Inhibition of Antigen Presentation in Multiple Sclerosis

描述(由申请人提供)：MHC第二类区域是多发性硬化症(MS)和许多其他自身免疫性疾病的主要易感基因，表明多肽呈递到CD4T细胞在发病机制中至关重要。该项目的主要目标是开发治疗多发性硬化症和其他自身免疫性疾病的新策略，这些疾病以MHC II类抗原递呈通路为靶点。这项计划是PI的实验室和哈佛神经变性与修复中心(HCNR)的合作努力，该中心已经在我们的学术环境中建立了一个药物发现计划。在之前的资助期间，我们开发了一种新的实时多肽结合分析方法，并鉴定了几组调节MHC II类多肽结合的小分子。其中最有趣的一组化合物是一种名为J10的小分子，它将多肽负荷量加速了70倍以上。这种小分子在功能上与人类白细胞抗原-DM有相似之处，人类白细胞抗原-DM是一种催化多肽加载到MHC第二类分子的蛋白质，位于晚期的内体隔室。基于MHC II类的治疗药物被低效地装载到MHC分子上，因为它们在到达人类白细胞抗原-DM隔室之前，会长时间暴露在内体蛋白酶中。J10在广泛的pH范围内具有活性，表明它可能使治疗药物在早期的内体隔室或细胞表面与MHC II类结合。新的数据表明，J10的衍生物在体内是活性的，它显著增强了多肽的展示。此外，J10催化剂与多肽C-末端的共价连接大大提高了多肽在体外和体内的载药效率。我们的假设是，基于MHC II类分子的治疗效果可以通过催化快速与MHC II类分子结合的小分子来显著增强。下一个资助期的目标是确定它们的精确作用机制，并在人源化的多发性硬化症小鼠模型中测试治疗应用。在目标1中，我们将研究这些小分子加速多肽负荷的结构和功能机制。我们将通过候选区域的定点突变来定位J10的结合部位，并将J10与DR/多肽复合物共结晶，以用于X射线结晶学结构测定。在目标2中，我们将检验三种不同类别的基于MHC II类药物(Copaxone、耐受性自肽、MHC II类抑制剂)的治疗效果是否可以通过联合给药或共价连接J10来增加。该计划的药物化学部分将继续为这些药物合成具有更高活性和类药物特性的衍生物。公共卫生相关性治疗自身免疫性疾病的一些治疗方法需要将治疗药物与MHC II类分子结合。一个主要的问题是，将这些化合物装载到MHC II类结合部位是低效的，因为加速多肽装载的天然催化剂仅限于较晚的内体隔室。我们已经发现了可以显著加速多肽与MHC II类分子结合的小分子，现在将研究它们的作用机制以及在人源化的MS小鼠模型中的治疗应用。

DESCRIPTION (provided by applicant): The MHC class II region is a principal susceptibility locus for multiple sclerosis (MS) and many other autoimmune diseases, indicating that peptide presentation to CD4 T cells is critical in the pathogenesis. The major goal of this project is develop novel strategies for the treatment of MS and other autoimmune diseases that target the MHC class II antigen presentation pathway. This program is a collaborative effort between the PI's lab and the Harvard Center for Neurodegeneration and Repair (HCNR) that has established a drug discovery program within our academic environment. During the previous funding period, we developed a novel real time peptide binding assay and identified several groups of small molecules that modulate peptide binding by MHC class II. The most interesting group of compounds is represented by a small molecule termed J10 that accelerates peptide loading more than 70-fold. This small molecule has functional similarities with HLA-DM, the protein that catalyzes loading of peptides onto MHC class II molecules in a late endosomal compartment. MHC class II-based therapeutics are inefficiently loaded onto MHC molecules because they are exposed to endosomal proteases for extended periods of time before they reach the HLA-DM compartment. J10 is active over a broad pH range, indicating that it may enable MHC class II binding of therapeutics in early endosomal compartments or at the cell surface. New data demonstrate that a J10 derivative is active in vivo and that it substantially enhances peptide display. Furthermore, covalent attachment of the J10 catalyst to the C-terminus of peptides substantially increases the efficiency of peptide loading both in vitro and in vivo. Our hypothesis is that the efficacy of MHC class II-based therapeutics can be substantially enhanced with a small molecule that catalyzes rapid binding to MHC class II molecules. The goals for the next funding period are to define their precise mechanisms of action and to test therapeutic applications in humanized mouse models of MS. In Aim 1, we will investigate the structural and functional mechanisms by which these small molecules accelerate peptide loading. We will map the J10 binding site by site-directed mutagenesis of candidate regions and co-crystallize J10 with DR/peptide complexes for structure determination by X-ray crystallography. In Aim 2, we will examine whether the therapeutic efficacy of three different classes of MHC class II-based therapeutics (Copaxone, tolerogenic self-peptides, MHC class II inhibitors) can be increased by co- administration or covalent attachment of J10. The medicinal chemistry component of this program will continue to synthesize derivatives with improved activity and drug-like properties for these. PUBLIC HEALTH RELEVANCE A number of therapeutic approaches for the treatment of autoimmune diseases require binding of the therapeutic to MHC class II molecules. A major issue is that loading of these compounds into the MHC class II binding site is inefficient because the natural catalyst that accelerates peptide loading is confined to a late endosomal compartment. We have discovered small molecules that substantially accelerate peptide binding to MHC class II molecules and will now examine their mechanism of action as well as therapeutic applications in humanized mouse models of MS.