Abstract
Recent work has demonstrated pro-oncogenic functions of the transcription factor CCAAT box/enhancer-binding protein β (C/EBPβ) in various tumors, implicating C/EBPβ as an interesting target for the development of small-molecule inhibitors. We have previously discovered that the sesquiterpene lactone helenalin acetate, a natural compound known to inhibit NF-κB, is a potent C/EBPβ inhibitor. We have now examined the inhibitory mechanism of helenalin acetate in more detail. We demonstrate that helenalin acetate is a significantly more potent inhibitor of C/EBPβ than of NF-κB. Our work shows that helenalin acetate inhibits C/EBPβ by binding to the N-terminal part of C/EBPβ, thereby disrupting the cooperation of C/EBPβ with the co-activator p300. C/EBPβ is expressed in several isoforms from alternative translational start codons. We have previously demonstrated that helenalin acetate selectively inhibits only the full-length (liver-enriched activating protein* (LAP*)) isoform but not the slightly shorter (LAP) isoform. Consistent with this, helenalin acetate binds to the LAP* but not to the LAP isoform, explaining why its inhibitory activity is selective for LAP*. Although helenalin acetate contains reactive groups that are able to interact covalently with cysteine residues, as exemplified by its effect on NF-κB, the inhibition of C/EBPβ by helenalin acetate is not due to irreversible reaction with cysteine residues of C/EBPβ. In summary, helenalin acetate is the first highly active small-molecule C/EBPβ inhibitor that inhibits C/EBPβ by a direct binding mechanism. Its selectivity for the LAP* isoform also makes helenalin acetate an interesting tool to dissect the functions of the LAP* and LAP isoforms.