Chemical Inhibitors of Autophagins for Autophagy modulation

描述（由申请人提供）：自噬因子是自噬所需的一类胞质半胱氨酸蛋白酶，自噬是一种进化保守的过程，细胞分解代谢蛋白质和细胞器以产生底物，在营养剥夺时维持ATP的产生。这种自食过程在发育和疾病中起着重要作用。例如，自噬可以促进肿瘤的进展，使癌细胞能够在营养贫乏和缺氧的环境中存活——比如在快速生长的恶性病变中心发现的血管供应不足的环境。另外，据报道，在某些情况下，非凋亡性肿瘤细胞死亡需要自噬机制的组成部分，这表明自噬可能发挥肿瘤抑制作用。在病毒和细胞内细菌攻击细胞的情况下，自噬也被证明是一种宿主防御机制，促进细胞内病原体的清除。自噬还可以清除不溶性蛋白质，防止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，唯一可用的药物是3-甲基腺嘌呤，需要毫摩尔浓度。需要针对自噬机制的特定成分的化学物质，作为研究工具来解决自噬在发育和疾病中的作用问题。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）方法来筛选抑制Autophagin 1 （ATG4B）的化合物。HTS实验利用可切割形式的磷脂酶A2 (PLA2)，其表达为融合蛋白，自噬蛋白底物LC3/ATG8附加在其n端。在PLA2的n端添加序列会抑制该酶的活性。通过蛋白酶去除n端延伸的切割，然后恢复酶活性，构成蛋白酶测定的基础。总之，这些努力将产生有效的化学探针，用于研究各种生物环境中的自噬。

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.