PROJECT SUMMARY/ABSTRACT The goal of this proposal is to identify agents that reduce the levels and accumulation of alpha-synuclein (αSyn) that underlies the pathogenesis of Parkinson's disease (PD). The premise of this proposal is supported by numerous reports including point mutations or duplication/triplication of the SNCA (Synuclein Alpha) gene that results in αSyn increases leading to autosomal-dominant early-onset PD. The finding that the promoter for the SNCA gene is hypomethylated in PD resulting in increased expression1 and that over-expression of the gene is a factor contributing to onset of PD2-4 suggests reducing the levels of αSyn is a promising target for therapeutic intervention5. Furthermore, reduction of αSyn levels using agents such as β2-adrenoreceptor (β2AR) agonists has been reported to be neuroprotective in both cell line and rodent models6. An epidemiological analysis of a Norwegian population revealed individuals using β2AR agonist, salbutamol for asthma, have a reduced risk of developing PD6. The Lewy-related pathology (LRP), primarily comprised of αSyn, is not restricted to PD and has been found in a subset of autopsied Alzheimer's disease (AD) brains7-9 and brains of Dementia with Lewy Bodies (DLB) patients. Apolipoprotein E4, a risk factor for AD, is also a risk factor for PD and is associated with earlier onset of PD10-12. A recent study also suggests there is a link between higher levels of αSyn in the CSF and early stages of development of cognitive decline in AD13. Current therapeutics for PD provide only symptomatic relief by modulating dopaminergic neurotransmission, but do not stop or slow the degenerative processes underlying PD pathogenesis, there is an urgent need for the development of disease-modifying compounds capable of slowing or halting PD progression. To address this need in PD as well as LRP in AD and DLB, in this proposal we will perform high throughput screening (HTS) of the UCLA 200K compound library to identify hits that lower intracellular αSyn levels; these hits will be prioritized by potency, drug-like properties, and brain permeability for further analysis. Compounds previously reported to lower αSyn such as β2AR agonists, will also undergo evaluation and prioritization. In Aim 1, an AlphaLISA that recognizes a broad range of αSyn conformations will be optimized and used for HTS to identify compounds that reduce intracellular αSyn in SK-N-MC human neuroepithelioma cells6; hits would then validated and dose-response determined in secondary assays and mouse primary dopaminergic neurons. In Aim 2, brain permeability of compounds such as β2AR agonists, hits prioritized from Aim 1, and analogs would be assessed by in vitro Parallel Artificial Membrane Permeability Analysis and in vivo pharmacokinetics (PK) analysis. Optimal compounds with good drug-like properties determined by in silico StarDrop analysis and in vitro ADME-T assays would be evaluated in mechanistic studies. In Aim 3, the best comp...