WCK 5107 (Zidebactam) and WCK 5153 Are Novel Inhibitors of PBP2 Showing Potent "β-Lactam Enhancer" Activity against Pseudomonas aeruginosa, Including Multidrug-Resistant Metallo-β-Lactamase-Producing High-Risk Clones

Abstract

Zidebactam and WCK 5153 are novel β-lactam enhancers that are bicyclo-acyl hydrazides (BCH), derivatives of the diazabicyclooctane (DBO) scaffold, targeted for the treatment of serious infections caused by highly drug-resistant Gram-negative pathogens. In this study, we determined the penicillin-binding protein (PBP) inhibition profiles and the antimicrobial activities of zidebactam and WCK 5153 against Pseudomonas aeruginosa, including multidrug-resistant (MDR) metallo-β-lactamase (MBL)-producing high-risk clones. MIC determinations and time-kill assays were conducted for zidebactam, WCK 5153, and antipseudomonal β-lactams using wild-type PAO1, MexAB-OprM-hyperproducing (mexR), porin-deficient (oprD), and AmpC-hyperproducing (dacB) derivatives of PAO1, and MBL-expressing clinical strains ST175 (bla_VIM-2) and ST111 (bla_VIM-1). Furthermore, steady-state kinetics was used to assess the inhibitory potential of these compounds against the purified VIM-2 MBL. Zidebactam and WCK 5153 showed specific PBP2 inhibition and did not inhibit VIM-2 (apparent K_i [K_iapp] > 100 μM). MICs for zidebactam and WCK 5153 ranged from 2 to 32 μg/ml (amdinocillin MICs > 32 μg/ml). Time-kill assays revealed bactericidal activity of zidebactam and WCK 5153. LIVE-DEAD staining further supported the bactericidal activity of both compounds, showing spheroplast formation. Fixed concentrations (4 or 8 μg/ml) of zidebactam and WCK 5153 restored susceptibility to all of the tested β-lactams for each of the P. aeruginosa mutant strains. Likewise, antipseudomonal β-lactams (CLSI breakpoints), in combination with 4 or 8 μg/ml of zidebactam or WCK 5153, resulted in enhanced killing. Certain combinations determined full bacterial eradication, even with MDR MBL-producing high-risk clones. β-Lactam-WCK enhancer combinations represent a promising β-lactam "enhancer-based" approach to treat MDR P. aeruginosa infections, bypassing the need for MBL inhibition.

Keywords: Gram-negative bacteria; PBP2 inhibition; Pseudomonas aeruginosa; WCK 5107; WCK 5153; bicyclo-acyl hydrazide; penicillin-binding proteins; time-kill curves; zidebactam; β-lactam enhancer.

FIG 1

Chemical structures of ZID (zidebactam; WCK 5107) and WCK 5153.

FIG 2

Killing curves measured in terms of reduction of viable CFU per milliliter over time for wild-type strain PAO1 at a 10⁷ inoculum. The concentrations tested were 1 and 4 μg/ml of cefepime (FEP) (1× and 4× MIC); 1, 4, and 8 μg/ml of zidebactam (ZID) (0.25×, 1×, and 2× MIC) and WCK 5153 (0.5×, 2×, and 4× MIC); and combinations of 1 and 4 μg/ml of FEP (1× and 4× MIC) with 0.25× to 1× MICs of ZID or WCK 5153. Mean values for three experiments ± the standard deviations are shown.

FIG 3

Results of LIVE/DEAD staining performed on wild-type PAO1. (A) Images obtained at 2 h of incubation with 1× MICs of cefepime (FEP), meropenem (MEM), zidebactam (ZID), and WCK 5153 and with 1× to 0.25× MIC of cefepime-zidebactam or cefepime-WCK 5153. (B) Images obtained at 8 h of incubation with 4× MICs of cefepime, zidebactam, and WCK 5153 and with 4× to 0.5× MICs of cefepime-zidebactam or cefepime-WCK 5153. Live cells are stained green, and dead cells are stained red.

FIG 4

Results of the killing curves measured in terms of reduction of viable CFU per milliliter over time for the AmpC-hyperproducing PAO1 dacB mutant (PAdB). The concentrations tested were 8 and 16 μg/ml of cefepime (FEP) (0.5× and 1× MIC), 8 and 16 μg/ml of aztreonam (ATM) (0.03× and 0.06× MIC), and 16 and 32 μg/ml of piperacillin (PIP) (0.06× and 0.125× MIC), at susceptible and intermediate CLSI breakpoint concentrations in combination with 8 μg/ml of zidebactam (ZID) or WCK 5153. Standalone drugs were tested at the maximum concentration used for combinations. Mean values for three experiments ± the standard deviations are shown. The dashed line represents the limit of detection.

FIG 5

Results of the killing curves measured in terms of reduction of viable CFU per milliliter over time for the MexAB-OprM-hyperproducing PAO1 mexR mutant (PAOMxR). Cefepime (FEP) (8 and 16 μg/ml), aztreonam (ATM) (8 and 16 μg/ml), piperacillin (PIP) (16 and 32 μg/ml), imipenem (IPM) (4 and 8 μg/ml), meropenem (MEM) (4 and 8 μg/ml), and doripenem (DOR) (4 and 8 μg/ml) were tested at susceptible and intermediate CLSI breakpoint concentrations in combination with 8 μg/ml of zidebactam (ZID) or WCK 5153. Standalone drugs were tested at the maximum concentration used for combinations. Mean values for three experiments ± the standard deviations are shown. The dashed line represents the limit of detection.

FIG 6

Results of the killing curves measured in terms of reduction of viable CFU per milliliter over time for the XDR ST111 (VIM-1) isolate. Cefepime (FEP) (8 and 16 μg/ml), aztreonam (ATM) (8 and 16 μg/ml), piperacillin (PIP) (16 and 32 μg/ml), imipenem (IPM) (4 and 8 μg/ml), meropenem (MEM) (4 and 8 μg/ml), and doripenem (DOR) (4 and 8 μg/ml) were tested at susceptible and intermediate CLSI breakpoint concentrations in combination with 8 μg/ml of zidebactam (ZID) or WCK 5153. Standalone drugs were tested at the maximum concentration used for combinations. Mean values for three experiments ± the standard deviations are shown. The dashed line represents the limit of detection.

FIG 7

Results of the killing curves measured in terms of reduction of viable CFU per milliliter over time for the XDR ST175 (VIM-2) isolate. Cefepime (FEP) (8 and 16 μg/ml), aztreonam (ATM) (8 and 16 μg/ml), piperacillin (PIP) (16 and 32 μg/ml), imipenem (IPM) (4 and 8 μg/ml), meropenem (MEM) (4 and 8 μg/ml), and doripenem (DOR) (4 and 8 μg/ml) were tested at susceptible and intermediate CLSI breakpoint concentrations in combination with 8 μg/ml of zidebactam (ZID) or WCK 5153. Standalone drugs were tested at the maximum concentration used for combinations. Mean values for three experiments ± the standard deviations are shown. The dashed line represents the limit of detection.