Synthesis and Assay of Vibrio Quorum Sensing Inhibitors

Our research focuses on designing, synthesizing, and analyzing potential inhibitors of Vibrio bacteria that cause disease in marine animals and humans. Our previous studies identified thiophene sulfonamide compounds as potent inhibitors of pathogenicity in Vibrio species. We aim to develop compounds for the treatment of Vibrio disease.

We have synthesized a panel of thiophene sulfonamide molecules active against several Vibrio species. We have solved the x-ray crystal structure of a thiophene sulfonamide molecule in the binding pocket of the target Vibrio, and we determined the thiophene sulfonamide mechanism of action in blocking the protein target and preventing pathogenicity in vivo. This protocol is designed with the novice researcher in mind.

Every step was optimized for simplicity and error reduction, while still allowing for teaching concepts and the scientific method. Our protocol bridges microbiology, organic chemistry, structure modeling, and drug design in a novel teaching-based experimental system. Novel therapeutics that target Vibrio bacteria are needed to treat the increasing cases of vibriosis in humans and aquaculture.

Because our compounds are not antibiotics and don't kill cells, they have the potential to treat disease without generating resistance. We will focus on optimizing the inhibitors of Vibrio species that do not have a therapeutically relevant inhibitor yet. We'll also explore the chemical space afforded by substituted aromatic sulfonyl chloride starting materials.

To begin, obtain the lysogeny broth or LB medium and add the antibiotics to the medium. Aliquot five milliliters of the medium into sterile test tubes with caps. Inoculate the media with Escherichia coli strains expressing plasmids taken from freezer stocks and incubate the culture in a shaker overnight.

Next, prepare a 10 millimolar thiophene sulfonamide solution in DMSO and vortex to mix thoroughly. Back dilute the cultures one to 100 times in 10 milliliters of media taken in 15 milliliter conical tubes. Vortex briefly to mix.

Add cultures to the appropriate wells of a 96-well plate. After mixing, prepare a dilution series from rows A to E, transferring 50 microliters of the solution each time. Cover the plate with microporous tape and incubate it.

After removing the tape, using a plate reader, read the optical density at 600 nanometers GFP and mCherry. Finally, record and plot the data as normalized fluorescence per cell. Data for three thiophene sulfonamide compounds suggested that compounds 1A and 3B were each inhibitory to LuxR/HapR to different extents, whereas 2B did not have any activity.