Discovery and validation of a novel inhibitor of HYPE-mediated AMPylation

Ali Camara; Heerak Chugh; Alyssa George; Lukas Dolidze; Kevin Ryu; Katrina J Holly; Daniel P Flaherty; Seema Mattoo

doi:10.1016/j.cstres.2024.04.001

Discovery and validation of a novel inhibitor of HYPE-mediated AMPylation

Cell Stress Chaperones. 2024 Apr 9;29(3):404-424. doi: 10.1016/j.cstres.2024.04.001. Online ahead of print.

Authors

Ali Camara¹, Heerak Chugh², Alyssa George¹, Lukas Dolidze¹, Kevin Ryu¹, Katrina J Holly³, Daniel P Flaherty³, Seema Mattoo⁴

Affiliations

¹ Department of Biological Sciences, Purdue University, West Lafayette, IN, USA.
² Department of Biological Sciences, Purdue University, West Lafayette, IN, USA; Drug Discovery and Development Laboratory, Department of Chemistry, University of Delhi, Delhi, India.
³ Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, IN, USA.
⁴ Department of Biological Sciences, Purdue University, West Lafayette, IN, USA; Purdue Institute for Integrative Neuroscience, Purdue University, West Lafayette, IN, USA; Purdue Institute for Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN, USA. Electronic address: smattoo@purdue.edu.

Abstract

Adenosyl monophosphate (AMP)ylation (the covalent transfer of an AMP from Adenosine Triphosphate (ATP) onto a target protein) is catalyzed by the human enzyme Huntingtin Yeast Interacting Partner E (HYPE)/FicD to regulate its substrate, the heat shock chaperone binding immunoglobulin protein (BiP). HYPE-mediated AMPylation of BiP is critical for maintaining proteostasis in the endoplasmic reticulum and mounting a unfolded protein response in times of proteostatic imbalance. Thus, manipulating HYPE's enzymatic activity is a key therapeutic strategy toward 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 provided 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.

Keywords: AMPylation/adenylylation; BiP/GRP78/HSPA5; Fluorescence polarization; HYPE/FICD; High-throughput screen; Small molecule inhibitor.