Identification of novel inhibitors of SARS-CoV-2 main protease (Mpro ) from Withania sp. by molecular docking and molecular dynamics simulation.

Since December 2019, coronavirus disease (COVID-19) has claimed the lives of millions of people across the globe. To date, no medicine is available for the responsible virus SARS-CoV-2. 3CLpro, that is, 3-chymotrypsin-like protease, the main protease (Mpro ), has an important role in cleaving pp1a and pp1ab polyproteins. This Mpro serves as an important target in drug designing against COVID-19. Herein, the study includes the investigation, screening, and identification of potent leads from (Withania sps.), against SARS-CoV-2, using virtual screening, molecular docking, and molecular dynamics (MD) simulations. Seventy-three natural compounds from this important medicinal plant were screened. The Binding affinity was used to identify the most probable target to inhibit the Mpro , compounds 27-hydroxywithanolide F (W32, -11.5 kcal/mol), withanolide A (W56, -11.4 kcal/mol), and withacoagulin H (W30, -11.1 kcal/mol) showed highest binding energy. Lipinski's rule, followed by drug-likability and likeness screening, resulted in 36 molecules. Further, MD simulation of 50 ns predicted withacoagulin H possessing strong binding affinity and hydrogen-bonding interactions with the active site. The binding free energy calculation showed the most negative energy of withacoagulin H (-63.463 KJ/mol) compared to other selected compounds. The study also compared the bonding energy of already reported repurposed and newly synthesized drugs. Further, absorption, distribution, metabolism, and excretion predictions were made to found a good balance of potency. Hence the following screened compounds from Withania sps. could serve as the potential leads for drug development against COVID-19.

The Bioactive Potential of Culturable Fungal Endophytes Isolated From the Leaf of Catharanthus roseus (L.) G. Don.

INTRODUCTION: Endophyte is considered a source of natural bioactive secondary metabolites that provides an array of bioactive lead compounds. The present study was aimed to determine the antimicrobial and anti-inflammatory potential of fungal endophytes isolated from Catharanthus roseus. METHODS: A total of seven fungal endophytes crude extract were screened against bacterial pathogens. Of these, Curvularia geniculata CATDLF7 crude extract exhibited the most potent inhibitory activity against bacterial pathogens. Hence, CATDLF7 crude extract was subjected to chromatographic separation. This purification leads to the isolation of six pure compounds (1PS - 6PS). Of these, 3PS was found to be a major constituent and most effective against clinical isolates of methicillin- resistant Staphylococcus aureus (MRSA) with minimum inhibitory concentration (MIC) values ranging from 100 to 200 µg/ml. Based on the spectroscopic data, 3PS was characterized as α,ß- dehydrocurvularin. This compound also showed synergistic interaction with norfloxacin and reduced its MIC up to 32-folds with a fractional inhibitory concentration index (FICI) of 0.09. RESULTS: To understand the possible antibacterial mechanism of action, α,ß-dehydrocurvularin alone (100 µg/ml) exhibited efflux pump inhibitory potential by 0.84 fold decreasing in ethidium bromide (EtBr) fluorescence. In addition, α,ß-dehydrocurvularin inhibited inflammatory cytokines TNF-α and IL-6 production, which is further validated by molecular docking scores -4.921 and -5.641, respectively, for understanding orientation and binding affinity. CONCLUSION: Overall, the results highlighted identifying bioactive compound α,ß-dehydrocurvularin, which could be used as an antimicrobial and anti-inflammatory agent.

A clerodane diterpene from Polyalthia longifolia as a modifying agent of the resistance of methicillin resistant Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus infections are raising serious concern across the world. The effectiveness of conventional drugs is continuously decreasing due to global emergence of multidrug resistance (MDR) and therefore, new resistance-modifying agents (RMAs) are highly needed. HYPOTHESIS: Clerodane diterpene 16α-hydroxycleroda-3,13(14)-Z-dien-15,16-olide (CD) from leaves of Polyalthia longifolia (Sonn.) Thwaites (Annonaceae) as RAM will be useful in improving the current treatment strategies for staphylococcal infections. STUDY DESIGN: In the present study, we determine the resistance-modifying activity of CD using clinical isolates of MRSA. Further, the influence of CD on innate immune response was also evaluated in vitro and in vivo. The nature of potential interactions was determined by fractional inhibitory concentration indices (FICIs) calculated from microdilution assays and time-kill curves. RESULTS: The result of in vitro combination study showed that CD significantly reduced MIC of fluoroquinolones up to 16-folds (FICI 0.315-0.500), while in S. aureus infected Swiss albino mice model, combination of CD with norfloxacin, significantly (p<0.01, p<0.001) lowered the systemic microbial burden in blood, liver, kidney, lung and spleen tissues in comparison to CD, norfloxacin alone as well as untreated control. Flow cytometry analysis clearly showed that CD significantly inhibited EtBr efflux and extended post-antibiotic effect. In qRT-PCR analysis, CD alone as well as in combination, significantly modulated the expression of various efflux pump genes including norA up to 2-fold in clinical isolate MRSA-ST2071. Further, the in vitro combination study of the CD (10, 5, 2.5µg/ml) along with the norfloxacin (10µg/ml) depicted a significant decline in the pro-inflammatory cytokines, IL6 and TNF-α. In septic shock mice model, CD did not exhibit any significant changes in the level of pro-inflammatory cytokines. CONCLUSION: This is the first report on drug resistance-modifying potential of CD through inhibition of MDR efflux pump.

Glycolipids: isolated from Oplismenus burmannii induce glucose uptake in L6-GLUT4myc myotube cells.

Bioactivity guided separation of combined n-hexane and chloroform extracts of Oplismenus burmannii resulted in the isolation and characterization of five new glycoglycerolipids, (2S)-1,2,6'-tri- O-hexadecanoyl-3-O-ß-D-galactopyranosyl glycerol (1a), (2S)-1,2,6'-tri-O-[(9Z,12Z)-octadeca-9,12- dienoyl]-3-O-ß-D-galactopyranosyl glycerol (1b), (2S)-1,6'-di-O-[(9Z,12Z)-octadeca-9,12-dienoyl]-3- O-ß-D-galactopyranosyl glycerol (2b), (2S)-1,6'-di-O-[(9Z,12Z,15Z)-octadeca-9,12,15-trienoyl]-3-O-ß-D-galactopyranosyl glycerol (2c), and (2S)-1,2-di-O-[(9Z,12Z)-octadeca-9,12-dienoyl]-3-O-(6- sulpho-α-D)-quinovopyranosyl glycerol (3b) along with five known glycoglycerolipids (1c, 2a, 3a, 3c and 4), a cerebroside (5), three monoacylglycerols (6a-c) and α-linoleic acid (7). The isolated compounds, 1-5 were in-vitro tested for their antihyperglycemic potential in terms of increase in 2-deoxyglucose uptake in L6-GLUT4myc myotube cells. The results showed that compounds, 1-5 were showing 1.52 (P<0.05), 1.50 (P<0.05), 1.28, 1.49 (P<0.05) and 1.50 (P<0.05) fold increase in the glucose uptake at concentration of 10 µg/mL and 1.71 (P<0.001), 1.74 (P<0.001), 1.50 (P<0.05), 1.76 (P<0.001) and 1.74 (P<0.001) fold increase in the glucose uptake at concentration of 25 µg/mL respectively. However, standard drug Rosiglitazone increases the glucose uptake by 1.59 fold at the concentration of 10µM. Further work on optimization of the anti-diabetic lead is under progress.

RP-HPLC-DAD method for the identification of two potential antioxidant agents namely verminoside and 1-O-(E)-caffeoyl-ß-gentiobiose from Spathodea campanulata leaves.

A simple and reliable high-performance liquid chromatographic method was successfully developed for the study of fingerprint chromatograms of extract and fractions from the leaves of Spathodea campanulata (SC) using verminoside (1) and 1-O-(E)-caffeoyl-ß-gentiobiose (2) as marker compounds. Antioxidant activity of SC was determined by using free radical of 2,2-diphenyl-1-picryl-hydrazyl-hydrate as an experimental model. The docking study of selected target, tyrosinase and ligands (ascorbic acid, compounds 1 and 2) was performed through Autodock Vina v0.8. Fingerprints of methanol, chloroform, ethylacetate, n-butanol and water extracts could resolve 13, 11, 22, 16 and 5 peaks, respectively. Extract, fractions and compounds 1 and 2 previously isolated from SC displayed remarkable antioxidant activity with radical-scavenging activity ranging from 2.5 to 6.7 µg/mL. In silico study identified compounds 1 and 2 as potential inhibitors of tyrosinase correlating with the observed antioxidant activity in vitro.

Bioactivity-guided isolation of antiplasmodial constituents from Conyza sumatrensis (Retz.) E.H. Walker.

Conyza sumatrensis (Retz.) E.H. Walker (Cs) leaves are used for traditional treatment of malaria in Cameroon. However, the antimalarial activity of the leaf constituents of this plant is still unexplored. The aim of our investigation was to evaluate the antiplasmodial activity of some bioactive constituents from Cs leaves. Compounds were isolated from Cs leaves and structurally elucidated using extensive spectroscopic analysis. The in vitro antiplasmodial activity of the extracts and pure compounds were evaluated on chloroquine-sensitive strain (NF54) of Plasmodium falciparum. The in vivo assay was done by administering seven doses of extracts in mice infected with Plasmodium berghei K173 through oral route. Cytotoxicity of pure compounds on murine macrophage cells was performed through [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] (MTT) test. Hemolysis and lactate dehydrogenase assays were also carried out using standard procedures. The in silico prediction of bioactive constituents was performed through Autodock Vina. Polarity-based extracts from Cs were found to be active against P. falciparum (NF54) and P. berghei (K173) in vitro and in vivo respectively. Further, bioactivity-guided isolation of n-hexane fraction yielded three compounds, (1), (2) and (3) with IC50 of 34, 17.9 and 18µg/ml, respectively, while the ethyl acetate fraction afforded the fourth compound with an IC50 of 25µg/ml, indicating anti-malarial potential of Cs through PfLDH interaction without compromising normal cell growth. This study reports for the first time, the antiplasmodial activity of bioactive constituents from Cs and confirms its traditional use.