Mannosidase inhibitors as therapeutics for glycoprotein misfolding diseases

描述（由申请人提供）：本申请的长期目标是开发ER糖蛋白加工酶的选择性抑制剂，该酶在内质网（ER）的质量控制中起关键作用，作为糖蛋白错误折叠疾病的广泛治疗剂。这种酶，ER α-甘露糖苷酶I（ERManl），作为新合成糖蛋白的ER驻留的关键计时器，通过启动限速步骤，导致级联相互作用，最终导致靶向末端错误折叠的糖蛋白，以在称为“ER相关降解”（ERAD）的过程中逆转位至细胞质和蛋白酶体处置。许多功能丧失的人类遗传疾病是由引起延迟的蛋白质折叠动力学而不是产生末端错误折叠的多肽的突变引起的。ERAD靶向机制对不完全折叠的中间体的识别可导致潜在功能性糖蛋白的过早处置，并随后导致病理学。抑制ERAD中的速率决定步骤可以通过延迟ERAD并提供足够的时间来完成蛋白质折叠过程，为治疗糖蛋白错误折叠疾病提供广泛的治疗方法。然而，所有已知的早期甘露糖修剪步骤的抑制剂也具有不可接受的严重副作用。它们还抑制高尔基复合体中的聚糖加工α-甘露聚糖酶，并阻断细胞表面和分泌的糖蛋白上的复合型聚糖结构的成熟。因此，本申请的目标是鉴定选择性ERManl抑制剂，其可以起到延迟ERAD、挽救人类疾病中的ER蛋白折叠缺陷和保持高尔基体复合体中正常聚糖成熟的作用。我们组建的独特的跨学科团队利用了格鲁吉亚大学和贝勒医学院的研究人员之间正在进行的协同合作互动，这些研究人员具有选择性糖苷酶抑制剂合成（Boons），ER和高尔基甘露聚糖酶的生物化学和结构生物学（Moremen）以及基于细胞的人类糖蛋白错误折叠障碍检测的专业知识，α 1-抗胰蛋白酶缺乏症（Sifers）。合作者（Amicus）还将在已建立的溶酶体贮积病模型中评价有前景的先导化合物。提出了三个具体目标，包括1）具有对ERManl的选择性的α-甘露糖苷酶抑制剂的类似物的定向合理和组合合成，2）与详细的生物化学和结构分析相结合的高通量筛选，以评估抑制剂化合物在阻断ERManl而不是高尔基体聚糖成熟中的选择性和有效性，和3）基于细胞的测定，以评估甘露糖苷酶抑制剂在拯救突变体α 1-抗胰蛋白酶分泌和溶酶体酶靶向中的化学伴侣作用，而不阻断N-聚糖成熟。

DESCRIPTION (provided by applicant): 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, ERalpha-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 loss-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 alpha-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, alpha1-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 alpha-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 alpha1-antitrypsin secretion and lysosomal enzyme targeting and without blockage of N-glycan maturation.