Activity of the β-Lactamase Inhibitor LN-1-255 against Carbapenem-Hydrolyzing Class D β-Lactamases from Acinetobacter baumannii

Abstract

The number of infections caused by Gram-negative pathogens carrying carbapenemases is increasing, and the group of carbapenem-hydrolyzing class D β-lactamases (CHDLs) is especially problematic. Several clinically important CHDLs have been identified in Acinetobacter baumannii, including OXA-23, OXA-24/40, OXA-58, OXA-143, OXA-235, and the chromosomally encoded OXA-51. The selection and dissemination of carbapenem-resistant A. baumannii strains constitutes a serious global threat. Carbapenems have been successfully utilized as last-resort antibiotics for the treatment of multidrug-resistant A. baumannii infections. However, the spread of OXA carbapenemases is compromising the continued use of these antimicrobials. In response to this clinical issue, it is necessary and urgent to design and develop new specific inhibitors with efficacy against these enzymes. The aim of this work was to characterize the inhibitory activity of LN-1-255 (a 6-alkylidene-2-substituted penicillin sulfone) and compare it to that of two established inhibitors (avibactam and tazobactam) against the most relevant enzymes of each group of class D carbapenemases in A. baumannii The β-lactamase inhibitor LN-1-255 demonstrated excellent microbiological synergy and inhibition kinetics parameters against all tested CHDLs and a significantly higher activity than tazobactam and avibactam. A combination of carbapenems and LN-1-255 was effective against A. baumannii class D carbapenemases. Docking assays confirmed the affinity of LN-1-255 for the active site of these enzymes. LN-1-255 represents a potential new β-lactamase inhibitor that may have a significant role in eradicating infections caused by A. baumannii isolates carrying CHDLs.

Keywords: Acinetobacter baumannii; antimicrobial resistance; carbapenem-hydrolyzing class D β-lactamases; β-lactamase inhibitors.

FIG 1

β-Lactamase inhibitors used in this study.

FIG 2

Assessment of synergy between inhibitors. Pictures represent checkerboard assays using imipenem and inhibitors with cultures of A. baumannii ATCC 17978 and clinical isolate Ab1 (both carrying OXA-23). Pink wells indicate growth and blue wells indicate growth inhibition.

FIG 3

Plots of turnover numbers (t_n). Ratio of inhibitor concentration to enzyme concentration necessary to decrease the enzyme activity by 90%.

FIG 4

(A) Comparison of the OXA-type carbapenemases employed in this study, OXA-23 (gray), OXA-24 (yellow), OXA-51 (green), OXA-58 (pink), OXA-143 (blue), and OXA-235 (cyan), highlighting the side chain residues responsible of the major structural differences. The side chain of the catalytic serine is also shown. (B) Detailed view of the Ω-loop and neighbor turn involving β1-strand and α1-helix. Note the relevant differences in the arrangement and sequence of the Ω-loop among the enzymes studied. (C and D) Selected views of the tunnel-like entrance to the active site of OXA-24 (C) and OXA-23 (D) enzymes. The position of the Tyr/Phe and Met residues that are involved in this entrance are highlighted in green and red, respectively. The catalytic serine is shown in orange. Note how the active site of OXA-24 containing a Try residue is more closed than the OXA-23 one having a Phe residue.

FIG 5

Selected view of inactivation of OXA-23 (A and B) and OXA-58 (C and D) from A. baumannii by LN-1-255 (A and C) and avibactam (B and D) obtained by molecular docking studies. Hydrogen bonding and electrostatic interactions between the ligands and the enzymes are shown as dashed blue lines. Relevant side chain residues are shown and labeled.