Discovery of novel inhibitors of Bcl-xL using multiple high-throughput screening platforms.
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.
Qian
Jie
J
Department of Biological Screening, Abbott Laboratories, Global Pharmaceutical Research and Development, Abbott Park, IL 60064, USA. jie.qian@abbott.com
Voorbach
Martin J
MJ
Huth
Jeffrey R
JR
Coen
Michael L
ML
Zhang
Haichao
H
Ng
Shi-Chung
SC
Comess
Kenneth M
KM
Petros
Andrew M
AM
Rosenberg
Saul H
SH
Warrior
Usha
U
Burns
David J
DJ
D017209:Apoptosis / Q000235:genetics / Q000502:physiology; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D005454:Fluorescence Polarization; D020128:Inhibitory Concentration 50; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D004364:Pharmaceutical Preparations / Q000032:analysis*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors*; D017186:Titrimetry; D051020:bcl-X Protein
D002352:Carrier Proteins; D004364:Pharmaceutical Preparations; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D004121:Dimethyl Sulfoxide