Identification of Autophosphorylation Inhibitors of the Inositol-Requiring Enzyme 1 Alpha (IRE1α) by High-Throughput Screening Using a DELFIA Assay
Identification of Autophosphorylation Inhibitors of the Inositol-Requiring Enzyme 1 Alpha (IRE1α) by High-Throughput Screening Using a DELFIA Assay
复制标题
DOI：
10.1177/1087057112465647
复制
发表时间：
2013-03-01
影响因子：
--
通讯作者：
van Montfort, Rob L. M.
中科院分区：
文献类型：
--
作者：
Newbatt, Yvette;Hardcastle, Anthea;van Montfort, Rob L. M.
肌醇需要酶1 α（IRE1 α）是一种跨膜传感蛋白，具有激酶和核糖核酸酶活性，在未折叠蛋白反应（UPR）中起着至关重要的作用。内质网（ER）腔中的蛋白质错误折叠引发IRE 1 α的二聚化和随后的反式自磷酸化。这导致其核糖核酸内切酶（RNase）结构域的激活和转录激活因子XBP1 mRNA的剪接，最终产生与多发性骨髓瘤生存有关的活性XBP1（XBP1s）。以前，我们已经确定了人IRE1 α作为开发激酶抑制剂的靶点，该激酶抑制剂可以调节人细胞中的UPR，这与多发性骨髓瘤和其他分泌性恶性肿瘤特别相关。在这里，我们描述了一个384孔高通量筛选试验的开发和验证，使用DELFIA技术，是特定的IRE1 α自磷酸化。使用这种形式，筛选了2312种潜在激酶抑制剂的集中文库，并鉴定了几种新型IRE1 α激酶抑制剂支架，这些支架可能被开发用于治疗多发性骨髓瘤的新疗法。
Inositol-requiring enzyme 1 alpha (IRE1 alpha) is a transmembrane sensor protein with both kinase and ribonuclease activity, which plays a crucial role in the unfolded protein response (UPR). Protein misfolding in the endoplasmic reticulum (ER) lumen triggers dimerization and subsequent trans-autophosphorylation of IRE1 alpha. This leads to the activation of its endoribonuclease (RNase) domain and splicing of the mRNA of the transcriptional activator XBP1, ultimately generating an active XBP1 (XBP1s) implicated in multiple myeloma survival. Previously, we have identified human IRE1 alpha as a target for the development of kinase inhibitors that could modulate the UPR in human cells, which has particular relevance for multiple myeloma and other secretory malignancies. Here we describe the development and validation of a 384-well high-throughput screening assay using DELFIA technology that is specific for IRE1 alpha autophosphorylation. Using this format, a focused library of 2312 potential kinase inhibitors was screened, and several novel IRE1 alpha kinase inhibitor scaffolds were identified that could potentially be developed toward new therapies to treat multiple myeloma.