Phosphoproteins are key regulators of cancer-related events such as proliferation, invasion, angiogenesis and metastasis, and mutations in the enzymes that regulate phosphoproteins, the kinases and phosphatases, are known to play a direct role in several cancers. This proposal describes a novel profiling technology that can assess all of the signal transduction pathways in a sample quickly and with great exactness using a panel of specific phosphoprotein binding agents we call Bindingzymes. Any one of these Bindingzymes will bind only a few of the phosphoproteins in a sample, but the entire panel (""""""""Profiling Panel"""""""") will bind virtually all of the phosphoproteins in a sample. This Profiling Panel will allow for the quick and comprehensive identification of differences in phosphoproteins between samples. A novel aspect of the approach is that those Bindingzymes that detect changes between samples can be simply and immediately configured into a single assay and used in a cell-based screen to discover all drug candidates in a library which change the cancer cell profile to better resemble the normal cell profile. Drug candidates that act at any of the steps of the disease-causing pathways can be detected without prior knowledge of these pathways and of the relevant protein targets. This technology should have wide application in the discovery and development of cancer drugs. The scope of this Phase I grant application is the creation of a small number of Bindingzymes (""""""""Mini-panel"""""""") and its use in proof-of-concept experiments: detection of changes in phosphoprotein profiles from cells grown under general phosphatase-inhibiting and non-inhibiting conditions; upon antibody-induced T-cell activation; and shifting of a typical cancer cell profile towards its normal counterpart after incubation with a cancer drug known to function through intervention of signal transduction events (Gleevec(tm)). These proof-of-concept experiments will form the basis for commercial development of the comprehensive Profiling Panel, which will enable the virtually hypothesis-free comparison of samples and rapid establishment of high-throughput screens for the discovery of new cancer drugs. The Profiling Panel should also provide a powerful new tool for determining selectivity of drug candidates and in the prospective and retrospective analysis of patients in clinical trials. ? ? ? ? ? ?
