Discussion
Although activation of the Wnt/β -Catenin signalling pathway has been linked to melanogenesis in melanocytes, no Wnt-based vitiligo treatment has been developed nor have relevant small molecule drugs been reported. In this study, we identified two phenanthridine-derived Wnt-specific agonists, HCJA121 and HCJA404, as potent anti-vitiligo agents. These compounds exhibited potent promotion of melanin synthesis and tyrosinase activity in B16 cells. Subsequent assays showed that melanogenic proteins, MITF, TYR, TRP1, and 2, were increased at both the mRNA and protein levels in the presence of HCJA121 and HCJA404, suggesting that these compounds may act as upstream regulators that modulate the MITF-TYRP/TYR melanogenic pathway by affecting Wnt. This speculation was then proven through co-administration of these compounds. In this study, the ability of the compounds to promote the expression of melanogenic proteins disappeared after the administration of XAV939, a classic Wnt/β -Catenin inhibitor.
Analysis of the crystal structures of the axin protein and the structures of HCJA121 and HCJA404 revealed a potential ligand-binding pocket in DAX. Inside this pocket, there are two residues, LYS781 and LEU784, that could form contacts with HCJA121 and HCJA404 through electrostatic or hydrophobic interactions. The importance of LYS781 and LEU784 was further underscored by an in vitro reporter assay, which showed that the wild-type axin but not the mutant K781A or L784A could be induced by compounds to activate the Wnt target gene and, subsequently, the melanogenic genes. The mechanism can be concluded from the aforementioned results: HCJA121 and HCJA404 target axin via binding to LYS781 and LEU784 and subsequently potentiating the axin–LRP6 association to activate Wnt signalling transduction and eventually promote melanogenesis.
In vivo assays demonstrated that administration of the phenanthridine-derived compounds (even at a low dose) in HQ-treated C57BL/6 vitiligo mice could significantly restore skin lesions induced by HQ and improve vitiligo-related biochemistry indexes. The curative effects on vitiligo were better in the treatment group than in the control group. In addition, ICR mice were treated with HCJA121 and HCJA404 at a high dose for evaluating the safety of the compounds, and no abnormal change was observed in the body weight, organs, or blood samples of the mice.
In summary, this work demonstrates that phenanthridine compounds could potentiate the axin–LRP6 association to activate Wnt signalling transduction and eventually induce melanogenic protein expression by targeting the axin protein. These findings suggest a new therapeutic strategy for vitiligo. Importantly, HCJA121 and HCJA404 may represent potential candidates for vitiligo treatment.