Antiviral peptides inhibiting the main protease of SARS-CoV-2 investigated by computational screening and in vitro protease assay.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an important role in viral replication and transcription and received great attention as a vital target for drug/peptide development. Therapeutic agents such as small-molecule drugs or peptides that interact with the Cys-His present in the catalytic site of Mpro are an efficient way to inhibit the protease. Although several emergency-approved vaccines showed good efficacy and drastically dropped the infection rate, evolving variants are still infecting and killing millions of people globally. While a small-molecule drug (Paxlovid) received emergency approval, small-molecule drugs have low target specificity and higher toxicity. Besides small-molecule drugs, peptide therapeutics are thus gaining increasing popularity as they are easy to synthesize and highly selective and have limited side effects. In this study, we investigated the therapeutic value of 67 peptides targeting Mpro using molecular docking. Subsequently, molecular dynamics (MD) simulations were implemented on eight protein-peptide complexes to obtain molecular-level information on the interaction between these peptides and the Mpro active site, which revealed that temporin L, indolicidin, and lymphocytic choriomeningitis virus (LCMV) GP1 are the best candidates in terms of stability, interaction, and structural compactness. These peptides were synthesized using the solid-phase peptide synthesis protocol, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and authenticated by mass spectrometry (MS). The in vitro fluorometric Mpro activity assay was used to validate the computational results, where temporin L and indolicidin were observed to be very active against SARS-CoV-2 Mpro with IC50 values of 38.80 and 87.23 µM, respectively. A liquid chromatography-MS (LC-MS) assay was developed, and the IC50 value of temporin L was measured at 23.8 µM. The solution-state nuclear magnetic resonance (NMR) structure of temporin L was determined in the absence of sodium dodecyl sulfate (SDS) micelles and was compared to previous temporin structures. This combined investigation provides critical insights and assists us to further develop peptide inhibitors of SARS-CoV-2 Mpro through structural guided investigation.

Detection and absolute quantification of Lactiplantibacillus plantarum ATCC 202195 by quantitative real-time PCR.

IMPORTANCE: When administered for seven consecutive days shortly after birth, the probiotic bacterium Lactiplantibacillus plantarum ATCC 202195 plus fructooligosaccharide (FOS) was reported to reduce sepsis and lower respiratory tract infection events during early infancy in a randomized trial in India. Since probiotic effects are often strain specific, strain-level detection and quantification by routine molecular methods enables the monitoring of safety outcomes, such as probiotic-associated bacteremia, and allows for the quality of probiotic interventions to be assessed and monitored (i.e., verify strain identity and enumerate). Despite the potential clinical applications of L. plantarum ATCC 202195, an assay to detect and quantify this strain has not previously been described. Herein, we report the design of primer and probe sequences to detect L. plantarum ATCC 202195 and the development and optimization of a real-time PCR assay to detect and quantify the strain with high specificity and high sensitivity.

Clonal Blumea lacera (Burm. f.) DC. ameliorates diabetic conditions by modulating carbohydrate and lipid hydrolases: a combine in vivo experimental and chemico-biological interaction study.

Blumea lacera (Burm. f.) DC. is an aromatic annual herb that has traditionally been used to treat or protect against diabetes. Although it has infallible uses, its supply is limited due to its short lifespan. In this study, we aim to investigate the anti-diabetic potential of its micropropagated plants in type 2 diabetic mammalian (mouse) model and further expand the molecular mechanistic understanding of its activity. The water extract of the micropropagated plants was tested in mice with streptozotocin-induced diabetes. The extract effectively suppressed glucose levels prevented weight loss, and improved dyslipidemia in mice. Additionally, it improved liver injury as well as all investigated toxicity indicators, including serum glutamate-pyruvate transaminase, serum glutamic oxaloacetic transaminase, and serum anti-inflammatory marker C-reactive protein. The intramolecular interaction study revealed that the innate polyphenolic constituents of this plant more profoundly inhibited α-amylase, α-glucosidase, and lipase compared to the standard. The prolific bioactive compounds of the micropropagated plant could be attributed to these superior anti-diabetic effects, presumably via an elaborate inhibition of carbohydrate and lipid hydrolyzing enzymes. Thus, the obtained results provide solid experimental proof of the year-round utility of micropropagated plants as a standard source plant material of Blumea lacera (Burm. f.) DC. for drug research and therapeutic production.

Current Landscape of Natural Products against Coronaviruses: Perspectives in COVID-19 Treatment and Anti-viral Mechanism.

BACKGROUND: SARS-CoV-2 is a coronavirus, and the infection by SARS-CoV-2, termed as COVID-19, was first reported in Wuhan, China, at the end of 2019, and this outbreak became a pandemic in February of 2020. Till now, there is no effective drug or vaccine against this virus that can make a complete cure; however, a number of drugs are in trials. OBJECTIVES: In this review, we have focused on an alternative therapeutic approach using natural products utilizing the host anti-viral responses for resolving COVID-19 pathogenesis. METHODS: We have searched databases like PubMed, Scopus, Web of Science, and Google Scholar for articles related to natural products and viral diseases, with a specific focus on coronaviruses, as well as other RNA viruses and recent updates on the COVID-19 pandemic, and collected articles and reviewed them comprehensively. RESULTS: Scientific studies clarified the viral pathogenesis that involved viral entrance into host cells and anti-viral response inside the cells, which can be effectively targeted by numerous natural compounds from different sources. Many of these compounds can potentially target viral genomic material or protein machinery. Natural products that were found effective against other coronaviruses, especially SARS-CoV or MERS-CoV (which emerged in 2002 and 2012, respectively), might be effective against SARS-CoV-2 due to their structural similarities. CONCLUSION: COVID-19 pandemic is a global emergency thus, urgent drug development is necessary. Natural products can be the biggest source of drugs, as they have been found to be effective in other coronaviruses previously; however, time is required to establish the clinical success of these drugs for clinical applications.

Cordycepin Downregulates Cdk-2 to Interfere with Cell Cycle and Increases Apoptosis by Generating ROS in Cervical Cancer Cells: in vitro and in silico Study.

BACKGROUND: Cordycepin is a small molecule from medicinal mushroom Cordyceps, which has been reported for anticancer properties. OBJECTIVE: In this study, we have focused on the investigation of cordycepin effect on cervical cancer cells with further clarification of possible molecular mechanism. METHOD: We have used cell viability and cell counting assay for cytotoxic effect of cordycepin, flow cytometric assay of apoptosis and cell cycle, and quantitative PCR (qPCR) and Western blotting for the determination of target gene expression. Molecular docking and Molecular dynamics simulation were used for in silico analysis of cordycepin affinity to target protein(s). RESULTS: Treatment of cordycepin controlled SiHa and HeLa cervical cancer cell growth, increased the rate of their apoptosis, and interfered with cell cycle, specifically elongated S-phase. qPCR results indicated that there was a downregulation of cell cycle proteins CDK-2, CYCLIN-A2 and CYCLIN-E1 in mRNA level by cordycepin treatment but no significant change was observed in pro-apoptotic or antiapoptotic proteins. The intracellular reactive oxygen species (ROS) level in cordycepin treated cells was increased significantly, implying that apoptosis might be induced by ROS. Western blot analysis confirmed significant decrease of Cdk-2 and mild decrease of Cyclin-E1 and Cyclin-A2 by cordycepin, which might be responsible for regulating cell cycle. Molecular docking indicated high binding affinity of cordycepin against Cdk-2. Molecular dynamics simulation further confirmed that the docked pose of cordycepin-Cdk-2 complex remained within the binding pocket for 10 ns. CONCLUSION: Our study suggests that cordycepin is effective against cervical cancer cells, and regulating cell cycle via cell cycle proteins, especially downregulating Cdk-2, and inducing apoptosis by generating ROS are among the mechanisms of anticancer activities of cordycepin.

Molecular Recognition of Azelaic Acid and Related Molecules with DNA Polymerase I Investigated by Molecular Modeling Calculations.

Molecular recognition has central role on the development of rational drug design. Binding affinity and interactions are two key components which aid to understand the molecular recognition in drug-receptor complex and crucial for structure-based drug design in medicinal chemistry. Herein, we report the binding affinity and the nonbonding interactions of azelaic acid and related compounds with the receptor DNA polymerase I (2KFN). Quantum mechanical calculation was employed to optimize the modified drugs using B3LYP/6-31G(d,p) level of theory. Charge distribution, dipole moment and thermodynamic properties such as electronic energy, enthalpy and free energy of these optimized drugs are also explored to evaluate how modifications impact the drug properties. Molecular docking calculation was performed to evaluate the binding affinity and nonbonding interactions between designed molecules and the receptor protein. We notice that all modified drugs are thermodynamically more stable and some of them are more chemically reactive than the unmodified drug. Promise in enhancing hydrogen bonds is found in case of fluorine-directed modifications as well as in the addition of trifluoroacetyl group. Fluorine participates in forming fluorine bonds and also stimulates alkyl, pi-alkyl interactions in some drugs. Designed drugs revealed increased binding affinity toward 2KFN. A1, A2 and A3 showed binding affinities of -8.7, -8.6 and -7.9 kcal/mol, respectively against 2KFN compared to the binding affinity -6.7 kcal/mol of the parent drug. Significant interactions observed between the drugs and Thr358 and Asp355 residues of 2KFN. Moreover, designed drugs demonstrated improved pharmacokinetic properties. This study disclosed that 9-octadecenoic acid and drugs containing trifluoroacetyl and trifluoromethyl groups are the best 2KFN inhibitors. Overall, these results can be useful for the design of new potential candidates against DNA polymerase I.

Multiple receptor conformers based molecular docking study of fluorine enhanced ethionamide with mycobacterium enoyl ACP reductase (InhA).

A major limitation in current molecular docking method is that of failure to account for receptor flexibility. Herein we report multiple receptor conformers based molecular docking as a practical alternative to account for the receptor flexibility. Multiple (forty) conformers of Mycobacterium Enoyl ACP Reductase (InhA) are generated from Molecular Dynamics simulation and twenty crystallographic structures of InhA bound to different inhibitors are obtained from the Protein Data Bank. Fluorine directed modifications are performed to currently available anti-tuberculosis drug ethionamide. The modified drugs are optimized using B3LYP 6-31G (d,p) level of theory. Dipole moment, frontier orbital gap and thermodynamical properties such as electronic energy, enthalpy and Gibbs free energy of these optimized drugs are investigated. These drugs are subsequently docked against the conformers of InhA. Molecular docking against multiple InhA conformations show variation in ligand binding affinity and suggest that Ser94, Gly96, Lys165 and Ile194 amino acids play critical role on strong drug-InhA interaction. Modified drug N1 showed greater binding affinity compared to EN in most conformations. Structure of PDB ID: 2NSD and snapshot conformer at 5.5ns show most favorable binding with N1 compared to other conformers. Fluorine participates in forming fluorine bonds and contributes significantly in increasing binding affinity. Our study reveal that addition of trifluoromethyl group explicitly shows promise in improving thermodynamic properties and in enhancing hydrogen bonding and non-bonded interactions. Molecular dynamics (MD) simulation show that EN and N1 remained in the binding pocket similar to the docked pose of EN-InhA and E1-InhA complexes and also suggested that InhA binds to its inhibitor in inhibitor-induced folding manner. ADMET calculations predict modified drugs to have improved pharmacokinetic properties. Our study concludes that multiple receptor conformers based molecular docking can be an alternative to study the effect of receptor flexibility in ligand binding and fluorine directed modifications can improve drug efficacy.