Covalent DNA Binding Is Essential for Gram-Negative Antibacterial Activity of Broad Spectrum Pyrrolobenzodiazepines

Abstract

It is urgent to find new antibiotic classes against multidrug-resistant bacteria as the rate of discovery of new classes of antibiotics has been very slow in the last 50 years. Recently, pyrrolobenzodiazepines (PBDs) with a C8-linked aliphatic-heterocycle have been identified as a new broad-spectrum antibiotic class with activity against Gram-negative bacteria. The active imine moiety of the reported lead pyrrolobenzodiazepine compounds was replaced with amide to obtain the non-DNA binding and noncytotoxic dilactam analogues to understand the structure-activity relationship further and improve the safety potential of this class. The synthesised compounds were tested against panels of multidrug-resistant Gram-positive and Gram-negative bacteria, including WHO priority pathogens. Minimum inhibitory concentrations for the dilactam analogues ranged from 4 to 32 mg/L for MDR Gram-positive bacteria, compared to 0.03 to 2 mg/L for the corresponding imine analogues. At the same time, they were found to be inactive against MDR Gram-negative bacteria, with a MIC > 32 mg/L, compared to a MIC of 0.5 to 32 mg/L for imine analogues. A molecular modelling study suggests that the lack of imine functionality also affects the interaction of PBDs with DNA gyrase. This study suggests that the presence of N10-C11 imine moiety is crucial for the broad-spectrum activity of pyrrolobenzodiazepines.

Keywords: antibacterial drug discovery; antimicrobial resistance; broad-spectrum antibiotics; gram-negative bacteria; pyrrolobenzodiazepines.

Figure 1

Structure of broad-spectrum C8-linked pyrrolobenzodiazepine compounds 7 and 8 [7].

Scheme 1

Synthesis of dilactam-modified PBD core 1d. Reagents and conditions: (a) Oxalyl chloride, DMF, Et₃N, (S)-proline methyl ester, dry DCM, r.t., overnight; (b) Raney-Ni, H₂, EtOH, 40 psi and (if needed) toluene, reflux; (c) NaOH, MeOH, H₂O, r.t., overnight.

Scheme 2

Synthesis of final dilactam derivatives 7a and 8a. Reagents and conditions: (a) HCl 4M in dioxane, MeOH, r.t., 30 min and then EDCI, DMAP, DMF, r.t., overnight.

Figure 2

WI-38 cell % viability after 24 h exposition to synthesised drugs, compared to a control with no exposition to the drug (100% viability). Results reported in the chart as mean + SD expressed by error bar (5 repeats). One way ANOVA test was performed comparing results of dilactam compounds to imine-containing compounds (7a vs. 7 and 8a vs 8), * p < 0.05, *** p < 0.001.

Figure 3

(A) DNA binding of compounds 7 (grey) and 7a (yellow) within the DNA minor groove of Sequence 1; (B) Interaction of 7 (grey) and 7a (yellow) within the binding pocket of DNA gyrase A from Staphylococcus aureus, showing differences in site occupancy.