Membrane activity profiling of small molecule B. subtilis growth inhibitors utilizing novel duel-dye fluorescence assay.

Small molecule disruption of the bacterial membrane is both a challenge and interest for drug development. While some avoid membrane activity due to toxicity issues, others are interested in leveraging the effects for new treatments. Existing assays are available for measuring disruption of membrane potential or membrane permeability, two key characteristics of the bacterial membrane, however they are limited in their ability to distinguish between these properties. Here, we demonstrate a high throughput assay for detection and characterization of membrane active compounds. The assay distinguishes the effect of small molecules on either the membrane potential or membrane permeability using the fluorescent dyes TO-PRO-3 iodide and DiOC2(3) without the need for secondary assays. We then applied this assay to a library of 3520 synthetic molecules previously shown to inhibit growth of B. subtilis in order to determine the frequency of membrane activity within such a biologically active library. From the library, we found 249 compounds that demonstrated significant membrane activity, suggesting that synthetic libraries of this kind do not contain a plurality of membrane active molecules.

Discovery of novel cell wall-active compounds using P ywaC, a sensitive reporter of cell wall stress, in the model gram-positive bacterium Bacillus subtilis.

The emergence of antibiotic resistance in recent years has radically reduced the clinical efficacy of many antibacterial treatments and now poses a significant threat to public health. One of the earliest studied well-validated targets for antimicrobial discovery is the bacterial cell wall. The essential nature of this pathway, its conservation among bacterial pathogens, and its absence in human biology have made cell wall synthesis an attractive pathway for new antibiotic drug discovery. Herein, we describe a highly sensitive screening methodology for identifying chemical agents that perturb cell wall synthesis, using the model of the Gram-positive bacterium Bacillus subtilis. We report on a cell-based pilot screen of 26,000 small molecules to look for cell wall-active chemicals in real time using an autonomous luminescence gene cluster driven by the promoter of ywaC, which encodes a guanosine tetra(penta)phosphate synthetase that is expressed under cell wall stress. The promoter-reporter system was generally much more sensitive than growth inhibition testing and responded almost exclusively to cell wall-active antibiotics. Follow-up testing of the compounds from the pilot screen with secondary assays to verify the mechanism of action led to the discovery of 9 novel cell wall-active compounds.