Development of high-throughput assay of lethal factor using native substrate.

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Anthrax lethal factor (LF) is a key virulence factor responsible for anthrax cytotoxicity.
• LF is a zinc-dependent metalloprotease that cleaves mitogen-activated protein kinase kinase (MKK) family members.
• Existing high-throughput screening assays for LF inhibitors often use synthetic peptides, potentially missing inhibitors targeting substrate specificity.

Purpose of the Study:

• To develop a novel high-throughput assay for anthrax lethal factor (LF) activity.
• To utilize a native substrate, mitogen-activated ERK kinase 1 (MEK1), for improved inhibitor screening.
• To enhance assay specificity and sensitivity for discovering broad-spectrum anthrax inhibitors.

Main Methods:

• Developed a high-throughput assay for LF activity using immobilized mitogen-activated ERK kinase 1 (MEK1) on a glutathione-coated multiwell plate.
• Employed an enhanced chemiluminescence signal generated by an antibody against the C-terminal region of the native substrate.
• Compared the performance of the immobilized substrate assay with solution-phase assays.

Main Results:

• The assay system demonstrated increased specificity and sensitivity for detecting LF-catalyzed substrate hydrolysis compared to solution-phase assays.
• Immobilization of the native substrate (MEK1) enhanced the detection of LF activity.
• The developed assay is suitable for high-throughput screening.

Conclusions:

• The novel assay system utilizing immobilized native substrate (MEK1) provides a sensitive and specific method for screening anthrax lethal factor (LF) inhibitors.
• This approach may facilitate the discovery of a wider range of therapeutic agents against anthrax.
• The assay's reliance on native substrate characteristics could lead to inhibitors with improved efficacy and specificity.