
DOI: 10.1016/j.plaphy.2023.107915 -
Journal of Peptide Science : An... Sep 2000The revealing of the entire complement of protease and protease inhibitor sequences by the Human Genome Project will be of great importance to both academic and... (Comparative Study)
Comparative Study
The revealing of the entire complement of protease and protease inhibitor sequences by the Human Genome Project will be of great importance to both academic and pharmaceutical research. Although the finishing phase is not yet complete, a selection of secondary annotation sources and comparisons with completed model organism genomes already allow useful estimates to be made. Conservative extrapolation suggests a total of approximately 1.8% for human proteases. This is close to the figures for yeast (1.7%) and worm (1.8%) but lower than the fly (3.4%) which has a large trypsin-like protease content. Using estimates for the human proteome of between 40,000 and 60,000 genes would extrapolate to 700-1,100 proteases, compared with approximately 360 currently represented as GenBank mRNAs. Preliminary comparisons between domain annotations for predicted human gene products and completed proteins suggest the genomic protease family and mechanistic class distributions will broadly reflect those in the current transcript data. The protease:inhibitor ratio at the mRNA level is currently approximately 9:1, but genome annotation data indicate that inhibitory domains are more widespread than this ratio would indicate.
Topics: Databases, Factual; Endopeptidases; Expressed Sequence Tags; Genome, Human; Humans; Protease Inhibitors; Protein Structure, Tertiary
PubMed: 11016882
DOI: 10.1002/1099-1387(200009)6:9<453::AID-PSC284>3.0.CO;2-Z -
Bioorganic & Medicinal Chemistry Feb 2020An octahydroisochromene scaffold has been introduced into a known SARS 3CL protease inhibitor as a novel hydrophobic core to interact with the S2 pocket of the protease....
An octahydroisochromene scaffold has been introduced into a known SARS 3CL protease inhibitor as a novel hydrophobic core to interact with the S2 pocket of the protease. An alkyl or aryl substituent was also introduced at the 1-position of the octahydroisochromene scaffold and expected to introduce additional interactions with the protease. Sharpless-Katsuki asymmetric epoxidation and Sharpless asymmetric dihydroxylation were employed to construct the octahydroisochromene scaffold. The introductions of the P1 site His-al and the substituent at 1-position was achieved using successive reductive amination reactions. Our initial evaluations of the diastereo-isomeric mixtures (16a-d) revealed that the octahydroisochromene moiety functions as a core hydrophobic scaffold for the S2 pocket of the protease and the substituent at the 1-position may form additional interactions with the protease. The inhibitory activities of the diastereoisomerically-pure inhibitors (3a-d) strongly suggest that a specific stereo-isomer of the octahydroisochromene scaffold, (1S, 3S) 3b, directs the P1 site imidazole, the warhead aldehyde, and substituent at the 1-position of the fused ring to their appropriate pockets in the protease.
Topics: Benzopyrans; Coronavirus 3C Proteases; Dose-Response Relationship, Drug; Molecular Structure; Protease Inhibitors; Severe acute respiratory syndrome-related coronavirus; Structure-Activity Relationship
PubMed: 31926775
DOI: 10.1016/j.bmc.2019.115273 -
Journal of Biochemistry Aug 1992A proteinaceous protease inhibitor was isolated from the culture broth of Streptomyces lividans 66 by a series of purification steps (salting out by ammonium sulfate,... (Comparative Study)
Comparative Study
A proteinaceous protease inhibitor was isolated from the culture broth of Streptomyces lividans 66 by a series of purification steps (salting out by ammonium sulfate, ion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on Phenyl-Sepharose, and gel-filtration on Sephacryl S-200), and was named S. lividans protease inhibitor (SLPI). The purified SLPI existed in a dimeric form consisting of two identical subunits, each of which was composed of 107 amino acids. SLPI exhibited strong inhibitory activity toward subtilisin BPN'. These features were similar to those of protein protease inhibitors produced by other Streptomyces (SSI family inhibitor). In addition, SLPI was capable of inhibiting trypsin with an inhibitor constant (Ki) of about 10(-9) M. The primary structure of SLPI and location of two disulfide bridges were homologous to those of the other serine protease inhibitors of Streptomyces. The reactive site of SLPI was found to be Arg67-Glu68 from the sequence analysis of cleaved SLPI which was produced by acidification of subtilisin-SLPI complex. An Arg residue at the P1 site was consistent with the trypsin-inhibitory property of SLPI. Sequence comparison with other members of the SSI family revealed that amino acid replacements in SLPI were mainly localized on the surface of the SLPI molecule, and many of the amino acid residues in beta-sheets and hydrophobic core were well conserved.
Topics: Amino Acid Sequence; Binding Sites; Chromatography, Gel; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Molecular Sequence Data; Molecular Weight; Protease Inhibitors; Proteinase Inhibitory Proteins, Secretory; Proteins; Serine Proteinase Inhibitors; Streptomyces; Subtilisins; Trypsin Inhibitors
PubMed: 1356971
DOI: 10.1093/oxfordjournals.jbchem.a123878 -
Organic & Biomolecular Chemistry Jun 2016Aspartyl proteases are important pharmacological targets. Historically aspartyl proteases have been commonly targeted with transition state derived peptidomimetics. The... (Review)
Review
Aspartyl proteases are important pharmacological targets. Historically aspartyl proteases have been commonly targeted with transition state derived peptidomimetics. The strategy to develop aspartyl protease inhibitors has undertaken a dramatic paradigm shift in the last 10 years. The pharmaceutical industry in 2005 disclosed several scaffolds or "head groups" that prompted the field to move beyond peptidomimetic derived inhibitors. Since the discovery of the first amino heterocycle aspartyl protease inhibitor, the amino hydantoin, industry and academia have positioned themselves for a foothold on the new molecular space, designing a variety of related "head groups". Both the design and synthetic efforts involved in constructing these scaffolds are varied and complex. Here we highlight the synthetic strategies used to access these amino heterocycle scaffolds.
Topics: Aspartic Acid Endopeptidases; Chemistry Techniques, Synthetic; Drug Design; Heterocyclic Compounds; Protease Inhibitors
PubMed: 27143279
DOI: 10.1039/c5ob01842k -
Clinical Pharmacokinetics Mar 1997Since its introduction in 1987, zidovudine monotherapy has been the treatment of choice for patients with HIV infection. Unfortunately it has been established that the... (Review)
Review
Since its introduction in 1987, zidovudine monotherapy has been the treatment of choice for patients with HIV infection. Unfortunately it has been established that the beneficial effects of zidovudine are not sustained due to the development of resistant viral strains. This has led to the strategy of combination therapy, and in 1995 treatment with zidovudine plus didanosine, or zidovudine plus zalcitabine, was demonstrated to be more effective than zidovudine monotherapy in preventing disease progression and reducing mortality in patients with HIV disease. Recent work demonstrates an even greater antiviral effect from triple therapy with 2 nucleosides, zidovudine plus zalcitabine with the addition of saquinavir, a new protease inhibitor drug. The HIV protease enzyme is responsible for the post-translational processing of gag and gag-pol polyprotein precursors, and its inhibition by drugs such as saquinavir, ritonavir, indinavir and VX-478 results in the production of non-infectious virions. As resistance may also develop to the protease inhibitors they may be used in combination, and future strategies may well include quadruple therapy with 2 nucleoside analogues plus 2 protease inhibitors. Administration of protease inhibitors alone or in combination with other drugs does raise a number of important pharmacokinetic issues for patients with HIV disease. Some protease inhibitors (e.g. saquinavir) have kinetic profiles characterised by reduced absorption and a high first pass effect, resulting in poor bioavailability which may be improved by administrating with food. Physiological factors including achlorhydria, malabsorption and hepatic dysfunction may influence the bioavailability of protease inhibitors in HIV disease. Protease inhibitors are very highly bound to plasma proteins (> 98%), predominantly to alpha 1-acid glycoprotein. This may influence their antiviral activity in vitro and may also predispose to plasma protein displacement interactions. Such interactions are usually only of clinical relevance if the metabolism of the displaced drug is also inhibited. This is precisely the situation likely to pertain to the protease inhibitors, as ritonavir may displace other protease inhibitor drugs, such as saquinavir, from plasma proteins and inhibit their metabolism. Protease inhibitors are extensively metabolised by the cytochrome P450 (CYP) enzymes present in the liver and small intestine. In vitro studies suggest that the most influential CYP isoenzyme involved in the metabolism of the protease inhibitors is CYP3A, with the isoforms CYP2C9 and CYP2D6 also contributing. Ritonavir has an elimination half-life (t1/2 beta) of 3 hours, indinavir 2 hours and saquinavir between 7 and 12 hours. Renal elimination is not significant, with less than 5% of ritonavir and saquinavir excreted in the unchanged form. As patients with HIV disease are likely to be taking multiple prolonged drug regimens this may lead to drug interactions as a result of enzyme induction or inhibition. Recognised enzyme inducers of CYP3A, which are likely to be prescribed for patients with HIV disease, include rifampicin (rifampin) [treatment of pulmonary tuberculosis], rifabutin (treatment and prophylaxis of Mycobacterium avium complex), phenobarbital (phenobarbitone), phenytoin and carbamazepine (treatment of seizures secondary to cerebral toxoplasmosis or cerebral lymphoma). These drugs may reduce the plasma concentrations of the protease inhibitors and reduce their antiviral efficacy. If coadministered drugs are substrates for a common CYP enzyme, the elimination of one or both drugs may be impaired. Drugs which are metabolised by CYP3A and are likely to be used in the treatment of patients with HIV disease include the azole antifungals, macrolide antibiotics and dapsone; therefore, protease inhibitors may interact with these drugs. (ABSTRACT TRUNCATED)
Topics: Anti-HIV Agents; Biological Availability; Blood Proteins; HIV Infections; Half-Life; Humans; Metabolic Clearance Rate; Protease Inhibitors; Zidovudine
PubMed: 9084959
DOI: 10.2165/00003088-199732030-00003 -
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 -
Current Medicinal Chemistry 2024Proteases play important roles in the regulation of many physiological processes, and protease inhibitors have become one of the important drug classes. Especially... (Review)
Review
BACKGROUND
Proteases play important roles in the regulation of many physiological processes, and protease inhibitors have become one of the important drug classes. Especially because the development of protease inhibitors often starts from a substrate- based peptidomimetic strategy, many of the initial lead compounds suffer from pharmacokinetic liabilities.
OBJECTIVE
To reduce drug attrition rates, drug metabolism and pharmacokinetics studies are fully integrated into modern drug discovery research, and the structure-property relationship illustrates how the modification of the chemical structure influences the pharmacokinetic and toxicological properties of drug compounds. Understanding the structure- property relationships of clinically approved protease inhibitor drugs and their analogues could provide useful information on the lead-to-candidate optimization strategies.
METHODS
About 70 inhibitors against human or pathogenic viral proteases have been approved until the end of 2021. In this review, 17 inhibitors are chosen for the structure- property relationship analysis because detailed pharmacological and/or physicochemical data have been disclosed in the medicinal chemistry literature for these inhibitors and their close analogues.
RESULTS
The compiled data are analyzed primarily focusing on the pharmacokinetic or toxicological deficiencies found in lead compounds and the structural modification strategies used to generate candidate compounds.
CONCLUSION
The structure-property relationships hereby summarized how the overall druglike properties could be successfully improved by modifying the structure of protease inhibitors. These specific examples are expected to serve as useful references and guidance for developing new protease inhibitor drugs in the future.
Topics: Humans; Peptide Hydrolases; Protease Inhibitors
PubMed: 37031455
DOI: 10.2174/0929867330666230409232655 -
Microbiology (Reading, England) Jul 2012A low-molecular-mass aspartic protease inhibitor was isolated from a novel Penicillium sp. The inhibitor was purified to homogeneity, as shown by reversed-phase HPLC and...
A low-molecular-mass aspartic protease inhibitor was isolated from a novel Penicillium sp. The inhibitor was purified to homogeneity, as shown by reversed-phase HPLC and SDS-PAGE. The M(r) of the inhibitor was 1585 and the amino acid composition showed the presence of D, D, D, E, A, K, L, Y, H, I and W residues. The steady-state kinetic interactions of Aspergillus saitoi aspartic protease with the inhibitor revealed the reversible, competitive, time-dependent tight-binding nature of the inhibitor, with IC(50) and K(i) values of 1.8 and 0.85 µM, respectively. Fluorescence spectroscopy and circular dichroism analysis showed that inactivation of the enzyme was due to binding of the inhibitor to the active site. The inhibitor was found to inhibit mycelial growth and spore germination of Aspergillus fumigatus and Aspergillus niger in vitro with MIC values of 1.65 and 0.30 µg ml(-1), respectively. This study will potentially open the way towards the development of a tight-binding peptidic inhibitor against fungal aspartic proteases to combat human fungal infections.
Topics: Antifungal Agents; Aspartic Acid Proteases; Aspergillus fumigatus; Aspergillus niger; Chromatography, High Pressure Liquid; Circular Dichroism; DNA, Fungal; DNA, Ribosomal Spacer; Electrophoresis, Polyacrylamide Gel; Humans; Inhibitory Concentration 50; Kinetics; Molecular Sequence Data; Penicillium; Protease Inhibitors; Sequence Analysis, DNA; Spectrometry, Fluorescence
PubMed: 22493301
DOI: 10.1099/mic.0.058511-0