title: Identification of exosite-targeting inhibitors of anthrax lethal factor by high-throughput screening.
abstract: Protease inhibitor discovery has focused almost exclusively on compounds that bind to the active site. Inhibitors targeting protease exosites, regions outside of the active site that influence catalysis, offer potential advantages of increased specificity but are difficult to systematically discover. Here, we describe an assay suitable for detecting exosite-targeting inhibitors of the metalloproteinase anthrax lethal factor (LF) based on cleavage of a full-length mitogen-activated protein kinase kinase (MKK) substrate. We used this assay to screen a small-molecule library and then subjected hits to a secondary screen to exclude compounds that efficiently blocked cleavage of a peptide substrate. We identified a compound that preferentially inhibited cleavage of MKKs compared with peptide substrates and could suppress LF-induced macrophage cytolysis. This approach should be generally applicable to the discovery of exosite-targeting inhibitors of many additional proteases.
authors: Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520 USA.
authors: Ludovic
authors: L
authors: Bannwarth
authors: Allison B
authors: AB
authors: Goldberg
authors: Catherine
authors: C
authors: Chen
authors: Benjamin E
authors: BE
authors: Turk
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000378:metabolism*; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D057166:High-Throughput Screening Assays; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D011480:Protease Inhibitors / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D054852:Small Molecule Libraries / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011480:Protease Inhibitors; D054852:Small Molecule Libraries; C030325:anthrax toxin; D020929:Mitogen-Activated Protein Kinase Kinases; D008666:Metalloendopeptidases