During studies conducted to examine inhibitors of the kinase active site for the epidermal growth factor (EGF) receptor, there were three assay methods used. One was an assay carried out on a genetically modified variant of the protein that had a kinase active site and was water-soluble.
The second was a cell assay measuring the total tyrosine phosphorylation in the presence of epidermal growth factor, and the third one was an in vivo study on mice that had their backs grafted with tumors. There were specific rationales behind the design of each of these assays.
The first assay was designed to measure the enzymatic effect of the inhibitor. By testing the inhibitors on a water-soluble version of the receptor, its binding capacity and enzymatic capabilities were measured.
The second assay was designed to test of the inhibitor could prove to be competitive with EGF. While tyrosine phosphorylation happens in the presence of EGF, if the inhibitor is an effective competitor for the receptor, the degree of phosphorylation in the assay should reduce significantly.
The third assay, or the in vivo study on mice with tumors grafted on their backs, can be used to test whether or not the inhibitor is potent enough to have a tumor-suppression effect. By examining the results of this assay, the potential for the inhibitors to be used as anticancer drugs can be gauged.