ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 328 (2004) 131–138 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2003.12.034 Discovery of novel inhibitors of Bcl-xL using multiple high-throughput screening platforms Jie Qian, a,¤ Martin J. Voorbach, b JeVrey R. Huth, c Michael L. Coen, a Haichao Zhang, d Shi-Chung Ng, d Kenneth M. Comess, a Andrew M. Petros, c Saul H. Rosenberg, d Usha Warrior, a and David J. Burns a a Department of Biological Screening, Abbott Laboratories, Global Pharmaceutical Research and Development, Abbott Park, IL 60064, USA b Metabolic Disease Research, Abbott Laboratories, Global Pharmaceutical Research and Development, Abbott Park, IL 60064, USA c Department of Structural Biology, Abbott Laboratories, Global Pharmaceutical Research and Development, Abbott Park, IL 60064, USA d Cancer Research, Abbott Laboratories, Global Pharmaceutical Research and Development, Abbott Park, IL 60064, USA Received 16 October 2003 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 Xuorescence polarization, based on the binding of Xuorescein-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 M were identiWed. The second assay format, aYnity selection/mass spectrometry (ASMS), used ultraWltration to separate Bcl-xL binders from nonbinders in mixtures of 2400 compounds. The bound species were subsequently separated from the protein and analyzed by Xow injection elec- trospray mass spectrometry. Utilizing the ASMS format, 263,382 compounds were screened in duplicate and 29 binders with aYni- ties below 100 M were identiWed. Two novel classes of Bcl-xL inhibitors were identiWed by both methods and conWrmed to bind 13 C- labeled Bcl-xL using heteronuclear magnetic resonance spectroscopy.  2004 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Bcl-xL; Fluorescence polarization assay; AYnity selection/mass spectrometry The Bcl-2 family of proteins has been demonstrated to play a key role in the mechanism of apoptosis. There are three classes of proteins that play crucial roles in this complex process: the BH3 domain-only proteins, the proapoptotic proteins, and the antiapoptotic proteins. Bim, Bid, and Bad, the BH3 domain-only proteins, act as triggers to stimulate the multidomain proapoptotic pro- teins Bax and Bak [1]. Bcl-2 and Bcl-xL, by sequestering BH3 domain-only proteins, prevent apoptosis [2,3]. It is apparent that heterodimerization between members of the Bcl-2 family of proteins is key to the regulation of cell death [4]. Complex formation was also illustrated by structural studies using NMR and X-ray crystallography [5–10]. The structure of Bcl-xL consists of eight  helices [8]. A hydrophobic groove, formed by three Bcl-2 homology regions (BH1, BH2, and BH3) on the surface, binds tightly with the Bad 26-mer with a K D of »6 nM [11,12]. Interestingly, Bcl-xL can form an ion channel in synthetic lipid membrane, which led to the discovery that Bcl-xL regulates apoptosis by two distinct mecha- nisms, termed heterodimerization dependent and ion- channel dependent [13,14]. Dysregulation of these proteins results in cancer or degenerative conditions, depending on which apoptosis- related protein is dominant in the biochemical balance. Bcl-xL, a close homolog of Bcl-2, plays an important role in cancer and resistance of cancer cells to cytotoxic chemotherapeutic drugs and  irradiation [15–18]. The functional inhibition of Bcl-xL should restore the ¤ Corresponding author. Fax: 1-847-938-3780. E-mail address: jie.qian@abbott.com (J. Qian).