Discovery and Validation of a Novel Inhibitor of HYPE-mediated AMPylation

AMPylation–the covalent transfer of an AMP from ATP onto a target protein—is catalyzed by the human enzyme HYPE/FicD to regulate its substrate, the heat shock chaperone BiP. HYPE-mediated AMPylation of BiP is critical for maintaining proteostasis in the ER (endoplasmic reticulum) and mounting an UPR (unfolded protein response) in times of proteostatic imbalance. Thus, manipulating HYPE’s enzymatic activity is a key therapeutic strategy towards the treatment of various protein misfolding diseases, including neuropathy and early onset diabetes associated with two recently identified clinical mutations of HYPE. Herein, we present an optimized, fluorescence polarization-based, high-throughput screening (HTS) assay to discover activators and inhibitors of HYPE-mediated AMPylation. After challenging our HTS assay with over 30,000 compounds, we discovered a novel AMPylase inhibitor, I2.10. We also determined a low micromolar IC50 for I2.10 and employed biorthogonal counter-screens to validate its efficacy against HYPE’s AMPylation of BiP. Further, we report low cytotoxicity of I2.10 on human cell lines. We thus established an optimized, high-quality HTS assay amenable to tracking HYPE’s enzymatic activity at scale, and provide the first novel small-molecule inhibitor capable of perturbing HYPE-directed AMPylation of BiP in vitro. Our HTS assay and I2.10 compound serve as a platform for further development of HYPE-specific small-molecule therapeutics.