Identification of L-Cysteinamide as a Potent Inhibitor of Tyrosinase-Mediated Dopachrome Formation and Eumelanin Synthesis

Abstract

The purpose of this study is to identify amino acid derivatives with potent anti-eumelanogenic activity. First, we compared the effects of twenty different amidated amino acids on tyrosinase (TYR)-mediated dopachrome formation in vitro and melanin content in dark-pigmented human melanoma MNT-1 cells. The results showed that only L-cysteinamide inhibited TYR-mediated dopachrome formation in vitro and reduced the melanin content of cells. Next, the antimelanogenic effect of L-cysteinamide was compared to those of other thiol compounds (L-cysteine, N-acetyl L-cysteine, glutathione, L-cysteine ethyl ester, N-acetyl L-cysteinamide, and cysteamine) and positive controls with known antimelanogenic effects (kojic acid and β-arbutin). The results showed the unique properties of L-cysteinamide, which effectively reduces melanin content without causing cytotoxicity. L-Cysteinamide did not affect the mRNA and protein levels of TYR, tyrosinase-related protein 1, and dopachrome tautomerase in MNT-1 cells. L-Cysteinamide exhibited similar properties in normal human epidermal melanocytes (HEMs). Experiments using mushroom TYR suggest that L-cysteinamide at certain concentrations can inhibit eumelanin synthesis through a dual mechanism by inhibiting TYR-catalyzed dopaquinone synthesis and by diverting the synthesized dopaquinone to the formation of DOPA-cysteinamide conjugates rather than dopachrome. Finally, L-cysteinamide was shown to increase pheomelanin content while decreasing eumelanin and total melanin contents in MNT-1 cells. This study suggests that L-cysteinamide has an optimal structure that can effectively and safely inhibit eumelanin synthesis in MNT-1 cells and HEMs, and will be useful in controlling skin hyperpigmentation.

Keywords: L-cysteinamide; MNT-1 melanoma; eumelanin; melanin; normal human melanocytes; tyrosinase; viability.

Figure 1

Effects of twenty different amidated amino acids on tyrosinase (TYR)-mediated dopachrome formation in vitro, and the eumelanin content and viability of MNT-1 cells. In (a), TYR-mediated dopachrome formation was determined using a lysate of human embryonic kidney 293 cells constitutively expressing human TYR in the absence (the control) or presence of each substance at 0.2 mM. Data are presented as % of control (means ± SD, n = 4). ** p < 0.01 vs. control. In (b) and (c), MNT-1 cells were treated with vehicle (control) or each substance at 0.5 mM for 72 h. Melanin content (b) and cell viability (c) are presented as % of control (means ± SD, n = 4). ** p < 0.01 vs. control.

Figure 2

Effects of L-cysteinamide, L-cysteine, N-acetyl L-cysteine, glutathione, kojic acid, and β-arbutin on TYR-mediated dopachrome formation in vitro. (a) Time-dependent changes in A₄₇₅ of the TYR reaction mixture in the absence (control) or presence of L-cysteinamide or L-cysteine at 0.2 mM (n = 3). (b) TYR-mediated dopachrome formation determined in the absence (control) and presence of each substance at 0.2 mM (n = 4). Data are presented as % of control (means ± SD). Duncan’s multiple range test was performed to compare all group means to each other. Groups that share the same Greek letters (α, β, γ, δ, or ε) do not have significantly different means at the p < 0.05 level.

Figure 3

Effects of L-cysteinamide, L-cysteine, N-acetyl L-cysteine, glutathione, kojic acid, and β-arbutin on the morphology and pigmentation of MNT-1 cells. Cells were treated with each substance at 1.0 mM for 72 h and subjected to Fontana-Masson staining.

Figure 4

Effects of L-cysteinamide, L-cysteine, N-acetyl L-cysteine, glutathione, kojic acid, and β-arbutin on the melanin content and viability of MNT-1 cells. Cells were treated with vehicle (control) or each substance at the indicated concentrations for 72 h. Melanin content (a) and cell viability (b) are presented as % of control (means ± SD, n = 4). * p < 0.05 vs. control; ** p < 0.01 vs. control.

Figure 5

Effects of L-cysteinamide, L-cysteine ethyl ester, N-acetyl L-cysteinamide, and cysteamine on the melanin content and viability of MNT-1 cells. Cells were treated with vehicle (control) or each substance at the indicated concentrations for 72 h. Melanin content (a) and cell viability (b) are presented as % of control (means ± SD, n = 3). ** p < 0.01 vs. control.

Figure 6

Effects of L-cysteinamide, L-cysteine, N-acetyl L-cysteine, glutathione, kojic acid, and β-arbutin on the activity levels of TYR, and the mRNA and protein expression levels of TYR, tyrosinase-related protein-1 (TYRP1), dopachrome tautomerase (DCT). MNT-1 cells were treated with vehicle (control) or each substance at 1.0 mM for 24 h. (a) TYR activity was determined using cell lysates (n = 4). (b) The mRNA levels of TYR, TYRP1, and DCT were determined by qRT-PCR and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels (n = 3). (c) Their protein levels were quantified by Western blot and normalized to β-actin protein levels (n = 4). Typical blot images are shown. Data are presented as % of control (means ± SD). ** p < 0.01 vs. control.

Figure 7

Inhibition of mushroom TYR-mediated dopachrome formation by L-cysteinamide. Mushroom TYR-mediated dopachrome formation was measured at varying concentrations of L-dihydroxyphenylalanin (DOPA) and L-cysteinamide. Data are presented as mean ± SD (n = 4).

Figure 8

Ultraviolet-visible absorption spectrum changes during TYR-catalyzed reactions. The reaction mixture containing 2 mM L-DOPA and varied concentrations of L-cysteinamide (100, 300, or 500 μM) were incubated at 25 °C for the indicated time. Spectra after 5-min reaction are shown in (a). Time-dependent changes in A₄₇₅ and A₃₅₀ are shown in (b) and (c), respectively.

Figure 9

Copper-chelating activities of L-cysteinamide, kojic acid, and β-arbutin. Absorption spectra of 200 μM pyrocatechol violet (PCV) reacted with 200 μM CuSO₄ in the absence or presence of L-cysteinamide, kojic acid, or β-arbutin, each at 200 μM, are shown.

Figure 10

Effects of L-cysteinamide on the pheomelanin and eumelanin contents of MNT-1 cells. Cells were treated with vehicle (control) or 1 mM L-cysteinamide for 72 h. The contents of pheomelanin (a) and eumelanin (b) were separately estimated and used to calculate the total melanin contents (c) and the pheomelanin to total melanin ratios (d). Data are presented as means ± SD (n = 3). * p < 0.05 vs. control; ** p < 0.01 vs. control.

Figure 11

Effects of L-cysteinamide on the viability, the melanin content, the cellular TYR activity, and the mRNA expression levels of melanogenic enzymes in normal human epidermal melanocytes (HEMs). Cells treated with vehicle (control) or L-cysteinamide at the specified concentration for 72 h (a–c) or 24 h (d,e). Cells were subjected to Fontana-Masson staining (a), and assays to determine total melanin content (n = 4) (b) and viability (n = 4) (c). Cellular TYR activity was determined using cell lysates (n = 4) (d). The mRNA levels of TYR, TYRP1, and DCT were determined by qRT-PCR and normalized to GAPDH mRNA levels (n = 3) (e). Data are presented as % of control (means ± SD). ** p < 0.01 vs. control.

Figure 12

A tentative model for anti-eumelanogenic or anti-melanogenic action of L-cysteinamide. L-DOPA or L-tyrosine is oxidized by TYR to dopaquinone, which is spontaneously converted to cyclodopa and then oxidized to dopachrome. L-Cysteinamide binds the Cu²⁺ ion at the active site of TYR and inhibits its activity. When TYR is partially inhibited by moderate concentrations of L-cysteinamide, dopaquinone synthesis is reduced but still occurs at significant levels. The synthesized dopaquinone is quickly captured by the available L-cysteinamide to form DOPA-cysteinamide conjugates, thus reducing dopachrome formation. If TYR is completely inhibited by high concentrations of L-cysteinamide, no dopaquinone is synthesized, and thus neither DOPA-cysteinamide conjugates nor dopachrome can be produced. Overall, it is proposed that L-cysteinamide may prevent eumelanin synthesis through a dual mechanism by inhibiting TYR activity, thereby reducing dopaquinone synthesis, and by diverting dopaquinone to the formation of DOPA-cysteinamide conjugates, thereby reducing dopachrome formation. At very high concentrations, L-cysteinamide might block total melanin (both pheomelanin and eumelanin) synthesis by almost completely inhibiting TYR activity.