A new assay for the discovery of Bcl-XL inhibitors

The Bcl-2 family of antiapoptotic proteins is commonly over expressed in many types of human cancer and remains one of the few validated targets. Antiapoptotic family proteins such as Bcl-2 and Bcl-XL function, at least in part, by binding proapoptotic members such as Bax and Bak and thereby prevent...

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Bibliographic Details
Published in:Farmaco (Società chimica italiana : 1989) 2005-11, Vol.60 (11), p.938-943
Main Authors: Pisoni, Cristina, Cimoli, Guido, Resconi, Anna, Losi, Daniele, Lorenzetti, Rolando, Parodi, Silvio, Carrano, Lucia
Format: Article
Language:English
Subjects:
Anti-Bacterial Agents - isolation & purification
Anti-Bacterial Agents - pharmacology
Apoptosis
Apoptosis - drug effects
Bacteria - chemistry
bcl-X Protein - antagonists & inhibitors
bcl-X Protein - genetics
bcl-X Protein - metabolism
Bcl-Xl
Binding, Competitive
Biological Assay
Dimerization
Drug Design
Fluorescein
Fluorescent Dyes
Fungi - chemistry
Histidine
Humans
In Vitro Techniques
Ligands
Peptide Fragments
Proto-Oncogene Proteins
Recombinant Proteins - antagonists & inhibitors
Reproducibility of Results
Screening
Spectrometry, Fluorescence - methods
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Summary:The Bcl-2 family of antiapoptotic proteins is commonly over expressed in many types of human cancer and remains one of the few validated targets. Antiapoptotic family proteins such as Bcl-2 and Bcl-XL function, at least in part, by binding proapoptotic members such as Bax and Bak and thereby prevent release of the apoptotic cascade of events. “BH3-only” members of the family disrupt this interaction by binding, via their BH3 domain, to a hydrophobic pocket on the surface of the antiapoptotic members. Disruption of heterodimerization could be used to modulate cell death reinstating apoptosis in cancer cells. An affinity displacement assay based on Bcl-XL/BH3 interaction has been developed. This assay makes use of soluble His-tagged Bcl-XL and fluorescein tagged BH3. Binding is measured as fluorescence associated with magnetic beads. The assay was miniaturized to 96-well microtiter plates and can be employed in high throughput screening (HTS), in addition it is robust enough to be applied to microbial fermentation extracts.
ISSN:0014-827X
1879-0569
DOI:10.1016/j.farmac.2005.06.017