Screening for Inhibitors of the Integrated Stress Response

综合应激反应抑制剂的筛选

• 批准号: 7365462
• 负责人: DAVID RON
• 金额: $ 2.43万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-27 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7365462
• 关键词: Accounting; Affect; Apical; Biochemical Genetics; Biogenesis; Biological; Biological Assay; Cell Surface Receptors; Cell Survival; Cell physiology; Cells; Chemicals; Communities; Defect; Disease; Dose; Drug Delivery Systems; Endoplasmic Reticulum; Environment; Enzymes; Equilibrium; Event; Exhibits; Failure; Family; Functional disorder; Gene Activation; Gene Expression; Genetic; Guanine Nucleotide Exchange Factors; Heat shock proteins; Homeostasis; Hormones; Human; Kidney Diseases; Libraries; Lysosomal Storage Diseases; Malignant Neoplasms; Measurement; Measures; Membrane Transport Proteins; Mind; Modeling; Molecular Chaperones; Numbers; Organelles; PERK kinase; Pathway interactions; Peptide Initiation Factors; Pharmaceutical Preparations; Phase; Phenotype; Phosphorylation; Phosphotransferases; Physiological; Process; Protein Biosynthesis; Protein Kinase; Proteins; Quality Control; Reporting; Repression; Research; Resources; Role; Screening procedure; Series; Serine; Signal Transduction; Signal Transduction Pathway; Site; Stress; Structure; System; Testing; Therapeutic; Therapeutic Intervention; Toxic effect; Translations; Upper arm; Variant; base; biological adaptation to stress; cancer cell; clinically relevant; coping; design; genetic manipulation; high throughput screening; human disease; in vivo; inhibitor/antagonist; loss of function mutation; miniaturize; pressure; programs; protein folding; protein misfolding; prototype; response; small molecule; stress activated protein kinase; stress protein; tool; tumor

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.

描述（由申请人提供）：大量生物学上重要的蛋白质，例如激素、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。人们认为这一过程的缺陷和效率变化会导致重要的人类疾病。例如，错误折叠和降解导致溶酶体贮积病中酶的缺乏或某些肾脏疾病中膜转运蛋白的缺乏。内质网中的蛋白质折叠环境受到信号转导途径的调节，这些信号转导途径共同构成了内质网未折叠蛋白反应（UPR），该反应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基因操作表明，调节 ER 中的蛋白质折叠环境可能会产生重要的病理生理学后果：例如，有证据表明，放松 ER 中的质量控制可能会导致轻度错误折叠的蛋白质，而这些蛋白质在其他情况下具有功能，可以逃脱 ER 保留和降解并有助于基本的细胞功能，从而改善功能丧失突变的严重表型。其他研究表明，来自人类肿瘤的癌细胞特别依赖其 UPR 来生存，这表明 UPR 抑制剂可能对癌症具有选择性毒性。因此，调节 UPR 中信号传导的药理学探针的可用性将提供急需的工具来测试该途径作为蛋白质错误折叠和癌症疾病的治疗干预目标的适用性。 ER 应激激活蛋白激酶 PERK 磷酸化翻译起始因子 2a (eIF2a) 是 UPR 的一个众所周知且具有潜在可塑性的臂，被称为综合应激反应 (ISR)。针对 ISR 活性的稳健的基于细胞的检测方法已经开发出来。这些需要测量 PERK 激活和 eIF2a 磷酸化所伴随的翻译抑制程度，以及报告由 eIF2a 磷酸化启动的基因表达程序的活性的补充测定。所讨论的测定已被小型化并转换为适合小分子（“探针”）高通量筛选（HTS）的同质形式，当将其添加到细胞中时，会阻断 PERK 活性，损害 eIF2a 磷酸化所需的下游步骤，或将 eIF2a 磷酸化信号转化为基因表达的激活。已经开发出三级测定法来查明 HTS 发现的任何抑制分子的作用位点。使用这些测定的 HTS 活动预计会产生有效的细胞渗透小分子，在体内的各个点抑制 ISR。与倾向于产生相对不连续的剂量反应的遗传方法不同，小分子抑制剂预计具有连续的剂量反应关系和较长的单调阶段。研究界将利用这些特征来检验这样的假设：温和且部分抑制 ISR 可能会促进错误折叠蛋白质的分泌，并选择性地损害肿瘤模型的生存能力。近年来，我们对蛋白质获得适当结构的过程的了解显着增加，随着这种了解，干预蛋白质折叠过程达到治疗目的的前景也随之而来。这项研究重点关注细胞调节其在分泌室中折叠蛋白质的能力的一种途径，旨在鉴定通过抑制其关键成分之一（一种称为 PERK 的酶）来调节该途径中信号传导的药物样化合物。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用途，例如溶酶体贮积病和各种癌症。