Journal article
Inhibition of Pseudomonas aeruginosa ExsA DNA-Binding Activity by N-Hydroxybenzimidazoles
Antimicrobial agents and chemotherapy, Vol.60(2), pp.766-776
02/2016
DOI: 10.1128/AAC.02242-15
PMCID: PMC4750669
PMID: 26574012
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.
Details
- Title: Subtitle
- Inhibition of Pseudomonas aeruginosa ExsA DNA-Binding Activity by N-Hydroxybenzimidazoles
- Creators
- Anne E Marsden - Department of Microbiology, University of Iowa, Iowa City, Iowa, USAJessica M King - Department of Microbiology, University of Iowa, Iowa City, Iowa, USAM Ashley Spies - Department of Biochemistry and Program in Medicinal & Natural Products Chemistry, University of Iowa, Iowa City, Iowa, USAOak K Kim - Paratek Pharmaceuticals, Boston, Massachusetts, USATimothy L Yahr - Department of Microbiology, University of Iowa, Iowa City, Iowa, USA tim-yahr@uiowa.edu
- Resource Type
- Journal article
- Publication Details
- Antimicrobial agents and chemotherapy, Vol.60(2), pp.766-776
- Publisher
- United States
- DOI
- 10.1128/AAC.02242-15
- PMID
- 26574012
- PMCID
- PMC4750669
- ISSN
- 0066-4804
- eISSN
- 1098-6596
- Grant note
- AI055042 / NIAID NIH HHS R01 AI055042 / NIAID NIH HHS T32 AI07511 / NIAID NIH HHS P30 DK054759 / NIDDK NIH HHS
- Language
- English
- Date published
- 02/2016
- Academic Unit
- Microbiology and Immunology; Pharmaceutical Sciences and Experimental Therapeutics; Biochemistry and Molecular Biology; Medicinal and Natural Products Chemistry
- Record Identifier
- 9984001208202771
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