Neuroblastoma, a lethal pediatric tumor, has a <50% 5 year survival rate for high-risk cases (e.g., MYCN-amplified). Direct N-Myc targeting is unfeasible due to the lack of small-molecule binding pockets, making USP7 (a deubiquitinase that stabilizes N-Myc) a key alternative target. We identified USP7 inhibitors via drug repurposing, computer-aided drug design (CADD), and molecular simulations: a 7322-compound library (from 4 databases) underwent multistage virtual screening (HTVS/SP/XP docking + MM/GBSA) to obtain 24 hits, which were further validated by 100 ns molecular dynamics (MD), steered molecular dynamics (SMD), and umbrella sampling (US) simulations. Compound C24 (BML-284) stood out with high USP7 affinity (MM/PBSA: −35.36 ± 5.11 kcal/mol) and stability (dissociation energy: 33.34 kJ/mol, comparable to the positive control P 22077). In vitro experiments showed that BML-284 inhibited both MYCN-amplified and nonamplified NB cells (IC50: 0.6278–1.410 μmol/L). It also significantly suppressed colony formation, cell proliferation (reflected by reduced EdU+ cells), and migration, with all of these effects being statistically significant. Mechanistically, BML-284 downregulated the expression of USP7, N-Myc, and MDM2 and promoted apoptosis (evidenced by decreased BCL-2 and increased Cleaved PARP-N/PARP levels). Additionally, DARTS experiments confirmed its direct binding to USP7, with results showing statistical significance. In conclusion, BML-284 is a potent USP7 inhibitor that exerts anti-NB activity by regulating the USP7-N-Myc axis. This study not only provides a new candidate for high-risk NB treatment but also offers a framework for the development of USP7 inhibitors.
Liu et al. (2026) studied this question.