Bisthiazolidines: A Substrate-Mimicking Scaffold as an Inhibitor of the NDM-1 Carbapenemase

Abstract

Pathogenic Gram-negative bacteria resistant to almost all β-lactam antibiotics are a major public health threat. Zn(II)-dependent or metallo-β-lactamases (MBLs) produced by these bacteria inactivate most β-lactam antibiotics, including the carbapenems, which are "last line therapies" for life-threatening Gram-negative infections. NDM-1 is a carbapenemase belonging to the MBL family that is rapidly spreading worldwide. Regrettably, inhibitors of MBLs are not yet developed. Here we present the bisthiazolidine (BTZ) scaffold as a structure with some features of β-lactam substrates, which can be modified with metal-binding groups to target the MBL active site. Inspired by known interactions of MBLs with β-lactams, we designed four BTZs that behave as in vitro NDM-1 inhibitors with Ki values in the low micromolar range (from 7 ± 1 to 19 ± 3 μM). NMR spectroscopy demonstrated that they inhibit hydrolysis of imipenem in NDM-1-producing Escherichia coli. In vitro time kill cell-based assays against a variety of bacterial strains harboring blaNDM-1 including Acinetobacter baumannii show that the compounds restore the antibacterial activity of imipenem. A crystal structure of the most potent heterocycle (L-CS319) in complex with NDM-1 at 1.9 Å resolution identified both structural determinants for inhibitor binding and opportunities for further improvements in potency.

Keywords: NDM-1; antibiotic resistance; bisthiazolidines; inhibitors; metallo-β-lactamase.

Figure 1

Structure of NDM-1 (PDB code 3SPU). The enzyme contains a dinuclear metal center in the active site, comprising two Zn(II) ions: one with a tetrahedral coordination sphere (Zn1, coordinated to residues His116, His118, and His196 and a water/ hydroxide molecule) and one in a trigonal bipyramidally coordination sphere (Zn2, coordinated to residues Asp120, Cys221, and His263, a water molecule, and the water/hydroxide molecule). Zinc ions and water molecules are represented as gray and red spheres, respectively. The amino acid side chains are colored according to atom type. The protein main chain is color-ramped from the N-terminus (blue) to the C-terminus (red). This figure was generated using PyMol (www.pymol.org).

Figure 2

Carbapenem hydrolysis inhibition in E. coli cells by BTZs. (Left) Remaining imipenem concentration, as determined from the ¹H NMR spectrum, after a given incubation time with NDM-1-bearing E. coli cells, in the absence or in the presence of different concentrations of each bisthiazolidine. (Right) Determination of the IC₅₀ from the plots of the percentage of inhibition as a function of compound concentration using eq 2.

Figure 3

BTZs restore the in vitro activity of imipenem against NDM-1-producing K. pneumoniae (A), A. baumannii (B), and P. rettgeri (C). Bacteria were grown at sublethal concentrations of imipenem alone (4 μg/mL for K. pneumoniae and 16 μg/mL for A. baumannii and P. rettgeri) or in combination with 100 μg/mL of each compound. Viable cells were recovered at 100, 300, and 500 min. Results shown are the mean of three biological replicates ± SD.

Figure 4

Crystal structure of the NDM-1:L-CS319 complex. Inhibitor L-CS319 interacts with both zinc ions via its sulfhydryl group. The carboxylate group interacts with K224 through two water molecules (Wats). Zinc ions and water molecules are represented as gray and red spheres, respectively. Hydrogen bonds and zinc coordination bonds are shown as black dashes and hydrophobic interactions as gray dashes. Protein main chain is color-ramped from the N-terminus (blue) to the C-terminus (red). The figure was generated using PyMol (www.pymol.org).

Figure 5

Comparison of NDM-1:L-CS319 complex with other NDM-1 structures. Overlay of structure of NDM-1:L-CS319 complex (green) with structures of NDM-1: (a) unliganded NDM-1 (magenta, pdb 3SPU); (b) complex with hydrolyzed ampicillin (yellow, pdb 3Q6X); (c) complex with hydrolyzed meropenem (cyan, pdb 4EYL); (d) complex with L-captopril (salmon, pdb 4EXS). Carbon atoms are colored as above, other atoms as standard. Zinc ions (gray spheres) and active site residues from the NDM-1:L-CS319 complex structure are shown. This figure was generated using Pymol (www.pymol.org).

Scheme 1

Benzylpenicillin and Imipenem Substrates of NDM-1 and the Bisthiazolidine (BTZ) Scaffold