Discovery of novel new Delhi metallo-β-lactamases-1 inhibitors by multistep virtual screening

Abstract

The emergence of NDM-1 containing multi-antibiotic resistant "Superbugs" necessitates the needs of developing of novel NDM-1inhibitors. In this study, we report the discovery of novel NDM-1 inhibitors by multi-step virtual screening. From a 2,800,000 virtual drug-like compound library selected from the ZINC database, we generated a focused NDM-1 inhibitor library containing 298 compounds of which 44 chemical compounds were purchased and evaluated experimentally for their ability to inhibit NDM-1 in vitro. Three novel NDM-1 inhibitors with micromolar IC50 values were validated. The most potent inhibitor, VNI-41, inhibited NDM-1 with an IC50 of 29.6 ± 1.3 μM. Molecular dynamic simulation revealed that VNI-41 interacted extensively with the active site. In particular, the sulfonamide group of VNI-41 interacts directly with the metal ion Zn1 that is critical for the catalysis. These results demonstrate the feasibility of applying virtual screening methodologies in identifying novel inhibitors for NDM-1, a metallo-β-lactamase with a malleable active site and provide a mechanism base for rational design of NDM-1 inhibitors using sulfonamide as a functional scaffold.

Fig 1. Comparative analyses of 21 published NDM-1 X-ray crystal structures.

(A) Pairwise RMSD matrix table of NDM-1 structures superimposed with force realignment method and refine with Gaussian Weights in MOE. PDB codes for structures with hydrolyzed substrate in the active site are highlighted in red. (B) Superposition of the 22 NDM-1 structures. 3S0Z, 4GYU, 4GYQ, 3SPU, and 3Q6X2 are highlighted in thick line and colored as shown in the index panel. (C) The RMSD-residue index 3D waterfall plots of NDM-1 structures compared with 3Q6X structure. (D) Superimposition of the active site among the reported NDM-1 structures (without 3S0Z and NDM-1 mutants 4GYQ and 4GYU) showing the metal chelating residues (Oliver) and conserved water molecules (Red) in the active site of NDM-1 structures. Residues from 3Q6X are highlighted in green.

Fig 2. Docking of the hydrolyzed ampicillin in the active site of NDM-1.

(A) Molecular surface of NDM-1 (PDB 3Q6X) active site with docked hydrolyzed ampicillin. The structurally determined hydrolyzed ampicillin is shown in gray stick representation while docked poses are shown in colored stick. 2D ligand-protein interaction maps showing the detailed binding pattern of structurally determined hydrolyzed ampicillin (B), docked hydrolyzed ampicillin (C) and docked ampicillin (D) in the active site of 3Q6X. (E) Residue-ligand interaction energies between NDM-1 (3Q6X) and hydolyzed ampicillin (vdw_ref) or docked hydolyzed ampicillin (vdw_pose). The hydrolyzed ampicillin and NDM-1 residue interaction energies were calculated for the best pose (RMSD = 1.53 Å).

Fig 3. L-captopril docked in the active site of NDM-1.

2D ligand-protein interaction maps showing the detailed binding pattern analysis of structurally determined (A) and docked (B) L-captopril. (C) Molecular surface of NDM-1 active site with docked hydrolyzed ampicillin.

Fig 4. PLIF analysis of the docking process.

The interaction frequency of individual residue with the docking poses of (A) hydolyzed ampicillin; (B) 10 beta-lactams (ampicillin, cefepime, cefotaxime, ceftazidime, cefuroxime, faropenem, imipenem, meropenem, penicillin G, piperacillin); (C) 298 virtual hit compounds. Each columns of every residue are denoted by some of the following characters to indicate the interaction role of each residue: side chain hydrogen bond acceptor, backbone hydrogen bond donor, backbone hydrogen bond acceptor, solvent hydrogen bond, ionic attraction or surface contact to the atom of the residues.

Fig 5. Experimental validation of selected virtual screening hits.

(A) Percent inhibition of NDM-1 activity in the presence of 30 uM individual compounds. Data are presented as mean ± standard deviation (n = 4). (B) Dose-dependent inhibitions of NDM-1 by VNI-24, VNI-34 and VNI-41 against NDM-1. Each data point indicated the remaining activity of NDM-1 after incubated with inhibitors, and were presented as mean ± standard deviation (n = 4). (C) Structures of three active compounds.

Fig 6. Molecular dynamic profile of NDM-1 and VNI-41 complex.

(A) RMSD of two Zn²⁺, conserved H₂O, VNI-41, and active site (atoms shown in Fig. 1D) spanning the 2000 ps molecular dynamic simulation process; (B) The overall molecular surface of NDM-1 colored by activeLP (white, hydrophobic; blue, polar; red, H-Bonding); (C) The 2D interaction map VNI-41 in the active site of NDM-1 at 1500 ps after the MS reached equilibrium; (D) Molecular surface of NDM-1 around the VNI-41 binding site with a cutoff limit of 4.5 Å (black, hydrophilic; purple, neutral; green, lipophilic). VNI-41 and adjacent NDM-1 residues shown in stick representation with carbon atoms colored by green and dark yellow respectively.

Fig 7. Structure movements during molecular dynamic simulation process.

Overlaid snapshots of the ribbon diagrams of NDM-1 Cα atoms and VNI-41 compound around the active site before (A) (snapshots interval 10 ps, N = 10) and after (B) (snapshots interval 150 ps, N = 10) the system reached equilibrium. Surface analyse of the active site cavity was performed on apo NDM-1(C), NDM-1/hydrolyzed ampicillin (D) and NDM-1/NVI-41(E). Atoms in the active site active site cavity are highlighted in colored balls. (F) The ribbon diagrams showing the active site associated loops (L3, L6, L10) moving toward the ligand and contraction of the active site. The structure of apo NDM-1, NDM-1/hydrolyzed ampicillin and NDM-1/NVI-41 is colored in blue, green and black respectively.