Strategic Approaches to Overcome Resistance against Gram-Negative Pathogens Using β-Lactamase Inhibitors and β-Lactam Enhancers: Activity of Three Novel Diazabicyclooctanes WCK 5153, Zidebactam (WCK 5107), and WCK 4234

Abstract

Limited treatment options exist to combat infections caused by multidrug-resistant (MDR) Gram-negative bacteria possessing broad-spectrum β-lactamases. The design of novel β-lactamase inhibitors is of paramount importance. Here, three novel diazabicyclooctanes (DBOs), WCK 5153, zidebactam (WCK 5107), and WCK 4234 (compounds 1-3, respectively), were synthesized and biochemically characterized against clinically important bacteria. Compound 3 inhibited class A, C, and D β-lactamases with unprecedented k₂/ K values against OXA carbapenemases. Compounds 1 and 2 acylated class A and C β-lactamses rapidly but not the tested OXAs. Compounds 1-3 formed highly stable acyl-complexes as demonstrated by mass spectrometry. Crystallography revealed that 1-3 complexed with KPC-2 adopted a "chair conformation" with the sulfate occupying the carboxylate binding region. The cefepime-2 and meropenem-3 combinations were effective in murine peritonitis and neutropenic lung infection models caused by MDR Acinetobacter baumannii. Compounds 1-3 are novel β-lactamase inhibitors that demonstate potent cross-class inhibition, and clinical studies targeting MDR infections are warranted.

Figure 1.

Structures of DBOs used in this study. R¹ side chain is highlighted by a gray circle.

Figure 2.

Syntheses of (A) 1 and 2 and (B) 3.

Figure 3.

(A) Scheme representing the interactions of β-lactamases with DBOs. In this model, formation of the noncovalent complex, enzyme:inhibitor (E:I) is represented by the dissociation constant, K_d, which is equivalent to k₋₁/k₁. k₂ is the first order rate constant for the acylation step or formation of E-I. k₋₂ is the first order rate constant for the recyclization step or reformation of E:I. Reported rarely to date, some DBOs undergo a desulfation reaction; k₃ is the first order rate constant for desulfation to form E:I*, where I* is the desulfated DBO. The desulfated DBO may undergo complete hydrolysis; the hydrolysis, which forms free E and product (P) is represented by the first order rate constant k₄. (B) Chemical representation of (A) using avibactam. (C) Acyl-transfer mass spectrometry with 3 (left), 2 (center), and 1 (right). Each DBO was preincubated with KPC-2 at a 1:1 E:I ratio for 1 min (data in top panels in blue) and used for mass spectrometry; then, TEM-1 was added, incubated for 15 s or 5 min (data in center and bottom panels in red), and used for mass spectrometry.

Figure 4.

(A) Electron density of compounds 1–3 bound in the active site of KPC-2. Shown are unbiased omit |F_o|-|F_c| electron density with the ligand removed from refinement and map calculations. Left, 3 bound to KPC-2; center, 2 bound to KPC-2; right, 1 bound to KPC-2. Compounds 1–3 are shown in blue carbon atom ball-and-stick representation, and the protein is depicted in gray carbon atom stick representation. Electron density is contoured at the 3σ level, and data sets are from inhibitor-soaked KPC-2 crystals. The sulfate moiety of 3 was observed to be in two conformations (0.6 and 0.4 occupancy conformations labeled a and b, respectively). (B) Compounds 1–3 bound to the active site of KPC-2. Left, 3 bound to KPC-2; center, 2 bound to KPC-2; right, 1 bound to KPC-2. Hydrogen bonds are depicted as dashed lines; the distances for key hydrogen bonds are shown (in Å). The deacylation water molecule is labeled as W#1. The two conformations for the sulfate moiety in 3 are labeled similarly to those in panel A.

Figure 5.

Stereo figure of superimpositioning of DBO inhibitors in the active site of KPC-2. Depicted are 3 bound to KPC-2 molecule A (gray), 3 bound to KPC-2 molecule B (light green), 2 bound to KPC-2 molecule A (blue), 1 bound to KPC-2 molecule A (gold), avibactam bound to KPC-2 molecule A (dark green; PDB ID: 4ZBE). Structures used for the superimposition are from inhibitor-soaked KPC-2 crystals.

Figure 6.

Compound 3 bound to OXA-24. (A) Unbiased omit |F_o|-|F_c| electron density of 3:OXA-24 complex with the ligand removed from refinement and map calculations. Density is contoured at the 3σ level. (B) Interactions of 3 in the active site of OXA-24; the distances for key hydrogen bonds are shown (in Å). (C) Stereo figure depicting the superimpositioning of OXA-24 in complex with 3 (cyan 3 carbon atoms and gray protein carbon atoms) and in complex with avibactam (blue carbon atoms for avibactam and protein; PDB ID: 4WM9). In close proximity to the 3 ligand, a chloride ion in two alternate positions is present and labeled Cl1 and Cl2.

Figure 7.

A murine neutropenic lung infection model using a clinical isolate of A. baumannii SL06 carrying bla_OXA‑23 and bla_OXA‑51. The graphs represent the change in CFU/lung after different antibiotic treatments were administered as q2h. (A) Cefepime at 50 mg/kg (FEP), meropenem:cilastatin at a 1:1 ratio of 25 mg/kg (MEM:CLS), and imipenem:cilastatin at a 1:1 ratio of 25 mg/kg (IPM:CLS). (B) Cefepime-2 at 50 mg/kg and 8.33 mg/kg, respectively (FEP-2), meropenem:cilastatin at a 1:1 ratio of 25 mg/kg and 4.68 mg/kg of 3 (MEM:CLS-3), and imipenem:cilastatin at a 1:1 ratio of 25 and 18.75 mg/kg of relebactam (IPM:CLS-REL), respectively.