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）中经历其生物发生的早期步骤。这一过程的缺陷和效率的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贮积性疾病或某些肾脏疾病的膜转运蛋白缺乏酶。内质网中的蛋白质折叠环境受信号转导途径调控，这些信号转导途径共同构成内质网未折叠蛋白反应（UPR）， UPR对细胞器中错误折叠的蛋白质应激作出反应。对这些途径的相对粗糙的遗传操作表明，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，放松内质网的质量控制可能会使原本具有功能的轻度错误折叠的蛋白质逃脱内质网的保留和降解，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信号传导的药理学探针的可用性将为测试该途径作为蛋白质错误折叠疾病和癌症治疗干预靶点的适用性提供急需的工具。翻译起始因子2a （eIF2a）被内质网应激激活的蛋白激酶PERK磷酸化是UPR的一个很好的理解和潜在的可塑性分支，被称为综合应激反应（ISR）。已经开发出了基于细胞的ISR活性检测方法。这需要测量PERK激活和eIF2a磷酸化引起的翻译抑制的幅度，以及报告由eIF2a磷酸化引发的基因表达程序活性的补充分析。所讨论的实验已经小型化并转化为适合小分子（“探针”）的高通量筛选（HTS）的同质格式，当添加到细胞中时，这些小分子（“探针”）要么会阻断PERK活性，损害eIF2a磷酸化所需的下游步骤，要么会将eIF2a磷酸化信号转化为基因表达的激活。三级分析已经发展，以查明任何抑制分子的作用地点发现的HTS。使用这些试验的HTS活动有望产生有效的细胞渗透小分子，在体内的各个点抑制ISR。与遗传方法不同，遗传方法倾向于产生相对不连续的剂量反应，小分子抑制剂预计具有连续的剂量-反应关系，具有较长的单调相。研究人员将利用这些特征来验证一个假设，即对ISR的温和和部分抑制可能会促进错误折叠蛋白的分泌，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著增加，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也随之出现。这项研究的重点是细胞调节其在分泌区折叠蛋白质的能力的一种途径，旨在识别通过抑制该途径的关键成分之一（一种名为PERK的酶）来调节该途径信号的药物样化合物。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，如溶酶体贮积病和各种癌症方面具有潜在的效用。