title: Inhibition of Pseudomonas aeruginosa ExsA DNA-Binding Activity by N-Hydroxybenzimidazoles.
abstract: The Pseudomonas aeruginosa type III secretion system (T3SS) is a primary virulence determinant and a potential target for antivirulence drugs. One candidate target is ExsA, a member of the AraC family of DNA-binding proteins required for expression of the T3SS. A previous study identified small molecules based on an N-hydroxybenzimidazole scaffold that inhibit the DNA-binding activity of several AraC proteins, including ExsA. In this study, we further characterized a panel of N-hydroxybenzimidazoles. The half-maximal inhibitory concentrations (IC50s) for the tested N-hydroxybenzimidazoles ranged from 8 to 45 μM in DNA-binding assays. Each of the N-hydroxybenzimidazoles protected mammalian cells from T3SS-dependent cytotoxicity, and protection correlated with reduced T3SS gene expression in a coculture infection model. Binding studies with the purified ExsA DNA-binding domain (i.e., lacking the amino-terminal self-association domain) confirmed that the activity of N-hydroxybenzimidazoles results from interactions with the DNA-binding domain. The interaction is specific, as an unrelated DNA-binding protein (Vfr) was unaffected by N-hydroxybenzimidazoles. ExsA homologs that control T3SS gene expression in Yersinia pestis, Aeromonas hydrophila, and Vibrio parahaemolyticus were also sensitive to N-hydroxybenzimidazoles. Although ExsA and Y. pestis LcrF share 79% sequence identity in the DNA-binding domain, differential sensitivities to several of the N-hydroxybenzimidazoles were observed. Site-directed mutagenesis based on in silico docking of inhibitors to the DNA-binding domain, and on amino acid differences between ExsA and LcrF, resulted in the identification of several substitutions that altered the sensitivity of ExsA to N-hydroxybenzimidazoles. Development of second-generation compounds targeted to the same binding pocket could lead to drugs with improved pharmacological properties.
authors.affiliation: Department of Microbiology, University of Iowa, Iowa City, Iowa, USA.
authors.forename: Anne E
authors.initials: AE
authors.lastname: Marsden
authors.affiliation: Department of Microbiology, University of Iowa, Iowa City, Iowa, USA.
authors.forename: Jessica M
authors.initials: JM
authors.lastname: King
authors.affiliation: Department of Biochemistry and Program in Medicinal & Natural Products Chemistry, University of Iowa, Iowa City, Iowa, USA.
authors.forename: M Ashley
authors.initials: MA
authors.lastname: Spies
authors.affiliation: Paratek Pharmaceuticals, Boston, Massachusetts, USA.
authors.forename: Oak K
authors.initials: OK
authors.lastname: Kim
authors.affiliation: Department of Microbiology, University of Iowa, Iowa City, Iowa, USA tim-yahr@uiowa.edu.
authors.forename: Timothy L
authors.initials: TL
authors.lastname: Yahr
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000235:genetics; D001562:Benzimidazoles / Q000494:pharmacology*; D001665:Binding Sites; D002373:Cyclic AMP Receptor Protein / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000378:metabolism / Q000472:pathogenicity; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators / Q000037:antagonists & inhibitors* / Q000235:genetics; D000069296:Type III Secretion Systems / Q000037:antagonists & inhibitors*; D015010:Yersinia pestis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D001562:Benzimidazoles; D002373:Cyclic AMP Receptor Protein; D004268:DNA-Binding Proteins; C095093:ExsA protein, Pseudomonas aeruginosa; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D000069296:Type III Secretion Systems; C091090:Vfr protein, Pseudomonas aeruginosa; C075515:lcrF protein, Yersinia pestis