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

Abstract

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 DiOC(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 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.

Fluorescein Derivatives as Antibacterial Agents Acting via Membrane Depolarization

“…As seen in Figure 4, all of the three fluorescein derivatives caused bacterial membrane depolarization, with C10-FL being somewhat more effective than mitoFluo and tol-mitoFluo. To exclude the bacterial membrane permeabilization as a possible alternative reason for the depolarization, we measured the fluorescence response of the cell-impermeant nucleic acid stain TO-PRO-3 iodide [35]. As seen in Figure 5, all of the three fluorescein derivatives produced no detectable response of TO-PRO-3 iodide, whereas alamethicin, used as a positive control, exhibited a pronounced increase in the fluorescence.…”

“…The violation of the bacterial membrane integrity was evaluated by using the cell-impermeant nucleic acid stain TO-PRO-3 iodide [35]. cover glass on top.…”

Opossum Cathelicidins Exhibit Antimicrobial Activity Against a Broad Spectrum of Pathogens Including West Nile Virus

“…Four colonies of B. cereus (ATCC 10876), E. coli (ATCC 25922), and S. aureus (ATCC 6538) were inoculated into 5 mL LB broth and incubated at 37 • C for 5 h. Cells suspensions were centrifuged at 3000 × g and 25 • C, washed and resuspended in phosphate buffered saline (PBS) + (0.14 M NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , and 1.8 mM KH 2 PO 4 supplemented with 10 mM glucose and 0.5 mM MgCl 2 ; pH 7.4) to an OD 600 of 0.1. Concurrently, a blank PBS+ sample without cells and cells treated with Nisin (Sigma Aldrich) and vancomycin (Sigma Aldrich) were used as controls (36). TO-PRO-3 iodide (Sigma Aldrich) and DiOC 2 (3) (Sigma Aldrich) dyes were then added into the sample and controls to a final concentration of 625 nM and 10 µM, respectively.…”

“…Fluorescent dyes have been used to determine the integrity of membranes (36). The increased fluorescence intensity of TO-PRO-3 iodide and DiOC 2 (3) in cells indicates the penetration of dyes to the cytoplasm due to the damaged membrane and disruption of membrane potential.…”

Discovery of a Novel DNA Gyrase-Targeting Antibiotic through the Chemical Perturbation of Streptomyces venezuelae Sporulation

“…By screening antibiotics having known targets we report that compounds targeting the integrity of DNA confers a sporulation defect at sub-inhibitory concentrations, whereas compounds that target the ribosome, RNA polymerase, the cell wall, and the cell's electrochemical gradient have only a simple antibacterial effect. We leveraged this discovery by screening a small library of 3,705 synthetic compounds, previously demonstrated to have biological activity against Gram-positive bacteria (Czarny and Brown, 2016, McAuley et al., 2018), for compounds having the capacity to inhibit sporulation in Streptomyces venezuelae. Here we report that the most potent of these is an inhibitor of DNA gyrase identified as EN-7.…”