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Antimicrob Agents Chemother. 2015 Nov 16;60(2):766-76. doi: 10.1128/AAC.02242-15. Print 2016 Feb.

Inhibition of Pseudomonas aeruginosa ExsA DNA-Binding Activity by N-Hydroxybenzimidazoles.

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Department of Microbiology, University of Iowa, Iowa City, Iowa, USA.
Department of Biochemistry and Program in Medicinal & Natural Products Chemistry, University of Iowa, Iowa City, Iowa, USA.
Paratek Pharmaceuticals, Boston, Massachusetts, USA.
Department of Microbiology, University of Iowa, Iowa City, Iowa, USA


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.

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