Robust lanthanide-based assays for the detection of anti-apoptotic Bcl-2-family protein antagonists
Michele F Rega1, John C Reed, Maurizio Pellecchia
1Burnham Institute for Medical Research, 10901 North Torrey Pines Rd., La Jolla, CA 92037, USA.
Abstract:
Anti-apoptotic Bcl-2-family proteins (Bcl-2, Bcl-x(L), Bfl-1, Mcl-1, Bcl-W and Bcl-B) have been recently validated as drug discovery targets for cancer, owed to their ability to confer tumor resistance to chemotherapy or radiation. The anti-apoptotic activity of Bcl-2 proteins is due to their ability to heterodimerize with their pro-apoptotic counterparts (proteins such as Bad, Bim or Bid) via a conserved peptide region termed BH3. Thus, molecules that mimic pro-apoptotic BH3 domains represent a direct approach to overcoming the protective effects of anti-apoptotic proteins such as Bcl-2 and Bcl-x(L). Here, we report on the development and evaluation of two novel Lanthanide-based assays that are formatted for high-throughput screening of small molecules capable of antagonizing BH3-Bcl-2 interactions. The assay conditions, robustness and reproducibility (Z' factors) are described. These assays represent useful tools to enable further studies in the search for novel, safe and effective anti-cancer agents targeting Bcl-2-family proteins.
Insights
Novel lanthanide-based assays were developed to screen for small molecules that disrupt anti-apoptotic Bcl-2-family proteins. These assays aid in discovering new anti-cancer agents targeting Bcl-2 and Bcl-x(L) interactions.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Anti-apoptotic Bcl-2-family proteins promote cancer resistance to therapy.
- These proteins inhibit pro-apoptotic proteins via BH3 domain interactions.
- Targeting these interactions is a strategy for cancer drug development.
Purpose of the Study:
- To develop and evaluate novel lanthanide-based assays.
- To enable high-throughput screening of small molecules targeting BH3-Bcl-2 interactions.
- To identify novel anti-cancer agents.
Main Methods:
- Development of two novel lanthanide-based assays.
- Evaluation of assay conditions, robustness, and reproducibility (Z' factors).
- High-throughput screening format for small molecules.
Main Results:
- Successful development of two lanthanide-based assays.
- Characterization of assay performance, including Z' factors.
- Demonstration of assays as tools for screening BH3-Bcl-2 antagonists.
Conclusions:
- The developed assays are effective tools for screening.
- These assays facilitate the search for novel anti-cancer agents.
- Targeting Bcl-2-family proteins offers a promising therapeutic avenue.
