Thyroid Derived Peptide Presentation by HLA-DR in Thyroiditis

描述（由申请人提供）：我们的目标是通过剖析HLA II类分子与甲状腺肽相互作用诱导自身免疫性甲状腺疾病（AITD）的机制，设计针对自身免疫性甲状腺疾病（AITD）的靶向治疗。在上一个资助周期中，我们朝着这些目标取得了实质性进展：(1)我们已经证明，在HLA-DR肽结合袋（DRb1-Arg74）的β 74位置存在精氨酸是AITD发展的关键；(2)我们发现了4种甲状腺球蛋白（Tg）肽，它们与DRb1-Arg74口袋紧密结合。其中一个肽（Tg.2098）是一个主要的t细胞体；(3)我们发现了一个小分子和一个d肽，可以阻断Tg.2098与DRb1-Arg74的结合；(4)发现干扰素与Tg通过IRF-1介导的表观遗传相互作用；(5)我们（和其他人）发现TSHR是Graves病（GD）的一个主要基因，它与DRb1-Arg74在GD发病风险中具有协同作用。这种协同作用表明，与Tg类似，TSHR肽与DRb1-Arg74口袋之间存在相互作用。我们建议在这些发现的基础上确定DRb1-Arg74阻滞剂作为AITD的潜在新疗法。我们的假设是，致病性甲状腺肽与DRb1-Arg74口袋的结合及其向t细胞的呈递对AITD的病因至关重要。阻断多肽与MHC II的结合可用于治疗AITD。特异性目的：特异性目的1：鉴定结合DRb1-Arg74的致病性TSH受体（TSHR）肽。我们将使用：(1)分子建模：预测bbbb750个潜在的TSHR肽中哪些可以结合DRb1- Arg74口袋；(2)通过生化和质谱研究确认与DRb1-Arg74结合的肽；(3) t细胞研究，测试肽结合物是否能刺激GD小鼠模型和GD患者的t细胞。特异性目的2：筛选肽- DRb1-Arg74结合的小分子抑制剂（SMI）。我们将使用：(1)一个大型化合物库（约115,000）的计算机屏幕来识别与DRb1-Arg74口袋结合的潜在SMI；(2)高通量实验筛选（HTS）约11.5万个化合物库；(3)利用重组DRb1-Arg74进行体外结合抑制实验，并通过Tg/TSHR肽抑制t细胞活化来确定先导化合物。特异性目标3：鉴定和分析肽- drb1 - arg74结合的d -氨基酸肽阻断剂。我们将使用：(1)预测阻断DRb1-Arg74口袋与肽结合的d肽的硅筛选；(2)通过与重组DRb1-Arg74结合的抑制实验，体外证实了预测的d肽；(3) t细胞试验，检测预测的d肽阻滞剂是否能抑制自身免疫性甲状腺炎小鼠和AITD患者分离淋巴细胞的增殖反应。综上所述，这是多学科的转化

DESCRIPTION (provided by applicant): Our goal is to design targeted therapies for autoimmune thyroid diseases (AITD) by dissecting the mechanisms by which HLA class II molecules interact with thyroidal peptides to induce AITD. In the last grant cycle, we made substantial progress toward these goals: (1) We have shown that the presence of arginine at position beta 74 of the HLA-DR peptide binding pocket (DRb1-Arg74) is key to the development of AITD; (2) We identified 4 thyroglobulin (Tg) peptides that bind strongly to the DRb1-Arg74 pocket. One of the peptides (Tg.2098), is a major T-cellepitope;(3) We identified a small molecule and a D-peptide that block the binding of Tg.2098 to DRb1-Arg74; (4) We discovered epigenetic interactions between interferon & Tg mediated via IRF-1; (5) We (& others) showed that TSHR is a major Graves' disease (GD) gene that has synergism with DRb1-Arg74 in conferring risk for GD. This synergism suggests that, similar to Tg, there is interaction between TSHR peptides & DRb1-Arg74 pockets. We propose building on these findings to identify blockers of DRb1-Arg74 as potential new therapies for AITD. Our hypothesis is that binding of pathogenic thyroidal peptides to the DRb1-Arg74 pocket & their presentation to T-cells are critical to the etiology of AITD. Blocking the binding of peptides to MHC II may be used to treat AITD. Specific Aims: Specific Aim 1: To identify pathogenic TSH receptor (TSHR) peptides that bind to DRb1-Arg74. We will use: (1) Molecular modeling: to predict which of the > 750 potential TSHR peptides can bind to DRb1- Arg74 pockets; (2) Biochemical and Mass Spectrometry studies to confirm peptides that bind to DRb1-Arg74; (3) T-cell studies to test whether peptide binders can stimulate T-cells from a GD mouse model and from GD patients. Specific Aim 2: To screen for small molecule inhibitors (SMI's) of peptide - DRb1-Arg74 binding. We will use: (1) Computer screen of a large library of compounds (~ 115,000) to identify potential SMI's that bind to DRb1-Arg74 pockets; (2) High throughput experimental screening (HTS) of a library of ~ 115,000 compounds; (3) Confirmation of lead compounds by in vitro binding inhibition assays using recombinant DRb1-Arg74, and by inhibition of T-cell activation by Tg/TSHR peptides. Specific Aim 3: Identifying & analyzing D-amino acid peptide blockers of peptide-DRb1-Arg74 binding. We will use: (1) In silico screen of D-peptides that are predicted to block the DRb1-Arg74 pocket to peptide binding; (2) In vitro confirmation of predicted D-peptides by inhibition assays of binding to recombinant DRb1-Arg74; (3) T-cell assays to test whether predicted D-peptide blockers can inhibit proliferative responses of lymphocytes isolated from mice with auto immune thyroid it is and from patients with AITD. In summary, this multidisciplinary translational project builds directly on the knowledge gained in the previous grant period. Our goals are to develop novel treatment modalities for AITD using SMI and D-peptides to block peptide HCIIbinding. Our collaborative team has the capacity, experience, & expertise to achieve our aims. The main advantage of our approach is that it is actually capable of achieving the rapeutic specificity since only peptides that bind to Arg74+ pockets will be blocked. Our translational studies will hopefully lead to novel therapies for AITD.