Chemical Inhibitors of Autophagins for Autophagy modulation

用于自噬调节的自噬素化学抑制剂

• 批准号: 7929409
• 负责人: JOHN C REED
• 金额: $ 4.78万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7929409
• 关键词: 3-methyladenine; Address; Amino Acids; Animal Disease Models; Apoptotic; Autophagocytosis; Autophagosome; Bacteria; Biochemical; Biological; Biological Assay; Biology; Caliber; Catalytic Domain; Cell Culture Techniques; Cell Death; Cell Nucleus; Cells; Cellular Morphology; Chemicals; Chemistry; Chimeric Proteins; Collaborations; Computer Simulation; Cysteine; Cysteine Protease; Development; Disease; Eating; Environment; Enzymes; Eukaryota; Family; Fluorescence; Genes; Genomics; Goals; Green Fluorescent Proteins; Health; Homeostasis; Homologous Protein; Host Defense Mechanism; Housekeeping; Human; Human Genome; Hypoxia; In Vitro; Inclusion Bodies; Laboratories; Lesion; Malignant - descriptor; Mediating; Membrane; Methodology; Methods; Microscopy; Minor; Molecular Chaperones; N-terminal; Nerve Degeneration; Nutrient; Organelles; Oxidation-Reduction; Pathogenesis; Pathology; Pathway interactions; Peptide Hydrolases; Phospholipase A2; Play; Process; Production; Proteins; Recombinants; Reporter; Reporter Genes; Reporting; Research; Role; Screening procedure; Structure; System; Time; Tissues; Tumor Suppressor Proteins; United States National Institutes of Health; Vascular blood supply; Vesicle; Virion; Virus; Yeasts; base; cancer cell; cell type; cheminformatics; deprivation; enzyme activity; high throughput screening; inhibitor/antagonist; member; milligram; multicatalytic endopeptidase complex; neoplastic cell; novel; pathogen; prevent; process optimization; public health relevance; reconstitution; senescence; small molecule libraries; three dimensional structure; tool; transcription factor; tumor progression

DESCRIPTION (provided by applicant): Autophagins are a class of cytosolic cysteine proteases required for autophagy, which is an evolutionarily conserved process whereby cells catabolize proteins and organelles for purposes of generating substrates for sustaining ATP production during times of nutrient deprivation. This self-eating process plays important roles in the development and disease. For example, autophagy may promote progression of tumors, allowing cancer cells to survive nutrient poor and hypoxic environments - such as those found in the centers of rapidly growing malignant lesions that have outstripped their vascular supply. Alternatively, components of the autophagy machinery have been reported to be required for non-apoptotic tumor cell death in some circumstances, suggesting that autophagy might play a tumor suppressor role. Autophagy has also been documented as a host defense mechanism in the context of cellular attack by viruses and intracellular bacteria, promoting clearance of intracellular pathogens. Autophagy also removes insoluble proteins and safeguards against protein inclusion body disease associated with neurodegeneration. Chemical modulators of autophagy are essentially non-existent, with the only available agent, 3-methyladenine, requiring millimolar concentrations. A need exists for chemicals that target specific components of the autophagy machinery for use as research tools for addressing questions about the role of autophagy in development and disease. In this proposal, we have developed a High Throughput Screening (HTS) assay based on fluorescence intensity to screen for compounds that inhibit Autophagin 1 (ATG4B). The HTS assay utilizes a cleavable form of Phospholipase A2 (PLA2), which is expressed as a fusion protein with the Autophagin substrate LC3/ATG8 appended to its N-terminus. The addition of sequences to the N-terminus of PLA2 inhibits the activity of this enzyme. Cleavage by proteases removing the N-terminal extension then restores enzyme activity, constituting the basis for a protease assay. Together, these efforts will result in validated chemical probes for studying the autophagy in a variety of biological settings. PUBLIC HEALTH RELEVANCE: Our goal is to identify chemicals capable of modulating the Autophagins, which are intracellular cysteine proteases required for autophagy. Autophagy plays important roles in development and disease. Thus, these chemical inhibitors of Autophagin will serve as powerful research tools for exploring the role of autophagy in cell and organismal biology and pathology.

描述（由申请人提供）：自噬素是自噬所需的一类胞质半胱氨酸蛋白酶，自噬是一种进化上保守的过程，细胞通过该过程分解代谢蛋白质和细胞器，以产生在营养缺乏期间维持 ATP 产生的底物。这种自食过程在发育和疾病中起着重要作用。例如，自噬可能促进肿瘤的进展，使癌细胞能够在营养不良和缺氧的环境中生存——例如在快速生长的恶性病变中心发现的癌细胞，这些病变已经超出了其血管供应。另外，据报道，在某些情况下，自噬机制的组成部分是非凋亡肿瘤细胞死亡所必需的，这表明自噬可能发挥肿瘤抑制作用。自噬也被证明是病毒和细胞内细菌攻击细胞时的宿主防御机制，促进细胞内病原体的清除。自噬还可以去除不溶性蛋白质并预防与神经变性相关的蛋白质包涵体疾病。自噬的化学调节剂基本上不存在，唯一可用的试剂是 3-甲基腺嘌呤，需要毫摩尔浓度。需要针对自噬机制特定成分的化学品作为研究工具来解决有关自噬在发育和疾病中的作用的问题。在本提案中，我们开发了一种基于荧光强度的高通量筛选 (HTS) 测定法，以筛选抑制自噬素 1 (ATG4B) 的化合物。 HTS 测定利用可裂解形式的磷脂酶 A2 (PLA2)，它表达为融合蛋白，其 N 末端附加有自噬素底物 LC3/ATG8。在 PLA2 的 N 末端添加序列会抑制该酶的活性。蛋白酶切割去除 N 末端延伸，然后恢复酶活性，构成蛋白酶测定的基础。总之，这些努力将产生经过验证的化学探针，用于研究各种生物环境中的自噬。 公共健康相关性：我们的目标是确定能够调节自噬素的化学物质，自噬素是自噬所需的细胞内半胱氨酸蛋白酶。自噬在发育和疾病中发挥着重要作用。因此，这些自噬素的化学抑制剂将成为探索自噬在细胞和有机体生物学和病理学中的作用的强大研究工具。