A strategy for developing pan-dihydrofolate reductase (DHFR) inhibitors having a broad-spectrum antibiotic activity with an enhanced coverage against multiple drug-resistant species is delineated. Inspired by Trimethoprim (TMP), using a structure-guided approach we have developed a series of next generation Propargyl-Linked Antifolates (PLAs) that exhibited high potency against several of the trimethoprim-resistant plasmid-encoded DHFR enzymes including Staphylococcus aureus and Escherichia coli. In this study, we report an improved synthetic strategy for the rapid development of structurally diverse PLAs, and an in depth structure activity relationship (SAR) analysis against Escherichia coli using a comprehensive multidisciplinary approach involving state of the art microbial susceptibility assays, intracellular small molecule accumulation experiments, lipopolysaccharide (LPS) binding assay and drug-target residence time analysis, along with X-ray crystallography, that enhances our understanding about permeability and active efflux of small molecules across the lipid bilayers of these pathogens. Most of our PLAs show excellent enzyme potency against Escherichia coli DHFR and good antimicrobial activity against this pathogen. Further, they exhibit excellent synergy with sulfamethoxazole (SMX), even better than the standard TMP-SMX combination in Escherichia coli.

Propargyl-linked antifolates as effective antibacterial against Escherichia coli

ACS Spring 2022

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