Synthesis of USP5-IN-1 derivatives as novel USP5 inhibitors with potent activity against cholangiocarcinoma cells

USP5 is a crucial deubiquitinase involved in regulating various pathophysiological processes, including DNA damage repair, immune responses, pathological pain, and, most notably, oncogenesis. Despite the considerable clinical potential of USP5-targeted inhibitors, their development remains in the early stages. USP5-IN-1 (also known as compound 64) stands out from other USP5 inhibitors due to its reported high selectivity for USP5 and its specific co-crystal structure with the USP5 ZnF-UBD (PDB: 7MS7). However, the activity of USP5-IN-1 has only been validated in vitro through the inhibition of USP5-catalyzed cleavage of a di-ubiquitin substrate, with its cell membrane penetration ability and intracellular activity still unverified. In this study, we structurally modified USP5-IN-1 to enhance its cell membrane penetration and inhibitory activity against USP5, and synthesized fifteen USP5-IN-1 derivatives (compounds 1a-1j, 2a-2d, and 3a). Compared to USP5-IN-1, compounds 1a and 1h exhibited enhanced inhibitory effects on USP5 deubiquitinase activity, as well as on the proliferation and metastasis of cholangiocarcinoma cells. Mechanistically, 1a and 1h significantly inhibited the mTORC1 and Erk1/2 pathways in HCCC9810 cells, without affecting the activation of PKCα, β-catenin or FAK, a pattern consistent with the effects of direct USP5 knockdown. Furthermore, both of the compounds, along with USP5 knockdown, significantly induced cell cycle arrest, apoptosis and ferroptosis. In-silico studies revealed that compounds 1a and 1h had significantly lower binding free energies and larger octanol–water partition coefficients than USP5-IN-1, indicating stronger affinity for USP5 and improved cell membrane penetration. We believe compounds 1a and 1h are promising USP5 inhibitors and merit further investigation.