An Antibiotic Discovery Campaign Targeting VirF, the Main Transcriptional Regulator of Virulence in Shigella flexneri

Abstract

Shigella flexneri is a gram-negative enteropathogen that infects the human colonic epithelium. It is estimated that Shigella spp. infect 165 million people a year worldwide. Symptoms of shigellosis include bloody dysentery, dehydration, and ultimately death if the infection is not treated properly. The current recommended first-line treatment for shigellosis is ciprofloxacin; however, many new multi-drug resistant strains of Shigella have begun to emerge. The emergence of these strains, along with the lack of novel antibiotics in the drug pipeline, makes the need for new effective treatments for shigellosis a priority. Genetic knockout studies have shown that VirF, the main transcriptional activator of the Shigella pathogenesis cascade, is necessary for virulence, but not bacterial viability. We hypothesized that a novel anti-virulence therapy for shigellosis could be developed through the inhibition of VirF. To identify inhibitors of VirF, we performed a high-throughput screening (HTS) campaign testing over 140,000 small molecules and 20,000 natural product extracts using a Shigella-based, VirF-driven, β-galactosidase reporter assay. Following a series of confirmation screens, we identified five compounds from the HTS campaign that had VirF inhibitory properties. Using tissue culture-based models of the S. flexneri infection process, we were able to show that three of compounds were able to attenuate the virulence of S. flexneri, thereby, validating VirF as a target for the treatment of shigellosis. To further characterize the hits from the HTS campaign a series of established in vitro assays were adapted and optimized for VirF. An electrophoretic mobility shift assay and a fluorescence polarization assay were used to monitor VirF binding to the virB promoter, and a fluorescence intercalator displacement assay was used to determine if the hits could directly bind DNA. Using these assays, we were able to determine the mechanism of inhibition (blockade of VirF binding to the virB promoter) and preliminary structure-activity relationship trends for one of the hits, and report the first dissociation constant for VirF binding to the virB promoter (2.8 ± 1.0 μM).