A HTS assay to identify molecular probes for arylsulfatase A

鉴定芳基硫酸酯酶 A 分子探针的 HTS 测定

• 批准号: 8299724
• 负责人: Gustavo Henrique Boff Maegawa
• 金额: $ 4.1万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8299724
• 关键词: Affect; Arylsulfatases; Binding; Biological; Biological Assay; Blood - brain barrier anatomy; Cells; Chemicals; Defect; Development; Disease; Endoplasmic Reticulum; Enzymes; FDA approved; Functional disorder; Gangliosidoses GM2; Gaucher Disease; Gene Mutation; Genomics; Health; Hex A; Hydrolase; Incidence; Inherited; Institution; Libraries; Live Birth; Lysosomal Storage Diseases; Lysosomes; Membrane Proteins; Metabolic; Metabolic Diseases; Metachromatic Leukodystrophy; Modeling; Molecular Bank; Molecular Probes; Mutation; Nerve Degeneration; Neurons; Pathway interactions; Patients; Pharmaceutical Preparations; Positioning Attribute; Process; Production; Proteins; Research; Residual state; Resources; Screening procedure; Symptoms; System; Testing; United States National Institutes of Health; Validation; Work; base; cost; disease phenotype; drug candidate; enzyme activity; enzyme replacement therapy; experience; glucosylceramidase; high throughput screening; lysosomal proteins; mutant; nervous system disorder; novel; prevent; protein folding; protein misfolding; public health relevance; small molecule; tool; trafficking

DESCRIPTION (provided by applicant): Lysosomal storage diseases (LSDs) are a group of inherited metabolic conditions caused by defects in proteins critical for lysosomal function. While individually rare, their collective incidence is just over 1/7,000 live births, which makes the LSDs an important health problem. The majority of these diseases have a progressive neurodegenerative course, resulting in severe debilitation. Symptoms are not manifested unless there is more than 90% reduction in the residual activity of the deficient enzyme. Thus, an enzyme activity level above the critical threshold will prevent lysosomal storage and dysfunction, and can arrest, or reverse the disease process. Current therapies available for LSDs, such as enzyme replacement therapy (ERT) are only available to treatment of a few LSDs, and then only the non-neurological symptoms. Additionally, the approximate cost of over US$250,000/year/patient limits accessibility to ERT. For these reasons, therapies based on small molecules, which could cross the blood-brain barrier and be less expensive; appear to be an attractive approach. Small molecules can function as enzyme enhancement agents by binding, and stabilizing mutant misfolded proteins in the endoplasmic reticulum (ER), allowing them to evade the ER associated degradation (ERAD), and reaches the lysosome. Previously, we screened a small compound library of 1,040-FDA approved compounds and were able to identify and characterize two drugs that function as enzyme enhancement agents for two lysosomal enzymes: hexosaminidase A and glucocerebrosidase. My hypothesis is that small molecules may enhance residual activity found in patients with LSDs by direct interactions with the mutant enzyme, or by an indirect effect on different cellular pathways resulting in increased levels of the mutant and partially functional protein in the lysosomal compartment. I hypothesize that I can identify molecular probes that would restore arylsulfatase A (ASA), lysosomal enzyme deficient in metachromatic leukodystrophy (MLD), a neurodegenerative LSD for which a specific treatment is not available. To test this hypothesis, I aim to: (i) develop a robust and reproducible cell-based high throughput screening (HTS) assay to identify molecular probes that may function as enzyme enhancement agents for ASA; (ii) to configure a quantitative HTS assay for ASA, and carry out validation strategies for the "active" small molecules generated by the primary screening. I anticipate the submission of the screening project upon completion to the NIH Chemical Genomics Center for implementation. The candidate molecular probes generated by this screening will then be validated using different assays based on my experience previously acquired on the identification and characterization of enzyme enhancement agents for other lysosomal enzymes. The validated small molecules will be potential drug candidates to treat patients with MLD, and may also be used as tools to uncover novel biological pathways involved on the folding, maturation and trafficking of lysosomal proteins. PUBLIC HEALTH RELEVANCE: The development of high throughput screening assay (HTS) for arylsulfatase A (ASA) will allow the identification of molecular probes that, once characterized and validated, may be potential drug candidates to treat patients with metachromatic leukodystrophy, a lysosomal storage disease caused by deficiency of ASA. In addition, these compounds may be used as research tools to examine biological pathways involved on the folding, maturation and trafficking of lysosomal proteins.

描述（由申请方提供）：溶酶体贮积病（LSD）是一组由溶酶体功能关键蛋白缺陷引起的遗传性代谢疾病。虽然个别罕见，但其集体发病率略高于1/7，000活产，这使得LSD成为一个重要的健康问题。这些疾病中的大多数具有进行性神经变性过程，导致严重衰弱。除非缺陷酶的残余活性降低超过90%，否则症状不明显。因此，高于临界阈值的酶活性水平将防止溶酶体储存和功能障碍，并且可以阻止或逆转疾病过程。目前可用于LSD的疗法，如酶替代疗法（ERT）仅可用于治疗少数LSD，并且仅用于治疗非神经症状。此外，超过250，000美元/年/患者的费用限制了ERT的可及性。由于这些原因，基于小分子的治疗方法，可以穿过血脑屏障，并且价格较低，似乎是一种有吸引力的方法。小分子可以通过结合和稳定内质网（ER）中的突变错误折叠蛋白质来发挥酶增强剂的作用，使它们能够逃避ER相关降解（ERAD）并到达溶酶体。此前，我们筛选了一个包含1，040种FDA批准的化合物的小型化合物库，并能够鉴定和表征两种药物，它们可作为两种溶酶体酶的酶增强剂：氨基己糖苷酶A和葡糖脑苷脂酶。我的假设是，小分子可能通过与突变酶的直接相互作用，或通过对不同细胞途径的间接影响，导致溶酶体区室中突变蛋白和部分功能蛋白水平升高，从而增强LSD患者的残留活性。我假设，我可以确定分子探针，将恢复芳基硫酸酯酶A（阿萨），溶酶体酶缺乏异染性脑白质营养不良（MLD），神经退行性LSD的具体治疗是不可用的。为了验证这一假设，我的目标是：（i）开发一个强大的和可重复的基于细胞的高通量筛选（HTS）测定，以确定分子探针，可能作为阿萨的酶增强剂;（ii）配置阿萨的定量HTS测定，并进行验证策略的“活性”的小分子产生的初步筛选。我预计筛选项目完成后提交给NIH化学基因组学中心实施。然后，将根据我先前在其他溶酶体酶的酶增强剂鉴定和表征方面获得的经验，使用不同的试验对通过该筛选产生的候选分子探针进行验证。经验证的小分子将是治疗MLD患者的潜在候选药物，也可用作发现溶酶体蛋白折叠，成熟和运输的新生物学途径的工具。 公共卫生关系：芳基硫酸酯酶A（阿萨）的高通量筛选试验（HTS）的发展将允许鉴定分子探针，一旦表征和验证，可能是治疗异染性脑白质营养不良（一种由阿萨缺乏引起的溶酶体贮积病）患者的潜在候选药物。此外，这些化合物可用作研究工具来检查涉及溶酶体蛋白的折叠、成熟和运输的生物途径。