Compounds capable of generating singlet oxygen represent a source of artifactual data in scintillation proximity assays measuring phosphopeptide binding to SH2 domains.
We developed scintillation proximity assays (SPA) to discover compounds which inhibit phosphopeptide binding to Src homology 2 (SH2) domain proteins Grb2 and Syk. An assay artifact is reported here as a caveat to others. The SPA used an antibody to couple glutathione-S-transferase SH2 domain fusion proteins to scintillant beads coated with protein A. A pyrazoloquinolone and indolocarbazole inhibited [3H]phosphopeptide binding in both assays. Their potency in the SPA increased with prolonged (2 to 24 h) assay exposure to ambient light. They were inactive in absence of light and in an alternate binding assay. Both compounds absorbed visible light and generated singlet oxygen based on 2-methylfuran-trapping experiments. Their inhibitory activity was suppressed by the singlet oxygen scavengers sodium azide and dithiothreitol. The results suggest that compounds, not previously considered photosensitizers, generated enough singlet oxygen to damage oxidant-sensitive SPA components. Therefore, this SPA should be protected from light to minimize occurrence of false positives.
D048868:Adaptor Proteins, Signal Transducing; D016477:Artifacts; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000378:metabolism; D005189:False Positive Reactions; D051380:GRB2 Adaptor Protein; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008027:Light; D010100:Oxygen / Q000378:metabolism*; D010748:Phosphopeptides / Q000378:metabolism*; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D012588:Scintillation Counting / Q000379:methods / Q000592:standards*; D026082:Singlet Oxygen; D019311:Staurosporine / Q000031:analogs & derivatives / Q000494:pharmacology; D000072377:Syk Kinase; D013997:Time Factors; D014407:Tumor Cells, Cultured; D018909:src Homology Domains