Development of high-throughput assay of lethal factor using native substrate.
The design of inhibitors for anthrax lethal factor (LF) is currently of interest as an approach for the treatment of anthrax because LF plays a major role in the cytotoxicity of target cells. LF is a zinc-dependent metalloprotease that specifically cleaves the mitogen-activated protein kinase kinase (MKK) family. Current assay systems for the screening of LF inhibitor use the optimized synthetic peptide coupled with various kinds of fluorophores, enabling fast, sensitive, and robust assays suited to high-throughput screening. However, evidence suggests that the regions beside the cleavage site are also involved in specificity and proteolytic activity of LF. In the current study, we tried to develop a high-throughput assay for LF activity based on native substrate, mitogen-activated ERK kinase 1 (MEK1). The assay system relies on the enhanced chemiluminescence signal resulting from a specific antibody against the C-terminal region of native substrate. A glutathione-coated multiwell plate was used as a solid support to immobilize the native substrate by its N-terminal glutathione-S-transferase moiety. Immobilized substrate increases the specificity and sensitivity of LF-catalyzed substrate hydrolysis compared with the solution phase assay. This assay system might be used to discover a wide spectrum of anthrax inhibitors.
Kim
Joungmok
J
Department of Chemistry, College of Natural Science, Hanyang University, Seoul 133-791, Republic of Korea.
Choi
Min-Kyung
MK
Koo
Bon-Sung
BS
Yoon
Moon-Young
MY
D000942:Antigens, Bacterial / Q000032:analysis* / Q000378:metabolism; D001408:Bacillus anthracis / Q000737:chemistry / Q000201:enzymology; D001427:Bacterial Toxins / Q000032:analysis* / Q000378:metabolism; D006868:Hydrolysis; D006877:Hydroxamic Acids / Q000737:chemistry; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D013379:Substrate Specificity
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D006877:Hydroxamic Acids; D010455:Peptides; C492740:anthrax LF protease inhibitor; C030325:anthrax toxin; D048369:MAP Kinase Kinase 1