Nutrient-limited conditions reveal the activity of a minor groove binder, MGB-BP-3, against Escherichia coli

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Eskandari, Kaveh and Young, Louise C. and Blair, Jessica M.A. and Oravcova, Katarina and Scott, Fraser J. (2025) Nutrient-limited conditions reveal the activity of a minor groove binder, MGB-BP-3, against Escherichia coli. Other. bioRxiv. (https://doi.org/10.64898/2025.12.22.695926)

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Abstract

Background: Antimicrobial resistance demands new antibiotics, but conventional screening in nutrient-rich media often fails to predict in vivo efficacy, especially against Gram-negative bacteria. Minor groove binders (MGB), such as MGB-BP-3, are a promising new antibacterial with potent activity against Gram-positive bacteria but thought to have limited potency against Gram-negative bacteria. Objectives: This study evaluated the antibacterial activity of MGB-BP-3 under nutrient-limited, physiologically relevant conditions to better mimic in vivo environments. Methods: Minimum inhibitory concentrations (MICs) were determined against a panel of ESKAPEE pathogens in both rich (Cation Adjusted Mueller-Hinton Broth, CA-MHB) and nutrient-limited media (Roswell Park Memorial Institute, RPMI-1640 + 10% Luria-Bertani, LB). Synergy with efflux pump inhibitors and membrane permeabilizers was evaluated via checkerboard assays. Permeability was assessed using a fluorescent DNA-binding cell permeant (Hoechst 33342) accumulation assay. MICs were determined in the presence of different concentrations of ions, using both wild-type and efflux-compromised strains. In vivo efficacy was assessed using the Galleria mellonella infection model. Results: The activity of MGB-BP-3 against Gram-negative bacteria was improved by shifting the medium from rich to nutrient-limited conditions. Specifically, it showed potent activity against E. coli in nutrient-limited media (MIC 3.1 μM) but was inactive in rich media (MIC 700 μM). Synergy with efflux inhibitors occurred mainly in rich media, indicating barriers to intracellular accumulation were not present in nutrient-limited conditions. Increased permeability correlated with enhanced susceptibility and efflux-deficient strains were more susceptible, confirming the role of efflux pumps in resistance. MGB-BP-3 provided dose-dependent protection in G. mellonella larvae infected with E. coli or S. aureus. For example, at concentrations of 12.5 μM and 100 μM, MGB-BP-3 was able to protect the larvae by more than 70% over 5 days of treatment, respectively. Conclusions: Nutrient-limited media better reveal MGB-BP-3's activity against Gram-negative bacteria and align with in vivo efficacy, highlighting the need for host-mimicking conditions in antibiotic screening.

ORCID iDs

Eskandari, Kaveh, Young, Louise C. ORCID logoORCID: https://orcid.org/0000-0003-1757-6404, Blair, Jessica M.A., Oravcova, Katarina and Scott, Fraser J. ORCID logoORCID: https://orcid.org/0000-0003-0229-3698;
CORE (COnnecting REpositories)