Structure-Based Design of Small Molecules for Aspartylglucosaminuria

天冬氨酸氨基葡萄糖尿症（AGU）是一种由代谢紊乱引起的溶酶体贮积病， 糖蛋白降解AGU导致糖天冬酰胺在几乎所有人的溶酶体中积累， 细胞类型，具有严重的临床症状，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，一种水解糖基天冬酰胺所需的溶酶体酶。AGU拥有 迄今为止，已在全球范围内报告了26种不同的AGU等位基因，但仍然没有可用的治疗方法 疾病然而，在过去几年中，在查明和 AGU致病突变的表征。因此，开发一种有效的AGU疗法将使 这种遗传性疾病适合新生儿筛查，以便早期治疗。我们的晶体学研究 GA分析表明，成熟水解酶的催化中心被一个表面环（称为前体P-loop）所阻断。 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，从而阻止它们的自激活 水解酶的活性。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，但迄今为止受到阻碍， 由于不能获得足够量的高度纯化的人GA。尽管如此，我们还是克服了 这一障碍通过纯化和结晶细菌GA，这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，具有类似于甘氨酸结构的小分子可以增强自蛋白水解， AGU突变体水解酶活性。基于我们最近在GA自动处理方面的进展，我们建议在 该应用1）表征AGU突变的分子发病机制; 2）推进我们的结构 GA自身蛋白水解激活机制的研究; 3）研究AGU的结构后果 突变;和4）开发小分子以刺激AGU分子的自动加工。宽，长- 本申请的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。

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.