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Updated: Aug 24, 2026
Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System
Published on: December 21, 2019
Discovery of novel inhibitors of Bcl-xL using multiple high-throughput screening platforms
Jie Qian1, Martin J Voorbach, Jeffrey R Huth
1Department of Biological Screening, Abbott Laboratories, Global Pharmaceutical Research and Development, Abbott Park, IL 60064, USA. jie.qian@abbott.com
Abstract:
Bcl-xL is a member of the Bcl-2 family of proteins that are implicated to play a vital role in several diseases including cancer. Bcl-xL suppresses apoptosis; thus the inhibition of Bcl-xL function could restore the apoptotic process. To identify antagonists of Bcl-xL function, two ultra-high-throughput screens were implemented. An activity assay utilized fluorescence polarization, based on the binding of fluorescein-labeled peptide [the BH3 domain of BAD protein (F-Bad 6)] to Bcl-xL. A 384-well plate assay with mixtures of 10 drug compounds per well, combined with a fast plate reader, resulted in a throughput of 46,080 data points/day. Utilizing this screening format, 370,400 compounds were screened in duplicate and 425 inhibitors with an IC(50) below 100 microM were identified. The second assay format, affinity selection/mass spectrometry (ASMS), used ultrafiltration to separate Bcl-xL binders from nonbinders in mixtures of 2400 compounds. The bound species were subsequently separated from the protein and analyzed by flow injection electrospray mass spectrometry. Utilizing the ASMS format, 263,382 compounds were screened in duplicate and 29 binders with affinities below 100 microM were identified. Two novel classes of Bcl-xL inhibitors were identified by both methods and confirmed to bind (13)C-labeled Bcl-xL using heteronuclear magnetic resonance spectroscopy.
Insights
Researchers screened hundreds of thousands of compounds to find new inhibitors of Bcl-xL, a protein involved in cancer. Two novel classes of Bcl-xL inhibitors were identified, offering potential therapeutic strategies.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Bcl-xL, a member of the Bcl-2 protein family, plays a critical role in disease, particularly cancer.
- Bcl-xL inhibits apoptosis, making its inhibition a potential strategy to restore programmed cell death in cancer treatment.
Purpose of the Study:
- To identify antagonists of Bcl-xL function through ultra-high-throughput screening.
- To discover novel classes of Bcl-xL inhibitors for potential therapeutic applications.
Main Methods:
- Two distinct ultra-high-throughput screening methods were employed: fluorescence polarization and affinity selection/mass spectrometry (ASMS).
- The fluorescence polarization assay screened 370,400 compounds using a fluorescein-labeled peptide (F-Bad 6) binding assay.
- The ASMS assay screened 263,382 compounds using ultrafiltration and mass spectrometry to identify Bcl-xL binders.
Main Results:
- The fluorescence polarization screen identified 425 inhibitors with IC(50) below 100 microM.
- The ASMS screen identified 29 binders with affinities below 100 microM.
- Both screening methods identified two novel classes of Bcl-xL inhibitors, confirmed by heteronuclear magnetic resonance spectroscopy.
Conclusions:
- Ultra-high-throughput screening effectively identified novel Bcl-xL inhibitors.
- The identified compounds represent promising leads for developing new cancer therapeutics by targeting Bcl-xL.
- The study validated two complementary screening approaches for identifying protein inhibitors.
