Protease Inhibitory Activity Research Articles

Soybean plants under simultaneous signals of drought and Anticarsia gemmatalis herbivory trigger gene expression and metabolic pathways reducing larval survival

Climate changes are promoting increases in global temperatures, variability in precipitation, and more frequent insect pest outbreaks. Understanding the molecular mechanisms for the tolerance of both, biotic and abiotic stresses, is critical for the development of improved genotypes and sustainability of productivity under climate change. Thus, we examine the regulatory cascades and metabolic pathways triggered by both drought and insect attack signals. Profiles from the genotypes drought-tolerant (EMBRAPA 48) and resistant to A. gemmatalis infestation (IAC17) were compared with one sensitive to drought/insect attack (BR16). EMBRAPA 48 and IAC17 plants under drought were less susceptible to insect attack, promoting lower caterpillar survival. Survival reductions were not dependent on the drought-tolerance or insect-resistance phenotypes, despite being more pronounced for IAC17. Furthermore, metabolites profiles, gene expression and enzymatic assays lead us to conclude that drought signal by itself was not enough to explain the survival reductions. Protease inhibition activities and expressions of LOX and PI genes correlated with ABA levels, indicating that JA-signalling was potentialized by ABA to enhance the production of deterrent metabolites. Thus, increased ABA levels during drought treatment may be acting synergistically to induce cascades responsive to JA. Thus, regulatory molecular hub(s) integrating signals may be a target to the genetic engineering of plants with multiple tolerances to environmental stresses.

Cannabis Extract Has a Positive-Immunostimulating Effect through Proteolytic System and Metabolic Compounds of Honey Bee (Apis mellifera) Workers.

Simple SummaryThe aim of our study was to test the immunostimulating effect of a diet with hemp extract on the resistance of the honey bee (Apis mellifera). The experiment compared the effect of supplementation between the bees receiving the extract in the form of a mixture with sugar syrup and on the strip with the extract, compared to the bees that had no contact with substance. In order to determine this effect, the biochemical indicators were analyzed: the proteolytic system (proteases, protease inhibitors, total protein concentration) responsible for the fight against pathogens/parasites, biomarkers (ALT, AST, ALP), and the basic components of metabolism (glucose and urea concentrations). Parameters were determined in the hemolymph of 2- and 7-day-old workers. Hemp extracts caused an increase in the protein concentrations. Regardless of the method of administration, proteases decreased. Protease inhibitors increased, except supplementation on strips where the activity decreased. The biomarker activities increased in the control group and workers feeding extract in syrup and decreased in workers supplemented with the extract on strips. The results of the metabolic component were as follows: glucose and urea concentrations indicate that the extract will not adversely affect metabolic changes in the insect’s organism. Hemp extract improves the natural immunity of bees.In the study, we assessed the effect of hemp extract on activities of resistance parameters and the metabolic compound concentration in adult workers’ hemolymph. Bees were divided into the following groups: (1) control group fed with mixture of sugar and water-glycerine solution, (2) experimental group with pure sugar syrup and inside with cotton strips soaked with hemp extract, (3) experimental group with a mixture of sugar syrup with hemp extract. Hemp extracts caused an increase in the protein concentrations and reduced the protease activities regardless of the administration method. The protease inhibitor activities were decreased only in the group that received hemp extract on the strips. The biomarker activities (ALP, ALT, AST) increased from the control group and workers feeding extract in syrup and decreased in workers supplemented with the extract on strips. In young, 2-day-old workers, the glucose concentration was higher in the groups feeding with the extract than in the control. Hemp extract influenced an increase in urea concentrations in workers’ hemolymph in comparison with the control. The hemp supplementation positively influences the immune system of workers, and the appropriate method of administration may be adapted to the health problems of bees.

QSAR Study for Prediction of HIV-1 Protease Inhibitor Using the Gravitational Search Algorithm–Neural Network (GSA-NN) Methods

Human immunodeficiency virus (HIV) is a virus that infects an immune cell and makes the patient more susceptible to infections and other diseases. HIV is also a factor that leads to acquired immune deficiency syndrome (AIDS) disease. The active target that is usually used in the treatment of HIV is HIV-1 protease. Combining HIV-1 protease inhibitors and reverse-transcriptase inhibitors in highly active antiretroviral therapy (HAART) is typically used to treat this virus. However, this treatment can only reduce the viral load, restore some parts of the immune system, and failed to overcome the drug resistance. This study aimed to build a QSAR model for predicting HIV-1 protease inhibitor activity using the gravitational search algorithm-neural network (GSA-NN) method. The GSA method is used to select molecular descriptors, while NN was used to develop the prediction model. The improvement of model performance was found after performing the hyperparameter tuning procedure. The validation results show that model 3, containing seven descriptors, shows the best performance indicated by the coefficient of determination (r2) and cross-validation coefficient of determination (Q2) values. We found that the value of r2 for train and test data are 0.84 and 0.82, respectively, and the value of Q2 is 0.81.

Macrolactin A as a Novel Inhibitory Agent for SARS-CoV-2 Mpro: Bioinformatics Approach.

COVID-19 is a disease that puts most of the world on lockdown and the search for therapeutic drugs is still ongoing. Therefore, this study used in silico screening to identify natural bioactive compounds from fruits, herbaceous plants, and marine invertebrates that are able to inhibit protease activity in SARS-CoV-2 (PDB: 6LU7). We have used extensive screening strategies such as drug likeliness, antiviral activity value prediction, molecular docking, ADME, molecular dynamics (MD) simulation, and MM/GBSA. A total of 17 compounds were shortlisted using Lipinski’s rule in which 5 compounds showed significant predicted antiviral activity values. Among these 5, only 2 compounds, Macrolactin A and Stachyflin, showed good binding energy of −9.22 and −8.00 kcal/mol, respectively, within the binding pocket of the Mpro catalytic residues (HIS 41 and CYS 145). These two compounds were further analyzed to determine their ADME properties. The ADME evaluation of these 2 compounds suggested that they could be effective in developing therapeutic drugs to be used in clinical trials. MD simulations showed that protein–ligand complexes of Macrolactin A and Stachyflin with the target receptor (6LU7) were stable for 100 nanoseconds. The MM/GBSA calculations of Mpro–Macrolactin A complex indicated higher binding free energy (−42.58 ± 6.35 kcal/mol). Dynamic cross-correlation matrix (DCCM) and principal component analysis (PCA) on the residual movement in the MD trajectories further confirmed the stability of Macrolactin A bound state with 6LU7. In conclusion, this study showed that marine natural compound Macrolactin A could be an effective therapeutic inhibitor against SARS-CoV-2 protease (6LU7). Additional in vitro and in vivo validations are strongly needed to determine the efficacy and therapeutic dose of Macrolactin A in biological systems.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12010-021-03608-7.

Inhibition of the 3CL Protease and SARS-CoV-2 Replication by Dalcetrapib.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) 3CL protease is a promising target for inhibition of viral replication by interaction with a cysteine residue (Cys145) at its catalytic site. Dalcetrapib exerts its lipid-modulating effect by binding covalently to cysteine 13 of a cholesteryl ester transfer protein. Because 12 free cysteine residues are present in the 3CL protease, we investigated the potential of dalcetrapib to inhibit 3CL protease activity and SARS-CoV-2 replication. Molecular docking investigations suggested that dalcetrapib-thiol binds to the catalytic site of the 3CL protease with a delta G value of −8.5 kcal/mol. Dalcetrapib inhibited both 3CL protease activity in vitro and viral replication in Vero E6 cells with IC50 values of 14.4 ± 3.3 μM and an EC50 of 17.5 ± 3.5 μM (mean ± SD). Near-complete inhibition of protease activity persisted despite 1000-fold dilution after ultrafiltration with a nominal dalcetrapib-thiol concentration of approximately 100 times below the IC50 of 14.4 μM, suggesting stable protease–drug interaction. The inhibitory effect of dalcetrapib on the SARS-CoV-2 3CL protease and viral replication warrants its clinical evaluation for the treatment of COVID-19.

Methods to improve efficacy of orally administered bioactive peptides using bovine colostrum as an exemplar.

BackgroundOral administration of bioactive peptides has potential clinical advantages, but its applicability is limited due to gastric and pancreatic enzyme proteolysis.ObjectiveTo examine whether the co-packaging of bovine colostrum (BC), a rich source of IgG, immune and growth factors, with the food additives trehalose (carbohydrate), stearine (fat), casein (protein present in BC) or soy flour (plant based with high protease inhibitory activity) enhances the stability of BC against digestion.DesignSamples alone and in combination (BC+ 10% wt/wt trehalose, stearine, casein or soy) were exposed to HCl/pepsin, followed by trypsin and chymotrypsin (“CT”). Assessment of proliferation used gastric AGS cells (Alamar blue), IgG function measured bovine IgG anti-E.coli binding and ELISAs quantified growth factor constituents. In vivo bioassay assessed ability of BC alone or with soy to reduce injury caused by dextran sodium sulphate (DSS, 4% in drinking water, 7 days, test products started 2 days prior to DSS).ResultsProliferative activity of BC reduced 61% following HCl/pepsin and CT exposure. This was truncated 50% if soy was co-present, and also protected against loss of total IgG, IgG E.coli binding, TGFβ, lactoferrin and EGF (all P<0.01 vs BC alone). Co-packaging with trehalose was ineffective in preventing digestion whereas casein or stearine provided some intermediate protective effects. Rats given BC alone showed beneficial effects on weight gain, disease activity index, tissue histology and colonic MPO. Soy alone was ineffective. BC+ soy combination showed the greatest benefit with a dose of 7 mg/kg (6.4 BC + 0.6 soy flour) having the same degree of benefit as using 20 mg/kg BC alone.ConclusionSoy, and to a lesser extent casein, enhanced the biostability of BC against digestive enzymes. Co-packaging of BC with other food products such as soy flour could result in a decreased dose being required, improving cost-effectiveness and patient compliance.

Synergistic effect of serine protease inhibitors and a bothropic antivenom in reducing local hemorrhage and coagulopathy caused by Bothrops jararaca venom

Snakebite accidents are a public health problem that affects the whole world, causing thousands of deaths and amputations each year. In Brazil, snakebite envenomations are caused mostly by snakes from the Bothrops genus. The local symptoms are characterized by pain, swelling, ecchymosis, and hemorrhages. Systemic disturbances can lead to necrosis and amputations. The present treatment consists of intravenous administration of bothropic antivenom, which is capable of reversing most of the systemic symptoms, while presenting limitations to treat the local effects, such as hemorrhage and to neutralize the snake venom serine protease (SVSP). In this context, we aimed to evaluate the activity of selective serine protease inhibitors (pepC and pepB) in combination with the bothropic antivenom in vivo. Further, we assessed their possible synergistic effect in the treatment of coagulopathy and hemorrhage induced by Bothrops jararaca venom. For this, we evaluated the in vivo activity in mouse models of local hemorrhage and a series of in vitro hemostasis assays. Our results showed that pepC and pepB, when combinated with the antivenom, increase its protective activity in vivo and decrease the hemostatic disturbances in vitro with high selectivity, possibly by inhibiting botropic proteases. These data suggest that the addition of serine protease inhibitor to the antivenom can improve its overall potential.

Anti-Inflammatory, Antiallergic, and COVID-19 Main Protease (Mpro) Inhibitory Activities of Butenolides from a Marine-Derived Fungus Aspergillus terreus.

In December 2020, the U.K. authorities reported to the World Health Organization (WHO) that a new COVID-19 variant, considered to be a variant under investigation from December 2020 (VUI-202012/01), was identified through viral genomic sequencing. Although several other mutants were previously reported, VUI-202012/01 proved to be about 70% more transmissible. Hence, the usefulness and effectiveness of the newly U.S. Food and Drug Administration (FDA)-approved COVID-19 vaccines against these new variants are doubtfully questioned. As a result of these unexpected mutants from COVID-19 and due to lack of time, much research interest is directed toward assessing secondary metabolites as potential candidates for developing lead pharmaceuticals. In this study, a marine-derived fungus Aspergillus terreus was investigated, affording two butenolide derivatives, butyrolactones I (1) and III (2), a meroterpenoid, terretonin (3), and 4-hydroxy-3-(3-methylbut-2-enyl)benzaldehyde (4). Chemical structures were unambiguously determined based on mass spectrometry and extensive 1D/2D NMR analyses experiments. Compounds (1–4) were assessed for their in vitro anti-inflammatory, antiallergic, and in silico COVID-19 main protease (Mpro) and elastase inhibitory activities. Among the tested compounds, only 1 revealed significant activities comparable to or even more potent than respective standard drugs, which makes butyrolactone I (1) a potential lead entity for developing a new remedy to treat and/or control the currently devastating and deadly effects of COVID-19 pandemic and elastase-related inflammatory complications.

In Silico Activity Identification of Cyclo Peptide Alkaloids from Zizyphus Spina-Christi Species Against Sars-Cov-2 Main Protease

The COVID-19 has spread worldwide and become an international pandemic. The promising target for drug discovery of COVID-19 was SARS-CoV-2 Main Protease (Mpro), that has been successfully crystallized along with its inhibitor. The discovery of peptide-based inhibitors may present better options than small molecules for inhibitor SARS-CoV-2 Mpro. Natural compounds have such a wide potential and still few explored, Zizyphus spina-christi is one of the medicinal plants that have many pharmacological activities and contains a peptide compound from alkaloids class, i.e. cyclopeptide alkaloids, that is interesting to explore as SARS-CoV-2 Mpro inhibitor. The compound structure was drawn and optimized using density functional theory 3-21G method. The protein chosen was the high resolution of SARS-CoV-2 MPro receptor (1.45 Å) with PDB ID: 6WNP, in complex with boceprevir. Molecular docking simulation was performed using Autodock4 with 100 numbers of GA run, the validation methods assessed by RMSD calculation. Furthermore, the prediction of pharmacological activity spectra was carried out using the PASS Prediction server. The results showed RMSD value was 1.98 Å, this docking method was valid. The binding energy of all compounds showed better results than the native ligand (Boceprevir). The in silico PASS prediction results indicated that all compounds showed antiviral activity. Some compounds showed protease inhibitory activity, i.e Ambiphibine-H, Franganine, and Mauritine-A, and the highest Pa (Predicted activity) value showed by Mauritine-A compounds. It can be concluded that the cyclopeptide compounds of Zizyphus spina-christi were indicated to have a potential as COVID-19 therapy targeting SARS-CoV-2 Mpro.

Comparison of COVID-19 Virus Main Protease Inhibition Activities of Phenolic Acids By Molecular Docking

The Covid-19 pandemic is new challenge all around the world. This pandemic provides an emergency development of vaccines and drugs against this virus. As main proteases of CoV-19 have essential roles in the transmission and virulence of the virus. So, this enzyme has been considered as a critical target to inhibit COVID-19. Natural compounds are well known as rich sources of antiviral drugs due to their structural diversity and safety. In this study, we have screened thirteen phenolic acids (Cinnamic acid and Hydroxy benzoic derivatives) to compare the potential inhibitory activity of these molecules against COVID-19 protease. Systematic molecular docking simulation was done using AutoDock 4.2.6 to achieve the binding affinities and interactions between phenolic acids and protease. Cinnamic acids are better protease inhibitors in comparison with benzoic acid derivatives. Among Cinnamic acid derivatives, Rosmarinic acid exhibited the minimum Gibbs binding energy and highest docking scores. Also, Chorgenic > Ellagic > Ferulic acid could more effective respectively . According to obtained results, Rosmarinic acid has formed strong hydrogen bonding interaction with His163A, Ser144A, Cys145A, Gly143A and Thr26A, while the propyl cyclohexadione of Rosmarinic acid formed hydrophobic interaction with residue Gly 143A. It seems that Cinnamic acid derivatives of phenolic acid, particularly Rosmarinic acid could be efficient SARS-CoV 3CLpro inhibitors. In the next step, it is necessary to survey the effect of these natural products in inhibition of SARS-CoV replication through cell culture and in vitro assays. This study will improve preclinical knowledge about potential of natural compounds as SARS-CoV inhibitors.

Chemical composition of Callistemon citrinus (Curtis) Skeels aerial part essential oil and its pharmacological applications, neurodegenerative inhibitory, and genotoxic efficiencies.

Callistemon citrinus aerial part essential oil (CCEO) was analyzed for chemical compositions using GC/MS. Pharmacological activities such as neurodegenerative inhibitory, antioxidant, anti-inflammatory, antimicrobial, and acetylcholinesterase activities were evaluated using the standard methodologies. Genotoxicity was investigated using Allium cepa assay. GC/MS analysis identified 27 compounds; eucalyptol (55.40%) was the major component. Radical scavenging activity showed IC50 value of 16.71μg/mL. Protein denaturation assay showed IC50 value of 21.19μg/mL and 19.53μg/mL in protease inhibitor activity. MIC assay revealed antimicrobial potential of CCEO against microbial strains B. cereus at 2.00mg/mL, S. typhimurium at 4.50mg/mL, S. mutans at 2.50mg/mL, C. albicans at 4.00mg/mL, and S. cerevisiae at 4.50mg/mL concentrations. Mitotic index value (MI) of CCEO showed negligible genotoxicity with MI 17.25% close to distilled water 18.22%. Acetylcholinesterase strong inhibitory activity of CCEO was observed from IC50 =6.335μg/mL. CCEO could be a cheap and easy source for the extraction of the pure compound eucalyptol and possess various biological activities which increase its pharmacological value as well as its applicability in the field of flavor and fragrance industries. PRACTICAL APPLICATIONS: Eucalyptol is the major component of many pharmaceutical and food/flavor industries. The present investigation provides a new source for the isolation of pure compound eucalyptol in cost-effective way. Additionally, the essential oil could also be used for the pharmaceutical formulation of antioxidant, inflammation inhibitory, and neurodegenerative inhibitory drug preparation under the safety range concentration.

ANALYSIS OF MEDICINAL PROPERTIES OF VIGNA ACONITIFOLIA (MATKI)

Objective: The seeds and sprouts of matki bean (Vigna aconitifolia), a common food, contain abundant nutrients with biological activities. This work was focused to analyze and compare the medicinal properties of seeds and sprouts of matki beans, with the goal of providing scientific evidence for better application of this commonly used food as a medicine.&#x0D; Methods: Using Soxhlet apparatus, ethanol extracts of matki bean sprouts and seeds were prepared. Anti-inflammatory activity (inhibition of protein denaturation, inhibition of heat induced hemolysis, and inhibition of protease activity), antioxidant activity, and antihypertensive activity were determined for both sprout and seed extracts.&#x0D; Results: The anti-inflammatory activity of sprout ethanol extract was found to be highest. Sprout ethanol extract inhibited the activity angiotensin-I-converting enzyme (ACE1) which was maximum as compared to seed extracts. Same was observed for the antioxidant activity, sprout ethanol extract showed higher activity than the seed extract.&#x0D; Conclusion: The results of the present study will help us to use Vigna aconitifolia as a healthy herbal alternative for the existing chemical remedies for various disorders and rule out the issue of side effects and resistance.

Roles of Porphyromonas gulae proteases in bacterial and host cell biology.

Porphyromonas gulae, an animal-derived periodontal pathogen, expresses several virulence factors, including fimbria, lipopolysaccharide (LPS) and proteases. We previously reported that its invasive efficiency was dependent on fimbriae types. In addition, P. gulae LPS increased inflammatory responses via toll-like receptors. The present study was conducted to investigate the involvement of P. gulae proteases in bacterial and host cell biology. Porphyromonas gulae strains showed an ability to agglutinate mouse erythrocytes and also demonstrated co-aggregation with Actinomyces viscosus, while the protease inhibitors antipain, PMSF, TLCK and leupeptin diminished P. gulae proteolytic activity, resulting in inhibition of haemagglutination and co-aggregation with A. viscosus. In addition, specific proteinase inhibitors were found to reduce bacterial cell growth. Porphyromonas gulae inhibited Ca9-22 cell proliferation in a multiplicity of infection- and time-dependent manner. Additionally, P. gulae-induced decreases in cell contact and adhesion-related proteins were accompanied by a marked change in cell morphology from well spread to rounded. In contrast, inhibition of protease activity prevented degradation of proteins, such as E-cadherin, β-catenin and focal adhesion kinase, and also blocked inhibition of cell proliferation. Together, these results indicate suppression of the amount of human proteins, such as γ-globulin, fibrinogen and fibronectin, by P. gulae proteases, suggesting that a novel protease complex contributes to bacterial virulence.

Microglial cell secretion of serpin‐like trypsin‐inhibitory activity: potential regulation of proteinase‐activated receptor‐2 (PAR2)‐mediated inflammatory signalling

Microglial cells function as a relay station from the periphery to the central nervous system (CNS). In part, microglial cells signal to their microenvironment via cytokines and other agonists. We hypothesized that microglial cells can also secrete proteinases, known to signal via proteinase-activated receptors (PARs) in the CNS. PARs, which are members of the G protein-coupled receptor superfamily, are activated upon cleavage of their extracellular N-terminus to unmask a tethered receptor-activating sequence (PMID: 22453980). However, the role(s) of PARs and their activating proteinases in a microglial cell environment remains to be elucidated. Using an EOC 13.31 (ATCC® CRL-2468) mouse neonatal-derived microglial cell culture model (EOC cells), we evaluated the presence of functional PARs (RT-PCR and calcium signaling assays), and their secretion of proteinases, proteinase-inhibitors, or both. Cell-secreted proteinase activity was assessed 1. using a microtiter plate chromogenic substrate cleavage assay and 2. a PAR cleavage assay whereby proteolysis releases an N-terminal-luciferase signal from an N-Luc-tagged PAR (PMID: 26957205). Proteinase-inhibitory activity was monitored using the same assays by observing the ability of the EOC cell supernatants to block substrate cleavage caused by thrombin and trypsin in the microtiter plate or N-Luc release assay. We found that the EOC cells express mRNA for PARs 1 and 2, which are known to cause inflammatory responses. However, there was no calcium response upon stimulation of either PAR by their selective peptide agonists, TFLLR-amide and 2-furoyl-LIGRLO-amide for PARs 1 and 2, respectively. PAR-cleaving proteinase activity in the EOC cell supernatants was not detected by neither the microtiter nor the PAR N-Luc release assay. In contrast, the EOC cell supernatants contained a serpin-like trypsin inhibitor that blocked substrate cleavage by trypsin (but not thrombin) in both the microtiter plate and PAR2 N-Luc release assay. Further, the EOC supernatants blocked the ability of trypsin to cleave/activate PAR2 in a target HEK cell PAR2 calcium signaling assay. We conclude that in addition to secreting cytokines and other agonists, microglial cells produce a serpin-like trypsin inhibitor that may modulate the inflammatory activity of proteinases in the CNS microenvironment. We aim to identify the secreted trypsin inhibitor using a proteomic and activity-based probe labelling approach.

Anti-inflammatory target prediction of Gentianae Radix et Rhizoma based on network pharmacology and construction of a bioassay method for its quality control

Based on the heat-clearing and detoxifying effects of Gentianae Radix et Rhizoma, the network pharmacology is mainly used to predict the potential targets of Gentianae Radix et Rhizoma for anti-inflammatory activity and to perform the experimental verification. A method for detecting the biological potency of Gentianae Radix et Rhizoma based on verifiable targets has been established to provide a reference for improving the quality evaluation and control standards of Gentianae Radix et Rhizoma. High performance liquid chromatography can be used to construct chemical fingerprints of different batches of Gentianae Radix et Rhizoma. Constructing a component-target-disease network of Gentianae Radix et Rhizoma for its anti-inflammatory activity was applied to screen potential anti-inflammatory components and related targets of Gentianae Radix et Rhizoma, and to verify the target of Gentianae Radix et Rhizoma by using biological evaluation methods. Detecting the biological potency of different batches of Gentianae Radix et Rhizoma extracts was used to inhibit COX-2 enzyme activity based the verifiable target cyclooxygenase-2(COX-2). The results showed that different batches of Gentianae Radix et Rhizoma accorded with the pharmacopoeia testing regulations, and the chemical fingerprints have a high similarity(similarity&gt;0.93), suggesting that there is no significant difference in the characteristics of the chemical components. Based on network pharmacology predictions, 18 candidate targets were found to have potential direct interactions with the ingredients in Gentianae Radix et Rhizoma. Among them, the most important target is COX-2. Based on the experimental verification of recombinant human COX-2 protease activity inhibition, Gentianae Radix et Rhizoma can inhibit the COX-2 enzyme activity in a dose-dependent manner. It can function with a low concentration(0.75 mg·mL~(-1)), which preliminarily confirmed the accuracy of network pharmacology prediction. The biological potency detection method of Gentianae Radix et Rhizoma based on COX-2 inhibitory activity was optimized and established. The qualitative response parallel line method was used to calculate the biological potency of anti-inflammatory activity, which ranged from 23.04 to 46.60 U·mg~(-1). For network pharmacology prediction, it can screen and clarify the possible targets of traditional Chinese medicine rapidly, which can guide the establishment of a biological evaluation method for the quality of medicinal materials with related activities. Compared with chemical fingerprints, the biological potency testing can better detect quality fluctuations of traditional Chinese medicine.

Natural DENV-2 NS2B/NS3 protease inhibitors from Myristica cinnamomea King.

The NS2B/NS3 protease is crucial for the pathogenesis of the DENV. Therefore, the inhibition of this protease is considered to be the key strategy for the development of new antiviral drugs. In the present study, malabaricones C (3) and E (4), acylphenols from the fruits of Myristica cinnamomea King, have been respectively identified as moderate (27.33 ± 5.45 μM) and potent (7.55 ± 1.64 μM) DENV-2 NS2B/NS3 protease inhibitors, thus making this the first report on the DENV-2 NS2B/NS3 protease inhibitory activity of acylphenols. Based on the molecular docking studies, compounds 3 and 4 both have π-π interactions with Tyr161. While compound 3 has hydrogen bonding interactions with Gly151, Gly153 and Tyr161, compound 4 however, forms hydrogen bonds with Ser135, Asp129, Phe130 and Ile86 instead. The results from the present study suggests that malabaricones C (3) and E (4) could be employed as lead compounds for the development of new dengue antivirals from natural origin.

Discovery of anti-infective adipostatins through bioactivity-guided isolation and heterologous expression of a type III polyketide synthase

Antibiotic resistance and emerging viral pandemics have posed an urgent need for new anti-infective drugs. By screening our microbial extract library against the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the notorious ESKAPE pathogens, an active fraction was identified and purified, leading to an initial isolation of adipostatins A (1) and B (2). In order to diversify the chemical structures of adipostatins toward enhanced biological activities, a type III polyketide synthase was identified from the native producer, Streptomyces davawensis DSM101723, and was subsequently expressed in an E. coli host, resulting in the isolation of nine additional adipostatins 3–11, including two new analogs (9 and 11). The structures of 1–11 were established by HRMS, NMR, and chemical derivatization, including using a microgram-scale meta-chloroperoxybenzoic acid epoxidation-MS/MS analysis to unambiguously determine the double bond position in the alkyl chain. The present study discovered SARS-CoV-2 main protease inhibitory activity for the class of adipostatins for the first time. Several of the adipostatins isolated also exhibited antimicrobial activity against selected ESKAPE pathogens.

Anti-Arthritic and Anti-Inflammatory Potential of Spondias mangifera Extract Fractions: An In Silico, In Vitro and In Vivo Approach.

The fruits of Spondias mangifera (S. mangifera) have traditionally been used for the management of rheumatism in the northeast region of India. The present study explores the probable anti-arthritis and anti-inflammatory potential of S. mangifera fruit extract’s ethanolic fraction (EtoH-F). To support this study, we first approached the parameters in silico by means of the active constituents of the plant (beta amyrin, beta sitosterol, oleonolic acid and co-crystallised ligands, i.e., SPD-304) via molecular docking on COX-1, COX-2 and TNF-α. Thereafter, the absorption, distribution, metabolism, excretion and toxicity properties were also determined, and finally experimental activity was performed in vitro and in vivo. The in vitro activities of the plant extract fractions were evaluated by means of parameters like 1,1-Diphenyl-2- picrylhydrazyl (DPPH), free radical-reducing potential, albumin denaturation, and protease inhibitory activity. The in vivo activity was evaluated using parameters like COX, TNF-α and IL-6 inhibition assay and arthritis score in Freund Adjuvant (CFA) models at a dose of 400 mg/kg b.w. per day of different fractions (hexane, chloroform, alcoholic). The molecular docking assay was performed on COX-1, COX-2 and TNF-α. The results of in vitro studies showed concentration-dependent reduction in albumin denaturation, protease inhibitors and scavenging activity at 500 µg/mL. Administration of the S. mangifera alcoholic fraction at the abovementioned dose resulted in a significant reduction (p < 0.01) in arthritis score, paw diameters, TNF-α, IL-6 as compared to diseased animals. The docking results showed that residues show a critical binding affinity with TNF-α and act as the TNF-α antagonist. The alcoholic fraction of S. mangifera extract possesses beneficial effects on rheumatoid arthritis as well as anti-inflammatory potential, and can further can be used as a possible agent for novel target-based therapies for the management of arthritis.

Anti-inflammatory Activity of the Protein Z-Dependent Protease Inhibitor.

The protein Z (PZ)-dependent plasma protease inhibitor (ZPI) is a glycoprotein that inhibits factor XIa and, in the presence of PZ, FXa. Recently, ZPI has been shown to be an acute-phase protein (APP). As usually APPs downregulate the harmful effects of inflammation, we tested whether ZPI could modulate the increase of cytokines observed in inflammatory states. We observed that recombinant human ZPI (rhZPI) significantly decreases the levels of interleukin (IL)-1, IL-6, and tumor necrosis factor- α (TNF-α) induced by lipopolysaccharide (LPS) in a whole blood model. This inhibitory effect was unaffected by the presence of PZ or heparin. A ZPI mutant within the reactive loop center ZPI (Y387A), lacking anticoagulant activity, still had an anti-inflammatory activity. Surprisingly, rhZPI did not inhibit the synthesis of IL-6 or TNF-α when purified monocytes were stimulated by LPS, whereas the inhibitory effect was evidenced when lymphocytes were added to monocytes. The requirement of lymphocytes could be due to the synthesis of CCL5 (RANTES), a chemokine mainly produced by activated lymphocytes which is induced by rhZPI, and which can reduce the production of proinflammatory cytokines in whole blood. Lastly, we observed that the intraperitoneal injection of rhZPI significantly decreased LPS-induced IL-6 and TNF-α production in mouse plasma.

Unmasking of crucial structural fragments for coronavirus protease inhibitors and its implications in COVID-19 drug discovery

Fragment based drug discovery (FBDD) by the aid of different modelling techniques have been emerged as a key drug discovery tool in the area of pharmaceutical science and technology. The merits of employing these methods, in place of other conventional molecular modelling techniques, endorsed clear detection of the possible structural fragments present in diverse set of investigated compounds and can create alternate possibilities of lead optimization in drug discovery. In this work, two fragment identification tools namely SARpy and Laplacian-corrected Bayesian analysis were used for previous SARS-CoV PLpro and 3CLpro inhibitors. A robust and predictive SARpy based fragments identification was performed which have been validated further by Laplacian-corrected Bayesian model. These comprehensive approaches have advantages since fragments are straight forward to interpret. Moreover, distinguishing the key molecular features (with respect to ECFP_6 fingerprint) revealed good or bad influences for the SARS-CoV protease inhibitory activities. Furthermore, the identified fragments could be implemented in the medicinal chemistry endeavors of COVID-19 drug discovery.