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.