A SCREENING METHOD OF SH2 DOMAIN LIGANDS AND BLOCKERS USING A SOLID-PHASE BINDING

We have developed a high throughput screening method for SH2 domain bi nding ligands and blockers. This method measures directly the binding of a H-3-labeled phosphopeptide derived from the sequence around tyros ine317 in the human Shc (SpYVNVK) to the SH2 domain of Grb2, which is precoated as glutathione S-transferase fusion proteins on solid phase. The optimum concentration for the fusion protein coating was 300 ng/1 00 mu l/well for SH2 domain binding. Although an 8-h incubation at 4 d egrees C for the coating of fusion protein was required to reach a max imum binding, even a 2-h coating produced 84% of the maximum binding. Saturation of ligand peptide binding in our assay system was observed at 10 pmol/well for the SH2 domain. However, 2 pmol/well showed consis tent and reproducible results for the binding when the incubations wer e performed for 8 h at 4 degrees C. Competitive binding inhibition stu dies with various unlabeled phosphopeptides imply that the binding ass ay is highly specific to peptide sequences and able to screen possible ligands or blockers of signal transduction pathway mediated by Grb2 S H2 binding. In conclusion, our new method for SH2 domain binding is ea sy, rapid, and most of all inexpensive. These advantages over existing assay methods make this method especially suitable for a high through put application, such as the screening for anticancer drug candidates. (C) 1997 Elsevier Science Ireland Ltd.