Identification and characterization of UBA5 inhibitors

ABSTRACT UBA5 is a critical E1-activating enzyme in the UFMylation pathway, a post-translational modification process implicated in neurodegenerative diseases and cancers. In this study, we developed a high-throughput screening (HTS) assay to identify inhibitors of UBA5 from a library of blood-brain barrier (BBB)-permeable compounds. The assay, based on the AMP-Glo™ kit, enabled the identification of five novel UBA5 inhibitors belonging to three distinct chemical scaffolds with low micromolar IC 50 values. These inhibitors demonstrated selectivity for UBA5 over other E1 enzymes, such as UBA1, and showed efficacy in inhibiting endogenous UFMylation in HEK293T cells. The identified inhibitors not only provided valuable tools for studying UFMylation but also represent potential therapeutic candidates for diseases associated with dysregulated UFMylation, such as Alzheimer’s disease and cancer. Further optimization of these compounds is necessary to enhance their potency and pharmacokinetic properties. Our findings highlight UBA5 as a promising therapeutic target and pave the way for the development of selective inhibitors that can modulate UFMylation in human diseases.