Gram-negative synergy and mechanism of action of alkynyl bisbenzimidazoles

Jordan Chamberlin,Dev P Arya,Sandra Story,Nihar Ranjan,Geoffrey Chesser

doi:10.1038/s41598-019-48898-4

Abstract

Bisbenzimidazoles with terminal alkynyl linkers, selective inhibitors of bacterial topoisomerase I, have been evaluated using bacterial cytological profiling (BCP) to ascertain their mechanism of action and screened for synergism to improve Gram-negative bacterial coverage. Principal component analysis of high throughput fluorescence images suggests a dual-mechanism of action affecting DNA synthesis and cell membrane integrity. Fluorescence microscopy of bacteria challenged with two of the alkynyl-benzimidazoles revealed changes in the cellular ultrastructure that differed from topoisomerase II inhibitors including induction of spheroplasts and membrane lysis. The cytoskeleton recruitment enzyme inhibitor A22 in combination with one of the alkynyl-benzimidazoles was synergistic against Acinetobacter baumannii and Escherichia coli. Gram-positive coverage remained unchanged in the A22-alkynyl bisbenzimidazole combination. Efflux inhibitors were not synergistic, suggesting that the Gram-negative outer membrane was a significant barrier for alkynyl-bisbenzimidazole uptake. Time-kill assays demonstrated the A22-bisbenzimidazole combination had a similar growth inhibition curve to that of norfloxacin in E.coli. Bisbenzimidazoles with terminal alkynyl linkers likely impede bacterial growth by compromising cell membrane integrity and by interfering with DNA synthesis against Gram-positive pathogens and in the synergistic combination against Gram-negative pathogens including E. coli and multidrug-resistant A. baumanii.

Highlights

There is a dire need for new classes of antibiotics with novel mechanisms of action (MOA), for which there are currently few options
We have reported the synthesis and activity of several bisbenzimidazoles modified with the addition of terminal alkynyl linkers which effectively inhibited the growth of methicillin-resistant Staphylococcus aureus (MRSA) and expressed selectivity towards bacterial topoisomerase I8,9
Bacterial cytological profiling (BCP) relies on the concept that antibacterial compounds with distinct mechanisms of action produce distinct phenotypes in drug-exposed bacteria

Summary

Introduction

There is a dire need for new classes of antibiotics with novel mechanisms of action (MOA), for which there are currently few options. Antibiotic resistance, especially among Gram-negative bacteria, is rising rapidly, and the World Health Organization considers the development of novel targets to counter these pathogens extremely important[1]. One such potential target is bacterial topoisomerase I, an enzyme critical for DNA replication[2,3]. Support for such enzymes as viable targets in need of further development is the extended use of fluoroquinolones that target bacterial topoisomerase II and IV which has resulted in the emergence of resistant bacteria. Cytological profiling can be applied to predict the observed morphology for a specific molecular target, dubbed rapid inhibition profiling, which further demonstrates the utility of fluorescence-microscopy methods for mechanistic analysis[12]

Methods

Results

Conclusion