The in vitro and in vivo efficacy of novel metallo-β- lactamase inhibitors co-administered with meropenem to target CREs.
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Date
2022
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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.
Description
Doctoral Degree. University of KwaZulu-Natal, Durban.