Arthritic progression derives in part from inappropriate activation of pro-inflammatory genes. In turn these genes are activated by transcriptional regulators and signaling systems within immune system cells. To pharmacologically effect intracellular physiology, we have created retroviruses capable of expressing short peptides in a stable form within mammalian cells and have created libraries of up to 10/9 randomly generated unique peptides. Using retroviral library systems and in vivo selection stratagems we will select for peptides that block or activate either NFAT activation or NF-kappaB activation in T cells, macrophages, and B cells. These transcription factors, and the genes they regulate, are suitable pro-inflammatory targets for drug design. We will use these novel peptides to study the epistatic relationships of signaling molecules within these pathways, clone or purify the targets that interact with these peptides, dissect the signaling systems effected with the peptides, and use these tools to understand the activation of pro-inflammatory genes in T cells and macrophages. Selected peptides can be cloned by PCR and their primary structure determined. Epitope tagging of the peptide for immunoprecipitation or two-hybrid screening with a selected peptide as bait allows for characterization of the target (biochemistry). We plan to design peptide structures 1) for intracellular expression (subcellularly localized or not), 2) that are conformationally constrained that allow for the eventual conversion of selected peptides that peptideomimetic compounds, 3) with biased sequence for specifically effecting proteins with SH-3 domains, 4) with transmembrane domains that allow surface or intracellular membrane-proximal presentation of randomized regions on tethered structures to interfere with surface receptors/channels and receptor associated proteins involved in the activation of NFAT/NF kappaB (I-Crac or TCR for instance). Isolated peptides will be tested for their ability to inhibit or active the expression of target genes named and will be used to study the signaling pathways of the critical immune system regulators NFkappaB and NFAT.
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