A HTS assay to identify molecular probes for arylsulfatase A

描述(申请人提供)：溶酶体储存疾病(LSD)是一组遗传性代谢疾病，由对溶酶体功能至关重要的蛋白质缺陷引起。虽然个别情况很少见，但它们的总体发病率仅略高于7000名活产儿，这使得LSD成为一个重要的健康问题。这些疾病中的大多数都有进行性的神经退化过程，导致严重的虚弱。除非缺陷酶的残留活性降低90%以上，否则不会出现症状。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，并可以阻止或逆转疾病过程。目前可用于LSD的治疗方法，如酶替代疗法(ERT)，仅适用于少数LSD的治疗，然后仅适用于非神经系统症状。此外，超过250,000美元/年/患者的大约费用限制了急诊治疗的可及性。出于这些原因，基于小分子的疗法似乎是一种有吸引力的方法，因为它可以跨越血脑屏障，而且成本更低。小分子可以作为酶增强剂，结合并稳定内质网(ER)中突变的错误折叠蛋白，使它们避开内质网相关降解(ERAD)，到达溶酶体。此前，我们筛选了1,040-FDA批准的化合物的小型化合物文库，并能够识别和表征两种药物，它们作为两种溶酶体酶的酶增强剂：己糖胺酶A和葡萄糖脑苷酶。我的假设是，小分子可能通过与突变酶直接相互作用，或通过间接作用于不同的细胞途径，导致突变蛋白和溶酶体部分功能蛋白水平的增加，来增强LSD患者的残余活性。我假设我可以找到修复芳基硫酸酯酶A(ASA)的分子探针，这是一种在异色性脑白质营养不良(MLD)中缺失的溶酶体酶，是一种神经退行性LSD，没有特定的治疗方法。为了验证这一假设，我的目标是：(I)建立一种可靠和可重复性的基于细胞的高通量筛选(HTS)试验，以确定可作为ASA酶增强剂的分子探针；(Ii)为ASA建立一种定量HTS试验，并对初步筛选产生的“活性”小分子实施验证策略。我预计筛查项目完成后将提交给NIH化学基因组学中心实施。通过这次筛选产生的候选分子探针将根据我之前在其他溶酶体酶的酶增强剂的鉴定和特性方面获得的经验，使用不同的检测方法进行验证。经过验证的小分子将成为治疗MLD患者的潜在候选药物，也可能被用作揭示涉及溶酶体蛋白折叠、成熟和运输的新生物途径的工具。 公共卫生相关性：芳基硫酸酯酶A(ASA)高通量筛选试验(HTS)的发展将使分子探针的鉴定成为可能，一旦表征和验证，这些分子探针可能成为治疗异色性脑白质营养不良患者的潜在候选药物，异色性脑白质营养不良是一种由ASA缺乏引起的溶酶体储存性疾病。此外，这些化合物还可作为研究工具，用于研究与溶酶体蛋白折叠、成熟和运输有关的生物途径。

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.