Small molecule-induced oxidation of protein disulfide isomerase is neuroprotective
Small molecule-induced oxidation of protein disulfide isomerase is neuroprotective
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DOI：
10.1073/pnas.1500439112
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发表时间：
2015-04-28
影响因子：
11.1
通讯作者：
Stockwell, Brent R.
中科院分区：
文献类型：
--
作者：
Kaplan, Anna;Gaschler, Michael M.;Stockwell, Brent R.
蛋白质二硫键异构酶（PDI）是内质网中的伴侣蛋白，其在涉及蛋白质错误折叠的神经变性疾病的小鼠模型和患者的脑中上调。然而，PDI在这些疾病中的作用尚未完全了解。在这里，我们报告了可逆的、神经保护性的先导优化化合物（R14）的发现，其充当PDI的调节剂。使用类似于10，000种铅优化的化合物的高通量筛选来鉴定L0C14，以有效拯救表达突变亨廷顿蛋白的PC12细胞的活力，然后在体外筛选中评估化合物对PDI还原酶活性的影响。等温滴定量热法和荧光实验显示，与PDI的结合是可逆的，Kd为62 nM，表明LOC 14是迄今报道的最有效的PDI抑制剂。使用2D异源单量子相关NMR实验，我们能够映射的结合位点的LOC14作为相邻的活性位点，并观察到，LOC14的结合力PDI采取氧化构象。此外，我们发现LOC 14诱导的PDI氧化不仅在细胞培养中具有神经保护作用，而且在皮质纹状体脑切片培养中也具有神经保护作用。LOC 14在小鼠肝微粒体和血浆中表现出高稳定性，低固有微粒体清除率和低血浆蛋白结合率。这些结果表明，LOC14是一种有前途的先导化合物，以评估在动物疾病模型中调节PDI的潜在治疗效果。
Protein disulfide isomerase (PDI) is a chaperone protein in the endoplasmic reticulum that is up-regulated in mouse models of, and brains of patients with, neurodegenerative diseases involving protein misfolding. PDI's role in these diseases, however, is not fully understood. Here, we report the discovery of a reversible, neuroprotective lead optimized compound (LOC)14, that acts as a modulator of PDI. LOC14 was identified using a high-throughput screen of similar to 10,000 lead-optimized compounds for potent rescue of viability of PC12 cells expressing mutant huntingtin protein, followed by an evaluation of compounds on PDI reductase activity in an in vitro screen. Isothermal titration calorimetry and fluorescence experiments revealed that binding to PDI was reversible with a K-d of 62 nM, suggesting LOC14 to be the most potent PDI inhibitor reported to date. Using 2D heteronuclear single quantum correlation NMR experiments, we were able to map the binding site of LOC14 as being adjacent to the active site and to observe that binding of LOC14 forces PDI to adopt an oxidized conformation. Furthermore, we found that LOC14-induced oxidation of PDI has a neuroprotective effect not only in cell culture, but also in corticostriatal brain slice cultures. LOC14 exhibited high stability in mouse liver microsomes and blood plasma, low intrinsic microsome clearance, and low plasma-protein binding. These results suggest that LOC14 is a promising lead compound to evaluate the potential therapeutic effects of modulating PDI in animal models of disease.