Structure-Based Design of Small Molecules for Aspartylglucosaminuria

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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.