Structure-Based Design of Small Molecules for Aspartylglucosaminuria

描述（由申请人提供）：天冬氨酸氨基葡萄糖尿症（AGU）是一种溶酶体积存疾病，由糖蛋白降解代谢紊乱引起。AGU导致糖天冬酰胺在几乎所有细胞类型的溶酶体中积累，并伴有严重的临床症状，如进行性神经变性和智力迟钝、面部特征粗糙、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是一种水解糖天冬酰胺所需的溶酶体酶。AGU已在世界范围内报道，到目前为止发现了26种不同的AGU等位基因，但仍然没有治疗这种疾病的方法。然而，在过去几年中，在鉴定和表征AGU致病突变方面取得了重大进展。因此，一种有效的AGU治疗方法的发展将使这种遗传疾病能够通过新生儿筛查进行早期治疗。我们对GA的晶体学研究表明，一个表面环（称为前体p环）阻断了成熟水解酶的催化中心。因此，为了打开催化中心，需要进行自蛋白水解来去除这个p环。然而，AGU突变会导致GA前体的错误加工和错误靶向，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极大地增强我们对这些AGU突变的结构后果的理解，但迄今为止，由于无法获得足够数量的高纯度人类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，该细菌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突变体的自身蛋白水解和水解酶活性。基于我们在遗传基因自动处理方面的最新进展，我们建议在这一应用中：1)表征AGU突变的分子发病机制；2)推进GA自蛋白水解活化的结构和机理研究；3)研究AGU突变的结构后果；4)开发小分子来刺激AGU分子的自动加工。该应用的广泛、长期目标是开发小分子治疗药物，以改善AGU的错误加工和错误靶向缺陷，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.