Chemical Inhibitors of Autophagins for Autophagy modulation

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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.