title: Yeast-hybrid based high-throughput assay for identification of anthrax lethal factor inhibitors.
abstract: Inhibitors of anthrax lethal factor (LF) are currently being sought as effective therapeutics for the treatment of anthrax. Here we report a novel screening approach for inhibitors of LF, a yeast-hybrid-based assay system in which the expression of reporter genes from a Gal4 promoter is repressed by LF proteolytic activity. Yeast cells were co-transformed with LF and a chimeric transcription factor that contains an LF substrate sequence inserted between the DNA-binding and activation domains of Gal4. In the resulting yeast cells, LF cleaves the substrate, thus inactivating the chimeric Gal4 and resulting in lack of expression of reporter genes. Compounds that inhibit LF cleavage of its substrate are identified by changes in reporter gene activity. Relative to in vitro screens for inhibitors of LF proteolytic activity, this screen has the advantage of excluding compounds that are toxic or non-permeable to eukaryotic cells. Additionally, the screen has the advantage of being fast, easy and cheap because exogenous LF and substrate are not needed. An initial chemical library screen with this system has identified four candidate inhibitors which were confirmed to inhibit LF protease activity in an in vitro assay. Furthermore, FBS-00831, one of the compounds identified, protects Raw 264.7 macrophages from anthrax lethal toxin and the possible binding site on LF was also evaluated by molecular docking.
authors.affiliation: Department of Chemistry and Research Institute of Natural Sciences, Hanyang University, Seoul 133-761, South Korea.
authors.forename: Joungmok
authors.initials: J
authors.lastname: Kim
authors.forename: Hae-Chul
authors.initials: HC
authors.lastname: Park
authors.forename: Vinayakumar
authors.initials: V
authors.lastname: Gedi
authors.forename: Hye-Yeon
authors.initials: HY
authors.lastname: Park
authors.forename: Arthur G
authors.initials: AG
authors.lastname: Roberts
authors.forename: William M
authors.initials: WM
authors.lastname: Atkins
authors.forename: Moon-Young
authors.initials: MY
authors.lastname: Yoon
mesh_terms: D000818:Animals; D000881:Anthrax / Q000188:drug therapy*; D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000201:enzymology*; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors*; D004268:DNA-Binding Proteins / Q000378:metabolism; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays; D006801:Humans; D008264:Macrophages / Q000187:drug effects; D051379:Mice; D011480:Protease Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D011753:Pyrones / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D054852:Small Molecule Libraries; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D004268:DNA-Binding Proteins; C555825:FBS 00831; C052130:GAL4 protein, S cerevisiae; D011480:Protease Inhibitors; D011753:Pyrones; D029701:Saccharomyces cerevisiae Proteins; D054852:Small Molecule Libraries; D013438:Sulfhydryl Compounds; D014157:Transcription Factors; C030325:anthrax toxin