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NDM-1 and its variants are the most prevalent types of metallo-β-lactamases, hydrolyze almost all antibiotics of β-lactam group leading to multiple-drug resistance in bacteria. No inhibitor has yet been obtained for NDM-1 or other class of metallo-β-lactamases. Therefore, strategies to identify novel anti-β-lactamase agents with specific mechanisms of action are the need of an hour. In this study, we have reported the discovery of novel non-β-lactam inhibitors against NDM-1 by multi-step virtual screening approach. The potential for virtually screened drugs was estimated through in vitro cell assays. Five chemical compounds were finally purchased and evaluated experimentally for their efficacies to inhibit NDM-1 producing bacterial cells, in vitro. The dissociation constants (Kd), association constant (Ka), stoichiometry (n) and binding energies (ΔG) of compounds with the respective targets were determined using isothermal titration calorimetry (ITC). Molecular dynamic simulation carried out for 25 ns revealed that these complexes were stable throughout the simulation with relative RMSD in acceptable range. Moreover, Microbiological and kinetic studies further confirmed high efficacies of these inhibitors by reducing the minimum inhibitory concentration (MIC) and catalysis of antibiotics by β-lactamases in the presence of inhibitors. Therefore, we conclude that these potential inhibitors may be used as lead molecules for future drug candidates.

BACE-1 and GSK-3β both are potential therapeutic drug targets for Alzheimer's disease. Recently, both these targets received attention for designing dual inhibitors. Till now only two scaffolds (triazinone and curcumin) derivatives have been reported as BACE-1 and GSK-3β dual inhibitors. In our previous work, we have reported first in class dual inhibitor for BACE-1 and GSK-3β. In this study, we have explored other naphthofuran derivatives for their potential to inhibit BACE-1 and GSK-3β through docking, molecular dynamics, binding energy (MM-PBSA). These computational methods were performed to estimate the binding affinity of naphthofuran derivatives towards the BACE-1 and GSK-3β. In the docking results, two derivatives (NS7 and NS9) showed better binding affinity as compared to previously reported inhibitors. Hydrogen bond occupancy of NS7 and NS9 generated from MD trajectories showed good interaction with the flap residues Gln73, Thr72 of BACE-1 and Arg141, Thr138 residues of GSK-3β. MM-PBSA and energy decomposition per residue revealed different components of binding energy and relative importance of amino acid involved in binding. The results showed that the binding of inhibitors was majorly governed by the hydrophobic interactions and suggesting that hydrophobic interactions might be the key to design dual inhibitors for BACE1-1 and GSK-3β. Distance between important pair of amino acid residues indicated that BACE-1 and GSK-3β adopt closed conformation and become inactive after ligand binding. The results suggested that naphthofuran derivatives might act as dual inhibitor against BACE-1 and GSK-3β.

Withania somnifera, popularly known as Indian ginseng, is one of the most important medicinal plants. The plant is well studied in terms of its pharmaceutical activities and genes involved in biosynthetic pathways. However, not much is known about the regulatory mechanism of genes responsible for the production of secondary metabolites. The idea was to identify miRNA transcriptome responsible for the regulation of withanolide biosynthesis, specifically of root and leaf tissues individually. The transcriptome data of in vitro culture of root and leaf tissues of the plant was considered for miRNA identification. A total of 24 and 39 miRNA families were identified in root and leaf tissues, respectively. Out of these, 15 and 27 miRNA families have shown their involvement in different biological functions in root and leaf tissues, respectively. We report here, specific miRNAs and their corresponding target genes for corresponding root and leaf tissues. The target genes have also been analyzed for their role in withanolide metabolism. Endogenous root-miR5140, root-miR159, leaf-miR477, and leaf-miR530 were reported for regulation of withanolide biosynthesis.

Ashaka stem bark (Saraca asoca De Wilde) is one of the most important ingredients of many Ayurvedic preparations used in the indigenous system of medicine mostly for uterine problems. Due to the poor availability of true Ashoka, it is adulterated by other barks including Polyalthia longifolia which also bears the same common name. Pharmacognostic study has revealed some diagnostic features which facilitate detection of authentic and adulterated materials.

The discovery of NDM-1 and its variants has caused the emergence of antibiotic resistance in the community and hospital setting, causing major concern for health care across the globe. New Delhi Metallo-β-lactamase is known to hydrolyze almost all β-lactam antibiotics. Studies have shown the hydrolytic activates of NDM-1 and some of its variants, however a comparative study of these NDM variants has not been explored in detail. Hence, we proposed to check their catalytic activity by performing a comparative study between NDM-1 and its variants. The study was initiated to clone NDM variants (NDM-1, NDM-4, NDM-5, NDM-6 and NDM-7) followed by overexpression of the recombinant proteins to check their hydrolytic properties against β-lactam antibiotics. The minimum inhibitory concentration of carbapenems antibiotics for bla clone was found four fold increased, whereas no change was observed in the clones having other variants. The hydrolytic activity of carbapenem with NDM-5 variant was found to be augmented as per the kinetics parameter where Km was decreased and kcat, kcat/Km values increased as compared to the NDM-1. Molecular docking studies were employed to identify the variations in the binding ability among all NDM variants with imipenem or meropenem. Simulation studies at 100ns showed a good stability of NDM-5 with imipenem and meropenem as compared to NDM-1. CD spectroscopy data revealed significant changes in the secondary structure of NDM variants. We conclude that NDM-5 showed higher hydrolytic activity as compared to other variants. This study provides a comparative analysis of the severity of NDM producing strains.

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