ABSTRACT
Remarkable resistance of bacterial biofilms to high doses of antimicrobials and antibiotics is one of their main challenges. Encapsulation of proteolytic enzymes is one of the suggested strategies to tackle this problem. In this regard, the antibacterial and anti-biofilm activity of biocompatible hyaluronic acid- Lysine nanogels containing serratiopeptidase (SRP-loaded HA-Lys nanogel) was assessed against P. aeruginosa and S. aureus strains. SRP-loaded HA-Lys nanogel was prepared using dropping method and optimized by Box-Behnken experimental design. These formulations were studied for physical characterization, release profile, stability, bioactivity, and anti-biofilm effects. The particle size, polydispersity index (PDI), and surface charge were measured by Zetasizer Nano ZS. The average particle size and zeta potential of the optimum sample were 156 nm and -14.1 mV, respectively. SRP release showed an initial burst followed by sustained release and the highest release was around 77%. Enzyme biological activity data revealed the higher efficiency of free SRP compared to SRP-loaded HA-Lys nanogel. The time-kill assay showed that both forms of SRP-loaded HA-Lys nanogel and blank HA-Lys nanogel showed significant antimicrobial activity against examined bacteria in comparison to the free enzyme. The obtained results demonstrated improved anti-biofilm efficacy and down regulation of tested biofilm genes for both SRP-loaded HA-Lys nanogel 100% and blank HA-Lys nanogel 100% compared to SRP 100%.
Subject(s)
Hyaluronic Acid , Lysine , Polyethylene Glycols , Polyethyleneimine , Nanogels/chemistry , Hyaluronic Acid/chemistry , Lysine/pharmacology , Staphylococcus aureus/physiology , Peptide Hydrolases/pharmacology , Anti-Bacterial Agents/pharmacology , BiofilmsABSTRACT
The inflamed mucosa contains a complex assortment of proteases that may participate in wound healing or the development of inflammation-associated colon cancer. We sought to determine the role of protease-activated receptor 2 (PAR2) in epithelial wound healing in both untransformed and transformed colonic epithelial cells. Monolayers of primary epithelial cells derived from organoids cultivated from patient colonic biopsies and of the T84 colon cancer cell line were grown to confluence, wounded in the presence of a selective PAR2-activating peptide, and healing was visualized by live cell microscopy. Inhibitors of various signaling molecules were used to assess the relevant pathways responsible for wound healing. Activation of PAR2 induced an enhanced wound-healing response in T84 cells but not primary cells. The PAR2-enhanced wound-healing response was associated with the development of lamellipodia in cells at the wound edge, consistent with sheet migration. The response to PAR2 activation in T84 cells was completely dependent on Src kinase activity and partially dependent on Rac1 activity. The Src-associated signaling molecules, focal adhesion kinase, and epidermal growth factor receptor, which typically mediate wound-healing responses, were not involved in the PAR2 response. Experiments repeated in the presence of the inflammatory cytokines TNF and IFNγ revealed a synergistically enhanced PAR2 wound-healing response in T84s but not primary cells. The epithelial response to proteases may be different between primary and cancer cells and is accentuated in the presence of inflammatory cytokines. Our findings have implications for understanding epithelial restitution in the context of inflammatory bowel disease (IBD) and inflammation-associated colon cancer.NEW & NOTEWORTHY Protease-activated receptor 2 enhances wound healing in the T84 colon cancer cell line, but not in primary cells derived from patient biopsies, an effect that is synergistically enhanced in the presence of the inflammatory cytokines TNF and IFNγ.
Subject(s)
Colonic Neoplasms , Receptor, PAR-2 , Humans , Cell Line , Cell Movement , Colonic Neoplasms/metabolism , Cytokines/metabolism , Epithelial Cells/metabolism , Inflammation/metabolism , Peptide Hydrolases/metabolism , Peptide Hydrolases/pharmacology , Receptor, PAR-2/metabolismABSTRACT
PURPOSE: The PRESTIGIO Registry was established in 2017 to collect clinical, virological and immunological monitoring data from people living with HIV (PLWH) with documented four-class drug resistance (4DR). Key research purposes include the evaluation of residual susceptibility to specific antiretrovirals and the validation of treatment and monitoring strategies in this population. PARTICIPANTS: The PRESTIGIO Registry collects annual plasma and peripheral blood mononuclear cell samples and demographic, clinical, virological, treatment and laboratory data from PLWH followed at 39 Italian clinical centres and characterised by intermediate-to-high genotypic resistance to ≥1 nucleoside reverse transcriptase inhibitors, ≥1 non-nucleoside reverse transcriptase inhibitors, ≥1 protease inhibitors, plus either intermediate-to-high genotypic resistance to ≥1 integrase strand transfer inhibitors (INSTIs) or history of virological failure to an INSTI-containing regimen. To date, 229 people have been recorded in the cohort. Most of the data are collected from the date of the first evidence of 4DR (baseline), with some prebaseline information obtained retrospectively. Samples are collected from the date of enrollment in the registry. FINDINGS TO DATE: The open-ended cohort has been used to assess (1) prognosis in terms of survival or development of AIDS-related or non-AIDS-related clinical events; (2) long-term efficacy and safety of different antiretroviral regimens and (3) virological and immunological factors predictive of clinical outcome and treatment efficacy, especially through analysis of plasma and cell samples. FUTURE PLANS: The registry can provide new knowledge on how to implement an integrated approach to study PLWH with documented resistance to the four main antiretroviral classes, a population with a limited number of individuals characterised by a high degree of frailty and complexity in therapeutic management. Given the scheduled annual updates of PLWH data, the researchers who collaborate in the registry can send study proposals at any time to the steering committee of the registry, which evaluates every 3 months whether the research studies can be conducted on data and biosamples from the registry and whether they are aimed at a better understanding of a specific health condition, the emergence of comorbidities or the effect of potential treatments or experimental drugs that may have an impact on disease progression and quality of life. Finally, the research studies should aim to be inclusive, innovative and in touch with the communities and society as a whole. TRIAL REGISTRATION NUMBER: NCT04098315.
Subject(s)
Anti-HIV Agents , HIV Infections , HIV-1 , Humans , Reverse Transcriptase Inhibitors/therapeutic use , Reverse Transcriptase Inhibitors/pharmacology , HIV-1/genetics , Integrase Inhibitors/pharmacology , Integrase Inhibitors/therapeutic use , Peptide Hydrolases/pharmacology , Peptide Hydrolases/therapeutic use , Leukocytes, Mononuclear , Quality of Life , Retrospective Studies , HIV Infections/drug therapy , Anti-HIV Agents/therapeutic use , Registries , Italy , RNA-Directed DNA Polymerase/pharmacology , RNA-Directed DNA Polymerase/therapeutic useABSTRACT
BACKGROUND: Enzyme combinations, particularly phytase (PHY) with various carbohydrases and proteases, are utilized in commercial broiler production to enhance nutrient and energy bioavailability. OBJECTIVE: A feeding study was undertaken to determine whether the efficiency of an Escherichia coli-derived PHY and a feed enzyme complex (FEC) derived from Bacillus spp. containing carbohydrase and protease as main activities in broiler chickens is dependent on diet quality. A total of 900 male one-day-old broiler chickens (Ross 308) were assigned to a 2 × 3 factorial arrangement of the treatments with 2 different nutrient density diets, standard nutrient diet (SN diet) and a low-nutrient diet (LN diet; -100 kcal/kg for AMEn and -5% for crude protein [CP] and limiting amino acids), and 3 enzyme treatments (control [no enzymes], PHY and PHY + FEC). Each treatment group was composed of 6 replicates of 25 birds each. RESULTS: The LN diet caused a decrease in performance index, tibia length and diameter, tibia calcium content and jejunal villus surface area (VSA). The interaction effects between diet and enzyme supplementation were observed (p < 0.05) on overall average daily gain (ADG), performance index, tibia ash content and jejunal villus height (VH) and VSA, with the favourable benefits of PHY + FEC treatment being more pronounced in the LN diets. Regardless of dietary nutrient density, supplementation with PHY alone or combined with FEC enhanced (p < 0.05) final body weight, overall ADG and jejunal villus height (VH)/crypt depth, with the highest values observed in the PHY + FEC group. The PHY + FEC treatment also improved (p < 0.05) overall feed conversion ratio, apparent ileal digestibility of dry matter, organic matter, CP, and energy, and tibia phosphorus content compared to the control treatment. CONCLUSIONS: The results indicate that the simultaneous addition of PHY and FEC to the LN diets improved the growth rate, bone mineralization and gut morphology.
Subject(s)
6-Phytase , Dietary Supplements , Glycoside Hydrolases , Animals , Male , Chickens , 6-Phytase/metabolism , 6-Phytase/pharmacology , Peptide Hydrolases/pharmacology , Calcification, Physiologic , Escherichia coli , Digestion , Diet/veterinary , Nutrients , Animal Feed/analysisABSTRACT
How different dietary fibers including pectin, cellulose and lignin affect casein digestibility was studied using in vitro static protocols. Peptides' profile, free amino acids (AAs) content, casein-DF interactions and their influences on enzymatic activities of proteolytic enzymes were studied using combined techniques. Under gastric and intestinal digestive conditions, while pectin could reduce casein digestibility (with an averaged decrease of 12.15% and 7.83, respectively) through both depletion flocculation and hydrogen-binding interactions, lignin inhibited the digestion of casein straightly through reducing the enzymatic activity of proteolytic enzymes, thereby altering the production of free AAs. Although cellulose showed the least detrimental effects, it still significantly reduced the content of Thr, Glu, Val, Leu, Phe, Lys, and no Arg was released. Deeper insight into casein-DF interactions and their influences on casein digestibility improves the development of more effective forms of DF for improving AA homeostasis in individuals.
Subject(s)
Caseins , Lignin , Humans , Caseins/metabolism , Animal Feed/analysis , Digestion , Amino Acids/metabolism , Dietary Fiber/metabolism , Cellulose/pharmacology , Pectins/pharmacology , Peptide Hydrolases/pharmacologyABSTRACT
In the pursuit of novel antiretroviral therapies for human immunodeficiency virus type-1 (HIV-1) proteases (PRs), recent improvements in drug discovery have embraced machine learning (ML) techniques to guide the design process. This study employs ensemble learning models to identify crucial substructures as significant features for drug development. Using molecular docking techniques, a collection of 160 darunavir (DRV) analogs was designed based on these key substructures and subsequently screened using molecular docking techniques. Chemical structures with high fitness scores were selected, combined, and one-dimensional (1D) screening based on beyond Lipinski's rule of five (bRo5) and ADME (absorption, distribution, metabolism, and excretion) prediction implemented in the Combined Analog generator Tool (CAT) program. A total of 473 screened analogs were subjected to docking analysis through convolutional neural networks scoring function against both the wild-type (WT) and 12 major mutated PRs. DRV analogs with negative changes in binding free energy ( ΔΔ G bind ) compared to DRV could be categorized into four attractive groups based on their interactions with the majority of vital PRs. The analysis of interaction profiles revealed that potent designed analogs, targeting both WT and mutant PRs, exhibited interactions with common key amino acid residues. This observation further confirms that the ML model-guided approach effectively identified the substructures that play a crucial role in potent analogs. It is expected to function as a powerful computational tool, offering valuable guidance in the identification of chemical substructures for synthesis and subsequent experimental testing.
Subject(s)
HIV Infections , HIV Protease Inhibitors , HIV-1 , Humans , Darunavir/pharmacology , HIV Protease Inhibitors/pharmacology , HIV Protease Inhibitors/chemistry , Peptide Hydrolases/pharmacology , Molecular Docking Simulation , HIV Protease/chemistry , Drug DiscoveryABSTRACT
Neurogenesis is known to be closely associated with depression. We aimed to investigate whether a polypeptide monomer derived from pilose antler (polypeptide sequence LSALEGVFYP, PAP) exerts an antidepressant effect by influencing neurogenesis, and to elucidate the mechanism of its antidepressant action. Behavioral tests were performed to observe the antidepressant effect of PAP. Neurogenesis in the dentate gyrus (DG) region of hippocampus was observed by immunofluorescence. The expression of key proteins of Sentrin/SUMO-specific proteases 2 (SENP2)- Phosphoinositide-specific phospholipase C beta 4 (PLCß4) pathway was accessed by co-immunoprecipitation (Co-IP), and the calcium homeostasis associated proteins were observed via Western blot (WB). Subsequently, temozolomide (TMZ) pharmacologically blocked neurogenesis to verify the antidepressant effect of PAP on neurogenesis. The mechanism of PAP antidepressant effect was verified by constructing a sh-SENP2 virus vector to silence SENP2 protein. Finally, corticosterone (CORT)-induced PC12 cell model was used to verify whether PAP was involved in the process of deconjugated PLCß4 SUMOylated. The results showed that PAP improved depression-like behavior and neurogenesis induced by chronic unpredictable mild stimulation (CUMS). In addition, PAP acted on SENP2-PLCß4 pathway to deconjugate the SUMOylation of PLCß4 and affect calcium homeostasis. Pharmacological blockade of neurogenesis by TMZ treatment impaired the antidepressant efficacy of PAP. Knockout of SENP2 in the CUMS model attenuated the antidepressant response of PAP, and the impaired neurogenesis was not ameliorated by PAP treatment. In summary, PAP acted on the SENP2-PLCß4 signaling pathway to inhibit the SUMOylation of PLCß4 and maintain calcium homeostasis, thereby protecting neurogenesis and playing an antidepressant role.
Subject(s)
Depression , Peptide Hydrolases , Animals , Depression/drug therapy , Depression/etiology , Depression/metabolism , Phospholipase C beta/metabolism , Peptide Hydrolases/pharmacology , Calcium/metabolism , Antidepressive Agents/pharmacology , Antidepressive Agents/therapeutic use , Antidepressive Agents/metabolism , Signal Transduction , Peptides/pharmacology , Endopeptidases/metabolism , Endopeptidases/pharmacology , Hippocampus , Stress, Psychological/metabolism , Disease Models, AnimalABSTRACT
OBJECTIVE: The COVID-19 pandemic has been recognized as severe acute respiratory syndrome, one of the worst and disastrous infectious diseases in human history. Until now, there is no cure to this contagious infection although some multinational pharmaceutical companies have synthesized the vaccines and injecting them into humans, but a drug treatment regimen is yet to come. AIM: Among the multiple areas of SARS-CoV-2 that can be targeted, protease protein has significant values due to its essential role in viral replication and life. The repurposing of FDA-approved drugs for the treatment of COVID-19 has been a critical strategy during the pandemic due to the urgency of effective therapies. The novelty in this work refers to the innovative use of existing drugs with greater safety, speed, cost-effectiveness, broad availability, and diversity in the mechanism of action that have been approved and developed for other medical conditions. METHODS: In this research work, we have engaged drug reprofiling or drug repurposing to recognize possible inhibitors of protease protein 6M03 in an instantaneous approach through computational docking studies. RESULTS: We screened 16 FDA-approved anti-viral drugs that were known for different viral infections to be tested against this contagious novel strain. Through these reprofiling studies, we come up with 5 drugs, namely, Delavirdine, Fosamprenavir, Imiquimod, Stavudine, and Zanamivir, showing excellent results with the negative binding energies in Kcal/mol as - 8.5, - 7.0, - 6.8, - 6.8, and - 6.6, respectively, in the best binding posture. In silico studies allowed us to demonstrate the potential role of these drugs against COVID-19. CONCLUSION: In our study, we also observed the nucleotide sequence of protease protein consisting of 316 amino acid residues and the influence of these pronouncing drugs over these sequences. The outcome of this research work provides researchers with a track record for carrying out further investigational procedures by applying docking simulations and in vitro and in vivo experimentation with these reprofile drugs so that a better drug can be formulated against coronavirus.
Subject(s)
COVID-19 , Humans , Antiviral Agents , SARS-CoV-2 , Drug Repositioning/methods , Pandemics , Molecular Docking Simulation , Peptide Hydrolases/pharmacologyABSTRACT
Flavonoids are vital candidates to fight against a wide range of pathogenic microbial infections. Due to their therapeutic potential, many flavonoids from the herbs of traditional medicine systems are now being evaluated as lead compounds to develop potential antimicrobial hits. The emergence of SARS-CoV-2 caused one of the deadliest pandemics that has ever been known to mankind. To date, more than 600 million confirmed cases of SARS-CoV2 infection have been reported worldwide. Situations are worse due to the unavailability of therapeutics to combat the viral disease. Thus, there is an urgent need to develop drugs against SARS-CoV2 and its emerging variants. Here, we have carried out a detailed mechanistic analysis of the antiviral efficacy of flavonoids in terms of their potential targets and structural feature required for exerting their antiviral activity. A catalog of various promising flavonoid compounds has been shown to elicit inhibitory effects against SARS-CoV and MERS-CoV proteases. However, they act in the high-micromolar regime. Thus a proper leadoptimization against the various proteases of SARS-CoV2 can lead to high-affinity SARS-CoV2 protease inhibitors. To enable lead optimization, a quantitative structure-activity relationship (QSAR) analysis has been developed for the flavonoids that have shown antiviral activity against viral proteases of SARS-CoV and MERS-CoV. High sequence similarities between coronavirus proteases enable the applicability of the developed QSAR to SARS-CoV2 proteases inhibitor screening. The detailed mechanistic analysis of the antiviral flavonoids and the developed QSAR models is a step forward toward the development of flavonoid-based therapeutics or supplements to fight against COVID-19.
Subject(s)
COVID-19 , Middle East Respiratory Syndrome Coronavirus , Humans , SARS-CoV-2 , RNA, Viral , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Flavonoids/pharmacology , Flavonoids/therapeutic use , Flavonoids/chemistry , Protease Inhibitors , Peptide Hydrolases/pharmacology , Molecular Docking SimulationABSTRACT
The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a serious threat to human. Since there are still no effective treatment options against the new emerging variants of SARS-CoV-2, it is necessary to devote a continuous endeavor for more targeted drugs and the preparation for the next pandemic. Salvia miltiorrhiza and its active ingredients possess wide antiviral activities, including against SARS-CoV-2. Danshensu, as one of the most important active ingredients in Salvia miltiorrhiza, has been reported to inhibit the entry of SARS-CoV-2 into ACE2 (angiotensin-converting enzyme 2)-overexpressed HEK-293T cells and Vero-E6 cells. However, there is a paucity of information regarding its detailed target and mechanism against SARS-CoV-2. Here, we present Danshensu as a covalent inhibitor of 3-chymotrypsin-like protease (3CLpro) against SARS-CoV-2 by the time-dependent inhibition assay (TDI) and mass spectrometry analysis. Further molecular docking, site-directed mutagenesis, circular dichroism (CD) and fluorescence spectra revealed that Danshensu covalently binds to C145 of SARS-CoV-2 3CLpro, meanwhile forming the hydrogen bonds with S144, H163 and E166 in the S1 site. Structure-based optimization of Danshensu led to the discovery of the promising compounds with good inhibitory activity and microsomal stability in vitro. Due to Danshensu inhibiting lung inflammation in the mouse model, we found that Danshensu derivatives also showed better anti-inflammatory activity than Danshensu in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Thus, our study provides not only the clue of the efficacy of Salvia miltiorrhiza against SARS-CoV-2, but also a detailed mechanistic insight into the covalent mode of action of Danshensu for design of covalent inhibitors against SARS-CoV-2 3CLpro, highlighting its potential as a bifunctional molecule with antivirus and anti-inflammation.
Subject(s)
COVID-19 , Lactates , SARS-CoV-2 , Animals , Mice , Humans , Molecular Docking Simulation , Viral Nonstructural Proteins/chemistry , Antiviral Agents/chemistry , Peptide Hydrolases/pharmacology , Protease Inhibitors/pharmacologyABSTRACT
Background: Feed additives that increase nutrient availability in feeds have gained a lot of interest. Aim: An experiment was conducted to determine whether amylase, protease, and their combined supplementation affected broiler performance. Methods: Two hundred eighty broiler chicks were selected and distributed randomly into 28 replicate pens with four treatment groups and seven replicates under a completely randomized design. A total of four diets were developed, having 0, 100, 100, and 100 + 100 g of control (AP0), amylase (A1), protease (P1), and amylase + protease (AP1)/ton of feed, respectively. Four replicates of each treatment were fed each diet. Each diet was randomly allotted to each group. Ad-libitum feeding was provided to the birds. The feeding program had starter and finisher diets. Upon completion of the experiment, three birds from each pen were slaughtered to analyze the carcass characteristics and organ weight. Results: Differences were insignificant between 100 g/ton of amylase supplementation and FI, body weight gain (BWG), or feed conversion ratio (FCR) (p > 0.05). Supplementation with 10 0g/ton of protease did not significantly affect FI, BWG, and FCR (p > 0.05). Similarly, 100 + 100 g/ton of amylase + protease addition had no significant effect on FI, BWG, and FCR (p > 0.05). None of the treatments significantly affected carcass weight, abdominal fat percentage, dressing percentage, drumstick, wings, breast, and thigh weights (p > 0.05). In addition, there were no significant effects (p > 0.05) on the weight of the heart, liver, gizzard, and spleen. Conclusion: In conclusion, amylase, protease, and their combined supplementation at a rate of 100 g/ton of feed did not influence BWG, FI, FCR, carcass characteristics, or organ weight.
Subject(s)
Amylases , Chickens , Animals , Diet/veterinary , Dietary Supplements , Peptide Hydrolases/pharmacologyABSTRACT
Middle East Respiratory Syndrome (MERS) is a viral respiratory disease caused b a special type of coronavirus called MERS-CoV. In the search for effective substances against the MERS-CoV main protease, we looked into compounds from brown algae, known for their medicinal benefits. From a set of 1212 such compounds, our computer-based screening highlighted four-CMNPD27819, CMNPD1843, CMNPD4184, and CMNPD3156. These showed good potential in how they might attach to the MERS-CoV protease, comparable to a known inhibitor. We confirmed these results with multiple computer tests. Studies on the dynamics and steadiness of these compounds with the MERS-CoV protease were performed using molecular dynamics (MD) simulations. Metrics like RMSD and RMSF showed their stability. We also studied how these compounds and the protease interact in detail. An analysis technique, PCA, showed changes in atomic positions over time. Overall, our computer studies suggest brown algae compounds could be valuable in fighting MERS. However, experimental validation is needed to prove their real-world effectiveness.
Subject(s)
Coronavirus Infections , Middle East Respiratory Syndrome Coronavirus , Humans , Viral Proteins , Coronavirus Infections/drug therapy , Endopeptidases , Peptide Hydrolases/pharmacologyABSTRACT
The COVID-19 pandemic has spurred intense research efforts to identify effective treatments for SARS-CoV-2. In silico studies have emerged as a powerful tool in the drug discovery process, particularly in the search for drug candidates that interact with various SARS-CoV-2 receptors. These studies involve the use of computer simulations and computational algorithms to predict the potential interaction of drug candidates with target receptors. The primary receptors targeted by drug candidates include the RNA polymerase, main protease, spike protein, ACE2 receptor, and transmembrane protease serine 2 (TMPRSS2). In silico studies have identified several promising drug candidates, including Remdesivir, Favipiravir, Ribavirin, Ivermectin, Lopinavir/Ritonavir, and Camostat Mesylate, among others. The use of in silico studies offers several advantages, including the ability to screen a large number of drug candidates in a relatively short amount of time, thereby reducing the time and cost involved in traditional drug discovery methods. Additionally, in silico studies allow for the prediction of the binding affinity of the drug candidates to target receptors, providing insight into their potential efficacy. This study is aimed at assessing the useful contributions of the application of computational instruments in the discovery of receptors targeted in SARS-CoV-2. It further highlights some identified advantages and limitations of these studies, thereby revealing some complementary experimental validation to ensure the efficacy and safety of identified drug candidates.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/metabolism , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Antiviral Agents/metabolism , Pandemics , Peptide Hydrolases/pharmacologyABSTRACT
Our previous report established that RUNX family transcription factor 1 (RUNX1) promotes proliferation of mouse retinal microvascular endothelial cells (mRMECs) and exacerbates diabetic retinopathy (DR). However, the mechanism behind the upregulation of RUNX1 remains unclear. This study aims to investigate the possible correlation between histone SUMOylation and RUNX1 in DR, as well as the involved molecules. A mouse model of diabetes was induced by streptozotocin (STZ). These mice had increased retinal thickness and elevated production of inflammatory cytokines. Additionally, they showed elevated levels of SUMO1 and SUMO2/3, but reduced levels of SUMO specific peptidase 1 (SENP1) in retinal tissues. Co-immunoprecipitation and Western blot assays revealed that the RUNX1 protein was primarily modified by SUMO2/3, and SENP1 inhibited SUMO2/3 modification, thereby reducing RUNX1 expression. Overexpression of SENP1 alleviated symptoms in mice and alleviated inflammation. In vitro experiments demonstrated that the SENP1 overexpression suppressed the proliferation, migration, and angiogenesis of high-glucose-induced mRMECs. However, further overexpression of RUNX1 counteracted the alleviating effects of SENP1 both in vivo and in vitro. In conclusion, this study demonstrates that the downregulation of SENP1 in DR leads to SUMO2/3-dependent activation of RUNX1. This activation promotes proliferation of mRMECs and exacerbates DR symptoms in mice.
Subject(s)
Diabetes Mellitus , Diabetic Retinopathy , Animals , Mice , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Diabetes Mellitus/metabolism , Diabetic Retinopathy/metabolism , Endothelial Cells/metabolism , Peptide Hydrolases/metabolism , Peptide Hydrolases/pharmacology , Retina/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/pharmacologyABSTRACT
Antimicrobial resistance (AMR) is a global health threat. The dramatic increase of Methicillin-resistant Staphylococcus aureus (MRSA) infections emphasizes the need to find new anti-infective agents with a novel mode of action. The Caseinolytic protease (ClpP) is a central virulence factor in stress survival, virulence, and antibiotic resistance of MRSA. Here, we found ayanin, a flavonoid isolated from Callicarpa nudiflora, was an inhibitor of MRSA ClpP with an IC50 of 19.63 µM. Using quantitative real-time PCR, ayanin reduced the virulence of Staphylococcus aureus (S. aureus) by down-regulating the level of some important virulence factors, including agrA, RNAâ ¢, hla, pvl, psmα and spa. The results of cellular thermal shift assay and thermal shift assay revealed a binding between ayanin and ClpP. Molecular docking showed that ASP-168, ASN-173 and ARG-171 were the potential binding sites for ClpP binding to ayanin. ClpP mutagenesis study further indicated that ARG-171 and ASN-173 were the main active sites of ClpP. The affinity constant (KD) value of ayanin with ClpP was 3.15 × 10-5 M measured by surface plasmon resonance. In addition, ayanin exhibited a significant therapeutic effect on pneumonia infection induced by S. aureus in mice in vivo, especially in combination with vancomycin. This is the first report of ayanin with in vivo and in vitro efficacy against S. aureus infection. In conclusion, ayanin is a promising therapeutic agent to combat MRSA infections by targeting ClpP.
Subject(s)
Methicillin-Resistant Staphylococcus aureus , Staphylococcal Infections , Animals , Mice , Staphylococcus aureus , Peptide Hydrolases/pharmacology , Molecular Docking Simulation , Flavonoids/pharmacology , Flavonoids/therapeutic use , Staphylococcal Infections/drug therapy , Virulence Factors , Endopeptidases , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Microbial Sensitivity TestsABSTRACT
Mast cell granules are packed with proteases, which are released with other mediators by degranulating stimuli. Several of these proteases are targets of potentially therapeutic inhibitors based on hypothesized contributions to diseases, notably asthma and ulcerative colitis for ß-tryptases, heart and kidney scarring for chymases, and airway infection for dipeptidyl peptidase-I. Small-molecule and antibody-based ß-tryptase inhibitors showing preclinical promise were tested in early-phase human trials with some evidence of benefit. Chymase inhibitors were given safely in Phase II trials without demonstrating benefits, whereas dipeptidyl peptidase-I inhibitor improved bronchiectasis, in effects likely related to inactivation of the enzyme in neutrophils.
Subject(s)
Mast Cells , Peptide Hydrolases , Humans , Peptide Hydrolases/pharmacology , Tryptases , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/pharmacologyABSTRACT
Antimicrobial resistance by pathogenic bacteria has become a global risk to human health in recent years. The most promising approach to combating antimicrobial resistance is to target virulent traits of bacteria. In the present study, a biosurfactant derived from the probiotic strain Lactobacillus acidophilus was tested against three Gram-negative bacteria to evaluate its inhibitory potential on their biofilms, and whether it affected the virulence factors controlled by quorum sensing (QS). A reduction in the virulence factors of Chromobacterium violaceum (violacein production), Serratia marcescens (prodigiosin production) and Pseudomonas aeruginosa (pyocyanin, total protease, LasB elastase and LasA protease production) was observed at different sub-MIC concentrations in a dose-dependent manner. Biofilm development was reduced by 65.76%, 70.64% and 58.12% at the highest sub-MIC levels for C. violaceum, P. aeruginosa and S. marcescens, respectively. Biofilm formation on glass surfaces exhibited significant reduction, with less bacterial aggregation and reduced formation of extracellular polymeric materials. Additionally, swimming motility and exopolysaccharides (EPS) production were shown to be reduced in the presence of the L. acidophilus-derived biosurfactant. Furthermore, molecular docking analysis performed on compounds identified through gas chromatography-mass spectrometry (GC-MS) analysis of QS and biofilm proteins yielded further insights into the mechanism underlying the anti-QS activity. Therefore, the present study has clearly demonstrated that a biosurfactant derived from L. acidophilus can significantly inhibit virulence factors of Gram-negative pathogenic bacteria. This could provide an effective method to inhibit the formation of biofilms and QS in Gram-negative bacteria.
Subject(s)
Probiotics , Quorum Sensing , Humans , Virulence , Lactobacillus acidophilus/metabolism , Molecular Docking Simulation , Lactobacillus/metabolism , Anti-Bacterial Agents/chemistry , Biofilms , Virulence Factors , Gram-Negative Bacteria , Serratia marcescens/metabolism , Peptide Hydrolases/pharmacologyABSTRACT
Herein, we described for the first time, an efficient biogenic synthesis of APTs-AgNPs using acid protease from Melilotus indicus leaf extract. The acid protease (APTs) has an essential role in the stabilization, reduction, and capping of APTs-AgNPs. The crystalline nature, size, and surface morphology of APTs-AgNPs were examined using different techniques such as XRD, UV, FTIR, SEM, EDS, HRTEM, and DLS analysis. The generated APTs-AgNPs demonstrated notable performance as dual functionality (photocatalyst and antibacterial disinfection). By destroying 91 % of methylene blue (MB) in <90 min of exposure, APTs-AgNPs demonstrated remarkable photocatalytic activity. APTs-AgNPs also showed remarkable stability as a photocatalyst after five test cycles. Furthermore, the APTs-AgNPs was found to be a potent antibacterial agent with inhibition zones of 30(±0.5 mm), 27(±0.4 mm), 16(±0.1 mm), and 19(±0.7 mm) against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria, respectively, under both light and dark conditions. Furthermore, APTs-AgNPs effectively scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, demonstrating their potent antioxidant activity. The outcomes of this study thus demonstrates the dual functionality of APTs-AgNPs produced using the biogenic approach method as a photocatalyst and an antibacterial agent for effective microbial and environmental control.
Subject(s)
Metal Nanoparticles , Peptide Hydrolases , Peptide Hydrolases/pharmacology , Silver/pharmacology , Silver/chemistry , Metal Nanoparticles/chemistry , Plant Extracts/pharmacology , Plant Extracts/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Endopeptidases/pharmacology , Escherichia coli , Microbial Sensitivity TestsABSTRACT
COVID-19 has become a global public health crisis since its outbreak in China in December 2019. Currently there are few clinically effective drugs to combat SARS-CoV-2 infection. The main protein (Mpro), papain-like protease (PLpro) and RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 are involved in the viral replication, and might be prospective targets for anti-coronavirus drug development. Here, we investigated the antiviral activity of oridonin, a natural small-molecule compound, against SARS-CoV-2 infection in vitro. The time-of-addition analysis showed that oridonin efficiently inhibited SARS-CoV-2 infection by interfering with the genome replication at the post-entry stage. Mechanistically, the inhibition of viral replication by oridonin depends on the oxidation activity of α, ß-unsaturated carbonyl. Further experiments showed that oridonin not only effectively inhibited SARS-CoV-2 Mpro activity, but also had some inhibitory effects on PLpro-mediated deubiquitinating and viral polymerase-catalyzed RNA elongation activities at high concentrations. In particular, oridonin could inhibit the bat SARS-like CoV and the newly emerged SARS-CoV-2 omicron variants (BA.1 and BA.2), which highlights its potential as a pan-coronavirus antiviral agent. Overall, our data provide strong evidence that oridonin is an efficient antiviral agent against SARS-CoV-2 infection.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Peptide Hydrolases/pharmacology , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Protease Inhibitors/pharmacologyABSTRACT
The application of plant essential oil liposomes to prevent and control food safety risks caused by Campylobacter jejuni (C. jejuni) still faces challenges such as lack of targeting and low release rate. Here, a bacteria-targeted and protease-activated antibacterial liposome (ACCLPs) was successfully synthesized through encapsulation of clove essential oil (CEO) by film dispersion method, embedding of casein by freeze-thaw method, and conjugation of C. jejuni antibody on the liposome membrane by post-insertion method. The average particle size, the essential oil encapsulation rate, the casein mosaic rate, and the antibody coupling efficiency of ACCLPs were determined as185.87 nm,16.9%,70.1% and 87.5%, respectively. The modification with C. jejuni antibody could significantly improve the targeting of ACCLPs to C. jejuni. Controlled release experiments showed that the exocrine protease from C. jejuni could hydrolyze the embedded casein and perforation on the ACCLPs, thus leading to a bacteria-dependent CEO release and significant prolonging the antibacterial effects of ACCLPs. Application results of ACCLPs on C. jejuni-contaminated foods showed that ACCLPs could effectively inhibit C. jejuni in a variety of meat products, fruits and vegetables and extend their shelf life without significantly affecting food quality. The results above in this work would provide a new view for the development of high efficient liposome-based antibacterial system of plant essential oil.