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.

Neutralizing Carbapenem Resistance by Co-Administering Meropenem with Novel β-Lactam-Metallo-β-Lactamase Inhibitors

“…For both β-lactamases, the model predicts that the BP2_SS configuration has a greater binding free energy than BP2_SR. This is the opposite result to that which we observed in our previous work with BP1 [ 40 ]. It should be noted that BP2_SS configuration binds exclusively to a single zinc atom in VIM-2 yielding the largest binding value ( Figures S2 and S5 ).…”

“…Further investigation of BP2 indicated inhibitor-specific activity towards the NDM-1 and VIM-2 MBLs, as there was no reduction in glyoxylase activity. These findings correspond to MBLI chelators’ reports, BP1 [ 40 ] and ZN148 [ 30 ]. It is possible that BP2 ultimately removes the zinc ions from the active site, immobilizing the enzyme completely ( Figure 5 ).…”

“…The utilization of metal chelators in combination therapy to target Carbapenem-Resistant Enterobacterales proves to be a promising treatment strategy as observed in reports of di- and tris-picolylamine zinc chelators [ 49 , 50 , 51 ]. Our previous results also indicated this, that is, with BP1 [ 40 ]. This led us to investigate other derivatives of BP1 (cyclic amino acidic zinc chelators attached to a β-lactam moiety) to study other aspects for optimization of the derivatives/hits with regards to synthetic route viability, solubility, affinity, selectivity, efficacy/potency, metabolic stability, and oral bioavailability.…”

“…A plasma concentration of 1.93 µg/L was obtained with a dose of 10 mg/kg.b.w. This was far below the cytotoxicity concentrations seen in the cell viability assays, advocating for the safe increase in the treatment dose to 100 mg/kg.b.w, which allowed us to reach therapeutic concentrations ( Figure 6 ) in plasma without the possibility of toxicity [ 40 ]. In vivo efficacy studies were undertaken to assess the potency of BP2 in a murine infection model.…”

“…Hence, we envisaged that conjugation of the chelator to a known drug would enhance its properties. This was achieved by attaching a cyclic zinc chelator (NOTA-derived) to known β-lactam antibiotic cores to produce the BP-series of compounds [ 40 ] ( Figure 1 ). Based on the excellent results for BP1, herein we expand the scope of the most potent derivative, to BP2, which can also be used as a potential MBLI to restore the efficacy of carbapenems against MBLs.…”