Chemoproteomic Identification of ALDOA as the Glycolytic Target of α-Cembratriene-4,6-diol in Lung Cancer Cells

Tobacco (Nicotiana tabacum L.) is a globally important economic crop and a rich source of bioactive cembratriene-4,6-diol (CBT-diol). The unique macrocyclic scaffold of these compounds has prompted an extensive study of its bioactivity. Although CBT-diol has known antitumor activity, its specific molecular targets remain unclear, hindering the translational development of tobacco-based therapeutics. In this study, we identified ALDOA as the direct target of tobacco-derived α-CBT-diol against A549 lung cancer cells. Using activity-based proteomics and target validation methods, we show that α-CBT-diol binds to the ALDOA catalytic site with high affinity (Kd = 12.4 μM) and inhibits its activity. Definitively, studies in ALDOA-knockout cells confirmed that α-CBT-diol inhibits ALDOA activity, thereby suppressing tumor glycolysis and growth by reducing glucose uptake and lactate production. To the best of our knowledge, α-CBT-diol represents the first identified member of the tobacco cembranoid class that exerts antitumor effects by targeting ALDOA.