title: A new assay for the discovery of Bcl-XL inhibitors.
abstract: 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.
authors.affiliation: Vicuron Pharmaceuticals Italy S.r.l., via R. Lepetit 34, 21040 Gerenzano, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Pisoni
authors.forename: Guido
authors.initials: G
authors.lastname: Cimoli
authors.forename: Anna
authors.initials: A
authors.lastname: Resconi
authors.forename: Daniele
authors.initials: D
authors.lastname: Losi
authors.forename: Rolando
authors.initials: R
authors.lastname: Lorenzetti
authors.forename: Silvio
authors.initials: S
authors.lastname: Parodi
authors.forename: Lucia
authors.initials: L
authors.lastname: Carrano
mesh_terms: D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001419:Bacteria / Q000737:chemistry; D001667:Binding, Competitive; D001681:Biological Assay; D019281:Dimerization; D015195:Drug Design; D019793:Fluorescein; D005456:Fluorescent Dyes; D005658:Fungi / Q000737:chemistry; D006639:Histidine; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods; D051020:bcl-X Protein / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; C494811:BCL2L1 protein, human; C474007:Bax protein (53-86); D005456:Fluorescent Dyes; D008024:Ligands; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D051020:bcl-X Protein; D006639:Histidine; D019793:Fluorescein