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A Chemical Biology Approach to Model Pontocerebellar Hypoplasia Type 1B (PCH1B)
ACS Chemical Biology ( IF 4 ) Pub Date : 2018-08-24 00:00:00 , DOI: 10.1021/acschembio.8b00745
Liberty François-Moutal 1, 2 , Shahriyar Jahanbakhsh 3 , Andrew D. L. Nelson 4 , Debashish Ray 5 , David D. Scott 1, 2 , Matthew R. Hennefarth 3 , Aubin Moutal 1 , Samantha Perez-Miller 1, 2 , Andrew J. Ambrose 6 , Ahmed Al-Shamari 1 , Philippe Coursodon 1 , Bessie Meechoovet 7 , Rebecca Reiman 7 , Eric Lyons 4 , Mark Beilstein 4 , Eli Chapman 6 , Quaid D. Morris 5, 8, 9, 10 , Kendall Van Keuren-Jensen 7 , Timothy R. Hughes 5, 8 , Rajesh Khanna 1, 2 , Carla Koehler 3 , Joanna Jen 11 , Vijay Gokhale 12 , May Khanna 1, 2
Affiliation  

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.

中文翻译:

1B型桥小脑发育不全(PCH1B)的化学生物学方法

EXOSC3的突变与罕见的神经系统疾病,称为1B桥小脑发育不全(PCH1B)有关。EXOSC3是将RNA导入RNA外泌体(降解RNA的细胞机制)的三种假定的RNA结合结构帽蛋白之一。使用RNAcompete,我们鉴定了与EXOSC3结合的富含G的RNA基序。表面等离振子共振(SPR)和微尺度热泳(MST)表明,EXOSC3在低微摩尔范围内对长和短的富含G的RNA序列具有亲和力。尽管EXOSC3中的几个引起PCH1B的突变没有参与RNA竞争显示的特定RNA图案,但它们却表现出了与富含G的RNA较低的结合亲和力,如MST所示。为了检验这一假设,即EXOSC3突变体中RNA-蛋白质界面的修饰可能被小分子表型复制,我们进行了一项在计算机上筛选5万个小分子,并使用酶联免疫吸附测定(ELISAs)和MST评估分子抑制EXOSC3结合RNA的能力。我们确定了一个小分子,EXOSC3-RNA破坏(ERD)化合物3(ERD03),(i)在饱和传递差核磁共振(STD-NMR)中与EXOSC3特异性结合,(ii)破坏了EXOSC3-RNA的相互作用。浓度依赖性的方式,并且(iii)产生了PCH1B样表型,小脑减少了50%,斑马鱼胚胎中的脊柱弯曲异常。该化合物还诱导了斑马鱼RNA表达水平的改变,类似于针对EXOSC3的吗啉代所观察到的水平。据我们所知,这是通过合理设计获得的小分子的第一个例子,该小分子模拟了整个生物体内神经退行性疾病的异常发育作用。
更新日期:2018-08-24
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