Discovery, biological evaluation, and molecular modeling of novel alkylamine-based Cbl-b inhibitors

Cbl-b, an E3 ubiquitin ligase, is a critical negative regulator of T-cell activation and an attractive target for cancer immunotherapy. Current small-molecule inhibitors largely rely on hydrophobic π-π stacking interactions with the gatekeeper residue Tyr363, which restricts the structural diversity of Cbl-b inhibitors and hinders the discovery of inhibitors with novel scaffolds. This study reports the stepwise optimization of the cyclic carbamate lead compound 5, eventually leading to the discovery of novel, representative alkylamine-based Cbl-b inhibitors. Our optimization process comprised three stages: (1) conformational restriction via lactamization, which yielded initial hit 12 (IC₅₀ = 31.99 ± 3.88 μM); (2) hydrophobic cavity filling, which provided the improved analog 22 (IC₅₀ = 8.58 ± 0.25 μM); and (3) SeeSAR-guided scaffold hopping, which ultimately identified the representative lead compound 27 (IC₅₀ = 6.83 ± 0.51 μM). Molecular docking and molecular dynamics (MD) simulations confirmed that 27 binds to the TKB-LH interface and stabilizes the inactive conformation of Cbl-b. Notably, MD simulations revealed that 27 engages Tyr363 through a unique polar interaction mode dominated by hydrogen bonds and water bridges, a distinct departure from traditional hydrophobic stacking. This novel alkylamine scaffold provides a new approach for developing structurally diverse Cbl-b inhibitors.