Presentation Title: HYPE-mediated AMPylation as a novel therapeutic target for neurodegeneration
Author Name(s): Ali Camara
Author Department and School Affiliation: Department of Biological Sciences, Purdue University
Abstract: A major hallmark of Parkinson’s disease (PD) is the deposition of the intrinsically disordered protein α-synuclein (αSyn) into intracellular inclusions termed Lewy bodies. We previously reported that HYPE/FicD—the sole human homolog of the Fic family of adenylyltransferase enzymes— covalently adenylylates (AMPylates) αSyn in vitro. HYPE-mediated AMPylation ameliorates many of the neurotoxic phenotypes of αSyn implicated in the progression of PD, such as αSyn fibrillation and membrane permeability. These potentially cytoprotective phenomena conferred by HYPE’s adenylyltransferase activity make it an attractive therapeutic target. We therefore set out to screen both FDA-approved and proprietary small-molecule compound libraries towards the identification of novel manipulators of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore—Fl-ATP—we developed and optimized an efficient, robust assay which monitors HYPE autoAMPylation and is amenable to automated, high-throughput processing of diverse chemical libraries. Challenging our pilot screen with compounds from the LOPAC, Spectrum, MEGx, and NATx libraries yielded 0.3% and 1% hit rates for HYPE activators and inhibitors, respectively. Further, these hits were assessed for dose-dependency and validated via orthogonal biochemical AMPylation assays. We thus present a high-quality HTS assay suitable for tracking HYPE’s enzymatic activity, and the first small molecule manipulators of HYPE-mediated AMPylation.