Discovery of Potent and Selective Reversible Ubiquitin-Like Modifier Activating Enzyme 5 Inhibitors Targeting the UFMylation Pathway

Ubiquitin-like modifier activating enzyme 5 (UBA5), an E1 enzyme in the UFMylation pathway, is of interest as a therapeutic target in diseases such as cancer due to the critical role that UFMylation plays in cellular function. Strategic structure–activity relationship optimization of high-throughput screen hit 6, a Bruton's tyrosine kinase covalent inhibitor, was conducted by using both conventional and microscale library synthesis. This approach identified 49 and 50 as potent and selective noncovalent UBA5 inhibitors with significantly improved surface plasmon resonance and nanodifferential scanning fluorimetry biophysical profiles. Cellular target engagement for 49 and 50 was confirmed by a cellular thermal shift assay and translated to inhibition of UFMylation of UBA5 and UFC1 in retinal pigment epithelial-1 cells as well as reduction of UFMylation of the downstream protein UFMylation substrate RPL26. Furthermore, 49 and 50 demonstrated specificity for UBA5 among the other E1–E2 transesterification pathways. In this communication, we report the discovery and synthesis of potent and selective UBA5 inhibitors, providing valuable tool compounds for studying the UFMylation pathway.