Mannosidase inhibitors as therapeutics for glycoprotein misfolding diseases

这项应用的长期目标是开发ER糖蛋白的选择性抑制剂 在质量控制中起关键作用的加工酶在内质网(ER)中具有广泛的基础 糖蛋白错误折叠疾病的治疗。这种酶，ERα-甘露糖苷酶I(ERManl)，作为一种 通过启动限速步骤导致新合成的糖蛋白的内质网滞留的关键计时器 最终导致靶向末端错误折叠的糖蛋白的一系列相互作用 在被称为“内质网相关”的过程中，向细胞质的逆转和蛋白酶体的处理 许多丧失功能的人类遗传病是由突变引起的 延迟蛋白质折叠动力学，而不是产生末端错误折叠的多肽。认识到 ERAD靶向机制未完全折叠的中间体可能导致过早处置 潜在的功能糖蛋白，并随后导致病理。对速度决定的抑制 ERAD中的步骤可以为糖蛋白错误折叠的治疗提供一种广泛的治疗方法 通过延缓ERAD和提供足够的时间来完成蛋白质折叠过程，可以预防和控制疾病。所有的 然而，已知的早期甘露糖修剪步骤的抑制剂也有不可接受的严重副作用。 它们还抑制高尔基复合体中处理a-甘露糖苷酶的葡聚糖，并阻止复合体的成熟。 细胞表面的类型糖链结构和分泌的糖蛋白。因此，此应用程序的目标是 寻找选择性ERManl抑制剂，延缓ERAD，修复人类ER蛋白折叠缺陷 并且在高尔基复合体中保持正常的糖链成熟。 我们组建的独特的跨学科团队利用持续的协同效应 佐治亚大学和贝勒医学院研究人员之间的协作互动 在选择性糖苷酶抑制剂(Boons)的合成、生化和结构方面拥有专业知识 内质网和高尔基甘露糖苷酶的生物学及人糖蛋白的细胞分析 错折叠障碍，A1抗胰蛋白酶缺乏症(Sifers)。有希望的线索也将在已建立的 合作者的溶酶体储存疾病模型(Amicus)。提出了三个具体目标：1) 选择性α-甘露糖苷酶抑制剂类似物的定向有理组合合成 2)高通量筛选与详细的生化和结构分析相结合 评估抑制剂化合物阻断ERManl而不是高尔基多糖的选择性和有效性 成熟，以及3)基于细胞的分析，以评估甘露糖苷酶抑制剂的化学伴侣效应。 靶向和不阻断N-糖链的突变型A1-抗胰蛋白酶分泌和溶酶体酶的解救 成熟。

The long-term goals of this application are to develop selective inhibitors for an ER glycoprotein processing enzyme that plays a key role in quality control in the endoplasmic reticulum (ER) as broad-based therapeutics for glycoprotein misfolding diseases. This enzyme, ER a-mannosidase I (ERManl), acts as a key timer for ER residence for newly synthesized glycoproteins by initiating a rate-limiting step leading to a cascade of interactions that ultimately leads to the targeting of terminally misfolded glycoproteins for retrotranslocation to the cytoplasm and proteasomal disposal in a process known as "ER-associated degradation" (ERAD). Many ioss-of-function human genetic diseases result from mutations that cause delayed protein folding kinetics rather than generating terminally misfolded polypeptides. Recognition of the incompletely folded intermediates by the ERAD targeting machinery can lead to premature disposal of potentially functional glycoproteins and subsequently leads to pathology. Inhibition of the rate-determining steps in ERAD could provide a broad-based therapeutic approach for treatment of glycprotein misfolding diseases by delaying ERAD and providing sufficient time to complete the protein folding process. All of the known inhibitors of early mannose trimming steps, however, also have unacceptable serious side effects. They also inhibit glycan processing a-mannosidases in the Golgi complex and block maturation to complex type glycan structures on cell surface and secreted glycoproteins. Thus, the goals of this application are to identify selective ERManl inhibitors that can act to delay ERAD, rescue ER protein folding defects in human disease, and retain normal glycan maturation in the Golgi complex. The unique interdisciplinary team that we have assembled takes advantage of ongoing synergistic collaborative interactions between investigators at the University of Georgia and Baylor College of Medicine with expertise in the synthesis of selective glycosidase inhibitors (Boons), the biochemistry and structural biology of the ER and Golgi mannosidases (Moremen), and cell-based assays for a human glycoprotein misfolding disorder, a1-antitrypsin deficiency (Sifers). Promising leads will also be evaluated in established lysosomal storage disease models by collaborators (Amicus). Three specific aims are proposed including 1) the directed rational and combinatorial synthesis of analogs of a-mannosidase inhibitors with selectivity toward ERManl, 2) high-throughput screens combined with detailed biochemical and structural analysis to assess selectivity and effectiveness of the inhibitor compounds in blocking ERManl but not Golgi glycan maturation, and 3) cell-based assays to assess chemical chaperone effects of mannosidase inhibitors in the rescue of mutant a1-antitrypsin secretion and lysosomal enzyme targeting and without blockage of N-glycan maturation.