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Src homology‐2 domain binding assays by scintillation proximity and surface plasmon resonance
Author(s) -
Mandine Eliane,
Gofflo Dominique,
JeanBaptiste Véronique,
Sarubbi Edoardo,
Touyer Gaetan,
Deprez Pierre,
Lesuisse Dominique
Publication year - 2001
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/jmr.542
Subject(s) - surface plasmon resonance , proto oncogene tyrosine protein kinase src , ligand binding assay , affinities , sh2 domain , chemistry , assay sensitivity , ligand (biochemistry) , drug discovery , biochemistry , combinatorial chemistry , computational biology , biophysics , nanotechnology , biology , receptor , materials science , medicine , alternative medicine , pathology , nanoparticle
Various SH2 competitive binding assays, based on different techniques, have been described in the literature to identify and characterize SH2 ligands. The consideration that most reported methods show experimental limitations associated with assay parameters has prompted us to base our Src‐SH2 inhibitor discovery program on the use of two different assays. In this study, two conceptually different biochemical methods designed to discover Src‐SH2 inhibitors, respectively scintillation proximity assay (SPA) and surface plasmon resonance (SPR), have been evaluated and compared. For its high sensitivity and adaptability to automation SPA was chosen for high capacity screening (primary screen), whereas SPR was used for hits confirmation (secondary screening). However with the drastic improvement of inhibitor affinities, the limit of sensitivity was rapidly reached for the SPR assay based on the canonical pYEEI ligand. The substitution of the natural, monophosphorylated peptide ligand with a triphosphorylated peptide has allowed us to remarkably increase its sensitivity, so that molecules with nanomolar affinities could be easily differentiated in terms of IC 50 ranking. Such a new, improved SPR assay can be of great interest for the study of high affinity ligands of different SH2‐based drug targets. Copyright © 2001 John Wiley & Sons, Ltd.