Ip Philbert, Sharda Priya Roy, Cunningham Anna, Chakrabartty Sumon, Pande Vijay, Chakrabartty Avijit
Campbell Family Institute for Cancer Research, Ontario Cancer Institute/University Health Network, Department of Biochemistry, University of Toronto, TMDT 4-305, 101 College Street, Toronto, Ontario, CanadaM5G 1L7.
Department of Chemical and Systems Biology, School of Medicine, Stanford University, 269 Campus Drive, Stanford, CA, 94305-5174, USA.
Protein Eng Des Sel. 2017 Jun 1;30(6):431-440. doi: 10.1093/protein/gzx025.
In many cases of familial amyotrophic lateral sclerosis (ALS), mutant forms of the Cu,Zn superoxide dismutase protein (SOD1) misfold and aggregate in motor neurons. Monomers of the normally homodimeric SOD1 have been found in patient tissue, presymptomatic mouse models of ALS, and in vitro misfolding assays which suggests that monomerization might be an early step in the pathological SOD1 misfolding pathway. In this study, we targeted the dimer interface with small molecules that might act as chemical chaperones to stabilize the native dimer and prevent downstream misfolding and aggregation. We performed a computational screen with a library of ~4400 drugs and natural compounds that were docked to two pockets around the SOD1 dimer interface. Of the resultant hits, seven were tested for misfolding and aggregation inhibition activity with A4V mutant SOD1. Quercitrin, quercetin-3-β-d-glucoside (Q3BDG), and, to a markedly lesser extent, epigallocatechin gallate (EGCG) were found to combat misfolding and aggregation induced by hydrogen peroxide, a physiologically relevant stress, as assessed by a gel-based assay and 8-anilinonaphthalene-1-suflonic acid (ANS) fluorescence. Isothermal titration calorimetry (ITC) and a colourimetric assay determined that these molecules directly bind A4V SOD1. Based on these findings, we speculate that quercitrin and Q3BDG may be potential therapeutic inhibitors of misfolding and aggregation in SOD1-associated ALS.
在许多家族性肌萎缩侧索硬化症（ALS）病例中，铜锌超氧化物歧化酶蛋白（SOD1）的突变形式在运动神经元中错误折叠并聚集。在患者组织、ALS症状前小鼠模型以及体外错误折叠试验中均发现了正常情况下为同二聚体的SOD1的单体，这表明单体化可能是病理性SOD1错误折叠途径中的早期步骤。在本研究中，我们用可能作为化学伴侣来稳定天然二聚体并防止下游错误折叠和聚集的小分子靶向二聚体界面。我们用一个包含约4400种药物和天然化合物的文库进行了计算筛选，这些化合物对接至SOD1二聚体界面周围的两个口袋。在所得的命中物中，对七种进行了针对A4V突变型SOD1的错误折叠和聚集抑制活性测试。通过基于凝胶的试验和8-苯胺基萘-1-磺酸（ANS）荧光评估发现，槲皮苷、槲皮素-3-β-D-葡萄糖苷（Q3BDG）以及程度明显较低的表没食子儿茶素没食子酸酯（EGCG）可对抗由过氧化氢（一种生理相关应激）诱导的错误折叠和聚集。等温滴定量热法（ITC）和比色测定法确定这些分子直接结合A4V SOD1。基于这些发现，我们推测槲皮苷和Q3BDG可能是SOD1相关ALS中错误折叠和聚集的潜在治疗抑制剂。
