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Updated: Aug 16, 2026
Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System
Published on: December 21, 2019
A new assay for the discovery of Bcl-XL inhibitors
Cristina Pisoni1, Guido Cimoli, Anna Resconi
1Vicuron Pharmaceuticals Italy S.r.l., via R. Lepetit 34, 21040 Gerenzano, Italy.
Abstract:
The Bcl-2 family of antiapoptotic proteins is commonly over expressed in many types of human cancer and remains one of the few validated targets. Antiapoptotic family proteins such as Bcl-2 and Bcl-XL function, at least in part, by binding proapoptotic members such as Bax and Bak and thereby prevent release of the apoptotic cascade of events. "BH3-only" members of the family disrupt this interaction by binding, via their BH3 domain, to a hydrophobic pocket on the surface of the antiapoptotic members. Disruption of heterodimerization could be used to modulate cell death reinstating apoptosis in cancer cells. An affinity displacement assay based on Bcl-XL/BH3 interaction has been developed. This assay makes use of soluble His-tagged Bcl-XL and fluorescein tagged BH3. Binding is measured as fluorescence associated with magnetic beads. The assay was miniaturized to 96-well microtiter plates and can be employed in high throughput screening (HTS), in addition it is robust enough to be applied to microbial fermentation extracts.
Insights
Researchers developed a new assay to screen for drugs targeting antiapoptotic proteins like Bcl-2, which are overexpressed in cancer. This high-throughput screening method can help identify compounds that reinstate cancer cell death.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- The Bcl-2 protein family, particularly antiapoptotic members like Bcl-2 and Bcl-XL, are frequently overexpressed in human cancers.
- These proteins inhibit apoptosis by binding to proapoptotic proteins (e.g., Bax, Bak), thus preventing programmed cell death.
- BH3-only proteins disrupt this interaction, offering a potential therapeutic strategy to restore apoptosis in cancer cells.
Purpose of the Study:
- To develop a robust assay for screening compounds that disrupt the Bcl-XL/BH3 interaction.
- To enable high-throughput screening (HTS) for novel cancer therapeutics targeting the Bcl-2 family.
Main Methods:
- Development of an affinity displacement assay utilizing soluble His-tagged Bcl-XL and fluorescein-tagged BH3.
- Measurement of binding through fluorescence associated with magnetic beads.
- Miniaturization of the assay to 96-well microtiter plates for HTS compatibility.
Main Results:
- A functional affinity displacement assay was successfully developed.
- The assay is suitable for high-throughput screening (HTS) of potential anti-cancer agents.
- The assay demonstrates robustness for application with microbial fermentation extracts.
Conclusions:
- The developed Bcl-XL/BH3 affinity displacement assay is a valuable tool for identifying modulators of apoptosis.
- This assay facilitates the discovery of novel therapeutics aimed at reinstating cancer cell death by targeting antiapoptotic proteins.
