Structure-Based Design of Small Molecules for Aspartylglucosaminuria

描述（由申请方提供）：天冬氨酸氨基葡萄糖尿症（AGU）是一种由糖蛋白降解代谢紊乱引起的溶酶体贮积病。AGU导致糖天冬酰胺在几乎所有细胞类型的溶酶体中积累，具有严重的临床症状，例如进行性神经变性和智力迟钝、粗糙的面部特征、骨骼异常和结缔组织病变。AGU突变发生在糖基天冬酰胺酶（GA）基因中，GA是一种水解糖基天冬酰胺所需的溶酶体酶。AGU已在世界范围内报道，迄今已发现26种不同的AGU等位基因，但仍没有治疗该病的方法。然而，在过去的几年中，在AGU致病突变的鉴定和表征方面取得了重大进展。因此，开发一种有效的AGU治疗方法将使这种遗传性疾病适合新生儿筛查，以便早期治疗。我们对GA的晶体学研究表明，一个表面环（称为前体P-loop）阻断了成熟水解酶的催化中心。因此，为了打开催化中心，需要自体蛋白水解来去除该P环。尽管如此，AGU突变导致GA前体的错误加工和错误定位，从而阻止其水解酶活性的自激活。GA和AGU分子的高分辨率结构研究将大大提高我们对这些AGU突变的结构后果的理解，但迄今为止，由于无法获得足够量的高度纯化的人GA而受到阻碍。尽管如此，我们已经通过纯化和结晶细菌GA克服了这一障碍，细菌GA已被证明具有相同的结构特征，并使用相同的机制来自动激活其水解酶活性。我们的初步结果表明，具有类似甘氨酸结构的小分子可以增强AGU突变体的自蛋白水解和水解酶活性。基于我们最近在GA自动加工方面的进展，我们在本申请中提出：1）表征AGU突变的分子发病机制; 2）推进我们对GA自动蛋白水解激活的结构和机制研究; 3）研究AGU突变的结构后果;以及4）开发小分子以刺激AGU分子的自动加工。本申请的广泛、长期目标是开发小分子作为治疗剂以改善AGU错误处理和错误定位缺陷，从而减轻AGU患者及其家属的痛苦。

DESCRIPTION (provided by applicant): Aspartylglucosaminuria (AGU) is a lysosomal storage disease caused by a metabolic disorder in glycoprotein degradation. AGU results in accumulation of glycoasparagines in the lysosomes of virtually all cell types, with severe clinical symptoms such as progressive neurodegeneration and mental retardation, coarse facial features, skeletal abnormalities, and connective tissue lesions. AGU mutations occur in the gene for glycosylasparaginase (GA), a lysosomal enzyme required to hydrolyze glycoasparagines. AGU has been reported worldwide, with 26 different AGU alleles found so far, but still no treatment available for this disease. However, during the past few years, there has been significant progress in the identification and characterization of AGU causative mutations. Thus development of an effective AGU therapy would make this genetic disease amenable to newborn screening for an early treatment. Our crystallographic studies on GA reveal that a surface loop (named precursor P-loop) blocks the catalytic center of mature hydrolase. Autoproteolysis is thus required to remove this P-loop in order to open up the catalytic center. Nonetheless, AGU mutations cause misprocessing and mistargeting of GA precursors, thus prevents their autoactivation for the hydrolase activity. High-resolution structural studies of GA and AGU molecules will greatly enhance our understanding of the structural consequences of these AGU mutations but have so far been hampered by the inability to obtain sufficient amounts of highly purified human GA. Nonetheless, we have overcome this hurdle by purifying and crystallizing bacterial GA, which has been demonstrated to have identical structural features and use the same mechanism to autoactivate its hydrolase activity. Our preliminary results indicate that small molecules with structures similar to glycine can enhance autoproteolytic and hydrolase activity of AGU mutants. Building on our recent progress on GA autoprocessing, we propose in this application to 1) characterize molecular pathogenesis of AGU mutations; 2) push forward our structural and mechanistic studies of GA autoproteolytic activation; 3) study structural consequences of AGU mutations; and 4) develop small molecules to stimulate autoprocessing of AGU molecules. The broad, long- term objective of this application is to develop small molecules as therapeutics to ameliorate the AGU misprocessing and mistargeting defect and thus alleviate the suffering of AGU patients and their families.