A HTS assay to identify molecular probes for arylsulfatase A

描述（由申请人提供）：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，但它们的总体发病率刚刚超过1/7,000活产，这使得lsd成为一个重要的健康问题。这些疾病大多具有进行性神经退行性过程，导致严重的衰弱。除非缺陷酶的剩余活性降低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。目前用于lsd的治疗方法，如酶替代疗法（ERT）仅用于治疗少数lsd，然后仅用于非神经系统症状。此外，每位患者每年超过25万美元的费用限制了ERT的可及性。基于这些原因，基于小分子的治疗方法，可以穿过血脑屏障并且更便宜；似乎是一个有吸引力的方法。小分子可以作为酶增强剂，通过结合和稳定内质网（ER）中突变的错误折叠蛋白，使其逃避内质网相关降解（ERAD），并到达溶酶体。在此之前，我们筛选了1,040个fda批准的化合物的小化合物库，并能够识别和表征两种药物，作为两种溶酶体酶的酶增强剂：己糖氨基酶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，导致溶酶体室中突变和部分功能蛋白的水平增加，从而增强lsd患者中发现的残留活性。我假设我可以识别分子探针，可以恢复芳基磺化酶A (ASA)，异色性脑白质营养不良（MLD）的溶酶体酶缺陷，这是一种神经退行性LSD，没有特定的治疗方法。为了验证这一假设，我的目标是：(I)开发一种可靠且可重复的基于细胞的高通量筛选（HTS）试验，以鉴定可能作为ASA酶增强剂的分子探针；（ii）配置ASA的定量HTS分析，并对初筛产生的“活性”小分子进行验证策略。我期待筛选项目完成后提交给NIH化学基因组学中心实施。通过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表征方面获得的经验，使用不同的测定方法进行验证。这些经过验证的小分子将成为治疗MLD患者的潜在候选药物，也可能被用作揭示参与溶酶体蛋白折叠、成熟和运输的新生物学途径的工具。

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.