## 2 Results and Discussion
### Inhibition of Melanogenesis by Mimosine Dipeptides  
As mentioned above, we showed that a small library of mimosine dipeptides had strong tyrosinase inhibition _in vitro_. Thus, we hypothesized that these compounds could exhibit melanogenesis inhibition in B16F10 melanoma cells. First, we examined the effect of mimosine dipeptides on cell survival by the thiazolyl blue tetrazolium bromide (MTT) assay, and found that none of them exhibited any significant cytotoxicity up to 100 uM. To determine the anti-melanogenic activity, we measured their effect on the melanin content in B16F10 cells. Generally, mimosine dipeptides reduced the melanin content more effectively than mimosine. Four of them (Mi-l-Pro, Mi-d-Val, Mi-d-Trp, Mi-l-Trp) exhibited strong inhibition on melanin content, with IC\({}_{50}\) values ranging 109-116 uM, compared with mimosine (IC\({}_{50}\) around 226 uM) (Table 3). To examine the possible mechanism underlying their anti-melanogenic act, cellular tyrosinase activity was evaluated. The results indicated that all of them strongly inhibited intracellular tyrosinase activity with IC\({}_{50}\) values ranging from 145-194 uM when compared to mimosine (IC\({}_{50}\) around 388 uM) (Table 3). Mi-d-Trp and Mi-d-Val are the most potent inhibitors on melanin content and cellular tyrosinase in B16F10 melanoma cells (Figure 4). Taken together, their anti-melanogenic effect exerted by their cellular tyrosinase suppression in B16F10 cells. As shown in Table 3, all prepared mimosine dipeptides had more anti-melanogenic activity than mimosine. When the C-terminus of mimosine was converted to the amide form, anti-melanogenic activity became better than that of the free acid form, suggesting the importance of \(C\)-terminal amide form in mimosine-peptide conjugates.  
The RAC/CDC42-activated kinase 1 (PAK1) is responsible for a variety of diseases/disorders including cancers, neurofibromatosisis (NF), diabetes (type 2), hypertension, short lifespan and neurodegenerative diseases such as Alzheimer's (AD) and Huntington's disease. Since PAK1 is not  
Figure 4: Reduction of the most potent dipeptides on melanin content and cellular tyrosinase in B16F10 cell. (**A**) Mi-d-Trp; (**B**) Mi-d-Val. Cells were treated with various concentrations of tested compounds for 48 h. Results are expressed as percentage in comparison with control. Kojic acid (500 \(\upmu\)M) was used as positive control. The experiments were conducted with three replications per each treatment with two independent experiments, and the values are means \(\pm\) SE. Data have statistical significance at \(p\leq 0.01\).  
\begin{table}
\begin{tabular}{l r r} \hline \hline
**Compound** & \
\begin{tabular}{l} **IC\({}_{50}\) (\(\upmu\)M) for Cellular** \\ **Tyrosinase Inhibition** \\ \end{tabular} &
\begin{tabular}{l} **IC\({}_{50}\) (\(\upmu\)M) for Melanin** \\ **Content Inhibition** \\ \end{tabular} \\ \hline Mi-L-Phe & \(179.2\pm 1.3\)\({}^{\text{\text{\text{\text{\text{\text{\text{\text{\text{\text{\text{ \text{\text{\text{\text{\text{\text{\text{ \text{    \text{       }}}}}}}}}}}}}\) \\ Mi-D-Phe & \(19  
essential for the growth of normal cells, blocking PAK1, _per se_, does not cause any side effect [16]. Recently, we demonstrated that PAK1 is essential for the melanogenesis in skin cells such as the melanoma B16F10 [17]. Moreover, in our previous study, three mimosine tetrapeptides, which directly inhibit oncogenic/kinase PAK1 [13], are anti-melanogenic [18]. Thus, it is most likely that mimosine dipeptides also could inhibit melanogenesis by blocking PAK1. We are planning to test whether mimosine dipeptides inhibit PAK1 in B16F10 cells or not.