Screening for Inhibitors of the Integrated Stress Response

描述(申请人提供)：大量重要的生物蛋白质，如激素、酶和细胞表面受体，在内质网(ER)中经历其生物发生的早期步骤。这一过程的缺陷和效率变化被认为是导致人类重大疾病的原因之一。例如，错误折叠和降解是溶酶体储存疾病中缺乏酶或某些肾脏疾病中缺乏膜转运蛋白的原因。内质网中的蛋白质折叠环境受信号转导途径的调节，这些信号转导通路共同构成内质网未折叠蛋白应答(UPR)，对细胞器中错误折叠的蛋白质应激做出反应。对这些途径的相对粗略的遗传操作表明，调节内质网中的蛋白质折叠环境可以产生重要的病理生理后果：例如，有证据表明，放松内质网中的质量控制可能会允许轻度错误折叠的蛋白质逃避内质网的保留和降解，从而有助于基本的细胞功能，从而改善严重的功能丧失突变的表型。其他研究表明，来自人类肿瘤的癌细胞尤其依赖于它们的UPR生存，这表明UPR抑制剂可能对癌症具有选择性毒性。因此，在UPR中调节信号的药物探针的可用性将提供迫切需要的工具来测试该通路作为蛋白质错误折叠和癌症疾病治疗干预的靶点的适用性。翻译起始因子2a(EIF2a)被内质网应激激活蛋白激酶PERK磷酸化，是UPR的一个被广泛理解和潜在可塑性的臂，被称为整合应激反应(ISR)。已经开发出可靠的基于细胞的ISR活性分析方法。这些需要测量伴随着PERK激活和eIF2a磷酸化的翻译抑制的程度，以及报告由eIF2a磷酸化启动的基因表达程序的活性的补充试验。所讨论的分析已被小型化并转换为适合小分子(“探针”)的高通量筛选(HTS)的均一格式，当添加到细胞中时，将阻断PERK活性，损害eIF2a磷酸化所需的下游步骤，或将eIF2a磷酸化信号转换为激活基因表达的信号。已经开发出三级分析方法来精确定位HTS发现的任何抑制分子的作用部位。使用这些分析的HTS活动预计将产生有效的细胞穿透性小分子，在体内的不同时间点抑制ISR。与倾向于产生相对不连续的剂量反应的遗传方法不同，小分子抑制剂被预测具有长时间单调阶段的连续剂量-反应关系。研究界将利用这些特征来检验这样一种假设，即温和和部分抑制ISR可能会促进错误折叠的蛋白质的分泌，并选择性地损害肿瘤模型的生存能力。近年来，我们对蛋白质获得其适当结构的过程的了解显著增加，随着对这种理解的理解，干预蛋白质折叠过程以达到治疗目的的前景也随之而来。这项研究的重点是细胞调节其在分泌室折叠蛋白质的能力的一条途径，旨在识别通过抑制其关键成分之一--一种名为perk的酶来调节该途径中的信号传递的类药物化合物。如果成功，这项研究将告诉我们，PERK抑制剂是否具有治疗蛋白质错误折叠疾病的潜在效用，如溶酶体储存疾病和各种癌症。

DESCRIPTION (provided by applicant): A large number of biologically important proteins, such as hormones, enzymes and cell surface receptors undergo early steps of their biogenesis in the endoplasmic reticulum (ER). Defects and variations in efficiency of this process are believed to contribute to important human diseases. For example, misfolding and degradation accounts for lack of enzymes in lysosomal storage diseases or membrane transporters in certain kidney diseases. The protein folding environment in the ER is regulated by signal transduction pathways that together constitute the ER unfolded protein response (UPR), which responds to misfolded protein stress in the organelle. Relatively crude genetic manipulation of these pathways has shown that modulating the protein folding environment in the ER can have important pathophysiological consequences: For example, evidence suggests that loosening the quality control in the ER might allow mildly misfolded proteins that are otherwise functional to escape ER retention and degradation and contribute to essential cellular functions and thereby ameliorate severe phenotypes of loss-of-function mutations. Other studies show that cancer cells from human tumors are particularly reliant on their UPR for survival, suggesting that UPR inhibitors may have selective toxicity against cancer. Therefore, availability of pharmacological probes to modulate signaling in the UPR will provide much needed tools to test the suitability of the pathway as a target for therapeutic intervention in diseases of protein misfolding and cancer. Phosphorylation of translation initiation factor 2a (eIF2a) by the ER stress activated protein kinase PERK is a well understood and potentially malleable arm of the UPR that is referred to as the integrated stress response (ISR). Robust cell-based assays for activity of the ISR have been developed. These entail measurements of the magnitude of translation repression attendant upon PERK activation and eIF2a phosphorylation and a complementary assay that reports on the activity of the gene expression program that is initiated by eIF2a phosphorylation. The assays in question have been miniaturized and converted to a homogenous format suitable for high throughput screens (HTS) for small molecules ("probes") that when added to cells, would either block PERK activity, impair the downstream steps required for eIF2a phosphorylation or the conversion eIF2a phosphorylation signal to the activation of gene expression. Tertiary assays have been developed to pinpoint the site of action of any inhibitory molecules discovered by the HTS. An HTS campaign using these assays is expected to yield potent cell penetrant small molecules that inhibit the ISR at various points in vivo. Unlike the genetic approaches, which tend to produce relatively discontinuous dose responses, small molecule inhibitors are predicted to have continuous dose-response relationships with lengthy monotonic phases. These feature will be exploited by the research community to test the hypothesis that gentle and partial inhibition of the ISR might promote the secretion of otherwise misfolded proteins and selectively compromise the viability of tumor models. Our understanding of the processes by which proteins attain their proper structure has increased markedly in recent years and with that understanding come the prospects of intervening in the process of protein folding to therapeutic ends. This study focuses on one pathway by which cells regulate their capacity to fold proteins in the secretory compartment and is designed to identify drug-like compounds that modulate signaling in that pathway by inhibiting one of its key components, an enzyme called PERK. If successful, this study will tell us whether or not PERK inhibitors have potential utility in treating diseases of protein misfolding, such as lysosomal storage diseases and various cancers.