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1.
PLoS One ; 10(9): e0137410, 2015.
Article in English | MEDLINE | ID: mdl-26352269

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 Ki 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.


Subject(s)
AraC Transcription Factor/metabolism , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Shigella flexneri/metabolism , AraC Transcription Factor/genetics , Bacterial Proteins/genetics , Humans , Shigella flexneri/genetics , Transcriptional Activation , Virulence/genetics
2.
J Antibiot (Tokyo) ; 67(5): 379-86, 2014 May.
Article in English | MEDLINE | ID: mdl-24549153

ABSTRACT

VirF is an AraC-type transcriptional regulator responsible for activating the transcription of virulence genes required for the intracellular invasion and cell-to-cell spread of Shigella flexneri. Gene disruption studies have validated VirF as a potential target for an anti-virulence therapy to treat shigellosis by determining that VirF is necessary for virulence, but not required for bacterial viability. Using a bacteria-based, ß-galactosidase reporter assay we completed a high-throughput screening (HTS) campaign monitoring VirF activity in the presence of over 140,000 small molecules. From our screening campaign, we identified five lead compounds to pursue in tissue culture-based invasion and cell-to-cell spread assays, and toxicity screens. Our observations of activity in these models for infection have validated our approach of targeting virulence regulation and have allowed us to identify a promising chemical scaffold from our HTS for hit-to-lead development. Interestingly, differential effects on invasion versus cell-to-cell spread suggest that the compounds' efficacies may depend, in part, on the specific promoter that VirF is recognizing.


Subject(s)
Anti-Bacterial Agents/pharmacology , High-Throughput Screening Assays/methods , Interferon Regulatory Factors/drug effects , Shigella flexneri/drug effects , Viral Proteins/drug effects , Virulence Factors , Algorithms , Anti-Bacterial Agents/isolation & purification , Data Interpretation, Statistical , Genes, Reporter/genetics , HeLa Cells , Humans , Indicators and Reagents , Microbial Sensitivity Tests , Plasmids/genetics , Reproducibility of Results , Shigella flexneri/genetics , Shigella flexneri/growth & development , Virulence/genetics , beta-Galactosidase/genetics
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