The long term objective of this project is to develop potent, safe an specific pharmacological inhibitors for interferon-gamma that are useful in the treatment of septic hock and autoimmune diseases such as multiple sclerois and Type 1 diabetes. The focus of the research is an oligonucleotide based inhibitor that specifically blocks a multitude of interferon-gamma effects, including the induction of the major histocompatibility complex Class II DR and Class I and ICAM-1 proteins in several cell types. This lead oligonucleotide also blocks the significant synergy between interferon-gamma and tumor necrosis factor-aalpha in mixture. It inhibits the binding of interferon-gamma to its cell surface receptor complex and thereby blocks downstream signaling by receptor associated kinases. Because our preliminary results support the feasibility of constructing oligonucleotide-based inhibitors for interferon-gamma, we propose to identify the molecular mechanisms of this inhibitory activity and use the resulting information to engineer an even more potent inhibitor for interferon-gamma. Accordingly, the specific aims of this project are to: i) identify the amino acid residues and nucleic acid bases that interact at the site of action, ii) use the mechanistic information to synthesize more potent inhibitors for interferon-gamma, iii) characterize the potency, activity, specificity and selectivity more potent inhibitors for interferon-gamma, iii) characterize the potency, activity, specificity and selectivity profiles of the newly synthesized inhibitors, iv) determine the effects of these inhibitors on human peripheral blood derived immune cells and to test the hypothesis that these inhibitors will drive the immune system to favor a humoral or TH2-like response and, v) test the inhibitors in a generalized Shwartzman model for septic shock. We will use epitope disruption and lysine protection assays to identify the amino acids of interferon-gamma that constitute the binding site. Hydroxyl radical footprinting and dimethylsulfate protection assays will be used to identify the nucleic acid bases involved in binding. The mechanistic information will then be used to optimize the length, sequence and backbone composition of these interferon-gamma inhibitory oligonucleotides. These studies will provide critical mechanistic information on the pharmacological and molecular basis for interferon-gamma inhibitory oligonucleotide activity. More importantly, the results from this model system should be generalizable to the design of oligonucleotide inhibitors for other protein, particularly cytokines.
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