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 Sep 27;21(19):7128. doi: 10.3390/ijms21197128.


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.

Keywords: AMPylation/adenylylation; BiP/GRP78/HSPA5; HYPE/FICD; assay development; drug discovery; fluorescence polarization; high-throughput screening; post-translational modification; α-synuclein.

MeSH terms

  • Adenosine Triphosphate / analogs & derivatives
  • Adenosine Triphosphate / chemistry
  • Drug Evaluation, Preclinical
  • Endoplasmic Reticulum Chaperone BiP
  • Enzyme Inhibitors / chemistry*
  • Fluorescence Polarization
  • Humans
  • Membrane Proteins* / antagonists & inhibitors
  • Membrane Proteins* / chemistry
  • Molecular Docking Simulation*
  • Nucleotidyltransferases* / antagonists & inhibitors
  • Nucleotidyltransferases* / chemistry


  • Endoplasmic Reticulum Chaperone BiP
  • Enzyme Inhibitors
  • HSPA5 protein, human
  • Membrane Proteins
  • Adenosine Triphosphate
  • FICD protein, human
  • Nucleotidyltransferases