Journal
ANALYTICAL BIOCHEMISTRY
Volume 307, Issue 1, Pages 70-75Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/S0003-2697(02)00028-3
Keywords
apoptosis; Bcl-x(L); fluorescence polarization assay; bad peptide; BH3 domain
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Antiapoptotic protein Bcl-x(L) has been demonstrated to play a very important role in a variety of diseases such as cancer. Its biological function can be inhibited by proapoptotic proteins such Bak, Bad, and Bax by forming complexes mediated primarily by the Bcl-2 homology 3 (BH3) domain. To facilitate drug discovery for Bcl-x(L) inhibitors, we have developed and optimized a fluorescence polarization assay based on the interaction between Bcl-x(L) and BH3 domain peptides. We observed that the fluorescein-labeled Bad BH3 peptide [NLWAAQRYGRELRRMSDK(fluorescein)FVD or fluorescent Bad peptide] generates best overall results. Fluorescent Bad peptide interacts strongly with Bcl-x(L) with a K-d of 21.48 nM. The assay is stable over a 24-h period and can tolerate the presence of dimethyl sulfoxide up to 8%. By using a competition assay, several peptides derived from the BH3 region of Bak, Bad, Bax, and Bcl-2 were investigated. Bad and Bak BH3 peptides compete efficiently with IC50 values of 0.048 and 1.14 muM, respectively, while the peptides from the BH3 region of Bcl-2 and Bax compete weakly. A mutated Bak peptide, which has been shown to be inactive for binding to Bcl-x(L), did not compete. The relative binding order of the peptides (Bad > Bak > Bcl-2 > Bax > mutated Bak) correlates well with previously published results. When tested in high-throughput formats, the assay has a signal-to-noise ratio of 15.37 and a Z' factor of at least 0.73. The plate-to-plate variability for free peptide control and bound peptide control is minimal. This validates the assay not only for investigating the nature of Bcl-x(L)-peptide interaction, but also for high-throughput screening of Bcl-x(L) inhibitors. (C) 2002 Elsevier Science (USA). All rights reserved.
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