Discovery of CN-3 as a Next-Generation RET Inhibitor Potently Overcoming Multiple Mutations.

RET fusions and activating mutations drive multiple human cancers, while resistance mutations limit the efficacy of current selective RET inhibitors. Here, we report CN-3, a potent RET inhibitor active against clinically relevant mutants, including solvent-front (G810R/S/C), gatekeeper (V804M), hinge (Y806H), and catalytic loop (M918T) variants. CN-3 inhibited all tested RET mutants (IC50 < 5 nM) and selectively suppressed proliferation of RET-driven cellular models, including TT (IC50 = 2.48 ± 0.78 nM) and LC-2/ad (IC50 = 17.05 ± 4.90 nM) cells, as well as Ba/F3 cells expressing RET fusions or mutations, without affecting RET-independent or normal cells. Mechanistically, CN-3 blocked RET autophosphorylation and downstream SHC/AKT/ERK signaling, inducing G0-G1 arrest and apoptosis. In RET-driven xenografts, CN-3 showed dose-dependent antitumor efficacy with good tolerability. Kinase profiling revealed moderate selectivity, with off-target activity mainly restricted to a limited group of receptor tyrosine kinases. These results support CN-3 as a promising lead for next-generation RET-targeted therapies.