Identification of inhibitors of a bacterial sigma factor using a new high-throughput screening assay.

Autor:	El-Mowafi SA; Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA., Sineva E; Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA., Alumasa JN; Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA., Nicoloff H; Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA., Tomsho JW; Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA., Ades SE; Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA., Keiler KC; Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA kkeiler@psu.edu.
Jazyk:	angličtina
Zdroj:	Antimicrobial agents and chemotherapy [Antimicrob Agents Chemother] 2015 Jan; Vol. 59 (1), pp. 193-205. Date of Electronic Publication: 2014 Oct 20.
DOI:	10.1128/AAC.03979-14
Abstrakt:	Gram-negative bacteria are formidable pathogens because their cell envelope presents an adaptable barrier to environmental and host-mediated challenges. The stress response pathway controlled by the alternative sigma factor σ(E) is critical for maintenance of the cell envelope. Because σ(E) is required for the virulence or viability of several Gram-negative pathogens, it might be a useful target for antibiotic development. To determine if small molecules can inhibit the σ(E) pathway, and to permit high-throughput screening for antibiotic lead compounds, a σ(E) activity assay that is compatible with high-throughput screening was developed and validated. The screen employs a biological assay with positive readout. An Escherichia coli strain was engineered to express yellow fluorescent protein (YFP) under negative regulation by the σ(E) pathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify σ(E) pathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds σ(E), inhibits RNA polymerase holoenzyme formation, and inhibits σ(E)-dependent transcription in vitro. These results demonstrate that alternative sigma factors can be inhibited by small molecules and enable high-throughput screening for inhibitors of the σ(E) pathway. (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
Databáze:	MEDLINE
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