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以诱导AITD的机制来设计针对自身免疫性甲状腺疾病（AITD）的靶向疗法。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肽结合口袋（DRb 1-Arg 74）的β 74位存在精氨酸是AITD发展的关键;（2）我们鉴定了4种与DRb 1-Arg 74口袋强烈结合的甲状腺球蛋白（Tg）肽。一种肽（3）我们发现了一个小分子和一个D肽阻断了Tg.2098与DRb 1-Arg 74的结合：（4）我们发现了干扰素和Tg之间通过IRF-1介导的表观遗传相互作用;（5）TSHR是Graves病（GD）的一个主要基因，与DRb 1-Arg 74基因在GD发病中具有协同作用。这种协同作用表明，类似于Tg，TSHR肽和DRb 1-Arg 74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DRb 1-Arg 74阻断剂作为AITD的潜在新疗法。我们的假设是，致病性甲状腺肽与DRb 1-Arg 74口袋的结合及其对T细胞的呈递对AITD的病因至关重要。阻断肽与MHC II的结合可用于治疗AITD。具体目的：具体目的1：鉴定与DRb 1-Arg 74结合的致病性TSH受体（TSHR）肽。我们将用途：（1）分子建模：以预测> 750种潜在TSHR肽中的哪一种可以与DRb 1-Arg 74口袋结合;（2）生物化学和质谱研究以确认与DRb 1-Arg 74结合的肽;（3）T细胞研究以测试肽结合剂是否可以刺激来自GD小鼠模型和来自GD患者的T细胞。具体目的2：筛选肽-DRb 1-Arg 74结合的小分子抑制剂（SMI）。我们将用途：（1）大型化合物库的计算机屏幕（2）对约115，000种化合物的文库进行高通量实验筛选（HTS）;（3）通过使用重组DRb 1-Arg 74的体外结合抑制测定确认先导化合物，以及通过Tg/TSHR肽抑制T细胞活化。具体目标3：鉴定和分析肽-DRb 1-Arg 74结合的D-氨基酸肽阻断剂。我们将用途：（1）预测阻断DRb 1-Arg 74口袋与肽结合的D肽的计算机筛选;（2）通过与重组DRb 1-Arg 74结合的抑制试验体外确认预测的D肽;（3）T细胞试验，以测试预测的D肽阻断剂是否可以抑制从自身免疫甲状腺小鼠和AITD患者中分离的淋巴细胞的增殖反应。 总之，这一多学科的翻译 项目直接建立在前一个赠款期获得的知识基础上。我们的目标是开发新的AITD治疗模式，使用SMI和D-肽阻断肽HCII结合。 我们的合作团队有能力，经验和专业知识来实现我们的目标。 我们的方法的主要优点是它实际上能够实现rapeutic特异性，因为只有与Arg 74+口袋结合的肽才会被阻断。 我们的转化研究将有望为AITD带来新的治疗方法。 公共卫生相关性：自身免疫性甲状腺疾病（AITD），包括桥本甲状腺病和格雷夫斯病，是由易感基因和环境触发因素之间的相互作用引起的复杂疾病。我们发现，AITD的发病机制之一是HLA分子异常提呈甲状腺衍生肽到T淋巴细胞，然后形成对甲状腺的免疫应答。我们的研究目标是鉴定这些甲状腺衍生肽，并使用小分子和D-肽阻断它们与HLA分子的结合，作为AITD的新治疗方法。

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. PUBLIC HEALTH RELEVANCE: Autoimmune thyroid diseases (AITD), including Hashimoto's thyroid it is and Graves' disease are complex diseases caused by an interaction between susceptibility genes and environmental triggers. We have found that the one of the mechanisms for development of AITD is the abnormal presentation of thyroid derived peptides by HLA moleculestoT-lymphocytes that then forman immune response to the thyroid. The goal of our studies is to identify these thyroid derived peptides and to block their binding to HLA molecules using small molecules and D-peptides as a novel therapeutic approach to AITD.