HD Protein Interaction Based Drug Screening Assays

描述(由申请人提供)： 亨廷顿病(HD)是一种家族性致死性神经退行性疾病，由Htt蛋白表达扩大的聚谷氨酰胺束引起。目前还没有有效的治疗方法或治疗方法。目前发现HD潜在治疗方法的药物筛选工作包括使用体外聚集分析、基于细胞的和基于模型生物的Htt毒性分析。HD病理的确切分子细节尚不完全清楚，但很明显，扩展的Htt毒性的某些方面是由涉及突变Htt和其他细胞蛋白的异常蛋白质-蛋白质相互作用驱动的。突变的Htt与转录因子Sp1和转录共激活因子CBP的直接蛋白质-蛋白质相互作用被认为是HD的关键病理机制。到目前为止，已经描述了大约40个Htt的蛋白质相互作用伙伴，其中一些相互作用很可能也在HD病理中起到致病作用。鉴于蛋白质相互作用在HD病理中的突出作用，可以特异性破坏致病Htt蛋白相互作用的药物可能具有治疗益处。 这项建议的主要目标是开发适合高通量筛选(HTS)的基于细胞和体外蛋白质相互作用的分析方法。这些将被用来发现能够破坏Htt蛋白质-蛋白质相互作用的化合物。其中一种检测将是基于酵母的“反向双杂交”检测，采用可逆选择的报告基因。这种酵母试验被配置为Htt-蛋白质相互作用是致命的，因此可以用来筛选抑制致命性的化合物。第二个补充试验将是基于体外荧光共振能量转移(FRET)的蛋白质相互作用试验，该试验将用于在简单的二元体系中筛选能够抑制蛋白质相互作用的化合物。这些检测将同时用于筛选能够抑制Htt和10个不同的Huntingtin结合伙伴之间的蛋白质相互作用的化合物。这些合作伙伴中的两个将是CBP和SPL，另外八个将从正在进行的蛋白质相互作用发现计划中选择。在分析开发和验证后，将使用一个小的化学库(NINDS定制收集)进行初始筛选。这些筛查工具和HITS将在内部进一步开发，并通过与学术和基于基金会的HD治疗发现工作的合作来进一步开发。

DESCRIPTION (provided by applicant): Huntington's Disease (HD) is a familial fatal neurodegenerative disease caused by expression of expanded polyglutamine tracts in the (Htt) protein. Currently there are no effective cures or treatments for HD. Current drug screening efforts to discover potential therapeutics for HD include the use of in vitro aggregation assays, cell-based, and model organism based Htt toxicity assays. The precise molecular details of HD pathology are not fully understood, however it is clear that some aspects of expanded Htt toxicity are driven by aberrant protein-protein interactions involving the mutant Htt and other cellular proteins. Direct protein-protein interaction of mutant Htt with the transcription factor Spl, and with the transcriptional co-activator CBP have been implicated as key pathologic mechanisms in HD. To date, about 40 protein interaction partners for Htt have been described, and it is likely that some of these interactions will also have causative roles in HD pathology. Given the prominent role for protein interactions in HD pathology, it is likely that drugs which could specifically disrupt pathogenic Htt protein interactions could be of therapeutic benefit. The primary goal of this proposal is to develop cell-based and in vitro protein interaction assays suitable for high throughput screening (HTS). These will be used to discover compounds able to disrupt Htt protein-protein interactions. One assay will be a yeast-based "reverse two hybrid" assay employing a counter-selectable reporter gene. This yeast assay is configured such that Htt-protein interactions are lethal, and can thus be used to screen for compounds that suppress lethality. A complementary second assay will be an in vitro fluourescence resonance energy transfer (FRET)-based protein interaction assay which will be used to screen for compounds able to inhibit protein interactions in simple binary systems. These assays will be used in parallel to screen for compounds able to inhibit protein interaction between Htt and 10 different huntingtin binding partners. Two of these partners will be CBP and Spl, and eight others will be chosen from an on-going protein interaction discovery program. After assay development and validation, initial screens will be performed with a small chemical library (NINDS custom collection). These screening tools and hits will be further developed internally, as well as through collaborations with academic and foundation-based HD therapeutic discovery efforts.