Anti-vitiligo mechanism and the target protein of phenanthridine
derivatives
To facilitate the identification of the cellular target (or targets) of
HCJA121 and HCJA404, biotinylated derivatives were generated through
label-free quantitative proteomic approaches. Axin appeared to act as a
binding protein of HCJA121 and HCJA404, which was confirmed using a
pull-down assay. Next, to confirm the anti-vitiligo target protein of
the phenanthridine derivatives, we performed modelling studies for the
complexes axin–HCJA121 and axin–HCJA404. A possible binding site for1 and 2 on DAX, which has important implications on
the function of axin, has been investigated using the AutoDock docking
program (Morris et al., 2009) with the structure of DAX (PDB, 1WSP)
(Schwarz-Romond et al., 2007) as the receptor molecule. Nine conformers
of HCJA121 and 11 conformers of HCJA404 that were outputted from
AutoDock onto DAX were analysed; six (HCJA121) and five (HCJA404) of the
conformers were localized to the same site formed by the juxtaposition
of the residues from two neighbouring protomers. Subsequently, the model
with the lowest estimated free energy for binding (−9.35 kcal
mol-1 for HCJA121 and −8.42 kcal
mol-1 for HCJA404) was selected and modified slightly.
The modelled structure of the complexes of axin with HCJA121 and
HCJA404 revealed that LYS781 and LEU784 in the binding cavity could form
contacts with HCJA121 and HCJA404 through electrostatic or hydrophobic
interactions (Fig. 3a). To verify the importance of these residues in
the binding of compounds, DAX mutants carrying K781A and L784A mutations
were generated. As predicted by the model, in the two DAX mutants,
HCJA121 and HCJA404 exhibited significantly reduced activation of not
only Wnt target genes but also melanogenesis genes (Fig. 3b). Similar to
melanogenesis, melanin synthesis was clearly decreased in drug-treated
mutated cells (Fig. 3c). However, the effects of 8-MOP on melanogenesis
genes and melanogenesis in mutated cells appeared to be unaffected,
suggesting that compounds targeting the DAX domain of axin through
binding with LYS781 and LEU784 potentiate the axin–LRP6 association to
promote Wnt signalling transduction and eventually promote
melanogenesis.
These data suggest that HCJA121 and HCJA404 could promote melanogenesis
by activating the Wnt signalling pathway. We have previously suggested
an autoinhibition hypothesis, according to which the binding of
phenanthridine derivatives to the axin C terminus disrupts the
interaction with the axin N terminus and thus relieves the
autoinhibition of axin. Therefore, active axin binds LRP6 more easily
and thus promotes LRP6 phosphorylation when the Wnt signalling pathway
is activated 24. Hence, we presumed that these
anti-vitiligo phenanthridine derivatives could potentiate the axin–LRP6
association to influence melanin synthesis. To validate this hypothesis,
we performed co-immunoprecipitation (co-IP) of axin and LRP6 from the
lysates of HCJA121-treated B16 cells (using anti-axin1 and anti-LRP6
antibodies for immunoprecipitation); subsequently, the proteins were
separated by western blotting. First, anti-axin1 was employed to capture
the axin protein and axin-related complex. Next, LRP6 antibody was used
on the protein mixtures captured by the axin1 antibody for capturing
LRP6. The spot representing LRP6/anti-LRP6 from the HCJA121-treated
group was considerably larger than that from the DMSO-treated group at
the same loading quantity of the sample (Fig. 3d), which suggested that
axin and LRP6 exhibited strong association in the
phenanthridine-derivative-treated cells. The protein complex of axin and
LRP6 in drug-treated cells appeared to be more common than it was in the
control, which implies that phenanthridine derivatives could potentiate
the axin–LRP6 association.