A scintillation proximity assay for histone demethylases

Area of Science:

• Biochemistry
• Molecular Biology
• Epigenetics

Background:

• Histone modifications, including methylation and demethylation, are crucial for chromatin dynamics and gene expression regulation.
• Lysine demethylases (KDMs) remove methyl groups from histones, and their dysregulation is linked to diseases like cancer.
• Existing high-throughput assays for KDM inhibitors are often fluorescence-based.

Purpose of the Study:

• To develop a robust coupled scintillation proximity assay (SPA) for three key lysine demethylases (KDMs): KDM1A, KDM3A, and KDM4A.
• To optimize enzyme activities and determine kinetic parameters for these KDMs using the developed SPA.
• To establish a reliable assay format suitable for high-throughput screening of KDM inhibitors.

Main Methods:

• A coupled SPA was designed where KDMs demethylate methylated peptides.
• A protein methyltransferase (PMT) then methylates the resulting peptide using tritiated S-(5'-adenosyl)-l-methionine.
• Assay conditions were optimized, and kinetic parameters were determined for KDM1A, KDM3A, and KDM4A.

Main Results:

• The coupled SPA demonstrated robust performance for KDM1A, KDM3A, and KDM4A.
• Optimized enzyme activities and kinetic parameters were successfully determined.
• The assay achieved high Z' factors (0.70-0.80) in a 384-well format, indicating suitability for screening.

Conclusions:

• The developed coupled SPA is a reliable and efficient tool for screening KDM activities.
• This assay facilitates the discovery of novel inhibitors targeting KDMs.
• The findings support the development of KDM-targeted therapeutics for diseases such as cancer.