A Chemical Biology Approach to Model Pontocerebellar Hypoplasia Type 1B (PCH1B)
A Chemical Biology Approach to Model Pontocerebellar Hypoplasia Type 1B (PCH1B)
复制标题
DOI：
10.1021/acschembio.8b00745
复制
发表时间：
2018-10-01
影响因子：
4
通讯作者：
Khanna, May
中科院分区：
文献类型：
--
作者：
Francois-Moutal, Liberty;Jahanbakhsh, Shahriyar;Khanna, May
EXOSC3的突变被认为与一种罕见的神经疾病有关，这种疾病被称为桥小脑发育不全1B型(PCH1B)。EXOSC3是三种假定的RNA结合结构帽蛋白之一，它们引导RNA进入RNA外体，即降解RNA的细胞器。利用RNA竞争，我们鉴定了一个与EXOSC3结合的富含G的RNA基序。表面等离子体共振(SPR)和微尺度热电泳法(MST)表明，在EXOSC3的低微摩尔范围内，长和短的富含G的RNA序列都具有亲和力。虽然EXOSC3中几个导致PCH1B的突变不像RNA竞争那样与特定的RNA基序结合，但MST表明它们与富含G的RNA结合亲和力较低。为了验证EXOSC3突变体中RNA-蛋白质界面的修饰可能被小分子所复制的假设，我们对50,000个小分子进行了电子筛选，并使用酶联免疫吸附分析(ELISA)和MST来评估这些分子抑制EXOSC3与RNA结合的能力。我们鉴定了一个小分子，EXOSC3-RNA破坏(ERD)化合物3(ERD03)，它(1)在饱和转移差核磁共振(STD-核磁共振)中与EXOSC3特异性结合，(2)以浓度依赖的方式中断EXOSC3 RNA的相互作用，(3)在斑马鱼胚胎中产生PCH1B样表型，小脑减少50%，脊椎异常弯曲。该化合物还诱导斑马鱼RNA表达水平的改变，与吗啡针对EXOSC3观察到的水平相似。据我们所知，这是第一个通过合理设计获得的小分子的例子，它模拟了整个有机体中神经退行性疾病的异常发育效应。
Mutations of EXOSC3 have been linked to the rare neurological disorder known as Pontocerebellar Hypoplasia type 1B (PCH1B). EXOSC3 is one of three putative RNA-binding structural cap proteins that guide RNA into the RNA exosome, the cellular machinery that degrades RNA. Using RNAcompete, we identified a G-rich RNA motif binding to EXOSC3. Surface plasmon resonance (SPR) and microscale thermophoresis (MST) indicated an affinity in the low micromolar range of EXOSC3 for long and short G-rich RNA sequences. Although several PCH1B-causing mutations in EXOSC3 did not engage a specific RNA motif as shown by RNAcompete, they exhibited lower binding affinity to G-rich RNA as demonstrated by MST. To test the hypothesis that modification of the RNA-protein interface in EXOSC3 mutants may be phenocopied by small molecules, we performed an in-silico screen of 50 000 small molecules and used enzyme-linked immunosorbant assays (ELISAs) and MST to assess the ability of the molecules to inhibit RNA-binding by EXOSC3. We identified a small molecule, EXOSC3-RNA disrupting (ERD) compound 3 (ERD03), which (i) bound specifically to EXOSC3 in saturation transfer difference nuclear magnetic resonance (STD-NMR), (ii) disrupted the EXOSC3 RNA interaction in a concentration-dependent manner, and (iii) produced a PCH1B-like phenotype with a 50% reduction in the cerebellum and an abnormally curved spine in zebrafish embryos. This compound also induced modification of zebrafish RNA expression levels similar to that observed with a morpholino against EXOSC3. To our knowledge, this is the first example of a small molecule obtained by rational design that models the abnormal developmental effects of a neurodegenerative disease in a whole organism.