HTS to identify small molecules to disrupt abnormal huntingtin interactions in HD

HTS 鉴定小分子以破坏 HD 中异常的亨廷顿蛋白相互作用

• 批准号: 8997127
• 负责人: Nancy A Muma
• 金额: $ 51.29万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8997127
• 关键词: Affect; Amino Acids; Area; Binding; Biological; Biological Assay; Ca(2+)-Calmodulin Dependent Protein Kinase; Calcium Signaling; Calmodulin; Cause of Death; Cell Culture Techniques; Cell Line; Cells; Characteristics; Chemicals; Code; Disease; Disease Progression; Enzymes; Evaluation; Functional disorder; Future; Genes; Genetic screening method; Goals; Health; Huntington Disease; Huntington gene; In Vitro; Individual; Ion Channel; Lead; Measures; Metabolic Pathway; Modality; Modeling; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Patients; Peptides; Pharmaceutical Chemistry; Phosphotransferases; Proteins; Quality Control; Reporting; Screening Result; Technology; Testing; Toxic effect; Transgenic Mice; Transglutaminases; United States; autosomal dominant mutation; base; cheminformatics; cytotoxicity; drug development; high throughput screening; in vitro Assay; in vivo; in vivo Model; indexing; inhibitor/antagonist; interest; mouse model; mutant; new therapeutic target; polyglutamine; prevent; progressive neurodegeneration; protective effect; protein protein interaction; receptor; research clinical testing; response; scaffold; screening; small molecule; small molecule inhibitor; symptom treatment

 DESCRIPTION (provided by applicant): Huntington's disease (HD) affects one in every 10,000 people in the US, with a current estimate of ~30,000 patients in the US. HD is a progressive neurodegenerative disease caused by an autosomal dominant mutation coding for a polyglutamine expansion in huntingtin protein. Although the cause of the disease has been identified and genetic tests are available to identify those individuals who carry the mutation and will succumb to the disease, there is currently no therapy to slow or prevent the disease progression, only symptomatic treatments with limited impact. The purpose of the proposed studies is to identify biological probes which can be used for future drug development and eventual testing for their ability to prevent the progressive neurodegeneration in HD. We previously reported in vitro and in vivo proof of concept that disrupting the interaction of mutant huntingtin (mhtt) with calmodulin (CaM) is an outstanding target for treatment in models of HD. We demonstrated that disrupting the binding of mhtt to CaM, with a 46 amino acid peptide, consisting of amino acids 76-121 of CaM, was protective in HEK293 cells transiently expressing a mhtt protein construct, in neuronally differentiated SH- SY5Y cells stably expressing a mhtt protein construct and a transgenic mouse model of HD, R6/2 mice. The goal of the current proposal is to identify selective small molecule inhibitors to disrupt the binding of mhtt to CaM. We have developed and validated a HTS ready, AlphaScreen(r) assay to identify compounds which disrupt the binding of mhtt to CaM. Orthogonal screens will be used to test for selectivity using in vitro assays to test for disruption of the interaction of CaM with other proteins and a cel culture-based assay to evaluate cytotoxicity of the compounds. {Cheminformatic analysis will identify a set of prioritized chemotypes that will be subjected to iterative medicinal chemistry optimization.} Lastly, tertiary screens in a cell culture model of HD will be used to determine whether the chemical probes are neuroprotective against mhtt and disrupt mhtt-CaM interactions in cells. The goal of this project is to identify compounds that disrupt the binding o mhtt to CaM and do so selectively without inhibiting the function of CaM. The top compounds will further protect against both the deleterious effects of mhtt in neuronal cells and the transamidation of mhtt in neurons. The long-term objective of the project, outside of the scope of this proposal is to develop compounds that are protective against the neurodegeneration and other deleterious effects of mhtt in transgenic mouse models of HD and eventually lead to translational clinical testing for HD.

 描述（由申请人提供）：亨廷顿病（HD）影响美国每10，000人中就有一人，目前估计美国约有30，000名患者。HD是一种进行性神经退行性疾病，由编码亨廷顿蛋白中多聚谷氨酰胺扩增的常染色体显性突变引起。虽然这种疾病的原因已经确定，基因测试可以识别那些携带突变的人， 如果患者会死于这种疾病，目前还没有减缓或预防疾病进展的治疗方法，只有影响有限的对症治疗。拟议研究的目的是确定可用于未来药物开发的生物探针，并最终测试其预防HD进行性神经变性的能力。我们先前报道了在体外和体内的概念证明，破坏突变体的相互作用， 亨廷顿蛋白（mhtt）与钙调蛋白（CaM）是HD模型中治疗的突出靶点。我们证明了用由CaM的氨基酸76-121组成的46个氨基酸的肽破坏mhtt与CaM的结合在瞬时表达mhtt蛋白构建体的HEK 293细胞中、在稳定表达mhtt蛋白构建体的神经元分化的SH-SY 5 Y细胞和HD R6/2小鼠的转基因小鼠模型中是保护性的。目前的建议的目标是确定选择性的小分子抑制剂，破坏mhtt的钙调素的结合。我们已经开发并验证了HTS就绪的AlphaScreen（r）测定，以鉴定破坏mhtt与CaM结合的化合物。正交筛选将用于测试选择性，使用体外测定来测试CaM与其他蛋白质的相互作用的破坏，以及基于细胞培养的测定来评估化合物的细胞毒性。{化学信息学分析将识别一组优先化型，其将经受迭代药物化学优化。}最后，在HD的细胞培养模型中的三级筛选将用于确定化学探针是否对mhtt具有神经保护作用并破坏细胞中的mhtt-CaM相互作用。该项目的目标是鉴定破坏mhtt与CaM结合的化合物，并且选择性地这样做而不抑制CaM的功能。最好的化合物将进一步防止神经元细胞中的mhtt的有害作用和神经元中的mhtt转酰胺作用。该项目的长期目标（超出本提案范围）是开发能够在HD转基因小鼠模型中保护mhtt免受神经变性和其他有害影响的化合物，并最终导致HD的转化临床试验。