Src homology-2 domain binding assays by scintillation proximity and surface plasmon resonance

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 inhib itor discovery program on the use of two different assays. In this study, two conceptually different biochemical methods designed to d iscover Src-SH2 inhibitors, respectively scintillation proximity assay (SPA ) and surface plasmon resonance (SPR), have been evaluated and compared. Fo r its high sensitivity and adaptability to automation SPA was chosen for hi gh capacity screening (primary screen), whereas SPR was used for hits confi rmation (secondary screening). However with the drastic improvement of inhi bitor 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 all owed us to remarkably increase its sensitivity, so that molecules with nano molar affinities could be easily differentiated in terms of IC50 ranking. S uch a new, improved SPR assay can be of great interest for the study of hig h affinity ligands of different SH2-based drug targets. Copyright (C) 2001 John Wiley & Sons, Ltd.