Fluorescence-quenching screening of protein kinase C ligands with an environmentally sensitive fluorophore

A novel fluorescence-quenching screening method for protein kinase C (PKC) ligands was developed utilizing solvatochromic fluorophores. Solvatochromic dyes, highly sensitive to the presence or the absence of competitive ligands in their binding to the C1b domain of PKCδ (δC1b), were combined with a known pharmacophoric moiety of 1,2-diacylglycerol (DAG) lactones, PKC ligands. Addition of δC1b to the fluorescent compounds caused a gradual increase in the fluorescent intensity in proportion to the increase of δC1b. As a competitive ligand was added to the complex of δC1b domain and fluorescent compounds, a gradual decrease in the fluorescent intensity was observed. The relative binding affinities of known ligands were successfully determined by this fluorescent method and corresponded well to the K(i) values measured by a radioisotope method. These results indicate that washing, which is a laborious step in binding evaluations, is not required for this environmentally sensitive fluorophore based system. Screening with the system was performed for 2560 preselected library compounds with possible pharmacophores, and some lead compounds were found. This fluorescence-based method could be applied widely to known ligand-receptor combinations.