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1.
Laryngoscope ; 134(7): 3080-3085, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38214310

ABSTRACT

OBJECTIVE: This study aimed to evaluate the role of pepsin inhibitors in the inflammatory response and their effects on laryngeal mucosal integrity during gastroesophageal reflux (GERD) under in vivo conditions. METHODS: A surgical model of GERD was used, in which mice were treated with pepstatin (0.3 mg/kg) or darunavir (8.6 mg/kg) for 3 days. On the third day after the experimental protocol, the laryngeal samples were collected to assess the severity of inflammation (wet weight and myeloperoxidase activity) and mucosal integrity (transepithelial electrical resistance and paracellular epithelial permeability to fluorescein). RESULTS: The surgical GERD model was reproduced. It showed features of inflammation and loss of barrier function in the laryngeal mucosa. Pepstatin and darunavir administration suppressed laryngeal inflammation and preserved laryngeal mucosal integrity. CONCLUSION: Pepsin inhibition by the administration of pepstatin and darunavir improved inflammation and protected the laryngeal mucosa in a mouse experimental model of GERD. LEVEL OF EVIDENCE: NA Laryngoscope, 134:3080-3085, 2024.


Subject(s)
Disease Models, Animal , Gastroesophageal Reflux , Pepsin A , Animals , Mice , Gastroesophageal Reflux/drug therapy , Pepstatins/pharmacology , Laryngeal Mucosa/drug effects , Laryngeal Mucosa/pathology , Male , Inflammation/drug therapy , Inflammation/prevention & control
2.
Eur J Med Chem ; 267: 116178, 2024 Mar 05.
Article in English | MEDLINE | ID: mdl-38295686

ABSTRACT

Cathepsin D (CD) is overexpressed in several types of cancer and constitutes an important biological target. Pepstatin A, a pentapeptide incorporating two non-proteinogenic statin residues, is among the most potent inhibitor of CD but lacks selectivity and suffers from poor bioavailability. Eight analogues of Pepstatin A, were synthesized, replacing residues in P3 or P1 position by non-canonical (S)- and (R)-α-Trifluoromethyl Alanine (TfmAla), (S)- and (R)-Trifluoromethionine (TFM) or non-natural d-Valine. The biological activities of those analogues were quantified on isolated CD and Pepsin by fluorescence-based assay (FRET) and cytotoxicity of the best fluorinated inhibitors was evaluated on SKOV3 ovarian cancer cell line. (R)-TFM based analog of Pepstatin A (compound 6) returned a sub-nanomolar IC50 against CD and an increased selectivity. Molecular Docking experiments could partially rationalize these results. Stabilized inhibitor 6 in the catalytic pocket of CD showed strong hydrophobic interactions of the long and flexible TFM side chain with lipophilic residues of S1 and S3 sub-pockets of the catalytic pocket. The newly synthesized inhibitors returned no cytotoxicity at IC50 concentrations on SKOV3 cancer cells, however the compounds derived from (S)-TfmAla and (R)-TFM led to modifications of cells morphologies, associated with altered organization of F-actin and extracellular Fibronectin.


Subject(s)
Cathepsin D , Methionine/analogs & derivatives , Pepsin A , Pepstatins/pharmacology , Pepstatins/chemistry , Molecular Docking Simulation , Alanine
3.
ACS Chem Biol ; 18(4): 686-692, 2023 04 21.
Article in English | MEDLINE | ID: mdl-36920024

ABSTRACT

Aspartic proteases are a small class of proteases implicated in a wide variety of human diseases. Covalent chemical probes for photoaffinity labeling (PAL) of these proteases are underdeveloped. We here report a full on-resin synthesis of clickable PAL probes based on the natural product inhibitor pepstatin incorporating a minimal diazirine reactive group. The position of this group in the inhibitor determines the labeling efficiency. The most effective probes sensitively detect cathepsin D, a biomarker for breast cancer, in cell lysates. Moreover, through chemical proteomics experiments and deep learning algorithms, we identified sequestosome-1, an important player in autophagy, as a direct interaction partner and substrate of cathepsin D.


Subject(s)
Aspartic Acid Endopeptidases , Cathepsin D , Pepstatins , Photoaffinity Labels , Humans , Aspartic Acid Endopeptidases/chemistry , Cathepsin D/chemistry , Diazomethane , Pepstatins/chemistry , Pepstatins/pharmacology , Photoaffinity Labels/chemistry , Sequestosome-1 Protein/chemistry
4.
J Antibiot (Tokyo) ; 75(9): 519-522, 2022 09.
Article in English | MEDLINE | ID: mdl-35882959

ABSTRACT

A new pepstatin with a phenylacetyl group, pepstatin Pa (1), and its methyl ester (2) were isolated from Streptomyces varsoviensis DSM 40346. Their structures were determined by high-resolution mass spectrometry and nuclear magnetic resonance techniques. The absolute configuration was determined using the Marfey's method. Both pentapeptide products are inhibitors of pepsin and cathepsin D. Interestingly, the bacterial genome contains no biosynthetic gene cluster for the new pepstatin, suggesting an extrachromosomal origin of the biosynthetic genes.


Subject(s)
Aspartic Acid Proteases , Pepstatins , Streptomyces , Aspartic Acid Proteases/antagonists & inhibitors , Bacterial Proteins , Pepstatins/pharmacology , Protease Inhibitors , Streptomyces/chemistry
5.
Biomolecules ; 10(7)2020 07 06.
Article in English | MEDLINE | ID: mdl-32640672

ABSTRACT

The human retroviral-like aspartic protease 1 (ASPRV1) is a mammalian retroviral-like enzyme that catalyzes a critical proteolytic step during epidermal differentiation; therefore, it is also referred to as skin-specific aspartic protease (SASPase). Neutrophil granulocytes were also found recently to express ASPRV1 that is involved in the progression of acute chronic inflammation of the central nervous system, especially in autoimmune encephalomyelitis. Thus, investigation of ASPRV1 is important due to its therapeutic or diagnostic potential. We investigated the structural characteristics of ASPRV1 by homology modeling; analysis of the proposed structure was used for interpretation of in vitro specificity studies. For in-vitro characterization, activities of SASP28 and SASP14 enzyme forms were measured using synthetic oligopeptide substrates. We demonstrated that self-processing of SASP28 precursor causes autoactivation of the protease. The highest activity was measured for GST-SASP14 at neutral pH and at high ionic strength, and we proved that pepstatin A and acetyl-pepstatin can also inhibit the protease. In agreement with the structural characteristics, the relatively lower urea dissociation constant implied lower dimer stability of SASP14 compared to that of HIV-1 protease. The obtained structural and biochemical characteristics support better understanding of ASPRV1 function in the skin and central nervous system.


Subject(s)
Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/metabolism , Mutation , Neutrophils/metabolism , Aspartic Acid Endopeptidases/genetics , Enzyme Activation , Gene Expression Regulation, Enzymologic/drug effects , Humans , Hydrogen-Ion Concentration , Models, Molecular , Pepstatins/pharmacology , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Structure, Secondary , Structural Homology, Protein
6.
Fungal Biol ; 124(8): 700-707, 2020 08.
Article in English | MEDLINE | ID: mdl-32690251

ABSTRACT

The opportunistic pathogens comprising the Candida haemulonii complex (C. haemulonii, C. duobushaemulonii and C. haemulonii var. vulnera) are notable for their intrinsic resistance to different antifungal classes. Little is known about the virulence attributes in this emerging fungal complex. However, it is well-recognized that enzymes play important roles in virulence/pathogenesis of candidiasis. Herein, we aimed to identify aspartyl-type peptidases in 12 clinical isolates belonging to the C. haemulonii complex. All isolates were able to grow in a chemically defined medium containing albumin as the sole nitrogen source, and a considerable consumption of this protein occurred after 72-96 h. C. haemulonii var. vulnera isolates showed the lowest albumin degradation capability and the poorest growth rate. The measurement of secreted aspartyl peptidase (Sap) activity, using the cathepsin D fluorogenic substrate, varied from 91.6 to 413.3 arbitrary units and the classic aspartyl peptidase inhibitor, pepstatin A, significantly blocked the Sap released by C. haemulonii complex. No differences were observed in the Sap activity among the three fungal species. Flow cytometry, using a polyclonal antibody against Sap1-3 of C. albicans, detected homologous proteins at the surface of C. haemulonii complex (anti-Sap1-3-labeled cells ranged from 24.6 to 79.1%). Additionally, the immunoblotting assay, conducted with the same Sap1-3 antibody, recognized a protein of ∼50 kDa in all fungal isolates. A glimpse in the genome of these fungi revealed several potential proteins containing Sap1-3-like conserved domain. Altogether, our results demonstrated the potential of C. haemulonii species complex to produce Saps, an important virulence factor of Candida spp.


Subject(s)
Antifungal Agents/pharmacology , Candida/drug effects , Candida/enzymology , Candidiasis/microbiology , Dipeptidases/metabolism , Candida/classification , Candida albicans/drug effects , Candida albicans/enzymology , Drug Resistance, Multiple , Humans , Pepstatins/pharmacology , Protease Inhibitors/pharmacology , Sequence Analysis, Protein
7.
Oncol Rep ; 43(2): 625-634, 2020 02.
Article in English | MEDLINE | ID: mdl-31894333

ABSTRACT

While exploring new angiogenesis inhibitors from microbial metabolites, we recently isolated ahpatinins C, E, and G from a soil­derived Streptomyces sp. 15JA150. Ahpatinins C, E and G are known to have pepsin and renin inhibitory activities; however, their antiangiogenic activities and underlying molecular mechanisms have not been fully elucidated. In the present study, the antiangiogenic properties of ahpatinins C, E and G were investigated. The results revealed that the natural compounds significantly inhibited the vascular endothelial growth factor (VEGF)­induced proliferation, invasion, adhesion, and tube formation of human umbilical vein endothelial cells (HUVECs) without exhibiting any cytotoxicity. It was also revealed that ahpatinin E effectively suppressed the neovascularization of the chorioallantoic membranes in growing chick embryos. Notably, ahpatinins C, E, and G led to the downregulation of VEGF­induced activation of VEGF receptor 2 (VEGFR2) and its downstream signaling mediators, including AKT, ERK1/2, JNK, p38, and NF­κB, in HUVECs. Moreover, they reduced the expression of matrix metalloproteinase (MMP)­2 and MMP­9 in the HUVECs following stimulation with VEGF. Furthermore, ahpatinins C, E, and G reduced the tumor cell­induced invasion and tube forming abilities of HUVECs, as well as the expression of VEGF, by suppressing hypoxia­inducible factor­1α (HIF­1α) activity in U87MG glioblastoma cells. Collectively, the present findings indicated that ahpatinins C, E, and G may be used in anticancer therapy by targeting tumor angiogenesis through the inhibition of both VEGFR2 and HIF­1α pathways.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Biological Factors/pharmacology , Neovascularization, Physiologic/drug effects , Streptomyces/metabolism , Vascular Endothelial Growth Factor A/pharmacology , Animals , Cell Adhesion , Cell Movement/drug effects , Cell Proliferation/drug effects , Chick Embryo , Down-Regulation/drug effects , Human Umbilical Vein Endothelial Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Pepstatins/pharmacology , Signal Transduction/drug effects , Vascular Endothelial Growth Factor Receptor-2/metabolism
8.
Int J Biol Macromol ; 139: 199-212, 2019 Oct 15.
Article in English | MEDLINE | ID: mdl-31374272

ABSTRACT

In the pursuit of industrial aspartic proteases, aspergillopepsin A-like endopeptidase from the fungi Aspergillus niger, was identified and cultured by solid state fermentation. Conventional chromatographic techniques were employed to purify the extracellular aspartic protease to apparent homogeneity. The enzyme was found to have single polypeptide chain with a molecular mass of 50 ±â€¯0.5 kDa. The optimum pH and temperature for the purified aspartic protease was found to be 3.5 and 60 °C respectively. The enzyme was stable for 60 min at 50 °C. The purified enzyme had specific activity of 40,000 ±â€¯1800 U/mg. The enzyme had 85% homology with the reported aspergillopepsin A-like aspartic endopeptidase from Aspergillus niger CBS 513.88, based on tryptic digestion and peptide analysis. Pepstatin A reversibly inhibited the enzyme with a Ki value of 0.045 µM. Based on homology modeling and predicted secondary structure, it was inferred that the aspartic protease is rich in ß-structures, which was also confirmed by CD measurements. Interaction of pepstatin A with the enzyme did not affect the conformation of the enzyme as evidenced by CD and fluorescence measurements. Degree of hydrolysis of commercial substrates indicated the order of cleaving ability of the enzyme to be hemoglobin > defatted soya flour > gluten > gelatin > skim milk powder. The enzyme also improved the functional characteristics of defatted soya flour. This aspartic protease was found to be an excellent candidate for genetic manipulation for biotechnological application in food and feed industries, due to its high catalytic turn over number and thermostability.


Subject(s)
Aspartic Acid Proteases/chemistry , Aspergillus niger/enzymology , Pepstatins/chemistry , Protease Inhibitors/chemistry , Aspartic Acid Proteases/antagonists & inhibitors , Aspartic Acid Proteases/isolation & purification , Aspartic Acid Proteases/metabolism , Aspergillus niger/classification , Aspergillus niger/genetics , Catalysis , Chromatography, Liquid , Enzyme Stability , Hydrogen-Ion Concentration , Hydrolysis , Molecular Weight , Pepstatins/pharmacology , Phylogeny , Protease Inhibitors/pharmacology , Protein Binding , Structure-Activity Relationship , Tandem Mass Spectrometry , Temperature
9.
Int J Mol Sci ; 20(6)2019 Mar 16.
Article in English | MEDLINE | ID: mdl-30884823

ABSTRACT

Triple-negative breast cancers (TNBCs) are more aggressive than other breast cancer (BC) subtypes and lack effective therapeutic options. Unraveling marker events of TNBCs may provide new directions for development of strategies for targeted TNBC therapy. Herein, we reported that Annexin A1 (AnxA1) and Cathepsin D (CatD) are highly expressed in MDA-MB-231 (TNBC lineage), compared to MCF-10A and MCF-7. Since the proposed concept was that CatD has protumorigenic activity associated with its ability to cleave AnxA1 (generating a 35.5 KDa fragment), we investigated this mechanism more deeply using the inhibitor of CatD, Pepstatin A (PepA). Fourier Transform Infrared (FTIR) spectroscopy demonstrated that PepA inhibits CatD activity by occupying its active site; the OH bond from PepA interacts with a CO bond from carboxylic acids of CatD catalytic aspartate dyad, favoring the deprotonation of Asp33 and consequently inhibiting CatD. Treatment of MDA-MB-231 cells with PepA induced apoptosis and autophagy processes while reducing the proliferation, invasion, and migration. Finally, in silico molecular docking demonstrated that the catalytic inhibition comprises Asp231 protonated and Asp33 deprotonated, proving all functional results obtained. Our findings elucidated critical CatD activity in TNBC cell trough AnxA1 cleavage, indicating the inhibition of CatD as a possible strategy for TNBC treatment.


Subject(s)
Annexin A1/genetics , Cathepsin D/genetics , Molecular Docking Simulation , Triple Negative Breast Neoplasms/drug therapy , Apoptosis/drug effects , Autophagy/drug effects , Catalytic Domain/drug effects , Cathepsin D/antagonists & inhibitors , Cell Lineage/drug effects , Cell Lineage/genetics , Cell Movement/drug effects , Cell Proliferation/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , MCF-7 Cells , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Pepstatins/pharmacology , Spectroscopy, Fourier Transform Infrared , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathology
10.
J Cell Biochem ; 120(6): 10662-10669, 2019 06.
Article in English | MEDLINE | ID: mdl-30652348

ABSTRACT

Earlier studies showed that the oxidant menadione (MD) induces apoptosis in certain cells and also has anticancer effects. Most of these studies emphasized the role of the mitochondria in this process. However, the engagement of other organelles is less known. Particularly, the role of lysosomes and their proteolytic system, which participates in apoptotic cell death, is still unclear. The aim of this study was to investigate the role of lysosomal cathepsins on molecular signaling in MD-induced apoptosis in U937 cells. MD treatment induced translocation of cysteine cathepsins B, C, and S, and aspartic cathepsin D. Once in the cytosol, some cathepsins cleaved the proapoptotic molecule, Bid, in a process that was completely prevented by E64d, a general inhibitor of cysteine cathepsins, and partially prevented by the pancaspase inhibitor, z-VAD-fmk. Upon loss of the mitochondrial membrane potential, apoptosome activation led to caspase-9 processing, activation of caspase-3-like caspases, and poly (ADP-ribose) polymerase cleavage. Notably, the endogenous protein inhibitor, stefin B, was degraded by cathepsin D and caspases. This process was prevented by z-VAD-fmk, and partially by pepstatin A-penetratin. These findings suggest that the cleaved Bid protein acts as an amplifier of apoptotic signaling through mitochondria, thus enhancing the activity of cysteine cathepsins following stefin B degradation.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , BH3 Interacting Domain Death Agonist Protein/genetics , Cystatin B/genetics , Gene Expression Regulation, Neoplastic , Lysosomes/drug effects , Vitamin K 3/pharmacology , Amino Acid Chloromethyl Ketones/pharmacology , Apoptosis/genetics , Apoptosomes/drug effects , Apoptosomes/metabolism , BH3 Interacting Domain Death Agonist Protein/metabolism , Caspase 3/genetics , Caspase 3/metabolism , Caspase 9/genetics , Caspase 9/metabolism , Cathepsin B/antagonists & inhibitors , Cathepsin B/genetics , Cathepsin B/metabolism , Cathepsin C/antagonists & inhibitors , Cathepsin C/genetics , Cathepsin C/metabolism , Cathepsin D/antagonists & inhibitors , Cathepsin D/genetics , Cathepsin D/metabolism , Cathepsins/antagonists & inhibitors , Cathepsins/genetics , Cathepsins/metabolism , Cystatin B/metabolism , Humans , Leucine/analogs & derivatives , Leucine/pharmacology , Lysosomes/metabolism , Membrane Potential, Mitochondrial/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Pepstatins/pharmacology , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Protease Inhibitors/pharmacology , Proteolysis/drug effects , Signal Transduction , U937 Cells
11.
Chem Biol Interact ; 298: 137-145, 2019 Jan 25.
Article in English | MEDLINE | ID: mdl-30576621

ABSTRACT

In this study, we investigated the effects of synthetic 6'-benzyloxy-4-bromo-2'-hydroxychalcone on viabilities of seven human leukaemia cells. It was cytotoxic against U-937, HL-60, K-562, NALM-6, MOLT-3 cells, and also against Bcl-2-overexpressing U-937/Bcl-2 cells and P-glycoprotein-overexpressing K-562/ADR, but had no significant cytotoxic effects against quiescent or proliferating human peripheral blood mononuclear cells. This chalcone is a potent apoptotic inducer in human leukaemia U-937 cells. Cell death was (i) mediated by the activation and the cleavage of initiator and executioner caspases and poly(ADP-ribose) polymerase; (ii) prevented by the pan-caspase inhibitor z-VAD-fmk, and by the selective caspase-3/7, -6 and -8 inhibitors, and by a cathepsins B/L inhibitor; (iii) associated with the release of mitochondrial proteins, including cytochrome c and Smac/DIABLO; (iv) accompanied by dissipation of the mitochondrial membrane potential, (v) partially blocked by the inhibition of p38MAPK and (vi) mostly abrogated by catalase. In conclusion, the synthetic chalcone is cytotoxic against several types of human leukaemia cell with apoptosis being induced by activation of the extrinsic pathway and the generation of reactive oxygen species.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Caspase 8/metabolism , Chalcone/analogs & derivatives , Chalcone/pharmacology , Leukemia/drug therapy , Reactive Oxygen Species/metabolism , Antineoplastic Agents/chemical synthesis , Apoptosis/physiology , Cathepsin D/antagonists & inhibitors , Cell Line, Tumor , Cell Survival/drug effects , Humans , Leukemia/metabolism , Leukemia/pathology , Membrane Potential, Mitochondrial/drug effects , Pepstatins/pharmacology
12.
Future Med Chem ; 10(1): 5-26, 2018 01.
Article in English | MEDLINE | ID: mdl-29235371

ABSTRACT

AIM: Cathepsin D, one of the attractive targets in the treatment of breast cancer, has been implicated in HIV neuropathogenesis with potential proteolytic effects on chemokines. Methodology/result: Diverse modeling tools were used to reveal the key structural features affecting the inhibitory activities of 78 pepstatin A analogs. Analyses were performed to investigate the stability, rationality and fluctuation of the analogs. Results showed a clear correlation between the experimental and predicted activities of the analogs as well as the variation in their activities relative to structural modifications. CONCLUSION: The insight gained from this study offers theoretical references for understanding the mechanism of action of cathepsin D and will aid in the design of more potent and clinically-relevant drugs. Graphical abstract [Formula: see text].


Subject(s)
Cathepsin D/antagonists & inhibitors , Pepstatins/pharmacology , Protease Inhibitors/pharmacology , Quantitative Structure-Activity Relationship , Cathepsin D/metabolism , Dose-Response Relationship, Drug , Humans , Models, Molecular , Molecular Structure , Pepstatins/chemical synthesis , Pepstatins/chemistry , Protease Inhibitors/chemical synthesis , Protease Inhibitors/chemistry , Structure-Activity Relationship
13.
J Exp Clin Cancer Res ; 36(1): 67, 2017 05 12.
Article in English | MEDLINE | ID: mdl-28499442

ABSTRACT

BACKGROUND: Chemotherapy resistance is one of the major factors contributing to mortality from human epithelial ovarian cancer (EOC). Identifying drugs that can effectively kill chemotherapy-resistant EOC cells would be a major advance in reducing mortality. Glycosylated antitumour ether lipids (GAELs) are synthetic glycolipids that are cytotoxic to a wide range of cancer cells. They appear to induce cancer cell death in an apoptosis-independent manner. METHODS: Herein, the effectiveness of two GAELs, GLN and MO-101, in killing chemotherapy-sensitive and -resistant EOC cells lines and primary cell samples was tested using monolayer, non-adherent aggregate, and non-adherent spheroid cultures. RESULTS: Our results show that EOC cells exhibit a differential sensitivity to the GAELs. Strikingly, both GAELs are capable of inducing EOC cell death in chemotherapy-sensitive and -resistant cells grown as monolayer or non-adherent cultures. Mechanistic studies provide evidence that apoptotic-cell death (caspase activation) contributes to, but is not completely responsible for, GAEL-induced cell killing in the A2780-cp EOC cell line, but not primary EOC cell samples. CONCLUSIONS: Studies using primary EOC cell samples supports previously published work showing a GAEL-induced caspase-independent mechanism of death. GAELs hold promise for development as novel compounds to combat EOC mortality due to chemotherapy resistance.


Subject(s)
Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , Drug Resistance, Neoplasm/drug effects , Glycolipids/pharmacology , Apoptosis/drug effects , Carcinoma, Ovarian Epithelial , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Humans , Neoplasms, Glandular and Epithelial/metabolism , Ovarian Neoplasms/metabolism , Pepstatins/pharmacology
14.
ACS Comb Sci ; 19(3): 181-192, 2017 03 13.
Article in English | MEDLINE | ID: mdl-28199790

ABSTRACT

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-µm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing.


Subject(s)
Combinatorial Chemistry Techniques/instrumentation , DNA/chemistry , Drug Evaluation, Preclinical/instrumentation , Lab-On-A-Chip Devices , Base Sequence , Cathepsin D/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Equipment Design , Gene Library , Humans , Microspheres , Pepstatins/chemistry , Pepstatins/pharmacology
15.
Nat Commun ; 8: 14240, 2017 01 20.
Article in English | MEDLINE | ID: mdl-28106035

ABSTRACT

Pepstatin is a potent peptidyl inhibitor of various malarial aspartic proteases, and also has parasiticidal activity. Activity of pepstatin against cultured Plasmodium falciparum is highly variable depending on the commercial source. Here we identify a minor contaminant (pepstatin butyl ester) as the active anti-parasitic principle. We synthesize a series of derivatives and characterize an analogue (pepstatin hexyl ester) with low nanomolar activity. By selecting resistant parasite mutants, we find that a parasite esterase, PfPARE (P. falciparum Prodrug Activation and Resistance Esterase) is required for activation of esterified pepstatin. Parasites with esterase mutations are resistant to pepstatin esters and to an open source antimalarial compound, MMV011438. Recombinant PfPARE hydrolyses pepstatin esters and de-esterifies MMV011438. We conclude that (1) pepstatin is a potent but poorly bioavailable antimalarial; (2) PfPARE is a functional esterase that is capable of activating prodrugs; (3) Mutations in PfPARE constitute a mechanism of antimalarial resistance.


Subject(s)
Antimalarials/pharmacology , Drug Resistance , Esterases/genetics , Mutation , Pepstatins/pharmacology , Plasmodium falciparum/drug effects , Plasmodium falciparum/enzymology , Protozoan Proteins/genetics , Esterases/antagonists & inhibitors , Esterases/metabolism , Plasmodium falciparum/genetics , Prodrugs/pharmacology , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/metabolism
16.
Fish Physiol Biochem ; 43(1): 127-136, 2017 Feb.
Article in English | MEDLINE | ID: mdl-27531133

ABSTRACT

Dietary nucleic acids (NAs) were important nutrients. However, the digestion of NAs in stomach has not been studied. In this study, the digestion of NAs by enzymes from fish stomach was investigated. The snakehead pepsins (SP) which were the main enzymes in stomach were extracted and purified. The purity of SP was evaluated by SDS-PAGE and HPLC. The snakehead pepsin 2 (SP2) which was the main component in the extracts was used for investigating the protein and NAs digestion activity. SP2 could digest NAs, including λ DNA and salmon sperm DNA. Interestingly, the digestion could be inhibited by treatment of alkaline solution at pH 8.0 and pepstatin A, and the digestion could happen either in the presence or absence of hemoglobin (Hb) and BSA as the protein substrates. Similarly, the stomach enzymes of banded grouper also showed the NAs digestion activity. NAs could be digested by the stomach enzymes of snakehead and banded grouper. It may be helpful for understanding both animal nutrition and NAs metabolic pathway.


Subject(s)
DNA/metabolism , Fish Proteins/metabolism , Pepsin A/metabolism , Perciformes/metabolism , Stomach/enzymology , Animal Nutritional Physiological Phenomena , Animals , Cattle , Digestion/drug effects , Fish Proteins/chemistry , Fish Proteins/isolation & purification , Hemoglobins/pharmacology , Pepsin A/chemistry , Pepsin A/isolation & purification , Pepstatins/pharmacology , Serum Albumin, Bovine/pharmacology
17.
EBioMedicine ; 9: 278-292, 2016 Jul.
Article in English | MEDLINE | ID: mdl-27333034

ABSTRACT

Although several ADAMs (A disintegrin-like and metalloproteases) have been shown to contribute to the amyloid precursor protein (APP) metabolism, the full spectrum of metalloproteases involved in this metabolism remains to be established. Transcriptomic analyses centred on metalloprotease genes unraveled a 50% decrease in ADAM30 expression that inversely correlates with amyloid load in Alzheimer's disease brains. Accordingly, in vitro down- or up-regulation of ADAM30 expression triggered an increase/decrease in Aß peptides levels whereas expression of a biologically inactive ADAM30 (ADAM30(mut)) did not affect Aß secretion. Proteomics/cell-based experiments showed that ADAM30-dependent regulation of APP metabolism required both cathepsin D (CTSD) activation and APP sorting to lysosomes. Accordingly, in Alzheimer-like transgenic mice, neuronal ADAM30 over-expression lowered Aß42 secretion in neuron primary cultures, soluble Aß42 and amyloid plaque load levels in the brain and concomitantly enhanced CTSD activity and finally rescued long term potentiation alterations. Our data thus indicate that lowering ADAM30 expression may favor Aß production, thereby contributing to Alzheimer's disease development.


Subject(s)
ADAM Proteins/metabolism , Amyloid beta-Peptides/metabolism , Cathepsin D/metabolism , ADAM Proteins/antagonists & inhibitors , ADAM Proteins/genetics , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amino Acid Sequence , Animals , Brain/metabolism , Brain/pathology , Cathepsin D/chemistry , Cell Line, Tumor , Down-Regulation/drug effects , HEK293 Cells , Humans , Lysosomes/metabolism , Macrolides/pharmacology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Fluorescence , Patch-Clamp Techniques , Pepstatins/pharmacology , RNA Interference , RNA, Small Interfering/metabolism
18.
Sci Rep ; 6: 27112, 2016 06 07.
Article in English | MEDLINE | ID: mdl-27271556

ABSTRACT

Acute kidney injury (AKI) is an abrupt reduction in kidney function caused by different pathological processes. It is associated with a significant morbidity and mortality in the acute phase and an increased risk of developing End Stage Renal Disease. Despite the progress in the management of the disease, mortality rates in the last five decades remain unchanged at around 50%. Therefore there is an urgent need to find new therapeutic strategies to treat AKI. Lysosomal proteases, particularly Cathepsin D (CtsD), play multiple roles in apoptosis however, their role in AKI is still unknown. Here we describe a novel role for CtsD in AKI. CtsD expression was upregulated in damaged tubular cells in nephrotoxic and ischemia reperfusion (IRI) induced AKI. CtsD inhibition using Pepstatin A led to an improvement in kidney function, a reduction in apoptosis and a decrease in tubular cell damage in kidneys with nephrotoxic or IRI induced AKI. Pepstatin A treatment slowed interstitial fibrosis progression following IRI induced AKI. Renal transplant biopsies with acute tubular necrosis demonstrated high levels of CtsD in damaged tubular cells. These results support a role for CtsD in apoptosis during AKI opening new avenues for the treatment of AKI by targeting lysosomal proteases.


Subject(s)
Acute Kidney Injury/metabolism , Cathepsin D/metabolism , Kidney Tubules/cytology , Nephrosis/complications , Reperfusion Injury/complications , Acute Kidney Injury/drug therapy , Acute Kidney Injury/etiology , Acute Kidney Injury/physiopathology , Animals , Apoptosis , Cell Line , Disease Models, Animal , Folic Acid/adverse effects , Humans , Kidney Function Tests , Kidney Tubules/drug effects , Kidney Tubules/enzymology , Male , Mice , Nephrosis/chemically induced , Nephrosis/drug therapy , Nephrosis/enzymology , Pepstatins/administration & dosage , Pepstatins/pharmacology , Reperfusion Injury/drug therapy , Up-Regulation
19.
Sci Rep ; 6: 23869, 2016 Mar 31.
Article in English | MEDLINE | ID: mdl-27029611

ABSTRACT

The widespread presence of pepsin-like enzymes in eukaryotes together with their relevance in the control of multiple biological processes is reflected in the large number of studies published so far for this family of enzymes. By contrast, pepsin homologs from bacteria have only recently started to be characterized. The work with recombinant shewasin A from Shewanella amazonensis provided the first documentation of this activity in prokaryotes. Here we extend our studies to shewasin D, the pepsin homolog from Shewanella denitrificans, to gain further insight into this group of bacterial peptidases that likely represent ancestral versions of modern eukaryotic pepsin-like enzymes. We demonstrate that the enzymatic properties of recombinant shewasin D are strongly reminiscent of eukaryotic pepsin homologues. We determined the specificity preferences of both shewasin D and shewasin A using proteome-derived peptide libraries and observed remarkable similarities between both shewasins and eukaryotic pepsins, in particular with BACE-1, thereby confirming their phylogenetic proximity. Moreover, we provide first evidence of expression of active shewasin D in S. denitrificans cells, confirming its activity at acidic pH and inhibition by pepstatin. Finally, our results revealed an unprecedented localization for a family A1 member by demonstrating that native shewasin D accumulates preferentially in the cytoplasm.


Subject(s)
Bacterial Proteins/metabolism , Cytoplasm/enzymology , Pepsin A/metabolism , Shewanella/enzymology , Amino Acid Sequence , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Biological Evolution , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Conserved Sequence , Cytoplasm/ultrastructure , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Hydrogen-Ion Concentration , Kinetics , Pepsin A/antagonists & inhibitors , Pepsin A/chemistry , Pepsin A/genetics , Pepstatins/pharmacology , Peptide Library , Proteolysis , Proteome/genetics , Proteome/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Shewanella/drug effects , Shewanella/genetics , Shewanella/ultrastructure , Substrate Specificity
20.
Sci Rep ; 6: 20101, 2016 Feb 02.
Article in English | MEDLINE | ID: mdl-26831567

ABSTRACT

During chronic kidney disease (CKD) there is a dysregulation of extracellular matrix (ECM) homeostasis leading to renal fibrosis. Lysosomal proteases such as cathepsins (Cts) regulate this process in other organs, however, their role in CKD is still unknown. Here we describe a novel role for cathepsins in CKD. CtsD and B were located in distal and proximal tubular cells respectively in human disease. Administration of CtsD (Pepstatin A) but not B inhibitor (Ca074-Me), in two mouse CKD models, UUO and chronic ischemia reperfusion injury, led to a reduction in fibrosis. No changes in collagen transcription or myofibroblasts numbers were observed. Pepstatin A administration resulted in increased extracellular urokinase and collagen degradation. In vitro and in vivo administration of chloroquine, an endo/lysosomal inhibitor, mimicked Pepstatin A effect on renal fibrosis. Therefore, we propose a mechanism by which CtsD inhibition leads to increased collagenolytic activity due to an impairment in lysosomal recycling. This results in increased extracellular activity of enzymes such as urokinase, triggering a proteolytic cascade, which culminates in more ECM degradation. Taken together these results suggest that inhibition of lysosomal proteases, such as CtsD, could be a new therapeutic approach to reduce renal fibrosis and slow progression of CKD.


Subject(s)
Cathepsin D/antagonists & inhibitors , Chloroquine/pharmacology , Lysosomes/enzymology , Pepstatins/pharmacology , Renal Insufficiency, Chronic/enzymology , Animals , Cathepsin D/metabolism , Collagen/biosynthesis , Dipeptides/pharmacology , Disease Models, Animal , Extracellular Matrix/metabolism , Extracellular Matrix/pathology , Female , Fibrosis , Humans , Lysosomes/pathology , Mice , Myofibroblasts/enzymology , Myofibroblasts/pathology , Renal Insufficiency, Chronic/drug therapy , Renal Insufficiency, Chronic/pathology
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