Discovery of novel inhibitors of Bcl-xL using multiple high-throughput screening platforms

• DOI: 10.1016/j.ab.2003.12.034
• Publication Date: 2004-04-17T14:17:16Z
• Authors: Jie Qian; Martin J Voorbach; Jeffrey R Huth; Michael L Coen; Haichao Zhang; Shi-Chung Ng; Kenneth M Comess; Andrew M Petros; Saul H Rosenberg; Usha Warrior; David J Burns
• Keywords: 0301 basic medicine; Magnetic Resonance Spectroscopy; Titrimetry; bcl-X Protein; Apoptosis; Fluorescence Polarization; Binding, Competitive; Mass Spectrometry; 3. Good health; Inhibitory Concentration 50; 03 medical and health sciences; Pharmaceutical Preparations; Proto-Oncogene Proteins c-bcl-2; Combinatorial Chemistry Techniques; Dimethyl Sulfoxide; Carrier Proteins

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.