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Abstract
Shigella spp. are among the main causative agents of acute diarrheal illness and claim more than 1 million lives per year worldwide. There are multiple bacterial genes that control the pathogenesis of Shigella, but the virF gene may be the most important. This gene, located on the primary pathogenicity island of Shigella, encodes VirF, an AraC-family transcriptional activator that is responsible for initiating the pathogenesis cycle in Shigella. We have previously shown that it is possible to attenuate the virulence of Shigella flexneri via small molecule inhibition of VirF. In this study, we probed the mechanism of action of our small molecule inhibitors of VirF. To enable these studies, we have developed a homologous and efficient expression and purification system for VirF and have optimized two different in vitro VirF-DNA binding assays. We have determined that one of our HTS hit compounds inhibits VirF binding to DNA with a calculated K i similar to the effective doses seen in our transcriptional activation and virulence screens. This is consistent with inhibition of DNA binding as the mechanism of action of this hit compound. We have also screened 15 commercially sourced analogs of this compound and deduced an initial SAR from the approximately 100-fold range in activities. Our four other HTS hit compounds do not inhibit DNA binding and yet they do block VirF activity. This suggests that multiple agents with different molecular mechanisms of inhibition of VirF could be developed. Pursuing hits with different mechanisms of action could be a powerful approach to enhance activity and to circumvent resistance that could develop to any one of these agents.
Highlights
The misuse of prescription antibiotics and the overuse of antibiotics in livestock feed have greatly contributed to the rapid increase in drug-resistant bacteria in the environment
We have previously reported the identification of five promising small molecule inhibitors of VirF from a high-throughput screening campaign and a series of follow-up assays, including tissue-culture based invasion and cell-tocell spread assays that model aspects of the infection process [27, 30]
We have developed a homologous and efficient expression and purification system for VirF and have optimized two different in vitro assays which monitor VirF binding to the DNA promoter region for VirB
Summary
The misuse of prescription antibiotics and the overuse of antibiotics in livestock feed have greatly contributed to the rapid increase in drug-resistant bacteria in the environment. At concentrations at or below their IC50s in the reporter assay, three of the compounds (19615, 144092, 153578) inhibited the spread of an active S. flexneri infection by approximately 75% in a tissueculture based plaque efficiency assay, and one of the compounds (144092) inhibited initial S. flexneri invasion by approximately 50% in a gentamicin protection assay These results, supported by similar results recently published by other groups [4, 5, 26], validate our approach by providing proof of principle that small molecules can attenuate virulence; the mechanism by which our compounds inhibit the VirF transcriptional activation process remained unclear (see Fig 1). We screened a series of analogs of 19615 and have deduced an initial structure-activity relationship that we are using as a basis to further optimize our inhibitor and work towards achieving our goal of developing a novel therapy for treating shigellosis
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