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A generic high-throughput screening assay for kinases: protein kinase a as an example
Rommel Mallari1, Elissa Swearingen, Wei Liu
1Lead Discovery Department, AGY Therapeutics, Inc., 290 Utah Ave., South San Francisco, CA 94080, USA.
Journal of Biomolecular Screening
|July 8, 2003
Summary
A novel scintillation proximity assay was developed for high-throughput screening of protein kinase inhibitors. This assay uses a biotinylated peptide captured on beads, showing high signal-to-noise ratios for drug discovery.
Area of Science:
- Biochemistry
- Assay Development
- Drug Discovery
Background:
- Protein kinases are crucial drug targets.
- High-throughput screening (HTS) is essential for identifying kinase inhibitors.
- Existing HTS assays may have limitations.
Purpose of the Study:
- To develop a generic, robust HTS assay for protein kinases.
- To optimize and validate the assay for screening chemical libraries.
- To assess the assay's performance using key statistical parameters.
Main Methods:
- Utilized scintillation proximity assay (SPA) technology.
- Employed a biotinylated (33)P-peptide product captured on polylysine Ysi beads via avidin.
- Optimized assay conditions and validated in 384-well microplates.
Main Results:
- Demonstrated linear increase in scintillation signal with avidin concentration.
- Achieved signal-to-noise ratios of 5- to 9-fold in pilot screens.
- Observed Z' factors ranging from 0.6 to 0.8, indicating assay robustness.
Conclusions:
- The developed SPA is suitable for HTS of protein kinase inhibitors.
- The assay's design facilitates efficient product capture and signal generation.
- This method enables effective screening of chemical libraries for novel kinase-targeting drugs.