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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.