title: Fragment screening using capillary electrophoresis (CEfrag) for hit identification of heat shock protein 90 ATPase inhibitors.
abstract: CEfrag is a new fragment screening technology based on affinity capillary electrophoresis (ACE). Here we report on the development of a mobility shift competition assay using full-length human heat shock protein 90α (Hsp90α), radicicol as the competitor probe ligand, and successful screening of the Selcia fragment library. The CEfrag assay was able to detect weaker affinity (IC(50) >500 µM) fragments than were detected by a fluorescence polarization competition assay using FITC-labeled geldanamycin. The binding site of selected fragments was determined by co-crystallization with recombinant Hsp90α N-terminal domain and X-ray analysis. The results of this study confirm that CEfrag is a sensitive microscale technique enabling detection of fragments binding to the biological target in near-physiological solution.
authors.affiliation: Discovery, Selcia Ltd, Fyfield Business and Research Park, Ongar, UK. carol.austin@selcia.com
authors.forename: Carol
authors.initials: C
authors.lastname: Austin
authors.forename: Simon N
authors.initials: SN
authors.lastname: Pettit
authors.forename: Sharon K
authors.initials: SK
authors.lastname: Magnolo
authors.forename: Jonathan
authors.initials: J
authors.lastname: Sanvoisin
authors.forename: Wenjie
authors.initials: W
authors.lastname: Chen
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Wood
authors.forename: Lauren D
authors.initials: LD
authors.lastname: Freeman
authors.forename: Reuben J
authors.initials: RJ
authors.lastname: Pengelly
authors.forename: Dallas E
authors.initials: DE
authors.lastname: Hughes
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D055808:Drug Discovery; D004353:Drug Evaluation, Preclinical; D019075:Electrophoresis, Capillary; D024202:Electrophoretic Mobility Shift Assay; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D018942:Macrolides / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D054852:Small Molecule Libraries
chemical_list: D018841:HSP90 Heat-Shock Proteins; D018942:Macrolides; D054852:Small Molecule Libraries; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C035359:monorden