Thyroid Derived Peptide Presentation by HLA-DR in Thyroiditis

描述(申请人提供)：我们的目标是通过剖析人类白细胞抗原II类分子与甲状腺多肽相互作用诱发自身免疫性甲状腺疾病(AITD)的机制，设计针对AITD的靶向治疗。在上一个赠款周期中，我们在实现这些目标方面取得了实质性进展：(1)我们已经证明，精氨酸在人类白细胞抗原-DR多肽结合口袋(DRB1-Arg74)的β74位的存在是AITD发生的关键；(2)我们鉴定了4个与DRB1-Arg74口袋强烈结合的甲状腺球蛋白(TG)多肽。其中一个多肽(Tg.2098)是一种主要的T细胞表位；(3)我们鉴定了一个小分子和一个D-肽可以阻断Tg.2098与DRB1-Arg74的结合；(4)我们发现干扰素和TG之间通过IRF-1介导的表观遗传相互作用；(5)我们(和其他人)发现TSHR是一个主要的Graves病(GD)基因，与DRB1-Arg74在增加GD的风险方面具有协同作用。这种协同作用表明，与TG类似，TSHR多肽与DRB1-Arg74片段之间存在相互作用。我们建议在这些发现的基础上，确定DRB1-Arg74的阻滞剂是治疗AITD的潜在新疗法。我们的假设是，致病甲状腺多肽与DRB1-Arg74口袋的结合及其对T细胞的呈递对于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：鉴定与DRB1-Arg74结合的致病TSH受体(TSHR)多肽。我们将使用：(1)分子建模：预测&gt；750个潜在的TSHR多肽中的哪些可以与DRB1-Arg74口袋结合；(2)生化和质谱学研究，以确认与DRB1-Arg74结合的多肽；(3)T细胞研究，以测试多肽结合物是否能够刺激GD小鼠模型和GD患者的T细胞。特定目的2：筛选多肽-DRB1-Arg74结合的小分子抑制物(SMI)。我们将使用：(1)计算机筛选大型化合物库(~115,000)以确定与DRB1-Arg74口袋结合的潜在SMI；(2)高通量实验筛选(HTS)~115,000化合物库；(3)通过使用重组DRB1-Arg74进行体外结合抑制试验以及通过抑制TG/TSHR多肽对T细胞激活的抑制来确认先导化合物。具体目标3：鉴定和分析与DRB1-Arg74结合的D-氨基酸多肽阻滞剂。我们将使用：(1)在电子筛选中预计将阻断DRB1-Arg74口袋与肽结合的D-肽；(2)通过与重组DRB1-Arg74结合的抑制试验在体外证实预测的D-肽；(3)T细胞分析以测试预测的D-肽阻滞剂是否能抑制自身免疫性甲状腺疾病小鼠和AITD患者淋巴细胞的增殖反应。总而言之，这一多学科的翻译 该项目直接建立在前一个赠款期间获得的知识的基础上。我们的目标是开发新的治疗AITD的方法，使用SMI和D-肽来阻断多肽HCII结合。我们的协作团队有能力、经验和专业知识来实现我们的目标。我们方法的主要优点是，它实际上能够实现洗脱的特异性，因为只有与Arg74+结合的多肽才会被阻断。我们的翻译研究有望为AITD带来新的治疗方法。

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.