In Vitro and In Vivo Development of a β-Lactam-Metallo-β-Lactamase Inhibitor: Targeting Carbapenem-Resistant Enterobacterales

Abstract

β-lactams are the most prescribed class of antibiotics due to their potent, broad-spectrum antimicrobial activities. However, alarming rates of antimicrobial resistance now threaten the clinical relevance of these drugs, especially for the carbapenem-resistant Enterobacterales expressing metallo-β-lactamases (MBLs). Antimicrobial agents that specifically target these enzymes to restore the efficacy of last resort β-lactam drugs, that is, carbapenems, are therefore desperately needed. Herein, we present a cyclic zinc chelator covalently attached to a β-lactam scaffold (cephalosporin), that is, BP1. Observations from in vitro assays (with seven MBL expressing bacteria from different geographies) have indicated that BP1 restored the efficacy of meropenem to ≤ 0.5 mg/L, with sterilizing activity occurring from 8 h postinoculation. Furthermore, BP1 was nontoxic against human hepatocarcinoma cells (IC₅₀ > 1000 mg/L) and exhibited a potency of (K_iapp) 24.8 and 97.4 μM against Verona integron-encoded MBL (VIM-2) and New Delhi metallo β-lactamase (NDM-1), respectively. There was no inhibition observed from BP1 with the human zinc-containing enzyme glyoxylase II up to 500 μM. Preliminary molecular docking of BP1 with NDM-1 and VIM-2 sheds light on BP1's mode of action. In Klebsiella pneumoniae NDM infected mice, BP1 coadministered with meropenem was efficacious in reducing the bacterial load by >3 log₁₀ units' postinfection. The findings herein propose a favorable therapeutic combination strategy that restores the activity of the carbapenem antibiotic class and complements the few MBL inhibitors under development, with the ultimate goal of curbing antimicrobial resistance.

Keywords: Klebsiella pneumoniae; New Delhi metallo β-lactamase; Verona integron-encoded MBL; carbapenem-resistant Enterobacterales; human hepatocarcinoma cells; β-Lactam-Metallo-β-Lactamase Inhibitor.

Figure 1

Examples of recent nitrogen-bearing chelators as promising MBLIs.^,

Figure 2

Time-kill kinetic study of BP1 with meropenem at varying concentrations. K. pneumoniae NDM control (green circles) represents the bacterial growth curve unhindered by the addition of BP1 and meropenem. The meropenem-only test group (yellow circles) highlights the ineffectiveness of meropenem when administered without BP1. Sterilizing activity was achieved with BP1 at all concentrations of meropenem used by 24 h (blue, purple, and red circles).

Figure 3

Activity of human recombinant glyoxylase II in the presence of four inhibitors. All assays were conducted in triplicate. Normal glyoxylase II activity is denoted by the legend representing no inhibitor added. BP1 did not decrease the activity of glyoxylase II in comparison to the metal chelating agents EDTA and TPEN. NOTA a component of BP1, also did not reduce the activity of glyoxylase II.

Figure 4

3D structures of NDM1—BP1 and VIM2—BP1 complexes, respectively. BP1 compounds are shown in green. In NDM1, the Zn301 (grey sphere) is coordinated to three histidines, and a water molecule (HOH426) coordinates the two zinc ions, while Zn302 is coordinated to histidine, aspartate, and cysteine residues. In VIM2, the Zn405 is coordinated to three histidines, and Zn06 is coordinated to one histidine, one aspartate, and one cysteine residue.

Figure 5

Plasma BP1 and meropenem concentrations over the eight hour treatment period. BALB/c mice were infected i.m with K. pneumoniae NDM. Treatment was initiated 2 h post-infection with either S, M, or BP1. Four treatment doses were administered via IP. Data are presented as a mean ± SD (n = 6). Meropenem only—animals treated with meropenem only, meropenem (BP 1)—meropenem concentration when animals were treated with the combination of BP1 (meropenem)—BP1 concentration when animals were treated with the combination.

Figure 6

Efficacy of BP1 combination therapy over monotherapy in a murine thigh infection model. Neutropenic BALB/c mice were infected i.m with 0.1 mL of 10⁶–10⁸ cfu/mLof NDM producing K. pneumoniae. Four treatment doses, were administered via i.p over an 8 h period. The co-administration of BP1 and meropenem resulted in a significant decrease in K. pneumoniae NDM cfu/mL (yellow circles) in comparison to the S (green circles) and M (blue circles) treatments. This clearly indicates that BP1 + meropenem is a favorable treatment strategy.

Scheme 1. Synthetic Route of BP1