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Discovery of novel small-molecule activators of autophagy

Parkinson’s disease (PD) is the second most common neurodegenerative disease, affecting approximately 1 million patients in the United States today. PD is characterized by progressive gastrointestinal dysfunction followed by deterioration of dopaminergic neurons in the substantia nigra of the brain, leading to severe disability and reduced life expectancy. Currently, available treatments for PD only provide temporary symptomatic relief of the dopaminergic motor function, without any capacity to prevent or slow disease progression. The Kelly lab is keen to develop drug-like small-molecules to treat the underlying pathology of PD by exploiting activation of autophagy. Autophagy is the cellular mechanism employed by eukaryotes for degradation of protein aggregates (e.g., Lewy bodies, abnormal cytoplasmic aggregations composed primarily of the a-synuclein protein, that are a hallmark of PD), damaged organelles (e.g., mitochondria) too large for processing by the proteasome, as well as neurotoxic oxidized lipids.

The genetic evidence for autophagy activation being disease modifying in PD is extensive. The cellular load of aggregate-prone a-synuclein is central to disease pathogenesis, with gene triplication yielding a completely penetrant form of familial PD. Conversely, genetic inhibition of key autophagy effector proteins yields a PD-like neurodegenerative condition in mice, characterized by motor dysfunction and cellular build-up of a-synuclein. Genetic upregulation of autophagy has demonstrated activity in cell models of PD, thus we are confident that novel small-molecule activators will be similarly disease modifying. The most commonly used small molecule to activate autophagy, rapamycin, does so by inhibiting mTOR. However, mTOR is also a key regulator for cell growth and survival, motility, transcription, and protein synthesis. Thus, we seek novel small-molecules that activate autophagy through an mTOR-independent mechanism(s).

We are currently conducting a cell-based, high-content imaging, high-throughput screen, based on fluorescent monitoring of lysosomal lipid droplet clearance (lipophagy). Hit compounds confirmed to induce dose-dependent alterations in lipid droplet clearance in a panel of neuronal and glial cell lines will be further validated and compared to literature compounds for specificity, selectivity and activity in established autophagy assays, such as quantitative confocal microscopy studies of cells transfected with mRFP-GFP–tagged p62 to confirm altered autophagosome formation and western blot analysis of GluA1, and LC3-I to LC3-II conversion. Importantly, understanding the mode of action of these compounds will be critical to their translational advancement. Gene editing techniques, such as CRISPR-Cas9, will be used to generate cell lines with putative targets knocked out, validating the mechanism of action of each candidate small molecule.

We are also formulating novel autophagy activator screens to enable high-throughput screening to discover autophagy activators with unique mechanisms of action that are downstream of mTOR.

We believe that the novel small molecule activators of autophagy that we identify could serve as the basis for therapies that could profoundly alter the clinical management of PD and radically improve the quality of life of patients with PD and other neurodegenerative diseases.