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内质网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药物。这种酶，erα -甘露糖苷酶I (ERManl)，作为新合成糖蛋白内质网驻留的关键计时器，通过启动限速步骤，导致一系列相互作用，最终导致最终错误折叠的糖蛋白靶向逆转录到细胞质和蛋白酶体处理，这一过程被称为“内质网相关降解”（ERAD）。许多人类遗传疾病的功能丧失是由于突变导致蛋白质折叠动力学延迟，而不是产生最终错误折叠的多肽。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糖蛋白的过早处置，并随后导致病理。抑制ERAD的速率决定步骤可以通过延迟ERAD和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有不可接受的严重副作用。它们还抑制高尔基复合物中α -甘露糖苷酶的聚糖加工，并阻止细胞表面复杂型聚糖结构和分泌糖蛋白的成熟。因此，本应用的目的是确定选择性ERManl抑制剂，这些抑制剂可以延缓ERAD，挽救人类疾病中的ER蛋白折叠缺陷，并保持高尔基复合体中正常的聚糖成熟。我们组建的独特的跨学科团队充分利用了乔治亚大学和贝勒医学院研究人员在选择性糖苷酶抑制剂（Boons）的合成、内质网和高尔基甘露糖酶（Moremen）的生物化学和结构生物学以及人类糖蛋白错误折叠紊乱、α - 1抗胰蛋白酶缺乏症（Sifers）的细胞检测方面的专业知识。合作者（Amicus）也将在已建立的溶酶体贮积病模型中评估有希望的线索。他们提出了三个具体目标：1)定向合理和组合合成具有ERManl选择性的α -甘露糖苷酶抑制剂类似物；2)结合详细的生化和结构分析的高通量筛选，以评估抑制剂化合物在阻断ERManl而不是高尔基聚糖成熟方面的选择性和有效性。3)基于细胞的检测，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且不阻碍n -聚糖成熟方面的化学伴侣作用。

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.