TOPIC 229: PHASE I ERK DEV. OF MOLECULAR PHARMACODYNAMIC ASSAYS

Extracellular-signal-regulated kinase (ERK) is a key protein kinase of the MAP kinase pathway and plays a key role in cell proliferation, differentiation and migration. The MAPK cascade presents many targets for the development of novel and safer cancer therapies. Despite a lot of progress in the discovery of therapeutic agents, a pharmacodynamic assay for ERK is missing. In this proposed research, BioAssay Systems aims to develop a rapid, sensitive, robust and rigorous assay for ERK 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 ERK protein and isolate it from any potentially interfering enzymes such as other kinases and phosphatases. The captured ERK 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 ERK 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.