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

基本信息

批准号：
8886806
负责人：
Nancy A Muma
金额：
$ 50.52万
依托单位：
UNIVERSITY OF KANSAS LAWRENCE
依托单位国家：
美国
项目类别：
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/8886806
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 Drug Targeting Enzymes Evaluation Functional disorder Future Genes Genetic screening method Goals Huntington Disease 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 human Huntingtin protein in vitro Assay in vivo in vivo Model indexing inhibitor/antagonist interest mouse model mutant novel polyglutamine prevent progressive neurodegeneration protective effect protein protein interaction public health relevance receptor research clinical testing response scaffold screening small molecule

项目摘要

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) 在美国每 10,000 人中就有一人患有亨廷顿病，目前估计美国约有 30,000 名患者患有亨廷顿病，这是一种由多聚谷氨酰胺编码的常染色体显性突变引起的进行性神经退行性疾病。尽管亨廷顿蛋白的扩增已经确定，并且可以通过基因测试来识别携带突变的个体。会死于这种疾病，目前没有减缓或预防疾病进展的疗法，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和最终测试的生物探针。我们之前报道了破坏突变体相互作用的体外和体内概念证明。亨廷顿蛋白 (mhtt) 与钙调蛋白 (CaM) 是 HD 模型中治疗的杰出靶标。我们证明，用由 CaM 76-121 氨基酸组成的 46 个氨基酸肽破坏 mhtt 与 CaM 的结合具有保护作用。在瞬时表达 mhtt 蛋白构建体的 HEK293 细胞中、在稳定表达 mhtt 蛋白构建体的神经元分化的 SH-SY5Y 细胞和转基因小鼠中HD、R6/2 小鼠模型的目标是确定选择性小分子抑制剂来破坏 mhtt 与 CaM 的结合。 mhtt 与 CaM 的结合将用于测试选择性，使用体外测定来测试 CaM 与其他蛋白质相互作用的破坏，并使用基于细胞培养的测定来评估 mhtt 的细胞毒性。 {化学信息学分析将确定一组优先化学型，这些化学型将进行迭代药物化学优化。}最后，HD 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 和破坏具有神经保护作用。该项目的目标是识别破坏 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。将进一步防止 mhtt 在神经元细胞中的有害影响以及 mhtt 在神经元中的转酰胺基作用。该项目的长期目标（超出本提案的范围）是开发可预防神经变性和其他有害物质的化合物。 mhtt 对 HD 转基因小鼠模型的影响，并最终导致 HD 的转化临床测试。