Chemical Inhibitors of Autophagins for Autophagy modulation

描述（由申请人提供）：自噬蛋白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，细胞通过自噬分解代谢蛋白质和细胞器以产生底物，从而在营养缺乏期间维持ATP的产生。这种自我进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不良和缺氧的环境中存活-例如在快速生长的恶性病变中心发现的那些已经超过了它们的血管供应。或者，自噬机制的组成部分已被报道在某些情况下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的背景下的宿主防御机制，促进细胞内病原体的清除。自噬还去除不溶性蛋白质，并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。需要针对自噬机制的特定组分的化学品，以用作研究工具来解决自噬在发育和疾病中的作用问题。在这个提议中，我们开发了一种基于荧光强度的高通量筛选（HTS）测定来筛选抑制自噬蛋白1（ATG 4 B）的化合物。HTS测定利用磷脂酶A2（PLA 2）的可裂解形式，其表达为融合蛋白，自噬蛋白底物LC 3/ATG 8附加到其N末端。在PLA 2的N-末端添加序列可抑制该酶的活性。通过蛋白酶切割除去N-末端延伸，然后恢复酶活性，构成蛋白酶测定的基础。总之，这些努力将导致有效的化学探针，用于研究各种生物环境中的自噬。 公共卫生相关性：我们的目标是确定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有力研究工具。

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.