Kinetic assay for characterization of spleen tyrosine kinase activity and inhibition with recombinant kinase and crude cell lysates.

Spleen tyrosine kinase (Syk) is involved in the activation of cells implicated in allergic or autoimmune diseases and certain cancers. Therefore, Syk inhibitors may prove to be effective in treating diseases where Syk activity or expression is increased or deregulated. We developed a continuous and direct (noncoupled) fluorescence intensity assay for measuring Syk activity using purified recombinant enzyme or crude lysates generated from anti-immunoglobulin M (IgM) antibody-treated RAMOS cells. The assay is based on the chelation-enhanced fluorophore 8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline (referred to as Sox), which has been incorporated into a peptide substrate selected for robust detection of Syk activity. This homogeneous assay is simple to use, provides considerably more information, and has been adapted to a 384-well, low-volume microtiter plate format that can be used for the high-throughput identification and kinetic characterization of Syk inhibitors. The assay can be performed with a wide range of adenosine triphosphate (ATP) concentrations and, therefore, can be used to analyze ATP-competitive and ATP-noncompetitive/allosteric kinase inhibitors. Measurement of Syk activity in RAMOS crude cell lysates or immunoprecipitation (IP) capture formats may serve as a physiologically more relevant enzyme source. These Sox-based continuous and homogeneous assays provide a valuable set of tools for studying Syk signaling and for defining inhibitors that may be more effective in controlling disease.