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

 描述(申请人提供)：亨廷顿病(HD)在美国每10,000人中就有1人受到影响，目前美国估计约有30,000名患者。HD是一种进行性神经退行性疾病，由亨廷顿蛋白中编码多谷氨酰胺扩张的常染色体显性突变引起。尽管疾病的病因已经确定，基因测试也可以识别那些携带突变和 会死于这种疾病，目前还没有减缓或阻止疾病进展的治疗方法，只有对症治疗，效果有限。拟议研究的目的是确定可用于未来药物开发的生物探针，并最终测试其防止HD进行性神经变性的能力。我们之前报道了在体外和体内的概念证明，扰乱突变体的相互作用 亨廷顿蛋白(MHTT)和钙调蛋白(CaM)是治疗HD模型的优秀靶点。我们证明，用由CaM的76-121个氨基酸组成的46个氨基酸的多肽来破坏mHTT与CaM的结合，对瞬时表达mHTT蛋白结构的HEK293细胞、稳定表达mHTT蛋白结构的神经元分化的SH-SY5Y细胞和HD、R6/2转基因小鼠模型具有保护作用。目前提案的目标是寻找选择性小分子抑制剂来破坏mHTT与CaM的结合。我们已经开发并验证了一种HTS Ready AlphaScreen(R)测试，以确定破坏mHTT与CaM结合的化合物。将使用正交筛选来测试选择性，使用体外测试来测试CaM与其他蛋白质相互作用的中断，并使用基于细胞培养的测试来评估化合物的细胞毒性。{化学信息学分析将确定一组优先的化学类型，这些类型将受到迭代药物化学优化。}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具有神经保护作用，并破坏细胞中mHTT-CaM的相互作用。该项目的目标是确定破坏mHTT与CaM结合的化合物，并在不抑制CaM功能的情况下选择性地这样做。顶级化合物将进一步防止mHTT对神经细胞的有害影响和mHTT在神经元中的转胺化。该项目的长期目标是，在这项提案的范围之外，开发在转基因HD小鼠模型中防止mHTT的神经退化和其他有害影响的化合物，并最终导致HD的转译临床测试。

 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.