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Discovery of Small Molecule Inhibitors of Huntingtin Exon 1 Aggregation by FRET-Based High-Throughput Screening in Living Cells.
ACS Chemical Neuroscience ( IF 5 ) Pub Date : 2020-06-22 , DOI: 10.1021/acschemneuro.0c00226
Chih Hung Lo 1 , Nitin K Pandey 2 , Colin Kin-Wye Lim 1 , Zhipeng Ding 1 , Meixin Tao 2 , David D Thomas 3, 4 , Ralf Langen 2 , Jonathan N Sachs 1
Affiliation  

Huntington’s disease (HD) is the most common inherited neurodegenerative disorder and one of the nine polyglutamine (polyQ) diseases. HD is characterized by the pathological aggregation of the misfolded huntingtin exon 1 protein (Httex1) with abnormally long polyQ expansion due to genetic mutation. While there is currently no effective treatment for HD, inhibition of aggregate formation represents a direct approach in mediating the toxicity associated with Httex1 misfolding. To exploit this therapeutic window, we engineered two fluorescence resonance energy transfer (FRET) based biosensors that monitor the aggregation of Httex1 with different expanded Q-lengths (Q39 and Q72) in living cells. These FRET biosensors, together with a high-precision fluorescence lifetime detection platform, enable high-throughput screening of small molecules that target Httex1 aggregation. We found six small molecules that decreased the FRET of the biosensors and reduced Httex1-Q72-induced neuronal cytotoxicity in N2a cells with nanomolar potency. Using advanced SPR and EPR techniques, we confirmed that the compounds directly bind to Httex1 fibrils and inhibit aggregate formation. This strategy in targeting the Httex1 aggregates can be applicable to other proteins involved in polyQ related diseases.

中文翻译:

通过基于FRET的活细胞高通量筛选,发现Huntingtin外显子1聚集的小分子抑制剂。

亨廷顿舞蹈病(HD)是最常见的遗传性神经退行性疾病,是九种多谷氨酰胺(polyQ)疾病之一。HD的特征是因基因突变而导致折叠错误的亨廷顿外显子1蛋白(Httex1)的病理性聚集,并具有异常长的polyQ扩展。尽管目前尚无有效的HD治疗方法,但抑制聚集体形成是介导与Httex1错误折叠相关的毒性的直接方法。为了利用这个治疗窗口,我们设计了两个基于荧光共振能量转移(FRET)的生物传感器,它们监视具有不同扩展Q长度(Q39和Q72)的Httex1在活细胞中的聚集。这些FRET生物传感器以及高精度的荧光寿命检测平台,可以对靶向Httex1聚集的小分子进行高通量筛选。我们发现了六个小分子,它们降低了生物传感器的FRET,并降低了具有纳摩尔效价的N2a细胞中Httex1-Q72诱导的神经元细胞毒性。使用先进的SPR和EPR技术,我们证实了这些化合物直接结合到Httex1原纤维并抑制聚集体形成。这种针对Httex1聚集体的策略可适用于与polyQ相关疾病有关的其他蛋白质。
更新日期:2020-08-05
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