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

Autor: Ali Camara, Alyssa George, Evan Hebner, Anika Mahmood, Jashun Paluru, Seema Mattoo
Jazyk: angličtina
Rok vydání: 2020
Předmět:
Zdroj: International Journal of Molecular Sciences, Vol 21, Iss 19, p 7128 (2020)
Druh dokumentu: article
ISSN: 1422-0067
1661-6596
DOI: 10.3390/ijms21197128
Popis: 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.
Databáze: Directory of Open Access Journals
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