Development and Validation of a Fluorescence Technology for both Primary and Secondary Screening of Kinases That Facilitates Compound Selectivity and Site-Specific Inhibitor Determination

Abstract

The IQ^® Technology has been developed to serve as a homogeneous, universal detection platform for HTS of kinases and phosphatases. The technology is a direct, noncompetitive assay format that does not require antibodies or radioactive reagents to measure phosphorylation state. Fluorophore-labeled peptides are used as enzyme substrates, and kinase or phosphatase activity is quantitated by direct measurement of the phosphorylation state of the substrate. Phosphorylation is measured by the change in fluorescence intensity that occurs when a proprietary iron-containing compound binds specifically to phosphoryl groups on peptides. This change in observed fluorescence is proportional to the extent of phosphorylation of the fluorophore-labeled peptide. The technology provides a universal method that can be used with any peptide sequence and is insensitive to high concentrations of ATP. Inhibition at the ATP-binding site versus the phosphorylation site can be differentiated and compound selectivity identified using the same detection method as in the primary screen. The technology has been tested against a large number of detergents, organics, and other reagents found in reaction mixtures, and the detection method eliminates common issues associated with fluorescent and chromogenic compounds. The technology has been formatted for 96-, 384-, and 1,536-well microplate formats, and a representativeZ′ value of 0.7 was obtained. IC₅₀ values generated using this platform correlate with previously reported values, and screening of a small compound library was performed to evaluate the assay further.