AMP-activated protein kinase (AMPK) is a serine/threonine kinase that is a member of a metabolite sensing protein kinase family. AMPK is involved in controlling cellular metabolism and cell proliferation and when activated has been found to be a potent tumor suppressor. Despite a lot of progress in the discovery of therapeutic agents, a pharmacodynamic assay for AMPK is missing. In this proposed research, BioAssay Systems aims to develop a rapid, sensitive, robust and rigorous assay for AMPK activity in tissue extract. In the Phase I period, BioAssay Systems will select a suitable pan antibody and coat it onto 96-well plates. The immobilized antibody will capture all AMPK protein and isolate it from any potentially interfering enzymes such as other kinases and phosphatases. The captured AMPK remains active so that its activity will be directly measured using a fluorescence technology. At the end of the Phase I period, BioAssay Systems will have established experimental conditions and characterized assay performance including detection limit, reagent stability, reproducibility, variation and accuracy. The SOP of the research pharmacodynamic assay for AMPK will be delivered to NCI. In the Phase II period, BioAssay Systems will further validate the assay in various normal and multiple tumor tissues. Studies will be performed to correlate results in tumor versus surrogate tissues such as blood, serum or plasma. Correlation studies will also be compared in human versus laboratory animals (e.g., mouse and rat). This proposal presents a generic platform technology, the successful development and application of which will allow BioAssay Systems to rapidly develop pharmacodynamic assays for other important cancer targets such as kinases, phosphatases and proteases of interest to NCI.
