A target-guided drug repurposing strategy for antibacterial discovery

The scarcity of effective antibiotics against drug-resistant pathogens has intensified the need for alternative antibacterial development strategies. Drug repurposing (DR) offers a practical solution; herein, we present detailed experimental workflows and in vitro results to validate the feasibility of a DR-based strategy for new antibacterial development.

Using Vina-GPU, we performed molecular docking between 125 highly conserved bacterial essential proteins and 2,027 approved non-antibacterials drugs to predict drug-target interactions. As preliminary screening, 14 candidate drugs were tested for inhibitory activity against six bacterial strains (Escherichia coli MC4100, Pseudomonas aeruginosa PAO1, Acinetobacter baumannii ATCC 19606, Salmonella enterica Typhi CMCC 50071, Salmonella enterica Typhimurium ATCC 14028, and Klebsiella oxytoca ATCC 13182) at 100 μg/ml. Subsequently, lower-concentration assays (combined with Polymyxin B nonapeptide, PMBN) were conducted for preliminarily active drugs. The FLUOStar Omega was used to measure bacterial suspension optical density (OD₆₀₀) and calculate drug inhibition rates. Surface plasmon resonance (SPR), MD simulation, and folate rescue experiments were used to further validate the mechanism of the drug.

Among the 2,027 non-antibacterials drugs, several exhibited antibacterial activities. Additionally, 173 existing antibacterials were clustered into two groups based on their binding affinity similarity to the 125 essential proteins, with the two groups showing distinct antibacterial activities. Notably, multiple repurposed drugs inhibited the growth of multiple bacterial species.

Our study revealed that the combination of 8 μg/ml lifitegrast and PMBN can effectively inhibit six gram-negative bacteria. Folate rescue experiments showed that the preliminary antibacterial mechanism of lifitegrast is to inhibit the function of FolA. In addition, future research should explore the structure-activity relationship of lifitegrast and its impact on antibacterial activity.