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，如激素、酶和细胞表面受体，在内质网（ER）中经历其生物合成的早期步骤。这一过程效率的缺陷和变化被认为是导致重要人类疾病的原因。例如，错误折叠和降解导致溶酶体胆积病中缺乏酶或某些肾脏疾病中缺乏膜转运蛋白。ER中的蛋白质折叠环境由信号转导途径调节，这些信号转导途径共同构成ER未折叠蛋白反应（UPR），其响应于细胞器中错误折叠的蛋白质应激。这些途径的相对粗糙的遗传操作表明，调节ER中的蛋白质折叠环境可能具有重要的病理生理学后果：例如，有证据表明，放松ER中的质量控制可能允许轻度错误折叠的蛋白质，这些蛋白质在其他方面具有功能，以逃避ER保留和降解，并有助于基本的细胞功能，从而改善严重的功能丧失突变表型。其他研究表明，来自人类肿瘤的癌细胞特别依赖于它们的UPR存活，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疾病和癌症的治疗干预的靶点的适用性。翻译起始因子2a（eIF 2a）通过ER应激激活蛋白激酶PERK的磷酸化是UPR的一个众所周知的和潜在的可塑性臂，其被称为整合应激反应（ISR）。已经开发了用于ISR活性的稳健的基于细胞的测定。这些需要测量PERK激活和eIF 2a磷酸化后伴随的翻译抑制的程度，以及报告由eIF 2a磷酸化启动的基因表达程序的活性的补充测定。所讨论的测定已经小型化并转化为适用于小分子（“探针”）的高通量筛选（HTS）的均质形式，当将所述小分子（“探针”）加入细胞中时，所述小分子将阻断PERK活性，损害eIF 2a磷酸化所需的下游步骤或将eIF 2a磷酸化信号转化为基因表达的活化。已经开发了三级测定来精确定位HTS发现的任何抑制分子的作用位点。预期使用这些测定的HTS活动产生在体内不同点抑制ISR的有效细胞渗透小分子。与倾向于产生相对不连续的剂量反应的遗传方法不同，预测小分子抑制剂具有连续的剂量-反应关系，具有漫长的单调阶段。研究界将利用这些特征来检验这一假设，即温和和部分抑制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，我们对蛋白质获得其适当结构的过程的理解显着增加，并且随着这种理解，干预蛋白质折叠过程以达到治疗目的的前景。这项研究的重点是细胞调节其在分泌室中折叠蛋白质的能力的一种途径，并旨在确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抑制剂是否在治疗蛋白质错误折叠疾病方面具有潜在的效用，例如溶酶体贮积病和各种癌症。