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European Journal of Medicinal Chemistry Mar 2024Cathepsin D (CD) is overexpressed in several types of cancer and constitutes an important biological target. Pepstatin A, a pentapeptide incorporating two...
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.
Topics: Pepstatins; Cathepsin D; Molecular Docking Simulation; Pepsin A; Alanine; Methionine
PubMed: 38295686
DOI: 10.1016/j.ejmech.2024.116178 -
Mikrochimica Acta Jan 2024A simple and effective pepsin detection assay is reported based on a pepsin-susceptible peptide (PSP) reporter degradation strategy. PSP, which can be specifically...
A simple and effective pepsin detection assay is reported based on a pepsin-susceptible peptide (PSP) reporter degradation strategy. PSP, which can be specifically cleaved by pepsin, was modified with fluorescein isothiocyanate (FITC) and biotin at the N- and C-terminals to be used as a reporter for colorimetric detection of dipsticks. A universal lateral flow dipstick consisting of a streptavidin test line for biotin binding and a sample pad immobilized with a gold-labeled polyclonal (rabbit) anti-FITC antibody was used to verify PSP-based pepsin detection. When the PSP reporter reacts with pepsin in a tube, it cleaves into two fragments, and the cleaved fragments do not display any color on the test line. Therefore, the higher the concentration of pepsin is, the greater is the decrease in test line intensity (I) and the higher is the control line intensity (I). First, the PSP cleavage and dipstick assay conditions for pepsin detection was optimized. The ratio of color intensity (I/I) of PSP-based dipstick assay showed a linear relationship with log concentration of pepsin ranging between 4 and 500 ng/mL (R = 0.98, n = 6), with a limit of detection of 1.4 ng/mL. It also exhibited high specificity and good reproducibility. Finally, pepsin levels were quantified in saliva samples from healthy controls (n = 34) and patients with laryngopharyngeal reflux (LPR, n = 61). Salivary pepsin levels were higher in patients with LPR than in healthy controls. The salivary pepsin levels correlated with those measured using a conventional enzyme-linked immunosorbent assay kit. Therefore, this PSP-based dipstick assay is a convenient tool for assessing salivary pepsin levels.
Topics: Animals; Humans; Rabbits; Biotin; Colorimetry; Cross-Sectional Studies; Pepsin A; Prospective Studies; Reproducibility of Results; Saliva; Fluorescein; Peptides; Isothiocyanates
PubMed: 38294558
DOI: 10.1007/s00604-024-06192-9 -
Pediatric Gastroenterology, Hepatology... Jan 2024This study aimed to investigate the presence of autoantigens in the gastric juices of children.
PURPOSE
This study aimed to investigate the presence of autoantigens in the gastric juices of children.
METHODS
Gastric juice and serum samples were obtained from 53 children <15 years of age who underwent gastric endoscopy. Among these, 8, 22, and 23 participants were in the age groups 0-5, 6-10, and 11-15 years, respectively. These samples were analyzed using two-dimensional electrophoresis (2-DE), immunoblot analysis, and matrix-assisted laser desorption ionization-time of-flight mass spectrometry. Furthermore, we reviewed the histopathological findings and urease test results and compared them with the results of 2-DE and immunoblot analysis.
RESULTS
There were no statistically significant differences in urease test positivity, grades of chronic gastritis, active gastritis, or infiltration of the antrum and body among the three age groups. Three distinct patterns of gastric juice were observed on 2-DE. Pattern I was the most common, and pattern III was not observed below the age of 5 years. Histopathological findings were significantly different among active gastritis (=0.037) and infiltration (=0.060) in the gastric body. The immunoblots showed large spots at an approximate pH of 3-4 and molecular weights of 31-45 kDa. These distinct, large positive spots were identified as gastric lipase and pepsin A and C.
CONCLUSION
Three enzymes, which are normally secreted under acidic conditions were identified as autoantigens. Further investigation of the pathophysiology and function of autoantigens in the stomach is required.
PubMed: 38249638
DOI: 10.5223/pghn.2024.27.1.15 -
Marine Drugs Dec 2023Fish head byproducts derived from surimi processing contribute about 15% of the total body weight, which are beneficial to health because they contain essential...
Pepsin Hydrolysate from Surimi Industry-Related Olive Flounder Head Byproducts Attenuates LPS-Induced Inflammation and Oxidative Stress in RAW 264.7 Macrophages and In Vivo Zebrafish Model.
Fish head byproducts derived from surimi processing contribute about 15% of the total body weight, which are beneficial to health because they contain essential nutrients. In this study, olive flounder (OF) was the target species in order to maximize the byproduct utilization. In RAW 264.7 macrophages, the seven hydrolysates from OF head byproducts were examined for their inhibitory potential against inflammation and the oxidative stress induced by lipopolysaccharides (LPS). The pepsin hydrolysate (OFH-PH) demonstrated strong anti-inflammatory activity via the down-regulation of NO production, with an IC50 value of 299.82 ± 4.18 µg/mL. We evaluated the inhibitory potential of pro-inflammatory cytokines and PGE2 to confirm these findings. Additionally, iNOS and COX-2 protein expressions were confirmed using western blotting. Furthermore, the results from the in vivo zebrafish model demonstrated that OFH-PH decreased the LPS-elevated heart rate, NO production, cell death, and intracellular ROS level, while increasing the survival percentage. Hence, the obtained results of this study serve as a platform for future research and provide insight into the mediation of inflammatory disorders. These results suggest that OFH-PH has the potential to be utilized as a nutraceutical and functional food ingredient.
Topics: Animals; Zebrafish; Flounder; Lipopolysaccharides; Pepsin A; Inflammation; Perciformes; Oxidative Stress; Macrophages
PubMed: 38248649
DOI: 10.3390/md22010024 -
Langmuir : the ACS Journal of Surfaces... Jan 2024Proteolytic enzymes play a pivotal role in the industry. Still, because of denaturation, the extensive applicability at their level of best catalytic efficiency over a...
Chemically Bonded Pepsin via Its Inert Center to Diazo Functionalized Silica Gel through Multipoint Attachment Mode: A Way of Restoring Biocatalytic Sustainability over "Wider pH" Range.
Proteolytic enzymes play a pivotal role in the industry. Still, because of denaturation, the extensive applicability at their level of best catalytic efficiency over a more comprehensive pH range, particularly in alkaline conditions over pH 8, has not been fully developed. On the other hand, enzyme immobilization following a suitable protocol is a long pending issue that determines the conformational stability, specificity, selectivity, enantioselectivity, and activity of the native enzymes at long-range pH. As a bridge between these two findings, in an attempt at a freezing temperature 273-278 K at an alkaline pH, the diazo-functionalized silica gel (SG) surface has been used to rapidly diazo couple pepsin through its inert center, the -carbon of the phenolic -OH of surface-occupied Tyr residues in a multipoint mode: when all the various protein groups, viz., amino, thiol, phenol, imidazole, carboxy, etc., in the molecular sequence including those belonging to the active sites, remain intact, the inherent inbuilt interactions among themselves remain. Thereby, the macromolecule's global conformation and helicity preserve the status quo. The dimension of the SG-enzyme conjugate confirms as {Si(OSi) (HO)} {-O-Si(CH)-O-CH-N═N}·{pepsin}·HO; where the values of and have been determined respectively as 347 and 188. The material performs the catalytic activity much better at 7-8.5 than at pH 2-3.5 and continues for up to six months without any appreciable change.
Topics: Pepsin A; Silica Gel; Enzymes, Immobilized; Proteins; Hydrogen-Ion Concentration; Enzyme Stability
PubMed: 38240266
DOI: 10.1021/acs.langmuir.3c03113 -
The Laryngoscope Jul 2024This study aimed to evaluate the role of pepsin inhibitors in the inflammatory response and their effects on laryngeal mucosal integrity during gastroesophageal reflux...
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.
Topics: Animals; Mice; Gastroesophageal Reflux; Disease Models, Animal; Pepsin A; Pepstatins; Laryngeal Mucosa; Male; Inflammation
PubMed: 38214310
DOI: 10.1002/lary.31239 -
International Journal of Biological... Feb 2024The substantial nutritional content and diversified biological activity of plant-based nutraceuticals are due to polyphenolic chemicals. These chemicals are important...
Study of an inhibitory effect of plant polyphenolic compounds against digestive enzymes using bench-working experimental evidence predicted by molecular docking and dynamics.
The substantial nutritional content and diversified biological activity of plant-based nutraceuticals are due to polyphenolic chemicals. These chemicals are important and well-studied plant secondary metabolites. Their protein interactions are extensively studied. This relationship is crucial for the logical development of functional food and for enhancing the availability and usefulness of polyphenols. This study highlights the influence of protein types and polyphenols on the interaction, where the chemical bindings predominantly consist of hydrophobic interactions and hydrogen bonds. The interaction between polyphenolic compounds (PCs) and digestive enzymes concerning their inhibitory activity has not been fully studied. Therefore, we have examined the interaction of four digestive enzymes (α-amylase, pepsin, trypsin, and α-chymotrypsin) with four PCs (curcumin, diosmin, morin, and 2',3',4'-trihydroxychalcone) through in silico and in vitro approaches. In vitro plate assays, enzyme kinetics, spectroscopic assays, molecular docking, and simulations were performed. We observed all these PCs have significant docking scores and preferable interaction with the active site of the digestive enzymes, resulting in the reduction of enzyme activity. The enzyme-substrate binding mechanism was determined using the Lineweaver Burk plot, indicating that the inhibition occurred competitively. Among four PCs diosmin and morin has the highest interaction energy over digestive enzymes with IC value of 1.13 ± 0.0047 and 1.086 ± 0.0131 μM. Kinetic studies show that selected PCs inhibited pepsin, trypsin, and chymotrypsin competitively and inhibited amylase in a non-competitive manner, especially by 2',3',4'-trihydroxychalcone. This study offers insights into the mechanisms by which the selected PCs inhibit the enzymes and has the potential to enhance the application of curcumin, diosmin, morin, and 2',3',4'-trihydroxychalcone as natural inhibitors of digestive enzymes.
Topics: Molecular Docking Simulation; Pepsin A; Trypsin; Curcumin; Kinetics; Diosmin; Polyphenols; Flavonoids; alpha-Amylases; alpha-Glucosidases
PubMed: 38185307
DOI: 10.1016/j.ijbiomac.2024.129222 -
Protein Expression and Purification Apr 2024Human pepsinogens (mainly pepsinogen I and pepsinogen II) are the major inactive precursor forms of the digestive enzyme pepsin which play a crucial role in protein...
Human pepsinogens (mainly pepsinogen I and pepsinogen II) are the major inactive precursor forms of the digestive enzyme pepsin which play a crucial role in protein digestion. The levels and ratios of human pepsinogens have demonstrated potential as diagnostic biomarkers for gastrointestinal diseases, particularly gastric cancer. Nanobodies are promising tools for the treatment and diagnosis of diseases, owing to their unique recognition properties. In this study, recombinant human pepsinogens proteins were expressed and purified as immunized antigens. We constructed a VHH phage library and identified several nanobodies via phage display bio-panning. We determined the binding potency and cross-reactivity of these nanobodies. Our study provides technical support for developing immunodiagnostic reagents targeting human pepsinogens.
Topics: Humans; Pepsinogens; Single-Domain Antibodies; Gastric Mucosa; Pepsin A
PubMed: 38184161
DOI: 10.1016/j.pep.2024.106431 -
Brazilian Journal of Microbiology :... Mar 2024This study aimed to investigate the probiotic properties of Lactic Acid Bacteria (LAB) isolates derived from various milk sources. These isolates identified based on...
This study aimed to investigate the probiotic properties of Lactic Acid Bacteria (LAB) isolates derived from various milk sources. These isolates identified based on their morphological characteristics and 16S rRNA gene sequencing. Four strains of Lactococcus lactis and two strains of Weissella confusa were identified with over 96% 16S rRNA gene similarity according to the NCBI-BLAST results. The survival of the isolates was determined in low pH, pepsin, bile salts, and pancreatin, and their adhesion ability was assessed by in vitro cell adhesion assay, hydrophobicity, auto- and co-aggregation, and safety criteria were determined by hemolytic, gelatinase activities, and DNAse production ability tests. The results showed that the LAB isolates had different levels of resistance to various stress factors. L. lactis subsp. cremoris MH31 showed the highest resistance to bile salt, while the highest pH resistance was observed in L. lactis MH31 at pH 3.0. All the isolates survived in pepsin exposure at pH 3.0 for 3 h. The auto-aggregation test results showed that all strains exhibited auto-aggregation ranging from 84.9 to 91.4%. Co-aggregation percentage ranged from 19 - 54% and 17 - 57% against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, respectively. The hydrophobicity capacity of the LAB isolated ranged from 35-61%. These isolates showed different adhesion abilities to Caco-2 cells (81.5% to 92.6%). None of the isolates exhibited DNase, gelatinase and hemolytic activity (γ-hemolysis). All results indicate that these LAB strains have the potential to be used as probiotics.
Topics: Humans; Animals; Lactococcus lactis; RNA, Ribosomal, 16S; Caco-2 Cells; Milk; Pepsin A; Lactobacillales; Probiotics; Deoxyribonucleases; Gelatinases; Weissella
PubMed: 38158467
DOI: 10.1007/s42770-023-01208-7 -
Food Chemistry May 2024Application of nanomaterials (NMs) in agriculture poses an ingestion risk to humans and may affect the digestive process. Different fates of NMs with differential...
Application of nanomaterials (NMs) in agriculture poses an ingestion risk to humans and may affect the digestive process. Different fates of NMs with differential charges in the gastrointestinal tract should be considered. In this study, the interaction between three carbon dots (CDs) carried with different functional groups (-NH, -OH, and -COOH) and pepsin was analyzed through an in vitro digestion model. The results showed that CDs significantly reduced pepsin activity. Among them, CDs-NH had the greatest effect, following by CDs-OH, and CDs-COOH. Besides, molecular docking demonstrated the specific binding site of CDs to pepsin, while the most stable binding energy (-8.10 kcal/mol) was formed between CDs-NH and pepsin. Further, CDs formed a nanomaterial-protein crown structure with pepsin. The present study enriches the functional group properties of CDs in the digestion and provides new ideas for the potential human health of NMs.
Topics: Humans; Pepsin A; Carbon; Molecular Docking Simulation; Binding Sites; Digestion; Quantum Dots
PubMed: 38134824
DOI: 10.1016/j.foodchem.2023.138224