2-Mercaptomethyl Thiazolidines (MMTZs) Inhibit All Metallo-β-Lactamase Classes by Maintaining a Conserved Binding Mode

Abstract

Metallo-β-lactamase (MBL) production in Gram-negative bacteria is an important contributor to β-lactam antibiotic resistance. Combining β-lactams with β-lactamase inhibitors (BLIs) is a validated route to overcoming resistance, but MBL inhibitors are not available in the clinic. On the basis of zinc utilization and sequence, MBLs are divided into three subclasses, B1, B2, and B3, whose differing active-site architectures hinder development of BLIs capable of "cross-class" MBL inhibition. We previously described 2-mercaptomethyl thiazolidines (MMTZs) as B1 MBL inhibitors (e.g., NDM-1) and here show that inhibition extends to the clinically relevant B2 (Sfh-I) and B3 (L1) enzymes. MMTZs inhibit purified MBLs in vitro (e.g., Sfh-I, K_i 0.16 μM) and potentiate β-lactam activity against producer strains. X-ray crystallography reveals that inhibition involves direct interaction of the MMTZ thiol with the mono- or dizinc centers of Sfh-I/L1, respectively. This is further enhanced by sulfur-π interactions with a conserved active site tryptophan. Computational studies reveal that the stereochemistry at chiral centers is critical, showing less potent MMTZ stereoisomers (up to 800-fold) as unable to replicate sulfur-π interactions in Sfh-I, largely through steric constraints in a compact active site. Furthermore, in silico replacement of the thiazolidine sulfur with oxygen (forming an oxazolidine) resulted in less favorable aromatic interactions with B2 MBLs, though the effect is less than that previously observed for the subclass B1 enzyme NDM-1. In the B3 enzyme L1, these effects are offset by additional MMTZ interactions with the protein main chain. MMTZs can therefore inhibit all MBL classes by maintaining conserved binding modes through different routes.

Keywords: antibiotic resistance; carbapenemase; inhibitors; β-lactamases.

Figure 1.. MBL-catalyzed β-lactam breakdown and MBL inhibitors.

(A) Penicillin breakdown by MBLs, resulting in penicilloic acid product formation via a proposed high-energy tetrahedral intermediate. (B) Inhibitors based on proposed species in β-lactam hydrolysis. Left, bisthiazolidine (BTZ) scaffold, R=H/CH₃; middle, bicyclic boronates (e.g. taniborbactam, QPX7728); right, 2-mercaptomethyl thiazolidine (MMTZ) scaffold, R=H/CH₃. (C) 2-Mercaptomethyl thiazolidines studied here. The absolute R/S configurations of the chiral carbon centers (C-2 and C-4, labelled) are shown. L-anti-1a (brown), D-anti-1a (orange), L-anti-1b (green), L-syn-1b (cyan), D-anti-1b (purple) and D-syn-1b (pink). (C) is adapted from Rossi et al.

Figure 2.. Effect of MMTZs on imipenem minimum inhibitory concentrations of MBL-expressing E. coli.

The minimum inhibitory concentration of imipenem (IMI) +/− 100 μg/mL MMTZ was determined for the E. coli strain DH10B expressing NDM-1, Sfh-I or L1 (see Methods for details). At this concentration, in the absence of antibiotic, MMTZs do not have a detrimental effect on bacterial growth. An asterisk denotes the inhibitor had a four-fold effect on MIC (i.e. two dilutions). A two-fold effect (i.e. one dilution factor) is not considered significant. Results are the mode of three biological replicates.

Figure 3.. Interactions of MMTZs in the active site of L1 and Sfh-I.

Left, thiol-Zn interactions and hydrogen bonds in MMTZ:MBL complexes (distance labelled). Right, interactions with hydrophobic residues lining the active site (blue sticks). (A) L-anti-1a (brown) binding to Sfh-I; (B) D-syn-1b (pink) binding to L1.

Figure 4.. Sulfur-π interactions of MMTZs in MBL active sites.

Views from the active sites of MBLs with dashes showing the interaction (distances labelled) of the MMTZ sulfur with an active site tryptophan (blue sticks) in (A) NDM-1:L-anti-1b (PDB 6zyp), (B) NDM-1:D-syn-1b (PDB 6zyq), (C) Sfh-I:L-anti-1a (PDB 7bj9) and (D) L1:D-syn-1b (PDB 7bj8).

Figure 5.. QM/MM optimized structures of MMTZs and their analagous oxazolidines in MBLs.

Views from the active show overlays of QM/MM optimized complex structures for MMTZs (colored as in Figure 3) and their MMOZ analogues (cyan) bound in MBLs. NDM-1 simulations were performed previously. (A) NDM-1:L-anti-1a; (B) NDM-1:D-syn-1b; (C) Sfh-I:L-anti-1a; (D) L1:D-syn-1b.