Global contamination of NDM-1 has become a major threat to human health. Currently, there is no NDM-1 clinically inhibitor. The discovery and development of an inhibitor for inhibiting of NDM-1, as well as restituting and reinforcing the efficacy of the beta-lactam antibiotic against this enzyme would have a significant effect on human health and create obvious socio-economic benefits . These limitations and uttermost necessity inspired us to mine a new lead compound against NDM-1 from natural resources . The reconnaissance of novel non-covalent inhibitors of β-lactamases is an optimistic path to preserve and restore the efficacy of β-lactam antibiotics.
Against this study, different and frequent studies have investigated some chelator compounds of the zinc of NDM and MBL (26, 28), but some studies indicated that mutation in non-active site residues sequences over some MBLs could severely decrease the dependency of these enzymes on zinc ions, therefore, concentrating on the metal’s chelators are not proper option. Even in the starvation of zinc and utilization of EDTA as a metal chelator, MBLs was powerful enough to degrade beta-lactam rings [27–29].
Other studies examined the compounds affecting one type of the MBL subclasses alone. During those studies, no effort was made to find common pharmacophore and crucial residues playing a key role in the active site (residues conserved in the active site of all kinds of MBL) in all MBL classes . In this study, regarding the above-mentioned weakness, the crucial conserved residues (hotspots residues) playing a key role in the active site and common pharmacophore in all MBLs were detected.
Hotspots of the active site were recognized by our post-docking analyses and were mentioned by others [30, 31]. Hotspots of amino acids that could be employed more effectively with configured lead molecules against the binding site of NDM-1 were Ser217, Ser251, Asn220, Asp124, His250, Lys211, Ile35. Among them, Ile35 involved in the Van der Waals interactions to all complexes. Also, His189, Gly219, Asp124 and His122 significantly contributed to the hydrophobic interactions alongside Ile35. These amino acids played an essential role in stabilizing the complex via hydrogen bonds or hydrophobic contacts. Alignment on all types of NDM (NDM-1 - NDM30 Figure S4) showed that all the above-considered amino acids not only played a pivotal role in binding with the active site, but also conserved in all types of NDM. Therefore, they can be used for the pharmacophore modeling and ligand discovery in future studies.
Our precise 3D conformational alignment over the tertiary structure of all metallo-β-lactamases and BLDB database alignment on the second structure of these proteins proved that the above amino acids conserved in all metallo-β-lactamases (most likely play a role in the catalysis reaction in the active site). Ser251, Asn220, Asp124, His250, His189, Gly219 and His122 residues conserved in the B1 and B2 subclass and for all three subclasses Asp124, His250, His189 and His122 conserved. Hence there is a possibility for our ten natural compounds in binding with all MBL subclasses and inhibiting them (because of interaction with these residues)
The discussed ligands based on these common pharmacophores and key residues may inhibit all MBLs. It would be cost-benefit effective in drug design and can inhibit broad types of the enzymes.
An insight into NDM-1 structures showed that a water molecule situated between Zn 302 and Zn 303, performs as a nucleophile during hydrolysis of the β-lactam ring of antibiotics. Analyses of the hydrolysis process by NDM indicated that prominent part served by the aforementioned residues in maintaining the overall structure and function of NDM-1.
The NPASS database was used as the database, which had a complete collection of 35032 natural compounds. For this purpose, 35032 compounds were screened, and 1300 compounds were docked. Finally, among them, ten compounds were selected. Since all ten compounds had interactions with those critical residues, there is a possibility to inhibit all types of MBLs, more investigations and laboratory experiments are essential.
Two steps of structural screening were done to found the best ligands with high ability for inhibition of the NDM1. At first, captopril was used in structural screening and NPC120633 ligand was obtained. Then to catch a better ligand with lower binding free energy, second screening based on the similarity to NPC120633 was performed and ten ligands with lower binding energy than NPC120633 were found.
Multi-step molecular docking assisted in facilitating the virtual screening process by yielding insight into the ligand binding energy and docking binding affinity. Multiple molecular docking using the Autodock Vina (module of LigandScout) software and structural screening via the infiniSee software help to identify the best NPs that could perfectly inhibit NDM-1. SeeSAR and Schrödinger suite softwares were applied to confirm secondary virtual screening (secondary docking) results. Schrödinger and SeeSAR results approved the Autodock Vina results for ten final compounds. Validation of docking was confirmed by using Glide XP-docking and Glide SP-docking.
In this study, ten inhibitors had high affinity, and the docking score against NDM-1 and their binding revealed an exothermic pattern for Gibbs free energy and entropy changes. The value of MM-PBSA and MM-GBSA binding free energy was considerable for all compounds and also the results obtained from RMSD and RMSF plots exhibited that all ten compounds made strong and stable complexes with NDM-1. Thus, these compounds can compete with β–lactam antibiotics to bind with NDM-1 and survive them from hydrolyzes by this enzyme. These NPs are suggested as the potential lead to develop future drug candidates.