Cell based HTS for Rescue of Mutant Lysosomal Enzymes

基于细胞的高温超导技术拯救突变型溶酶体酶

• 批准号: 6900881
• 负责人: Don Joseph MAHURAN
• 金额: $ 12.49万
• 依托单位: HOSPITAL FOR SICK CHLDRN (TORONTO)
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-15 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6900881
• 关键词: Gaucher' s disease; Sandhoff disease; Tay Sachs disease; X ray crystallography; beta N acetylhexosaminidase; binding sites; bioassay; biotechnology; cell line; combinatorial chemistry; drug discovery /isolation; enzyme activity; enzyme inhibitors; fibroblasts; gene mutation; high throughput technology; human tissue; inborn lysosomal enzyme disorder; lysosomes; method development; molecular chaperones; peptide library; protein binding; small molecule

DESCRIPTION (provided by applicant): The majority of lysosomal storage diseases (LSD) exist as late-onset variants with neurological involvement. In Tay-Sachs and Gaucher disease the affected enzymes, Hexosaminidase and Glucocerebrosidase, are unable to cleave the terminal monosaccharides from gangliosides leading to their accumulation in the lysosome. We and others have shown that the residual activity of the affected enzymes can be augmented by pharmacological chaperones; small molecule compounds that specifically target and stabilize the mutant. We have further validated this approach and shown that high throughput screening (NTS) of drug-like small molecule libraries can be used to identify novel compounds which inhibit the activity of purified hexosaminidase and that can enhance enzyme activity in cells. These and other pharmacological chaperones achieve specificity by targeting the active site of the enzyme. However, effective in the enhancing the activity of the enzyme, as active site inhibitors and depending on dosage and duration, they could in the log-term potentially inhibit the activity of the enzyme to be rescued. Our goal is to identify other specific "chemical chaperones" that do not bind to functional sites on the mutant enzyme, i.e. are not inhibitory, but which also specifically bind to and stabilize the enzyme. Thus we aim to: 1) Develop cell based assays which continuously monitor the activity of lysosomal enzymes using 4-methylumbelliferone based substrates. 2) Use these cell based assays in a HTS for compounds which enhance the activity of the lysosomal enzyme in cells derived from patients with the lysosomal storage disease. 3) Select those compounds for further development, which enhance the activity of the mutant by specifically binding to the lysosomal enzyme, without compromising its activity. 4) Determine the area in the enzyme that binds each compound. In the future these results will be used to design more new effective compounds.

描述（由申请人提供）：大多数溶酶体储存疾病（LSD）作为具有神经系统参与的晚发变体存在。在Tay-Sachs和Gaucher疾病中，受影响的酶，己糖胺酶和葡萄糖脑苷酶无法从神经节剂中裂解末期单糖，导致它们在溶酶体中的积累。我们和其他人表明，药理学伴侣可以增强受影响酶的残留活性。小分子化合物专门靶向并稳定突变体。我们已经进一步验证了这种方法，并表明可以使用高吞吐量筛选（NTS），可用于鉴定抑制纯化的己糖胺酶活性的新型化合物，并可以增强细胞中的酶活性。这些和其他药理学伴侣通过靶向酶的活性位点实现特异性。然而，有效地增强酶的活性，作为活性部位抑制剂，并取决于剂量和持续时间，它们可以在对数期可能会抑制要营救的酶的活性。我们的目标是鉴定其他与突变酶上功能部位不结合的其他特定的“化学伴侣”，即不是抑制性的，而是特异性的，而是特异性的，它也特别结合并稳定酶。因此，我们的目的是：1）开发基于细胞的测定，该测定方法不断使用基于4-甲基固体酮的底物来连续监测溶酶体酶的活性。 2）在HTS中使用这些基于细胞的测定方法，以增强源自溶酶体储存疾病患者的细胞中溶酶体酶的活性。 3）选择这些化合物以进行进一步发育，从而通过特异性结合溶酶体酶来增强突变体的活性，而不会损害其活性。 4）确定结合每种化合物的酶中的面积。将来，这些结果将用于设计更多新的有效化合物。