The goal of this Phase I proposal is to identify novel regulators of transcription factor NFkappaB. The products of this Phase I discovery program will be targets utilized in a Phase II campaign to develop of small molecule inhibitors as potential new drug candidates for the treatment of cancer and inflammation diseases. To achieve the goal of this proposal, we will apply our patented Random Activation of Gene Expression (RAGE) technology to a unique NFkappaB reporter assay system. Athersys' plasmid-based RAGE technology is validated to provide unbiased, high-level activation of mammalian genes, irrespective of the normal genetic regulatory cues that dictate their temporal and tissue-specific expression patterns. Importantly, RAGE drives high-level expression of functional protein from any activated gene, large RAGE libraries can be generated to provide comprehensive gene activation, and RAGE-activated genes are sequence-tagged to facilitate their rapid characterization. Our assay design utilizes an engineered cell line that, in response to NFkappaB activation, expresses a chimeric reporter gene enabling selection for either positive or negative survival traits. Expression of this reporter is tightly regulated, being repressed during normal cell growth, but highly induced upon cellular activation of pathways culminating in NFkappaB signaling. Hence, a cell sustaining RAGE-activation of a gene that confers constitutive NFkappaB activation can be selected through its survival in drug-containing medium. There are two specific aims for this Phase I initiative, They are: 1.) Utilize our NFkappaB reporter cell line and family of retroviral RAGE vectors to generate 7 independent libraries, each representing greater than or equal too 1X activation-coverage of endogenous genes, select those libraries to identify clones expressing activated NFkappaB, and isolate and characterize the RAGE-activated gene from each NF(B vclone. And 2.) Utilize strategic cell lines to express the cDNAs of novel genes to verify and investigate their functions as regulators of NFkappaB activation. The successful outcome of this Phase I program will result in cloned and characterized genes expressing a diverse group of novel NFkappaB regulators. Some of these proteins will present important targets for the development of new drugs that treat cancer and immune diseases. A selection of these novel proteins will provide the resources (i.e., targets) needed to fuel a future Phase II initiative, in which we will conduct high-throughput screening of our proprietary compound libraries to identify and further develop lead-candidates for continued drug development.
