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Endoplasmic Reticulum Stress and Unfolded Protein Response in Neurodegenerative Diseases
Article

A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD

1
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA
2
William Henry Harrison High School, West Lafayette, IN 47906, USA
3
Purdue Institute for Integrative Neuroscience, Purdue University, West Lafayette, IN 47907, USA
4
Purdue Institute for Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN 47907, USA
*
Author to whom correspondence should be addressed.
These authors contributed equally.
Int. J. Mol. Sci. 2020, 21(19), 7128; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21197128
Received: 27 August 2020 / Revised: 24 September 2020 / Accepted: 25 September 2020 / Published: 27 September 2020
(This article belongs to the Special Issue Endoplasmic Reticulum Stress and Unfolded Protein Response 2.0)
The covalent transfer of the AMP portion of ATP onto a target protein—termed adenylylation or AMPylation—by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in endoplasmic reticulum homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification of small-molecule activators and inhibitors of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore—Fl-ATP—we developed and optimized an efficient, robust assay that 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 resultant first small-molecule manipulators of HYPE-promoted autoAMPylation. View Full-Text
Keywords: HYPE/FICD; AMPylation/adenylylation; post-translational modification; BiP/GRP78/HSPA5; α-synuclein; fluorescence polarization; high-throughput screening; drug discovery; assay development HYPE/FICD; AMPylation/adenylylation; post-translational modification; BiP/GRP78/HSPA5; α-synuclein; fluorescence polarization; high-throughput screening; drug discovery; assay development
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MDPI and ACS Style

Camara, A.; George, A.; Hebner, E.; Mahmood, A.; Paluru, J.; Mattoo, S. A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD. Int. J. Mol. Sci. 2020, 21, 7128. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21197128

AMA Style

Camara A, George A, Hebner E, Mahmood A, Paluru J, Mattoo S. A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD. International Journal of Molecular Sciences. 2020; 21(19):7128. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21197128

Chicago/Turabian Style

Camara, Ali, Alyssa George, Evan Hebner, Anika Mahmood, Jashun Paluru, and Seema Mattoo. 2020. "A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD" International Journal of Molecular Sciences 21, no. 19: 7128. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21197128

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