The in vitro and in vivo efficacy of novel metallo-β- lactamase inhibitors co-administered with meropenem to target CREs.

Abstract

The evolution and phenotypic expression of metallo β-lactamase genes across the world has led to the escalated transmission rates of carbapenem resistance. The effect has crippled the already impaired healthcare system, with the emergence of COVID-19 exacerbating the crisis further. Our plight for a solution to combat antimicrobial resistance has not been greater. One strategy to tackle this non-susceptibility is the development of metallo-β-lactamase inhibitors that can neutralize the metallo-β-lactamase enzyme, thereby allowing the carbapenem antibiotic to elicit its function on the microorganism. Currently, there is no FDA-approved metallo-β-lactamase inhibitor to meet the clinical challenges of drug resistance. In a desperate need to find a candidate drug, research has been initiated into the discovery and development of biologically active inhibitors. Therefore, this thesis focuses on the advances made by our research group, the Catalysis and Peptide Research Unit, in developing novel β-lactam derived inhibitors; NOTA, NO3PY, BP- 1, 6,10 and 14, that re-sensitize the microbe to the efficacy of meropenem. The in vitro and in vivo activities of the initial chelators, NOTA and NO3PY, were evaluated as potential metallo-β-lactamase inhibitors (MBLIs) against metallo-β-lactamase (MBL) resistant bacteria. Time-kill studies showed that NOTA and NO3PY restored the efficacy of meropenem against all bacterial strains tested. A murine infection model was then used to study both metal chelators’ in vivo pharmacokinetics and efficacy. NO3PY displayed poor bioavailability at the selected doses using a validated LC-MS/MS method, therefore discouraging the in vivo efficacy evaluation. NOTA showed good bioavailability; hence, the in vivo efficacy was determined in a murine thigh infection model. The co-administration of meropenem and NOTA (100 mg/kg.bw each) significantly decreased the colony-forming units of K. pneumoniae NDM over an eight-hour treatment period. The findings suggested that chelators, such as NOTA, hold strong potential for use as an MBLI in treating CRE infections; however, further preclinical development was needed to improve the pharmacokinetic properties of these agents to increase their bioavailability and tissue distribution. With this information, our group derivatized NOTA by coupling it to a β-lactam to create the BP series of novel MBLIs. The results generated by the BP compounds have proven to interact synergistically with meropenem, by restoring the MIC of meropenem to therapeutically acceptable concentrations (< 2 mg/L) that concur with the breakpoints outlined by CLSI. In addition, the bactericidal activity of the re-sensitized meropenem was evident in the time-kill study over 24 hours. Cytotoxicity assays were further conducted to study the inhibitors, with an outcome in favor of safe administration in vivo. The metallo-β-lactamase inhibitors reported herein have demonstrated good potency against NDM-1 and VIM-2 metallo-β-lactamases with a Ki of 25-97μM. Since the BP compounds are metal chelators that function as metallo-β- lactamase inhibitors, it was important to determine the binding specificity of the BP compounds to a physiologically relevant zinc-harboring enzyme, glyoxylase II. At concentrations of up to 500 μM of BP, the activity of glyoxylase II remained unhindered. This confirmed the hypothesis of BP specificity to be exclusive to NDM-1 and VIM-2 metallo-β-lactamases. These findings prompted further interest in the binding exhibited by BP and led to additional studies to address the binding interactions of BP with the metallo-β-lactamases through quenching and computational experiments. Fluorescent quenching experiments investigating the Ka of BP indicated that a higher binding affinity was noted for NDM-1 compared to VIM-2 MBLs, thus implying a stronger interaction with NDM-1. Molecular docking and dynamic simulation experiments shed light on the BPs’ mode of action, showing the interaction of the chelators’ carboxylic moiety with the Zn 2+ ions in the MBLs structure. In favor of this BP series as functional inhibitors, in vivo efficacy was explored in a murine infection model (BP1 and BP10). In Klebsiella pneumoniae NDM infected mice, BP co-administered with meropenem was efficacious in reducing the bacterial load by > 3 log10 units’ post-infection, compared to meropenem monotherapy. These findings validate our strategy for derivatizing NOTA into the series of the BPs, as the bioavailability of NOTA, when coupled to a cephalosporin, improved the overall in vivo efficacy, and allowed the drug to be quantified in plasma under the same conditions previously used. This study clearly indicated the influence of the BP compounds in reducing the bacterial burden and the success of employing combination therapy as a treatment alternative. Moreover, the outcome of this preclinical development represents a solid foundation, whereby we can build on our existing knowledge. In aligning with our research goals of alleviating the threat of antimicrobial resistance, coupling β-lactams to a cyclic zinc chelator offers a safe and efficacious solution to meet the calamity that plagues our healthcare system.