Abstract
An impermeable outer membrane and multidrug efflux pumps work in concert to provide Gram-negative bacteria with intrinsic resistance against many antibiotics. These resistance mechanisms reduce the intracellular concentrations of antibiotics and render them ineffective. The natural product thiomarinol A combines holothin, a dithiolopyrrolone antibiotic, with marinolic acid A, a close analogue of mupirocin. The hybridity of thiomarinol A converts the mupirocin scaffold from inhibiting Gram-positive bacteria to inhibiting both Gram-positive and -negative bacteria. We found that thiomarinol A accumulates significantly more than mupirocin within the Gram-negative bacterium Escherichia coli, likely contributing to its broad-spectrum activity. Antibiotic susceptibility testing of E. coli mutants reveals that thiomarinol A overcomes the intrinsic resistance mechanisms that render mupirocin inactive. Structure-activity relationship studies suggest that the dithiolopyrrolone is a privileged moiety for improving the accumulation and antibiotic activity of the mupirocin scaffold without compromising binding to isoleucyl-tRNA synthetase. These studies also highlight that accumulation is required but not sufficient for antibiotic activity. Our work reveals a role of the dithiolopyrrolone moiety in overcoming intrinsic mupirocin resistance in E. coli and provides a starting point for designing dual-acting and high-accumulating hybrid antibiotics.