Development of a unique, improved alternative to the use of 32P-ATP and antibodies specific to the phospho-epitopes of the peptide subtrates in in vitro kinase assay of serine-threonine kinases is proposed. This novel assay approach for serine/threonine kinases uses ATP thiotriphosphate to thiophosphorylate substrates and a monoclonal antibody to detect the thiophosphate-epitope created in the kinase reaction. Initial research will involve optimization of ATP thiotriphosphate, enzyme, substrate, and antibody concentrations in the assay of three widely studied mitogen activated protein kinases (MAPKs), p38, JNK and ERK2. The system will be optimized using a colorimetric based detection system then converted to chemiluminescent-based detection for increased sensitivity. To validate the potential usefulness of the system to screen kinase inhibitors, we will assay p38 kinase in the presence of known inhibitors. The long-term objective of the research is to extend the system to the assay of other serine/threonine kinases and to commercialize kits facilitating the rapid, non-radioactive identification of compounds sought for potential use in the treatment of cancer, autoimmune, and neurological diseases.
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