This research aimed to determine the target protein and molecular mechanism of trans-(±)-kusunokinin (KU) derivatives ((±)-arctigenin (ARC) and trans-(±)-TTPG-B). Molecular docking was used to predict potential synthesized (±)-KU targets among 22 proteins. The (+)-TTPG-B bound HSP90α better than EC44, native (±)-KU and trans-(±)-ARC. In contrast, (−)-ARC bound PI3K more strongly than any other test compound. CSF1R and AKR1B1 were not supposed to be the target of (±)-TTPG-B and (±)-ARC, unlike native (±)-KU. The (+)-TTPG-B bound Tyr139 and Trp162 of HSP90α. Moreover, (−)-ARC bound PI3K via hydrogen bonds and π-π stacking at distinct amino acids, which was different from the other tested compounds. Using half of the IC₅₀ concentration, (±)-TTPG-B, (±)-KU and (±)-ARC enhanced cell cycle arrest at the G0/G1 phase after 12 h and 24 h on KKU-M213 (CCA) cells. The (±)-TTPG-B showed a stronger inhibitory effect than (±)-ARC and (±)-KU on HSP90α, PI3K, HSP90β, c-Myc, AKT, MEK1, CyclinB1, CyclinD1, and CDK1 for 24 and 48 h after treatment with the same concentration (0.015 µM). Thus, trans-(±)-TTPG-B, a newly synthesized compound, has pharmacological potential for development as a target therapy for CCA treatment.

Cardol (5-[8(Z), 11(Z)-pentadecadienyl] resorcinol) is a long chain phenolic lipid with reported antitumor properties, but its molecular targets and mechanisms of growth inhibition against hepatocellular carcinoma cells remain unclear. This study aimed to elucidate its cytostatic mechanism and molecular targets in Hep-G2 hepatocellular carcinoma cells. Cytotoxicity was assessed by MTT assay, revealing an IC50 value of 24.56 ± 0.31 µM, and morphological examination revealed cell rounding, shrinkage, and detachment indicative of stress induced death. Gene expression analysis by RT-qPCR after 48 h treatment with cardol at its IC50 value demonstrated significant upregulation of p21 accompanied by downregulation of Cyclin E, Cyclin A, and replication licensing genes (CDC6, MCM2, PCNA), indicative of checkpoint activation and G1/S transition arrest. Molecular docking across 35 cancer-related proteins identified CDK2 (ΔGbind = -7.84 kcal/mol; Ki ≈ 1.8 µM) and MEK1 (ΔGbind = -7.66 kcal/mol; Ki ≈ 2 µM) as high-affinity targets, with interaction residues overlapping those of reference inhibitors roscovitine and refametinib, respectively. Inhibition of CDK2 explains the G1/S arrest and repression of replication factors, whereas MEK1 inhibition suppressed ERK signaling and was likely associated with compensatory upregulation of STAT3, AKT, and c-MYC as part of a survival feedback loop. Consequently, these findings suggest that cardol acts as a multitarget modulator that suppresses proliferation through coordinated inhibition of CDK2-RB-E2F and MEK-ERK signaling, leading to cytostatic arrest that progresses toward cytotoxicity.