Protein Protease Research Articles

Evaluation of the anti-inflammatory and antioxidant properties and isolation and characterization of a new bioactive compound, 3,4,9-trimethyl-7-propyldecanoic acid from Vitex negundo

Ethnopharmacological relevanceHerbal medicines derived from plant extraction are affordable, more therapeutically effective, and have fewer side effects than contemporary medications. Vitex negundo L. (V. negundo). is a medicinal shrub, which contains numerous phytoconstituents. In ancient medicinal practices, V. negundo was primarily prescribed as an analgesic and anti-inflammatory drug.Aim of the study: This study aims to evaluate the anti-inflammatory and antioxidant characteristics of crude extracts from V. negundo leaves, including those derived from petroleum ether (P), methanol (M), and aqueous (A) solvents. Additionally, the research seeks to identify the specific bioactive compounds responsible for these observed properties. Materials and methodsThe nitric oxide scavenging study was performed to evaluate the V. negundo crude extract's ability to function as a nitric oxide scavenger. Protein denaturation and proteinase inhibition experiments were employed to study the ability of extracts to suppress proteolysis and inhibit the enzymes that cause tissue injury. The membrane-stabilizing potency of plant extracts were examined through the process of heat-induced hemolysis. The ability of the extracts to neutralize free radicals showed a dose-dependent response, and the aqueous extract exhibited substantially higher activity in both FRAP and DPPH. The GC-MS analysis of V. negundo extracts revealed a vast array of pharmacologically active metabolites. Based on this Bioassay-guided fractionation approach, the optimal extract was selected for the potent molecule isolation and characterization. ResultsThe findings demonstrated that the aqueous extract of V. negundo exhibited markedly superior radical scavenging and anti-inflammatory capabilities compared to the other two extracts. Furthermore, a new molecule, 3,4,9-trimethyl-7-propyldecanoic acid was isolated from this extract, and its chemical structure was successfully determined. ConclusionThis study revealed that the aqueous extract of V. negundo demonstrated notably stronger in vitro anti-inflammatory and antioxidant properties in comparison to the methanol and petroleum ether extracts. The identified active compound, 3,4,9-trimethyl-7-propyldecanoic acid is likely responsible for the extract's free radical scavenging and anti-inflammatory effects. Furthermore, conducting both in vitro and in vivo studies is crucial to substantiate the potential of this active constituent for the development of an anti-inflammatory drug derived from V. negundo.

Development of an Anti-HER2 Single-Chain Variable Antibody Fragment Construct for High-Yield Soluble Expression in Escherichia coli and One-Step Chromatographic Purification.

Although single-chain variable fragment (scFv) is recognized as a highly versatile scaffold of recombinant antibody fragment molecules, its overexpression in Escherichia coli often leads to the formation of inclusion bodies. To address this issue, we devised and tested four different constructs, named v21, v22, v23 and v24, for producing anti-human epidermal growth factor receptor 2 (HER2) scFv. Among them, the v24 construct obtained from N-terminal fusion of maltose-binding protein (MBP) and subsequent tobacco etch virus protease (TEV) was identified as the most efficient construct for the production of anti-HER2 scFv. Aided by an MBP tag, high-yield soluble expression was ensured and soluble scFv was liberated in cells via autonomous proteolytic cleavage by endogenously expressed TEV. The isolated scFv containing a C-terminal hexahistidine tag was purified through a one-step purification via nickel-affinity chromatography. The purified scFv exhibited a strong (nanomolar Kd) affinity to HER2 both in vitro and in cells. Structural and functional stabilities of the scFv during storage for more than one month were also assured. Given the great utility of anti-HER2 scFv as a basic platform for developing therapeutic and diagnostic agents for cancers, the v24 construct and methods presented in this study are expected to provide a better manufacturing system for producing anti-HER2 scFv with various industrial applications.

Integrating In Vitro and In Silico Approaches to Assess Monotheca buxifolia Plant Extract against Rhipicephalus (Boophilus) microplus and Sarcoptes scabiei.

Tick and mite infestations pose significant challenges to animal health, agriculture, and public health worldwide. The search for effective and environmentally friendly acaricidal agents has led researchers to explore natural alternatives. In this study, we investigated the acaricidal potential of the Monotheca buxifolia plant extract against Rhipicephalus microplus ticks and Sarcoptes scabiei mites. Additionally, we employed a computational approach to identify phytochemicals from the extract that could serve as drug candidates against these ectoparasites. The contact bioassay results demonstrated that the M. buxifolia plant extract exhibited significant efficacy against R. microplus and S. scabiei, with higher concentrations outperforming the positive control acaricide permethrin in terms of mite mortality. Time exposure to the extract also showed a positive correlation with better lethal concentration (LC50 and LC90) values. Similarly, the adult immersion test revealed a notable inhibition of tick oviposition via the plant extract, especially at higher concentrations. The two-protein primary structure, secondary structure and stability were predicted using the Expasy's ProtParam server, SOPMA and SUSUI server, respectively. Using Homology modeling, the 3D structure of the protein was obtained and validated through the ERRAT server, and active sites were determined through the CASTp server. The docking analysis revealed that Alpha-Amyrenyl acetate and alpha-Tocopherol exhibited the highest docking scores for S. scabiei and R. microplus aspartic protease proteins, respectively. These phytochemicals demonstrated strong binding interactions, suggesting their potential as acaricidal drug candidates. In conclusion, the M. buxifolia plant extract displayed significant acaricidal activity against R. microplus and S. scabiei. Moreover, the computational approach identified promising phytochemicals that could serve as potential drug candidates for controlling these ectoparasites.

Effect of Electrostatic Interaction between Collagen and Enzymes on Permeation of Protease into the Pelt during Leather Bating Process

The enzymatic delimed pelts bating process using proteases is critical to improving the overall performance of the leather. Bating effectiveness is determined not only by the properties but also by the permeation behavior of the proteases. Imperfect methods to control protease permeation often results in uneven distribution of enzyme proteins in the pelts, leading to excessive enzymolysis of the surface layer and inadequate opening-up of the inner layer. In this study, the relative size of proteases and delimed pelts were analyzed, the permeation behavior of fluorescein-labeled proteases in the pelt was observed using a confocal laser scanning microscope (CLSM), and the effect of electrostatic interaction between protease and collagen proteins on the permeation of protease into the pelt was investigated. The results showed that, after dehairing, liming and deliming operations, the enzyme can easily permeate into the pelts due to the formation of large cavities and interfibrillar gaps. The permeation of protease within the delimed pelt is significantly influenced by the affinity (electrostatic interactions) between the collagen and protease proteins. The isoelectric point (pI) of the protease protein, the collagen and the pH of the solution directly influence the electrostatic properties and interactions. When the enzyme and collagen are similarly charged (electrostatic repulsion), the enzyme can easily permeate into the pelts; when the enzyme and collagen are oppositely charged (electrostatic attraction), the permeation of the enzyme into the inner layer is difficult, resulting in the accumulation of protease on the grain and excessive hydrolysis of the grain layer. Therefore, the established permeation regulation mechanism of protease based on electrostatic interactions between enzyme and collagen could serve as an important basis for the selection of protease and the regulation of the enzymatic bating process.

A MERS-CoV antibody neutralizes a pre-emerging group 2c bat coronavirus.

The repeated emergence of zoonotic human betacoronaviruses (β-CoVs) dictates the need for broad therapeutics and conserved epitope targets for countermeasure design. Middle East respiratory syndrome (MERS)-related coronaviruses (CoVs) remain a pressing concern for global health preparedness. Using metagenomic sequence data and CoV reverse genetics, we recovered a full-length wild-type MERS-like BtCoV/li/GD/2014-422 (BtCoV-422) recombinant virus, as well as two reporter viruses, and evaluated their human emergence potential and susceptibility to currently available countermeasures. Similar to MERS-CoV, BtCoV-422 efficiently used human and other mammalian dipeptidyl peptidase protein 4 (DPP4) proteins as entry receptors and an alternative DPP4-independent infection route in the presence of exogenous proteases. BtCoV-422 also replicated efficiently in primary human airway, lung endothelial, and fibroblast cells, although less efficiently than MERS-CoV. However, BtCoV-422 shows minor signs of infection in 288/330 human DPP4 transgenic mice. Several broad CoV antivirals, including nucleoside analogs and 3C-like/Mpro protease inhibitors, demonstrated potent inhibition against BtCoV-422 in vitro. Serum from mice that received a MERS-CoV mRNA vaccine showed reduced neutralizing activity against BtCoV-422. Although most MERS-CoV-neutralizing monoclonal antibodies (mAbs) had limited activity, one anti-MERS receptor binding domain mAb, JC57-11, neutralized BtCoV-422 potently. A cryo-electron microscopy structure of JC57-11 in complex with BtCoV-422 spike protein revealed the mechanism of cross-neutralization involving occlusion of the DPP4 binding site, highlighting its potential as a broadly neutralizing mAb for group 2c CoVs that use DPP4 as a receptor. These studies provide critical insights into MERS-like CoVs and provide candidates for countermeasure development.

Luteolin is a potential inhibitor of COVID-19: An in silico analysis.

The severe respiratory syndrome 2019 novel coronavirus disease (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread explosively, raising global health concerns. Luteolin shows antiviral properties, but its effect on SARS-CoV-2 and the associated mechanisms are not elucidated. We used network pharmacology, molecular docking and molecular dynamics to provide potential molecular support of luteolin (3,4,5,7-tetrahydroxyflavone) (LUT) against COVID-19. We employed network pharmacology, molecular docking, and molecular dynamics techniques to investigate how LUT affected COVID-19. Several databases were queried to determine potential target proteins related to LUT and COVID-19. Protein-protein interaction network was constructed, and core targets were filtered by degree value. Following that, functional enrichment was conducted. Molecular docking was utilized to ensure LUT was compatible with core target proteins. Finally, molecular dynamics was used to analyze the effects of the LUT on the optimal hub target. A total of 64 potential target genes for treating COVID-19 were identified, of which albumin, RAC-alpha serine/threonine-protein kinase, caspase-3, epidermal growth factor receptor, heat shock protein HSP 90-alpha, and mitogen-activated protein kinase 1 might be the most promising. In addition, molecular docking results showed that LUT could interact with SARS-CoV-2 major protease 3CL. LUT can bind to the active sites of 3CL protease and mitogen-activated protein kinase 1, showing an anti-SARS-CoV-2 potential.

Co-folding and RNA activation of poliovirus 3Cpro polyprotein precursors

Positive-strand RNA viruses use long open reading frames to express large polyproteins that are processed into individual proteins by viral proteases. Polyprotein processing is highly regulated and yields intermediate species with different functions than the fully processed proteins, increasing the biochemical diversity of the compact viral genome while also presenting challenges in that proteins must remain stably folded in multiple contexts. We have used circular dichroism spectroscopy and single molecule microscopy to examine the solution structure and self-association of the poliovirus P3 region protein composed of membrane binding 3A, RNA priming 3B (VPg), 3Cpro protease, and 3Dpol RNA-dependent RNA polymerase proteins. Our data indicate that co-folding interactions within the 3ABC segment stabilize the conformational state of the 3C protease region, and this stabilization requires the full-length 3A and 3B proteins. Enzymatic activity assays show that 3ABC is also an active protease, and it cleaves peptide substrates at rates comparable to 3Cpro. The cleavage of a larger polyprotein substrate is stimulated by the addition of RNA, and 3ABCpro becomes 20-fold more active than 3Cpro in the presence of stoichiometric amounts of viral cre RNA. The data suggest that co-folding within the 3ABC region results in a protease that can be highly activated toward certain cleavage sites by localization to specific RNA elements within the viral replication center, providing a mechanism for regulating viral polyprotein processing.

A systematic analysis of the subtilase gene family and expression and subcellular localization investigation of anther-specific members in maize

Subtilases (SBTs), also known as Subtilisin-like serine proteases, are extracellular alkaline protease proteins. SBTs function in all stages of plant growth, development and stress responses. Maize (Zea mays L.) is a crop widely used worldwide as food, feed, and industrial materials. However, information about the members and their functions of the SBT proteins in maize is lacking. In this study, we identified 58 ZmSBT genes from the maize genome and conducted a comprehensive investigation of ZmSBTs by phylogenetic, gene duplication event, gene structure, and protein conserved motif analyses. The ZmSBT proteins were phylogenetically classified into seven groups, and collinearity analysis indicated that many ZmSBTs originate from tandem or segmental duplications. Structural and homolog protein comparison revealed ZmSBTs have conserved protein structures with reported subtilase proteins, suggesting the conserved functions. Further analysis showed that ZmSBTs are expressed in different tissues, and many are responses to specific abiotic stress. Analysis of the anther-specific ZmSBT genes showed their expression peaked at different developmental stages of maize anthers. Subcellular localization analysis of selected maize ZmSBTs showed they are located in different cellular compartments. The information provided in this study is valuable for further functional study of ZmSBTs.

Chromatography Scrutiny, Molecular Docking, Clarifying the Selectivities and the Mechanism of [3 + 2] Cycloloaddition Reaction between Linallol and Chlorobenzene-Nitrile-oxide.

Employing the Molecular Electron Density Theory, [3 + 2] cycloaddition processes between 4-chlorobenzenenitrileoxide and linalool, have been applied using the DFT/B3LYP/6-311(d,p) method, activation, reaction energies and the reactivity indices are calculated. In an investigation of conceptual DFT indices, LIL-1 will contribute to this reaction as a nucleophile, whilst NOX-2 will participate as an electrophile. This cyclization is regio, chemo and stereospecific, as demonstrated by the reaction and activation energies, in clear agreement with the experiment's results, in addition, ELF analysis revealed that the mechanism for this cycloaddition occurs in two steps. Furthermore, a docking study was conducted on the products studied, and the interaction with the protein protease COVID-19 (PDB ID: 6LU7), our results indicate that the presence of the -OH group increases the affinity of these products, moreover, adsorption study by chromatography was made on silica gel as support; our outcome reveals that the -OH group creates an intramolecular hydrogen bond in the product P2, while in the product P3 will create a hydrogen bond with the silica gel which makes the two products P2 and P3 are very easy to separate by chromatography, this result is in excellent agreement with the Rf retention value. The study might provide a fundamental for developing natural anti-viral compound in promoting human health.

Structure based exploration of potential lead molecules against the extracellular cysteine protease (EcpA) of Staphylococcus epidermidis: a therapeutic halt

Nosocomial infection caused by Staphylococcus epidermidis is one of the most widely spread diseases affecting the world’s population. No strategies have been developed to overcome this infection and inhibit its spread in immunocompromised patients or patients with indwelling medical devices. EcpA is an extracellular cysteine protease protein involved in biofilm formation on medical devices. Thus, blocking this mechanism may be viable for developing a drug against S. epidermidis. The current research aimed to find new, potent inhibitors that could stop the S. epidermidis EcpA protein from functioning. This study attempted to identify the most promising drug candidates using structure-based virtual screening (SBVS) from libraries of natural ligands. The top-scored molecules were shortlisted based on their IC50 values and pharmacophore properties and further validated through density functional theory (DFT) studies. We found five inhibitors using virtual screening, and the results indicate that these drugs had the highest energy binding potential towards the EcpA targets when compared to the reference molecule E-64, a known cysteine protease inhibitor. In order to evaluate the binding conformational stability of protein-ligand complexes, molecular dynamics (MD) simulations were performed in triplicate for 100 ns, revealing the significant stability of anticipated molecules at the docked site. Furthermore, principal component analysis and binding free energy calculations were performed to understand the dynamics and stability of the complexes. The current study indicated that these compounds looked to be suitable novel inhibitors of the EcpA protein and pave the path for further discovery of novel inhibitors of EcpA. Communicated by Ramaswamy H. Sarma

Identification of potential antiviral compounds from Egyptian Red Sea soft corals against Middle East respiratory syndrome coronavirus

The ongoing threat of Middle East respiratory syndrome coronavirus (MERS-CoV) underscores the importance of developing effective antiviral treatments. Current research was conducted to identify potential antiviral compounds from soft corals: Sinularia leptoclados, Sarcophyton ehrenbergi, Nephthea sp., Sarcophyton glaucum and Sarcophyton regulare. The antiviral activities of soft corals extracts were evaluated against MERS-CoV. Gas chromatography-mass spectrometry (GC-MS) was used to identify bioactive compounds. The molecular docking was performed to examine the identified compounds for their binding potentials towards three pathogenic factors of MERS-CoV: main protease, spike and non-structural protein 16/10 complex. The methanolic extract of soft coral Sarcophyton regulare exhibited the most promising activity with 50% inhibitory concentration (IC50) of 4.29 µg/ml and selective index (SI) of 112.2. Among the identified compounds in the active fraction, the molecular docking showed that two fatty acid esters: hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester and octadecanoic acid, 2-hydroxy-1 (hydroxymethyl) ethyl ester had promising docking scores.

5‐Arylidene‐2,4‐thiazolidinediones as Cysteine Protease Inhibitors against Leishmania Donovani

AbstractA series of 5‐arylidene‐2,4‐thiazolidinediones were synthesized using Knoevenagel condensation and evaluated for their anti‐leishmanial activity against L. donovani promastigotes and axenic amastigotes. Among the compounds tested, three were the most active, with IC50 values of 0.82–1.42 μM against L. donovani promastigotes and 0.69–1.19 μM against L. donovani amastigote. (Z)‐5‐(4‐(trifluoromethyl)benzylidene)thiazolidine‐2,4‐dione was the most prominent among all the tested compounds and demonstrated better anti‐leishmanial properties when compared to the standard drug miltefosine (1.26 μM against L. donovani promastigotes and 1.17 μM against L. donovani amastigotes). It was insignificantly toxic compared to the standard miltefosine in THP‐1 human monocytic cells. It was further evaluated for its in vitro cysteine protease (papain) inhibitory activity using Z‐RR‐AMC fluorogenic peptide substrate. It demonstrated promising inhibitory activity with the IC50 value of 3.42 μM. In silico docking studies also supported that the (Z)‐5‐(4‐(trifluoromethyl)benzylidene)thiazolidine‐2,4‐dione is bound to cysteine protease proteins′ catalytic active binding site. Anti‐leishmanial properties of this class of compounds have been evaluated for the first time, and (Z)‐5‐(4‐(trifluoromethyl)benzylidene)thiazolidine‐2,4‐dione emerged as a lead molecule from the library of compounds tested. This may serve as a template for further drug discovery in Leishmania.

Anti-inflammatory, remorin-like protein from green marine Macroalga Caulerpa sertularioides (S.G.Gmel.) M.Howe

The most prevalent natural source of hydrocolloids, cosmetics, medications, and nutraceuticals is marine seaweed (macroalgae). Numerous bioactivities, including antiviral, anticancer, anti-inflammatory, and immunomodulatory characteristics, have been found in bioactive substances such as polyphenols and sulfated and non-sulfated polysaccharides. As a result, new start-up projects and industries based on seaweed are emerging in all regions of the world with abundant marine biodiversity. In this current investigation, the anti-inflammatory activity of two different marine macroalgae Caulerpa racemosa (CR) and Caulerpa sertularioides (CS) was evaluated. Consequently, CS demonstrated more anti-inflammatory and antioxidant effects at a lower dose than CR. The IC50 value for DPPH inhibition was 456.1 μg/mL, and 180.9 μg/mL for CS and CR respectively. A similar result was obtained in the case of protein denaturation (PD), membrane stabilization (MS), and protease inhibition (PI) anti-inflammatory assays with 127.2 μg/mL, 135.5 μg/mL, and 71.88 μg/mL for CR, and 66.78 μg/mL, 88.96 μg/mL, and 59.54 μg/mL for CS respectively. Based on the SDS-PAGE, the molecular weight of lectin responsible for the anti-inflammatory activity was determined as 17 kDa. Protein mass fingerprinting was performed for the particular lectin by in-gel trypsin digestion, MALDI-MS analysis, and Mascot peptide mass fingerprinting. Because of this, the unidentified lectin protein was discovered to be a remorin-like protein that shared 65% of its sequence with the remorin-like protein of Aegilops tauschii subsp. tauschii. Therefore, it is the hitherto report on the presence of remorin-like protein from the green macroalga Caulerpa sertularioides.

Medicinal plants with antiviral effect: A review

Several phytochemicals exhibited high level of antiviral activity. Medicinal plant possessed antiviral activity via many mechanisms included inhibition of viral replication, inhibition of the assembly of intracellular infectious virus particles, inhibition of viral infectivity, inhibition of RNA polymerase, DNA polymerase, viral neuraminidase, protease, reverse transcriptase and viral protein expression and many other mechanisms. The current review discuss the medicinal plants with antiviral activity with their mechanisms of action.

Drug repurposing of FDA-approved anti-viral drugs via computational screening against novel 6M03 SARS-COVID-19.

The COVID-19 pandemic has been recognized as severe acute respiratory syndrome, one of the worst anddisastrous infectious diseases in human history. Until now, there is no cure to this contagious infection althoughsome multinational pharmaceutical companies have synthesized the vaccines and injecting them into humans, buta drug treatment regimen is yet to come. Among the multiple areas of SARS-CoV-2 that can be targeted, protease protein has significant values due to itsessential role in viral replication and life. The repurposing of FDA-approved drugs for the treatment of COVID-19has been a critical strategy during the pandemic due to the urgency of effective therapies. The novelty in this workrefers to the innovative use of existing drugs with greater safety, speed, cost-effectiveness, broad availability, anddiversity in the mechanism of action that have been approved and developed for other medical conditions. In this research work, we have engaged drug reprofiling or drug repurposing to recognize possible inhibitors ofprotease protein 6M03 in an instantaneous approach through computational docking studies. We screened 16 FDA-approved anti-viral drugs that were known for different viral infections to be tested againstthis 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 energiesin Kcal/mol as - 8.5, - 7.0, - 6.8, - 6.8, and - 6.6, respectively, in the best binding posture. In silico studies allowedus to demonstrate the potential role of these drugs against COVID-19. In our study, we also observed the nucleotide sequence of protease protein consisting of 316 amino acid residuesand the influence of these pronouncing drugs over these sequences. The outcome of this research work providesresearchers with a track record for carrying out further investigational procedures by applying docking simulationsand in vitro and in vivo experimentation with these reprofile drugs so that a better drug can be formulated againstcoronavirus.

Deciphering the role of fucoidan from brown macroalgae in inhibiting SARS-CoV-2 by targeting its main protease and receptor binding domain: Invitro and insilico approach

The current study investigated the role of fucoidan from Padina tetrastromatica and Turbinaria conoides against 3-chymotrypsin like protease (3CLpro) and receptor binding domain (RBD) spike protein of SARS-CoV-2 using an invitro and computational approach. The 3CLpro and RBD genes were successfully cloned in pET28a vector, expressed in BL-21DE3 E. coli rosetta cells and purified by ion exchange affinity and size exclusion chromatography. Fucoidan extracted from both biomass using green approach, subcritical water, was found to inhibit 3CLpro of SARS-CoV-2 with an IC50 value of up to 0.35 mg mL−1. However, fucoidan was found to be inactive against the RBD protein. Molecular docking studies demonstrated that fucoidan binds to the active sites of 3CLpro with an affinity of −5.0 kcal mol−1. In addition, molecular dynamic simulations recorded stabilized interactions of protein-ligand complexes in terms of root mean square deviation, root mean square fluctuation, the radius of gyration, solvent accessible surface area and hydrogen bond interaction. The binding energy of fucoidan with 3CLpro was determined to be −101.821 ± 12.966 kJ mol−1 using Molecular Mechanic/Poisson-Bolt-Boltzmann Surface Area analysis. Fucoidan satisfies the Absorption, Distribution, Metabolism, and Excretion (ADME) properties, including Lipinski's rule of five, which play an essential role in drug design. According to the toxicity parameters, fucoidan does not exhibit skin sensitivity, hepatotoxicity, or AMES toxicity. Therefore, this work reveals that fucoidan from brown macroalgae could act as possible inhibitors in regulating the function of the 3CLpro protein, hence inhibiting viral replication and being effective against COVID-19.

Molecular Docking Studies : Activity of Natural Compounds as SAR-CoV-2 Inhibitors in Papain-like protease (PL pro)

SARS-CoV-2 is a respiratory virus often referred to as COVID-19. The challenge of finding SARS-CoV-2 antivirals also attracted many researchers, one of which was by utilizing natural compounds. The purpose of this study was to analyze the potential activity of herbal plants in Indonesia as anti-COVID-19, specifically tasked with inhibiting the target protein, PLpro using the in-silico method. This research is an insilico approach with molecular docking studies. Docking simulation in this study was used to analyze the activity of natural herbal plant compounds against the target protein, namely PLpro. In addition, the docking method is also able to examine the interaction of compounds contained in plants with target receptors seen from a computational-based visual approach. So, the results obtained in this study are that when viewed from the value of the gibbs energy produced, the compound that has the best activity to inhibit the PLpro target protein (SARS CoV-2) is Luteolin. The Gibbs energy generated in the Luteolin docking simulation is -7.3 kcal/mol. The Gibbs energy value produced by the Luteolin ligand is the most negative or the smallest value of the 14 tested ligands. This means that the bond formed between the Papain-like protease (PLpro) protein and the Luteolin ligand is the most stable among the others. In addition to the Luteonin compound, there are also several compounds that have quite good potential when viewed from the amount of gibbs energy owned, namely Mangostin, quercetin compounds and Myricetin compounds with gibbs energy values of -7.1 kcal / mol, -7.0 kcal / mol and -7.0 kcal / mol. Based on these results, it can be concluded that several compounds from typical Indonesian plants such as Luteolin compounds (Compounds found in Celery plants (Apium graveolens)), Mangostin compounds (Compounds found in Mangosteen plants (Garcinia mangostana)), Quercetin compounds (Compounds found in Citrus plants (Citrus aurantium)) and Myricetin compounds (Compounds found in Clove plants (Syzygium aromaticum)) have the potential to be developed as anti-COVID-19.

QTL-seq Identifies Genomic Regions Associated with Resistance to Dirty Panicle Disease in Rice

Dirty panicle disease is one of the most important diseases that can cause yield losses in rice production. Despite the severity of the disease, the molecular basis of resistance to the pathogen is poorly understood. Using QTL-seq with an F2 population, we identified three genomic regions on chromosomes 1, 9, and 10, namely qDP1, qDP9, and qDP10. These regions are significantly associated with resistance to dirty panicle disease caused by two fungal pathogens, Bioplaris oryzae and Cirvularia lunata. qDP1 was significantly associated only with resistance to B. oryzae, whereas qDP9 and qDP10 were significantly associated with both B. oryzae and C. lunata. We also developed KASP markers for each QTL detected and validated them in the F2 population. The markers were able to explain phenotypic variation in a range of 5.87–15.20%. Twelve potential candidate genes with annotated functions as resistance-related genes were proposed. These candidate genes include those encoding RLK, MATE, WAK, NBS-LRR, subtilisin-like protease, and ankyrin repeat proteins. The results of this study provide insights into the genetic mechanism of dirty panicles in rice and will be useful for future breeding programs for dirty panicle resistance. This is the first report of QTLs associated with resistance to dirty panicle disease in rice.

Quality control assessment, toxicity profiling, and experimental validation of network pharmacology-predicted anti-inflammatory potential of Natsiatum herpeticum Buch.-Ham. Ex Arn.

Ethnopharmacological relevanceNatsiatum herpticum Buch.-Ham. Ex Arn., a least-explored plant, is being considered a wild edible plant by the Bankariya community of Nepal and the Mishing, Sonowal Kachari, and several ethnic groups in the north-east region of India. It is also used as a traditional remedy for the treatment of pain and inflammation-associated conditions like cuts and wounds, stomach ache, backache, and headache as a practice of a folkloristic system of medicine. In spite of several previous publications suggesting its use by different tribes, no documentation or scientific approaches have been made hitherto to validate its ethnopharmacological claims. Aim of studyThe study aimed at the botanical quality control assessment, toxicity profiling, and network pharmacology-assisted experimental validation of the anti-inflammatory potential of the aqueous extract of N. herpeticum to fill the lacunae in the current knowledge. Material and methodPlant material was authenticated using a classical taxonomical approach and DNA barcoding. The quality control methods, acute toxicity study, and repeated dose 28-day oral toxicity study were performed as per standard guidelines. QToF-MS analysis, drug-likeness properties, network pharmacology-based anti-inflammatory prediction, and in vitro assays were carried out. ResultsQuality control assessment was done for the plant. Toxicity studies revealed the aqueous extract to be non-toxic when consumed for short periods at low doses. Alterations in food and water intake, biochemical parameters, and alterations in liver histology (n = 2 female rats) implicate repeated exposure to high doses (2000 mg/kg) that may possess deleterious effects, particularly in hepatic tissues. 21 representative compounds (14 drug-like molecules) were detected by QToF-MS analysis and then subjected to network pharmacology to predict anti-inflammatory effects. It was found that an anti-inflammatory effect may be exerted by modulating inflammatory pathways involving genes such as TNF, PTGS2, EGFR, STAT3, PPARG, PTGER4, PPARA, NOS2, TRPV1, and JAK2. Further, in vitro studies demonstrated plant extract to possess a good anti-inflammatory effect with IC50 values of 98.76, 85.73, and 96.16 μg/ml in protein denaturation, proteinase inhibition, and haemolysis inhibition assays, respectively. ConclusionThe plant extract was found to be safer at acute dose but may cause potential liver toxicity on prolonged use. The anti-inflammatory property predicted by network pharmacology was further supported by the positive results of in vitro experiments. In summary, to further establish the toxicity profile of this edible plant and its anti-inflammatory properties, chronic toxicity study and in vivo experiments are required.

Screening of wild species and transcriptome profiling to identify differentially regulated genes in response to late blight resistance in potato.

Late blight (Phytophthora infestans) is a serious disease of potatoes. The aim of this study was to screen wild potato species and identify differentially expressed genes (DEGs) associated with late blight resistance. Wild potato species such as PIN45 (Solanum pinnatisectum), CPH62 (Solanum cardiophyllum), JAM07 (Solanum jamesii), MCD24 (Solanum microdontum), PLD47 (Solanum polyadenium), and cv. Kufri Bahar (control) were tested by artificial inoculation of P. infestans under controlled conditions. Transcriptomes of the leaf tissues (96h post-inoculation) were sequenced using the Illumina platform. Statistically significant (p < 0.05) DEGs were analyzed in wild species by comparison with the control, and upregulated (>2 log2 fold change, FC) and downregulated (<-2 log2 FC) genes were identified. DEGs were functionally characterized with Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Selected genes were validated by real-time PCR analysis to confirm RNA-seq results. We identified some upregulated genes associated with late blight resistance in wild species such as cytochrome P450, proline-rich protein, MYB transcription factor MYB139, ankyrin repeat-containing protein, and LRR receptor-like serine/threonine-protein kinase in PIN45; glucosyltransferase, fructose-bisphosphate aldolase, and phytophthora-inhibited protease 1 in CPH62; steroid binding protein and cysteine proteinase 3 in JAM07; glycine-rich cell wall structural protein 1 and RING finger protein in MCD24; and cysteine proteinase 3 and major latex protein in PLD47. On the other hand, downregulated genes in these species were snakin-2 and WRKY transcription factor 3 in PIN45; lichenase and phenylalanine ammonia-lyase 1 in CPH62; metallothionein and LRR receptor-like serine/threonine-protein kinase in JAM07; UDP-glucoronosyl/UDP-glucosyl transferase family protein and steroid binding protein in MCD24; and cytoplasmic small heat shock protein class I and phosphatase PLD47. Our study identified highly resistant wild potato species and underlying genes such as disease resistance, stress response, phytohormones, and transcription factors (e.g., MYB, WRKY, AP2/ERF, and AN1) associated with late blight resistance in wild potato species.