Cross-class metallo-β-lactamase inhibition by bisthiazolidines reveals multiple binding modes

Abstract

Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics and are unaffected by clinically available β-lactamase inhibitors (βLIs). Active-site architecture divides MBLs into three classes (B1, B2, and B3), complicating development of βLIs effective against all enzymes. Bisthiazolidines (BTZs) are carboxylate-containing, bicyclic compounds, considered as penicillin analogs with an additional free thiol. Here, we show both l- and d-BTZ enantiomers are micromolar competitive βLIs of all MBL classes in vitro, with Kis of 6-15 µM or 36-84 µM for subclass B1 MBLs (IMP-1 and BcII, respectively), and 10-12 µM for the B3 enzyme L1. Against the B2 MBL Sfh-I, the l-BTZ enantiomers exhibit 100-fold lower Kis (0.26-0.36 µM) than d-BTZs (26-29 µM). Importantly, cell-based time-kill assays show BTZs restore β-lactam susceptibility of Escherichia coli-producing MBLs (IMP-1, Sfh-1, BcII, and GOB-18) and, significantly, an extensively drug-resistant Stenotrophomonas maltophilia clinical isolate expressing L1. BTZs therefore inhibit the full range of MBLs and potentiate β-lactam activity against producer pathogens. X-ray crystal structures reveal insights into diverse BTZ binding modes, varying with orientation of the carboxylate and thiol moieties. BTZs bind the di-zinc centers of B1 (IMP-1; BcII) and B3 (L1) MBLs via the free thiol, but orient differently depending upon stereochemistry. In contrast, the l-BTZ carboxylate dominates interactions with the monozinc B2 MBL Sfh-I, with the thiol uninvolved. d-BTZ complexes most closely resemble β-lactam binding to B1 MBLs, but feature an unprecedented disruption of the D120-zinc interaction. Cross-class MBL inhibition therefore arises from the unexpected versatility of BTZ binding.

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

Fig. 1.

Architecture of B1, B2, and B3 metallo-β-lactamases. The common αββα-fold of representative MBLs is colored from the N (light blue) to C terminus (light red), and is shown alongside a close up view of the active-site (boxed). Zinc ions (gray) and water molecules (red) are represented as spheres. Zinc coordination bonds are shown as gray dashes, with the corresponding residues (labeled) shown as sticks. (A) B1 BcII (PDB ID code 4C09), (B) B2 Sfh-I (PDB ID code 3SD9), and (C) B3 L1 (PDB ID code 1SML).

Fig. 2.

Chemical structures of bisthiazolidine inhibitors. Bisthiazolidines 1a (l-CS319, blue), 1b (d-CS319, gray), 2a (l-VC26, orange), and 2b (d-VC26, cyan).

Fig. S1.

In vitro inhibition of B1 MBLs BcII and IMP-1 by the bisthiazolidines. The initial rate of hydrolysis of imipenem (0–1,500 µM) by 1 nM BcII and IMP-1 was evaluated in the absence and presence of varying concentrations of each bisthiazolidine. The lines show the fits to the competitive inhibition model, according to the parameters (K_i) presented in Table 1.

Fig. S2.

In vitro inhibition of Sfh-I, L1 and GOB-18 by the bisthiazolidines. The initial rate of hydrolysis of imipenem (0–1,500 µM) by 1 nM Sfh-I and GOB-18 was evaluated in the absence and presence of varying concentrations of each bisthiazolidine. The lines show the fits to the competitive inhibition model, according to the parameters (K_i) presented in Table 1.

Fig. 3.

Bisthiazolidines restore the in vitro activity of ticarcillin-clavulanate against a S. maltophilia clinical isolate. Bacteria were grown at sublethal concentrations of a mixture of ticarcillin (TIC, 64 µg/mL) and clavulanate (CLV, 2 µg/mL), or in combination with 100 µg/mL of each compound. Viable cells were recovered at 4, 8, and 12 h. Results shown are the mean of three biological replicates ± SD.

Fig. S3.

Bisthiazolidines restore the in vitro activity of imipenem against IMP-1, BcII, Sfh-I, and GOB-18–producing Escherichia coli. Bacteria were grown at sublethal concentrations of imipenem alone (0.25 µg/mL for BcII and GOB-18, 0.5 µg/mL for IMP-1 and 8 µg/mL for Sfh-I) 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.

Fig. S4.

Bisthiazolidines restore the in vitro activity of imipenem against a Stenotrophomonas maltophilia clinical isolate. Bacteria were grown at sublethal concentrations of a mixture of ticarcillin (TIC, 64 µg/mL) and clavulanate (CLV, 2 µg/mL) or in combination with 100 µg/mL of each bisthiazolidine. Viable cells were recovered at 4, 8, and 12 h. Results shown are the mean of three biological replicates ± SD.

Fig. S5.

Mode of inhibitor binding as defined by electron density maps calculated after removal of modeled ligand. Active sites of MBL:BTZ complexes (protein colors and representations as in Fig. 1), with the F_o–F_c density (green, contoured at 3σ) calculated from the final model after removal of the ligand and refinement in Phenix (L1, IMP-1, BcII) or Refmac (Sfh-I). Ligands colored as in Fig. 2. (A) B1 IMP-1 in complex with (Left) 2a and (Right) 1b. Noncontiguous density between the thiol group and bisthiazolidine ring in IMP-1:2a is because of the inherent flexibility of this chemical bond. (B) B1 BcII in complex with 1b. (C) B2 Sfh-I in complex with 1a. (D) B3 L1 in complex with (Left) 1a, (Center) 2b, and (Right) 1b.

Fig. 4.

l-bisthiazolidine binding to B1 (IMP-1) and B3 (L1) metallo-β-lactamases. Close-up view of IMP-1 and L1 active sites with bound l-BTZ (sticks, colored as in Fig. 2). Protein main chain is colored from the N (light blue) to C terminus (light red). Residues that interact with the zinc ions (Zn1 and Zn2, gray spheres) are shown as sticks (colored as main chain), whereas hydrophobic residues that stabilize BTZ binding are shown as gray sticks. IMP-1 K224 and L1 S223 (labeled) interact with the BTZ carboxylate. (A) 2a bound to B1 IMP-1. Protein–zinc interactions are shown as gray dashes and BTZ–protein or BTZ–zinc interactions are shown as yellow dashes. (B) 1a bound to B3 L1. Interactions shown as in A. (C) Superposition of IMP-1:2a (orange) and L1:1a (blue). BTZ–protein or BTZ–zinc interactions are shown as dashes colored orange (IMP-1:2a) or blue (L1:1a).

Fig. S6.

Comparison of l-BTZ binding in IMP-1 and L1 to NDM-1 and VIM-2. Interactions between BTZ (sticks) and protein side chains (sticks) or active site zincs (spheres) are shown as dashes, colored according to their respective structure. L1:1a (light blue) is superposed on (A) NDM:1a (dark blue, PDB ID code 4U4L) and (B) VIM-2:1a (dark blue, PDB ID code 4UA4). IMP-1:2a (orange) is superposed on (C) NDM-1:1a (dark blue, PDB ID code 4U4L) and (D) VIM-2:1a (dark blue, PDB ID code 4UA4).

Fig. S7.

Comparison of l-BTZ binding to IMP-1 and L1 with uncomplexed native structures. (A) Superposition of IMP-1:2a (chain C, orange) with the native, uncomplexed, di-zinc IMP-1 (chain C, green). There is a rearrangement of the W64 side chain in the L3 loop which undergoes a 1.7–2.6 ± 0.23 Å shift on 2a binding. (B) Superposition of L1:1a (blue) with native, uncomplexed L1 (green). The thiol group of l-BTZs bind in approximately the same position as the active site water (red sphere) in both B1 IMP-1 and B3 L1.

Fig. 5.

1a binding to Sfh-I and concomitant active-site conformational changes. Active-site zinc ions (gray spheres) and water molecules (red spheres) are labeled. (A) 1a (blue) bound in the active site of B2 Sfh-I (main chain colored as in Fig. 1). Hydrophobic residues, on the α3 region and within the active site, involved in binding 1a are shown as gray sticks. The 1a carboxylate bridges N233 and H196 (sticks). Residues involved in binding the active-site zinc ion (Zn2, gray sphere) and water (Wat2, red sphere) are represented as sticks. Interactions of 1a with the protein main chain or Zn2 are shown as yellow dashes. Protein–zinc and protein–water interactions are shown as gray dashes. (B) Superposition of Sfh-I:1a (blue) with unliganded, native Sfh-I (PDB ID code 3SD9, green). Interactions of H118 and H196 with the BTZ or Wat2 are shown as dashes.

Fig. 6.

1b binding to B3 MBL L1. A water molecule (red sphere) mediates the interaction of Y32 (sticks, labeled) with the d-BTZ carboxylate, but S223 (sticks, labeled), which binds 1a, is not involved. Hydrophobic and zinc-binding residues represented as Fig. 4. (A) 1b (gray) bound in the active site of B3 L1 (colored as in Fig. 4). Interactions shown as in Fig. 4. (B) Superposition of L1:1b (gray) with L1:1a (blue). Interactions between the BTZ and protein are shown as gray or blue dashes, according to their respective structures. (C) 2b (cyan) bound to B3 L1, represented as in B.

Fig. 7.

1b binding to B1 MBLs BcII and IMP-1. 1b bound in the active of B1 MBLs (A) IMP-1 and (B) BcII (colors and interactions as in Fig. 4). In both A and B, K224 binds the 1b carboxylate and the D120–Zn2 coordination is disrupted. (C) Superposition of IMP-1:1b (gray; zinc ions, light gray) with unliganded, native IMP-1 (green; zinc ions, dark gray). Binding causes movement of Zn2, and loss of the D120–Zn2 interaction. (D) Superposition of BcII:1b (gray; zinc ions, light gray) with unliganded, native BcII (PDB ID code 4C09 green; zinc ions, dark gray). Binding is similar to C, causing the same rearrangements within the active site.

Fig. S8.

Movement of the BcII L3 loop on binding 1b. Superposition of BcII:1b (gray) on native BcII (green) reveals movement of the L3 loop and rearrangement of the F61 side chain, moving it closer to the bound 1b (stick representation, transparent for clarity). Zinc ions (gray) and native nucleophilic water (red) shown as spheres.

Fig. S9.

Disruption of Asp120–Zn2 in IMP-1:1b and BcII:1b clearly defined by electron density. Active sites of B1 enzymes (colors and representations as in Fig. S5) with 1b (gray sticks) bound, shows disruption of the D120–Zn2 coordination, with 2F_o–F_c density contoured at 1.0σ (blue). Density for 1b is omitted for clarity. (A) 1b bound in the active site of IMP-1. (B) 1b bound in the active site of BcII.

Fig. 8.

Comparisons of the mode of binding between bisthizolidines and hydrolyzed antibiotics. Superpositions of (A) IMP-1:1a (orange; zinc ions, light gray) with NDM-1:hydrolyzed ampicillin (PDB ID code 3Q6X, green; zinc ions, dark gray); (B) L1:1a (blue; zinc ions, light gray) with L1:moxalactam (PDB 2AIO, green; zinc ions, dark gray); (C) L1:1b (gray; zinc ions, light gray) with L1:moxalactam (as in B); (D) Sfh-I:1a (gray; zinc ions, light gray) with CphA:biapenem (PDB ID code 1X8I, green; zinc ions, dark gray).

Fig. 9.

1b binding to B1 MBLs IMP-1 and BcII closely resembles binding of hydrolyzed antibiotic. Superpositions of NDM-1:ampicillin (green) with (A) IMP:1b and (B) BcII:1b (gray; zinc ions, light gray). The modes of binding and ligand-protein/zinc interaction distances are shown in schematic representation for (C) NDM-1:hydrolyzed ampicillin, (D) IMP-1:1b, and (E) BcII:1b.

Fig. S10.

Comparison of BTZ and captopril binding to MBLs. Superpositions of MBL:BTZ structures with MBL:captopril structures (pink). (A) IMP-1:2a (orange) with IMP-1:l-captopril (PDB ID code 4C1F). W64 (stick representation) on the L3 loop is positioned closer to the active site in captopril-bound IMP-1 than 2a-bound IMP-1. (B) IMP-1: 1b (gray) with IMP-1:d-captopril (PDB ID code 4C1G). (C) BcII: 1b (gray) with BcII:d-captopril (PDB ID code 4C1C). The L3 loop of BcII:d-captopril is not modeled because of poorly defined density. (D) L1: 1b (blue) with L1:d-captopril (PDB ID code 2FU8). (E) L1:1a (blue) with L1:d-captopril (PDB ID code 2FU8). (F) Sfh-I:1a (blue) with CphA:d-captopril (PDB ID code 2QDS).

Fig. S11.

Movement of the uncomplexed di-zinc IMP-1 L3 loop between chains in the asymmetric unit. Superposition of the four chains in the asymmetric unit of the uncomplexed, di-zinc IMP-1 structure reveals the L3 loop (boxed) of chain C (pink) is positioned differently to chains A (green), B (cyan), and D (yellow), which all adopt the same conformation. N and C termini are labeled.