Chemical Inhibitors of Autophagins for Autophagy modulation

描述(申请人提供)：自噬蛋白是一类需要自噬的胞浆半胱氨酸蛋白酶，这是一个进化保守的过程，细胞分解蛋白质和细胞器以产生底物，以在营养缺乏期间维持ATP的生产。这种自食过程在疾病的发生和发展中起着重要的作用。例如，自噬可能会促进肿瘤的进展，使癌细胞能够在营养贫乏和缺氧的环境中生存--比如在快速增长的恶性病变中心发现的那些已经超出了血管供应的环境。或者，据报道，在某些情况下，自噬机制的组成部分是非凋亡性肿瘤细胞死亡所必需的，这表明自噬可能起到了肿瘤抑制作用。自噬也被证明是病毒和细胞内细菌攻击细胞时的一种宿主防御机制，促进细胞内病原体的清除。自噬还可以去除不溶性蛋白质，防止与神经退行性变相关的蛋白质包涵体疾病。自噬的化学调节剂基本上是不存在的，唯一可用的试剂3-甲基腺嘌呤需要毫摩尔浓度。需要针对自噬机器的特定组件的化学物质用作研究工具，以解决有关自噬在发育和疾病中的作用的问题。在这项建议中，我们开发了一种基于荧光强度的高通量筛选(HTS)方法来筛选抑制自噬蛋白1(ATG4B)的化合物。HTS检测利用一种可切割形式的磷脂酶A2(PLA2)，该磷脂酶A2以融合蛋白的形式表达，并将自噬底物LC3/ATG8附加在其N端。在PLA2的N-末端增加序列会抑制该酶的活性。用去除N-末端延伸的蛋白酶进行切割，然后恢复酶的活性，这构成了蛋白酶分析的基础。总之，这些努力将产生有效的化学探针，用于在各种生物环境中研究自噬。 与公共卫生相关：我们的目标是识别能够调节自噬蛋白的化学物质，自噬蛋白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起着重要作用。因此，这些自噬蛋白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。

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.