PHARMACOLOGY OF HUMAN INTERFERON GAMMA INHIBITORS

该项目的长期目标是开发有效、安全和 干扰素-γ的特定药物抑制剂，对 败血症关节及多发性等自身免疫性疾病的治疗 硬化症和1型糖尿病。研究的重点是一个 基于寡核苷酸的抑制剂，专门阻断大量的 干扰素-伽马效应，包括诱发的主要 组织相容性复合体II类DR和I类及ICAM-1蛋白 几种细胞类型。这种铅寡核苷酸还阻断了有意义的 干扰素-γ与肿瘤坏死因子-α的协同作用 混合物。它抑制干扰素-γ与其细胞表面的结合 受体复合体，从而阻断受体的下游信号转导 相关的蛋白激酶。因为我们的初步结果支持 构建以寡核苷酸为基础的药物抑制剂的可行性 干扰素-γ，我们建议确定其分子机制。 抑制活性，并使用产生的信息来设计一个偶数 对干扰素-伽马的抑制作用更强。相应地，具体目标是 该项目的主要目的是：i)鉴定氨基酸残基和核酸 在作用部位相互作用的酸碱，II)使用机理 合成更有效的干扰素-γ抑制剂的信息，III) 更有效地表征其效力、活性、特异性和选择性 干扰素-伽马的抑制剂，III)表征其效力、活性、 新合成的抑制剂的特异性和选择性曲线， 四)确定这些抑制剂对人外周血的影响 并测试这些抑制物将 驱动免疫系统支持体液或类似TH2的反应，以及，v) 在感染性休克的广义Shwartzman模型中测试抑制剂。我们 将使用表位破坏和赖氨酸保护分析来鉴定 构成结合部位的干扰素-γ的氨基酸。羟基 自由基足迹和二甲基硫酸盐保护分析将用于 确定与结合有关的核酸碱基。机械论 然后将使用信息来优化长度、序列和主干 这些干扰素-伽马抑制寡核苷酸的组成。这些 研究将提供关键的机械信息关于 干扰素-γ抑制作用的药理学和分子基础 寡核苷酸活性。更重要的是，这个模型的结果 该系统应可推广到寡核苷酸抑制剂的设计 对于其他蛋白质，特别是细胞因子。

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.