Investigating the Antibacterial Efficiency and Mechanism of Indole- and Naphthalimide-Mediated Benzimidazoles: Membrane Damage, Metabolic Inactivation, and Oxidative Stress against Bacillus subtilis

Resistance by bacteria to available antibiotics is a threat to human health, which demands the development of new antibacterial agents. Considering the prevailing conditions, we have developed a series of naphthalimide/indole benzimidazoles with diverse amines and aryl rings to avoid the molecular framework of conventional drug molecules to overcome the cross-resistance issue. Most of the synthesized compounds, especially electron-withdrawing and halide substituents, show broad-spectrum activity against both Gram-positive and Gram-negative bacterial strains. Preliminary studies indicate that compounds IB-14 and NB-8 display excellent antibacterial activity against Bacillus subtilis, exceeding the performance of the marketed drug amoxicillin. In addition to the rapid bactericidal effect, both compounds significantly inhibit the formation of biofilm, lowering the development of drug resistance. Moreover, both compounds exhibit fast-bactericidal properties, thus shortening the time of treatment and also resisting the emergence of drug resistance up to 20 passages. Further, biofunctional evaluation reveals that both compounds effectively disrupt the membrane, causing the leakage of cytoplasmic contents and loss in metabolic activity. Both compounds efficiently induce the reactive oxygen species (ROS), leading to the oxidation of GSH to GSSG, decreasing the GSH activity of the cell, and causing oxidative damage to cells. DNA studies show that compounds significantly bind to DNA and form DNA-IB-14/NB-8 complexes that inhibit the replication of DNA and protein. The significant binding affinity of compounds with HSA suggests easy transport of the developed antibacterial candidates to the target site through the carrier protein. These findings suggest that both compounds have broad-spectrum and multitargeting potential as antibacterial agents and provide a new possibility to overcome the global issue of the development of multidrug resistance by bacteria toward conventional antibiotics.