Natural product PROTACylation: Development of Maslinic acid-based JAK2 degraders for cancer therapy.

Proteolysis-targeting chimeras (PROTACs) offer a catalytic strategy for sustained oncogenic protein suppression. Here, we applied a "natural product PROTACylation" approach, utilizing maslinic acid (MA) as the JAK2-targeting warhead and lenalidomide as the CRBN recruiter to generate a focused library of 18 compounds (SZ1-SZ18). Lead compound P4 (SZ15), featuring a 14-atom linker, demonstrated superior antiproliferative activity compared to the parent MA across multiple cancer cell lines, with IC50 values of 6.57 μM (MCF-7), 11.73 μM (HeLa), and 9.56 μM (A549). Structure-activity relationship analysis revealed that linker length critically modulates cytotoxicity and selectivity, with the optimized linker enabling productive ternary complex formation. Mechanistic studies confirmed that P4 induces time-dependent, proteasome-mediated degradation of JAK2, reaching maximal protein reduction at 48 h. Competitive inhibition experiments using excess MA or lenalidomide, along with MLN4924 treatment, validated the requirement for ternary complex formation and cullin-RING E3 ligase pathway dependence. Direct MA-JAK2 engagement was demonstrated through CETSA and DARTS assays, supporting MA's suitability as a POI ligand. Molecular docking revealed preferential binding of P4 to JAK2 over CRBN (-8.7 vs -7.7 kcal/mol), suggesting an initial JAK2 anchoring mechanism that facilitates catalytically competent ternary complex assembly. Functionally, P4 exhibits comprehensive antitumor activity in A549 cells, suppressing clonogenic growth and invasion while inducing robust apoptosis (≥80% at 20 μM) and G0/G1 phase arrest. P4 effectively downregulates the IL-6/JAK2/STAT3 signaling axis and simultaneously activates dual apoptotic pathways: intrinsic mitochondrial apoptosis (decreased Bcl-2, XIAP, survivin; increased Bax, cytochrome c, cleaved caspases) and ER stress-mediated apoptosis (elevated IRE1, BIP, ATF4, CHOP, p-JNK1, cleaved caspase-12). This work establishes P4 as a promising JAK2-targeting degrader whose anticancer efficacy derives from coordinated target elimination, oncogenic pathway suppression, and dual apoptotic pathway activation, validating natural product PROTACylation as a viable strategy for developing multifunctional anticancer therapeutics.