A network pharmacology-based approach to understand the mechanism of action of anti-mycobacterial activity of Acacia nilotica: a modelling and experimental study.

The rapid rise in drug-resistant tuberculosis poses a serious threat to public health and demands the discovery of new anti-mycobacterial agents. Medicinal plants are a proven potential source of bioactive compounds; however, identifying those responsible for the putative anti-mycobacterial action still remains a challenging task. In this study, we undertook a systematic network pharmacology approach to identify and evaluate anti-mycobacterial compounds from a traditional plant, Acacia nilotica, as a model system. The protein-protein interaction network revealed 17 key pathways in M. tuberculosis encompassing 40 unique druggable targets that are necessary for its growth and survival. The phytochemicals of A. nilotica were preferentially found to interfere with the cell division and cell wall biogenesis proteins, especially FtsZ and Mur. Notably, the compounds epigallocatechin, ellagic acid, chlorogenic acid, and D-pinitol were found to exhibit a potential polypharmacological effect against multiple proteins. Further, in vitro studies confirmed that the selected candidates, chlorogenic acid, and ellagic acid exhibited potent anti-mycobacterial activity (against M. smegmatis) with specific inhibition of purified M.tb FtsZ enzyme. Taken together, the present study demonstrates that network pharmacology combined with molecular docking can be utilized as an efficient approach to identify potential bioactive phytochemicals from natural products along with their mechanism of action. Hence, the compounds identified in this study can be potential lead candidates for developing novel anti-mycobacterial drugs, while the key proteins identified here can be potential drug targets.

High molecular weight heparin-induced angiogenesis mainly mediated via basic fibroblast growth factor-2- an in-vivo (CAM) and in-silico analysis.

Background: High-molecular weight heparin (HMWH), a molecule extensively used as an anticoagulant, shows concentration-dependent angiogenic and anti-angiogenic potential. So far, no studies have reported the interactive potential of HMWH with various pro-angiogenic growth factors under physiological conditions. Haence, we aimed to find the impact of major pro-angiogenic growth factors under HMWH induced angiogenesis. Methods: Chicken Chorioallantoic Membranes (CAMs) are incubated with various concentrations of HMWH. Semiquantitative PCR method was implemented to measure the changes in the transcription level of pro-angiogenic growth factors. The scanning electron microscopic technique is applied to find the morphological changes in CAM. Molecular docking and molecular dynamics simulation studies using NAMD and CHARMM force field discerned the heparin-binding mode with the pro-angiogenic growth factors. Results: HMWH can enhance the transcription level of major pro-angiogenic growth factors, significantly impacting FGF2 under 100 µM concentration. The in-silico analysis reveals that HMWH shows the highest binding affinity with FGF2. Further, molecular dynamics and interaction studies using 1 kDa Heparin against FGF2 showed that the former binds stably with the latter due to a strong salt bridge formation between the sulfate groups and arginine residues (ARG 119 and ARG109). Conclusion: The combined experimental and in-silico analysis results reveal that HMWH can interact with pro-angiogenic growth factors under micromolar concentration while inducing angiogenesis. This observation further supports the therapeutic benefits of HMWH as an angiogenic factor under such low concentration. This technique is used to replenish the blood supply to chronic wounds to speed healing and prevent unnecessary amputations.

Molecular docking analysis reveals the functional inhibitory effect of Genistein and Quercetin on TMPRSS2: SARS-COV-2 cell entry facilitator spike protein.

BACKGROUND: The Transmembrane Serine Protease 2 (TMPRSS2) of human cell plays a significant role in proteolytic cleavage of SARS-Cov-2 coronavirus spike protein and subsequent priming to the receptor ACE2. Approaching TMPRSS2 as a therapeutic target for the inhibition of SARS-Cov-2 infection is highly promising. Hence, in the present study, we docked the binding efficacy of ten naturally available phyto compounds with known anti-viral potential with TMPRSS2. The aim is to identify the best phyto compound with a high functional affinity towards the active site of the TMPRSS2 with the aid of two different docking software. Molecular Dynamic Simulations were performed to analyse the conformational space of the binding pocket of the target protein with selected molecules. RESULTS: Docking analysis using PyRx version 0.8 along with AutoDockVina reveals that among the screened phyto compounds, Genistein shows the maximum binding affinity towards the hydrophobic substrate-binding site of TMPRSS2 with three hydrogen bonds interaction ( - 7.5 kcal/mol). On the other hand, molecular docking analysis using Schrodinger identified Quercetin as the most potent phyto compound with a maximum binding affinity towards the hydrophilic catalytic site of TMPRSS2 ( - 7.847 kcal/mol) with three hydrogen bonds interaction. The molecular dynamics simulation reveals that the Quercetin-TMPRSS complex is stable until 50 ns and forms stable interaction with the protein ( - 22.37 kcal/mol of MM-PBSA binding free energy). Genistein creates a weak interaction with the loop residues and hence has an unstable binding and exits from the binding pocket. CONCLUSION: The compounds, Quercetin and Genistein, can inhibit the TMPRSS2 guided priming of the spike protein. The compounds could reduce the interaction of the host cell with the type I transmembrane glycoprotein to prevent the entry of the virus. The critical finding is that compared to Genistein, Quercetin exhibits higher binding affinity with the catalytic unit of TMPRSS2 and forms a stable complex with the target. Thus, enhancing our innate immunity by consuming foods rich in Quercetin and Genistein or developing a novel drug in the combination of Quercetin and Genistein could be the brilliant choices to prevent SARS-Cov-2 infection when we consider the present chaos associated with vaccines and anti-viral medicines.

Self-Assembled Inhalable Immunomodulatory Silk Fibroin Nanocarriers for Enhanced Drug Loading and Intracellular Antibacterial Activity.

In this study, a pH-induced self-assembly-based method has been developed to form silk fibroin nanoparticles (SFN-2) with a higher drug loading capacity (21.0 ± 2.1%) and cellular uptake than that of silk fibroin particles produced by a conventional desolvation method (SFN-1). Using the self-assembly method, rifampicin-encapsulated silk fibroin nanoparticles (R-SFN-2) were prepared with a size of 165 ± 38 nm at an optimum pH of 3.8. In silico analysis reveals that at acidic pH, the amino acid side chain charge neutralization of acidic residues, especially GLU64, promotes the formation of additional favorable interactions between the silk fibroin and the drug. The SFN-2 also possess a good aerosol property with a mass median aerodynamic diameter of 3.82 ± 0.71 µm and fine particle fraction of 64.0 ± 1.4%. These SFN-2 particles were selectively endocytosed by macrophages through clathrin- and caveolae-mediated endocytosis with a higher uptake efficiency (66.2 ± 2.1%) and were found to exhibit a sustained drug release in the presence of macrophage intracellular lysates. The cytokine and biomarker expression analyses revealed that SFN-2 could exhibit an immunomodulatory effect by polarizing the macrophages to an initial M1 phase and later M2 phase. Further, R-SFN-2 also exhibited an enhanced and sustained intracellular antibacterial activity against Mycobacterium smegmatis-infected macrophages than free rifampicin. Thus, the self-assembled silk fibroin particles with immunomodulatory action combined with a good aerosol and intracellular drug release property can be an attractive choice as a carrier for developing pulmonary drug delivery systems.

Arjunetin as a promising drug candidate against SARS-CoV-2: molecular dynamics simulation studies.

Stem and bark of the tree Terminalia arjuna Wight & Arn. (Combretaceae) has been documented to exhibit therapeutic properties like cardiotonic, anticancer, antiviral, antibacterial, antifungal, hypercholesterolemia, hypolipidemic, and anti-coagulant. Our previous studies have shown that, ethanolic extract of T. arjuna bark exhibits radical scavenging anti-oxidant activity and also effectively inhibited catalase activity. In this study, oleanane triterpenoids type compounds viz., oleanolic acid, arjunolic acid, arjunolitin, arjunetin were isolated from ethanolic bark extract as bio-active compound and their structures were elucidated using 1H, 13C NMR, HR-ESIMS, IR. Of the various compounds, Arjunetin showed significant inhibition of catalase activity as compared to the other compounds. Based on the structural similarity between arjunetin and current antiviral drugs, we propose that arjunetin might exhibit antiviral activity. Molecular docking and molecular dynamics studies showed that arjunetin binds to the binds to key targets of SARS-CoV-2 namely, 3CLpro, PLpro, and RdRp) with a higher binding energy values (3CLpro, -8.4 kcal/mol; PLpro, -7.6 kcal/mol and RdRp, -8.1 kcal/mol) as compared with FDA approved protease inhibitor drugs to Lopinavir (3CLpro, -7.2 kcal/mole and PLpro -7.7 kcal/mole) and Remdesivir (RdRp -7.6 kcal/mole). To further investigate this, we performed 200-500 ns molecular dynamics simulation studies. The results transpired that the binding affinity of Arjunetin is higher than Remdesivir in the RNA binding cavity of RdRp. Based on structural similarity between arjunetin and Saikosaponin (a known antiviral agents) and based on our molecular docking and molecular dynamic simulation studies, we propose that arjunetin can be a promising drug candidate against Covid-19.Communicated by Ramaswamy H. Sarma.

Preparation of Curdlan sulphate - Chitosan nanoparticles as a drug carrier to target Mycobacterium smegmatis infected macrophages.

In this study, curdlan sulphate - chitosan nanoparticles were prepared through polyelectrolyte complexing at a mass ratio of 2:1 respectively. The curdlan was produced by fermentation with Agrobacterium sp. ATCC 31750, which was then sulphated to form the polyanionic polymer. A first-line tuberculosis drug, Rifampicin and a phytochemical, DdPinitol, were encapsulated into Curdlan Sulphate (CS) - Chitosan Nanoparticles (C) (CSC NPs) of size 205.41 ± 7.24 nm. The drug release kinetics followed a Weibull model with initial burst release (48 % Rifampicin and 27 % d-Pinitol within 6 h), followed by a sustained release. The prepared CSC: d-PIN + RIF NPs was cytocompatible and entered the M.smegmatis infected macrophages through multiple endocytic pathways including clathrin, caveolae and macropinocytosis. They showed superior bactericidal activity (2.4-2.7 fold) within 4 h when compared to free drug Rifampicin (1.6 fold). The drug encapsulated CSC: RIF suppressed the pro-inflammatory gene (TNF-α by 3.66 ± 0.19 fold) and CSC: d-PIN + RIF increased expression of the anti-inflammatory gene (IL-10 by 13.09 ± 0.47 fold). Expression of TGF- ß1 gene also increased when treated with CSC: d-PIN + RIF (13.00 ± 0.19 fold) which provided the immunomodulatory activity of the encapsulated CSC NPs. Thus, curdlan sulphate - chitosan polyelectrolyte complex can be a potential nanocarrier matrix for intracellular delivery of multiple drugs.

Dual inhibitors of SARS-CoV-2 proteases: pharmacophore and molecular dynamics based drug repositioning and phytochemical leads.

SARS-related coronaviruses poses continual threat to humanity by rapidly mutating and emerging as severe pandemic outbreaks, including the current nCoV-19 pandemic. Hence a rapid drug repositioning and lead identification strategy are required to mitigate these outbreaks. We report a pharmacophore and molecular dynamics-based approach for drug repositioning and lead identification against dual targets (3CLp and PLp) of SARS-CoV-2. The pharmacophore model of 3CLp inhibitors was apolar with two aromatic and two H-bond acceptors, whereas that of PLp was relatively polar, bearing one aromatic and three H-bond acceptors. Pharmacophore-based virtual screening yielded six existing FDA-approved drugs and twelve natural products with both the pharmacophoric features. Among them are nelfinavir, tipranavir and licochalcone-D, which has shown better binding characteristics with both the proteases compared to lopinavir. The molecular dynamics revealed that the connecting loop (residues 176-199) of 3CLp is highly flexible, and hence, inhibitors should avoid high-affinity interactions with it. Lopinavir, due to its high affinity with the loop region, exhibited unstable binding. Further, the van der Waals size of the 3CLp inhibitors positively correlated with their binding affinity with 3CLp. However, the van der Waals size of a ligand should not cross a threshold of 572Å3, beyond which the ligands are likely to make high-affinity interaction with the loop and suffer unstable binding as observed in the case of lopinavir. Similarly, the total polar surface area of the ligands were found to be negatively correlated with their binding affinity with PLp.

Inhibitory activity of traditional plants against Mycobacterium smegmatis and their action on Filamenting temperature sensitive mutant Z (FtsZ)-A cell division protein.

The study was designed to assess whether plant extracts / phytochemical (D-Pinitol) synergistically combine with antituberculosis drugs and act on Mycobacterium smegmatis (M. smegmatis) as well as assess their mode of action on Mycobacterium tuberculosis (M.tb) Filamenting temperature sensitive mutant Z (FtsZ) protein. Resazurin microtitre plate assay (Checker board) was performed to analyze the activity of plant extracts against M. smegmatis. Synergistic behaviour of plant extracts / D-Pinitol with Isoniazid (INH) and Rifampicin (RIF) were determined by time-kill and checker board assays. Elongation of M. smegmatis cells due to this treatment was determined by light microscopy. The effect of Hexane methanol extract (HXM) plant extracts on cell viability was determined using PI/SYTO9 dual dye reporter Live/Dead assay. Action of HXM plant extracts / D-Pinitol on inhibition of FtsZ protein was done using Guanosine triphosphatase (GTPase) light scattering assay and quantitative Polymerase Chain Reaction (qPCR). The Hexane-methanolic plant extract of Acacia nilotica, Aegle marmelos and Glycyrrhiza glabra showed antimycobacterial activity at 1.56 ± 0.03, 1.32 ± 0.02 and 1.25 ± 0.03 mg/mL respectively and that of INH and RIF were 4.00 ± 0.06 µg/mL and 2.00 ± 0.04 µg/mL respectively. These plant extracts and major phytochemical exudate D-Pinitol was found to act synergistically with antimycobacterial drugs INH and RIF with an FIC index ~ 0.20. Time-Kill kinetics studies indicate that, these plant extracts were bacteriostatic in nature. D-Pinitol in conjunction with INH and RIF exhibited a 2 Log reduction in the growth of viable cells compared to untreated. Attempt to elucidate their mode of action through phenotypic analysis indicated that these plant extracts and D-Pinitol was found to interfere in cell division there by leading to an abnormal elongated cellular morphology. HXM extracts and D-Pinitol synergistically combined with the first line tuberculosis drugs, INH and RIF, to act on M. smegmatis. The increase in the length of M. smegmatis cells on treatment with D-Pinitol and HXM extract of the plants indicated that they hinder the cell division mechanism thereby leading to a filamentous phenotype, and finally leading to cell death. In addition, the integrity of the bacterial cell membrane is also altered causing cell death. Further gene expression analysis showed that these plant extracts and D-Pinitol hampers with function of FtsZ protein which was confirmed through in vitro inhibition of FtsZ-GTPase enzymatic activity.

Synthesis of novel spirobibenzopyrans as potent anticancer leads inducing apoptosis in HeLa cells.

Spirobibenzopyrans are an unexplored class of therapeutics. We report the anticancer activity of novel spirobibenzopyrans, synthesized by a one-pot reaction and extensively characterized. Structure of one of the spirobibenzopyran has been determined by the single crystal XRD technique. The in vitro anticancer activity of these derivatives across the NCI 60-cell line panel was evaluated and for the first time their mechanism of action against HeLa cells was probed via cell morphology analysis and cell cycle analysis. They were determined to be apoptosis inducers with cell cycle arrest in G0/G1 and S phase suggesting CDK-4 protein inhibition and the inhibition of DNA replication. The DNA inhibition was studied and confirmed using the alkaline comet assay for the compound CHX-4MO-SAL showing S phase inhibition. Further, conformity with the in silico Lipinski's score signify the potential of spirobibenzopyrans as anticancer leads.

Pharmacophore based approach to screen and evaluate novel Mycobacterium cell division inhibitors targeting FtsZ - A modelling and experimental study.

Tuberculosis, caused by Mycobacterium tuberculosis has been one of the primal afflictions to human, and owing to the current scenario of drug resistance, newer drugs, and alternate targets are required to mitigate the disease. FtsZ is a GTP hydrolyzing protein, conserved in prokaryotes that plays a central role in Z-ring formation during cell division cytokinesis stage. This study employs the use of pharmacophore models derived from two different datasets based on Mtb-FtsZ GTPase inhibition and whole cell antibacterial activity, to virtually screen and shortlist novel compounds from In-house small molecule library as Mtb-FtsZ inhibitors and evaluate their in-vitro and ex-vivo activity. The results revealed Piperine (IC50 = 21.2 ±â€¯0.7 µM), 4-Bromo di-methoxy coumarin (IC50 = 13.0 ±â€¯1.6 µM) and Di-ethyl amino methyl coumarin (IC50 = 19.4 ±â€¯1.1) as potent Mtb-FtsZ GTPase inhibitors which showed considerable antibacterial activity (84.0 ±â€¯2.6 µM, 56.0 ±â€¯4.3 µM and 108 ±â€¯7.1 µM respectively) against M. smegmatis. They appear to be bacteriostatic, as well as treatment with these compounds led to a 3× increase in cell length of M. smegmatis. Further these molecules also altered the FtsZ gene expression by 3-fold when compared to untreated. In addition compound Aloin, an Aloe exudate showed potent Mtb-FtsZ inhibition (IC50 = 16.7 ±â€¯0.4 µM) but exhibited poor anti-mycobacterial activity (>500 µM).

Ferulic acid derivative inhibits NorA efflux and in combination with ciprofloxacin curtails growth of MRSA in vitro and in vivo.

A series of ferulic acid (FA) derivatives were synthesized and evaluated for its ability to inhibit NorA efflux in methicillin resistant Staphylococcus aureus (MRSA), by in silico docking analysis. Based on prediction from glide scores and ability to reduce EtBr MIC, two of the ten derivatives S3- [4-((E)-2-(diethylcarbamoyl)vinyl)-2-methoxyphenyl acetate] and S6- [(E)-methyl 3-(4-((p-tolylcarbamoyl)methoxy)-3-methoxyphenyl)acrylate] were chosen as putative efflux pump inhibitors (EPI's). Time dependent accumulation studies revealed that S6 caused enhanced EtBr accumulation relative to standard NorA efflux inhibitor reserpine, in clinical isolate of MRSA (CIMRSA) and in NorA overexpressed strain of S. aureus (SA1199B). S6 also exhibited synergy with Ciprofloxacin (CPX) against NorA overexpressed strain (SA1199B) of S. aureus but not in NorA knock out strain (K1758). MIC reversal studies showed that S3 in CIMRSA and S6 in NorA overexpressed strain of S. aureus (SA1199B), caused a 4 fold reduction in CPX MIC. In vitro time kill studies revealed that both S3 and S6 with sub MIC of CPX caused a significant 4 log CFU decline in CIMRSA. A decline of >3 log fold CFU by time kill assay implies synergy between FA derivatives and CPX. When tested in vivo in infected muscle tissue of zebrafish both S3 and S6 with CPX caused >3.2 log decline in CIMRSA cell counts relative to CPX treatment alone. Of the two potent derivatives, S6 probably acts through NorA whereas S3 might exert its effect through pump other than NorA. Greater in vitro and in vivo efficiency of FA derivatives implies its potential to be used as an adjuvant along with CPX to curtail MRSA infection in higher animal models.