A Small Molecule Substrate Mimic to Inhibit Bacterial Hsp70 Chaperone Function

Given the rapid spread of antibiotic resistance worldwide, it is imperative to develop new strategies against pathogenic bacteria. Here, we report a small-molecule inhibitor of bacterial heat shock protein 70 (Hsp70), called DnaK, a central component of the bacterial protein chaperone network. Screening of an in-house pyridylamide-based peptidomimetic library identified M7 as an inhibitor of DnaK ATPase and substrate refolding activity with intrinsic fluorescence properties. Mechanistic studies revealed that M7 likely engages the substrate-binding pocket of DnaK. Treatment of bacterial cells with M7 led to the accumulation of σ-32, without affecting protein translation, consistent with impaired DnaK function. Microscopy images of Escherichia coli AS19 cells treated with M7 revealed filamentous growth, a phenotypic defect consistent with compromised DnaK function. Moreover, M7 effectively inhibited the growth of E. coli AS19 and some Gram-positive bacteria, including Staphylococcus aureus and Bacillus cereus. These findings highlight a promising class of peptidomimetic DnaK inhibitors as potential antibacterial agents, as well as a chemical biology tool to interrogate the broader network of chaperone-mediated protein folding.