Characterization of a small molecule inhibitor of melanogenesis that inhibits tyrosinase activity and scavenges nitric oxide (NO)

Background

Excessive melanin production and accumulation are characteristics of a large number of skin diseases, including melasma, and post-inflammatory hyperpigmentation. During our on-going search for new agents with an inhibitory effect on tyrosinase, we synthesized a new type of tyrosinase inhibitor, 4-(thiazolidin-2-yl)benzene-1,2-diol (MHY-794), which directly inhibits mushroom tyrosinase.

Methods

The inhibitory effect of MHY-794 on tyrosinase activity and nitric oxide (NO) scavenging activity was evaluated in cell free system. Additional experiments were performed using B16F10 melanoma cells to demonstrate the effects of MHY-794 in vitro. HRM2 hairless mice were used to evaluate anti-melanogenic effects of MHY-794 in vivo.

Results

MHY-794 effectively inhibited mushroom tyrosinase activity in cell free system. In silico docking simulation also supported the inhibitory effects of MHY-794 on mushroom tyrosinase. MHY-794 also proved to be effective at scavenging nitric oxide (NO), which serves as an important modulator in the melanogenesis signaling pathway. In addition, MHY-794 effectively inhibited SNP (NO donor)-induced melanogenesis by directly inhibiting tyrosinase and diminishing NO-mediated melanogenesis signaling in B16 melanoma cells. The anti-melanogenic effects of MHY-794 were further confirmed in HRM2 hairless mice. Ultraviolet light (UV) significantly up-regulated NO-mediated melanogenesis signaling in HRM2 hairless mice, but MHY-794 effectively inhibited both melanogenesis and diminished UV-induced NO-signaling.

Conclusions

Our results indicate that MHY-794 is highly effective at inhibiting NO-mediated melanogenesis in vitro and in vivo by direct NO scavenging and directly inhibiting tyrosinase activity, and suggest that MHY-794 be considered a new developmental candidate for the treatment of hyper-pigmentation disorders.

General significance

MHY-794, which showed great efficacy on NO-mediated melanogenesis by direct NO scavenging as well as direct inhibition of tyrosinase catalytic activity, might be utilized for the development of a new candidate for treatment of the hyper-pigmentation disorders.     

Up-regulation of tyrosinase gene by nitric oxide in human melanocytes

Ultraviolet light (UV) radiation causes skin-tanning, which is thought to be mediated by stimulating the release of melanogenic factors from keratinocytes as well as other cells. Nitric oxide (NO) has been reported to be generated after UV radiation and to stimulate melanocytes as one of the melanogens. In a previous experiment by another group on melanogenesis induced by NO, increases in both tyrosinase activity and tyrosinase protein levels were observed after daily stimulation of NO for 4 days. In the present study, we investigated tyrosinase gene expression within the first 24 hr of NO-induced melanogenesis. Tyrosinase mRNA expression was found to be induced 2 hr after a single treatment with S-nitroso-N-acetyl-L-arginine. An increase of tyrosinase activity was also detected time-dependently within the 24-hr period, accompanied by an increase of tyrosinase protein levels. The induction of mRNA expression was suppressed by a cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase (cGMP/PKG) inhibitor. These results suggest that the enhancement of tyrosinase gene expression via the cGMP pathway may be a primary mechanism for NO-induced melanogenesis.     

Essential role of nitric oxide in acute ischemic preconditioning: S-Nitros(yl)ation versus sGC/cGMP/PKG signaling?

Nitric oxide (NO) plays an important role in acute ischemic preconditioning (IPC). In addition to activating soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signaling pathways, NO-mediated protein S-nitros(yl)ation (SNO) has been recently shown to play an essential role in cardioprotection against ischemia–reperfusion (I/R) injury. In our previous studies, we have shown that IPC-induced cardioprotection could be blocked by treatment with either N-nitro-L-arginine methyl ester (L-NAME, a constitutive NO synthase inhibitor) or ascorbate (a reducing agent to decompose SNO). To clarify NO-mediated sGC/cGMP/PKG-dependent or -independent (i.e., SNO) signaling involved in IPC-induced cardioprotection, mouse hearts were Langendorff-perfused in the dark to prevent SNO decomposition by light exposure. Treatment with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, a highly selective inhibitor of sGC) or KT5823 (a potent and selective inhibitor of PKG) did not abolish IPC-induced acute protection, suggesting that the sGC/cGMP/PKG signaling pathway does not play an important role in NO-mediated cardioprotective signaling during acute IPC. In addition, treatment with ODQ in IPC hearts provided an additional protective effect on functional recovery, in parallel with a higher SNO level in these ODQ+IPC hearts. In conclusion, these results suggest that the protective effect of NO is not related primarily to activation of the sGC/cGMP/PKG signaling pathway, but rather through SNO signaling in IPC-induced acute cardioprotection.     

De novo tyrosinase inhibitor: 4-(6,7-Dihydro-5H-indeno[5,6-d]thiazol-2-yl)benzene-1,3-diol (MHY1556)

In this study, we have synthesized and studied de novo tyrosinase inhibitor, MHY1556, which showed significantly better efficacy than other pre-existing tyrosinase inhibitors in vitro experiments. The IC₅₀ value of MHY1556 was 0.50 μM which was significantly lower than that of kojic acid (IC₅₀ = 53.95 μM), which is a well-known tyrosinase inhibitor and was used as a positive control in this study. We predicted the tertiary structure of tyrosinase, simulated the docking with compound MHY1556 and confirmed that the compound strongly interacts with mushroom tyrosinase residues. Substitutions with a hydroxy group at both R1 and R3 of the phenyl ring indicated that these groups play a major role in the high binding affinity to tyrosinase, especially through the hydrogen bonding interaction of the hydroxyl group at R1 with GLY281. In addition, MHY1556 showed concentration-dependent inhibitory effects in melanin content assay where B16F10 melanoma cells were treated with α-melanocyte stimulating hormone (α-MSH), and also there is no significant cytotoxicity of this compound in cell viability assay conducted in B16F10 melanoma cells. The tyrosinase activity assay results with MHY1556 also support its potent inhibitory effects. Therefore, our data strongly suggest MHY1556 suppresses the melanogenesis via a tyrosinase inhibitory effect.     

Up‐regulation of Tyrosinase Gene by Nitric Oxide in Human Melanocytes

Ultraviolet light (UV) radiation causes skin‐tanning, which is thought to be mediated by stimulating the release of melanogenic factors from keratinocytes as well as other cells. Nitric oxide (NO) has been reported to be generated after UV radiation and to stimulate melanocytes as one of the melanogens. In a previous experiment by another group on melanogenesis induced by NO, increases in both tyrosinase activity and tyrosinase protein levels were observed after daily stimulation of NO for 4 days. In the present study, we investigated tyrosinase gene expression within the first 24 hr of NO‐induced melanogenesis. Tyrosinase mRNA expression was found to be induced 2 hr after a single treatment with S‐nitroso‐N‐acetyl‐ l ‐arginine. An increase of tyrosinase activity was also detected time‐dependently within the 24‐hr period, accompanied by an increase of tyrosinase protein levels. The induction of mRNA expression was suppressed by a cyclic guanosine 3′,5′‐monophosphate (cGMP)‐dependent protein kinase (cGMP/PKG) inhibitor. These results suggest that the enhancement of tyrosinase gene expression via the cGMP pathway may be a primary mechanism for NO‐induced melanogenesis.     

Tyrosinase inhibition and anti-melanin generation effect of cinnamamide analogues

Abnormal melanogenesis results in excessive production of melanin, leading to pigmentation disorders. As a key and rate-limiting enzyme for melanogenesis, tyrosinase has been considered an important target for developing therapeutic agents of pigment disorders. Despite having an (E)-β-phenyl-α,β-unsaturated carbonyl scaffold, which plays an important role in the potent inhibition of tyrosinase activity, cinnamic acids have not attracted attention as potential tyrosinase inhibitors, due to their low tyrosinase inhibitory activity and relatively high hydrophilicity. Given that cinnamic acids’ structure intrinsically features this (E)-scaffold and following our experience that minute changes in the chemical structure can powerfully affect tyrosinase activity, twenty less hydrophilic cinnamamide derivatives were designed as potential tyrosinase inhibitors and synthesised using a Horner-Wadsworth-Emmons reaction. Four of these cinnmamides (4, 9, 14, and 19) exhibited much stronger mushroom tyrosinase inhibition (over 90% inhibition) at 25 µM compared to kojic acid (20.57% inhibition); crucially, all four have a 2,4-dihydroxy group on the β-phenyl ring of the scaffold. A docking simulation using tyrosinase indicated that the four cinnamamides exceeded the binding affinity of kojic acid, and bound more strongly to the active site of tyrosinase. Based on the strength of their tyrosinase inhibition, these four cinnamamides were further evaluated in B16F10 melanoma cells. All four cinnamamides, without cytotoxicity, exhibited higher tyrosinase inhibitory activity (67.33 – 79.67% inhibition) at 25 μM than kojic acid (38.11% inhibition), with the following increasing inhibitory order: morpholino (9) = cyclopentylamino (14) < cyclohexylamino (19) < N-methylpiperazino (4) cinnamamides. Analysis of tyrosinase activity and melanin content in B16F10 cells showed that the four cinnamamides dose-dependently inhibited both cellular tyrosinase activity and melanin content and that their inhibitory activity at 25 μM was much better than that of kojic acid. The results of melanin content analysis well matched those of the cellular tyrosinase activity analysis, indicating that tyrosinase inhibition by the four cinnamamides is a major factor in the reduction of melanin production. These results imply that these four cinnamamides with a 2,4-dihydroxyphenyl group can act as excellent anti-melanogenic agents in the treatment of pigmentation disorders.     

A novel synthetic compound, (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-iminothiazolidin-4-one (MHY773) inhibits mushroom tyrosinase

As part of continued efforts for the development of new tyrosinase inhibitors, (Z)-5-(substituted benzylidene)-2-iminothiazolidin-4-one derivatives (1a – 1l) were rationally synthesized and evaluated for their inhibitory potential in vitro. These compounds were designed and synthesized based on the structural attributes of a β-phenyl-α,β-unsaturated carbonyl scaffold template. Among these compounds, (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-iminothiazolidin-4-one (1e, MHY773) exhibited the greatest tyrosinase inhibition (IC₅₀ = 2.87 μM and 8.06 μM for monophenolase and diphenolase), and outperformed the positive control, kojic acid (IC₅₀ = 15.59 and 31.61 μM). The kinetic and docking studies demonstrated that MHY773 interacted with active site of tyrosinase. Moreover, a melanin quantification assay demonstrated that MHY773 attenuates α-melanocyte-stimulating hormone (α-MSH) and 3-isobutyl-1-methylxanthine (IBMX)-induced melanin contents in B16F10 melanoma cells. Taken together, these data suggest that MHY773 suppressed the melanin production via the inhibition of tyrosinase activity. MHY773 is a promising for the development of effective pharmacological and cosmetic agents for skin-whitening.     

N-Methyl-D-aspartate receptor stimulation activates tyrosinase and promotes melanin synthesis in the ink gland of the cuttlefish Sepia officinalis through the nitric Oxide/cGMP signal transduction pathway. A novel possible role for glutamate as physiologic activator of melanogenesis

The tyrosinase-catalyzed conversion of l-tyrosine to melanin represents the most distinctive biochemical pathway in the ink gland of the cuttlefish Sepia officinalis; however, the molecular mechanisms underlying its activation have remained so far largely uncharted. In this paper we demonstrate for the first time that l-glutamate can stimulate tyrosinase activity and promote melanin synthesis in Sepia ink gland via the N-methyl-d-aspartate (NMDA) receptor/NO/cGMP signal transduction pathway. Incubation of intact ink glands with either l-glutamate or NMDA resulted in an up to 18-fold increase of tyrosinase activity and a more than 6-fold elevation of cGMP levels. Comparable stimulation of tyrosinase was induced by an NO donor and by 8-bromo-cGMP. An NMDA receptor antagonist, NO synthase (NOS) inhibitors, and a guanylate cyclase blocker suppressed NMDA-induced effects. Immunohistochemical evidence indicated that enhanced cGMP production was localized largely in the mature part of the ink gland. Increased de novo synthesis of melanin was demonstrated in NMDA- and NO-stimulated ink glands by a combined microanalytical approach based on spectrophotometric determination of pigment levels and high performance liquid chromatography quantitation of pyrrole-2,3, 5-tricarboxylic acid, a specific melanin marker, in melanosome-containing fractions. These results fill a longstanding gap in the understanding of the complex biochemical mechanisms underlying activation of melanogenesis in the mature ink gland cells of S. officinalis and disclose a novel physiologic role of the excitatory neurotransmitter glutamate mediated by the NMDA receptor/NO/cGMP signaling pathway.     

DKK3调控羊驼黑色素细胞中黑色素生成

Dickkopf-3 (DKK3),Wnt/β-catenin信号通路中一个重要的抑制因子,可能参与调控黑色素生成过程。本文研究了DKK3在羊驼黑色素细胞中黑色素生成的作用。在羊驼黑色素细胞中,过表达DKK3显著下调Wnt1,Lef1,Myc和黑色素生成相关基因MITF及其下游基因TYR,TYRP1和TYRP2的表达,在mRNA和蛋白质水平均明显下降(P<0.05);总黑素,褐黑素和真黑素的含量分别下降80.30%、72.17%和64.60% ( P <0.05)。相反,在羊驼黑色素细胞中转染siRNA-DKK3,一种小干扰RNA,可以显著上调Wnt1,Lef1,Myc和黑色素生成相关基因MITF及其下游基因TYR,TYRP1和TYRP2在mRNA和蛋白质水平的表达(P<0.05);总黑素、褐黑素和真黑素的含量分别增加1.65倍、1.25倍和1.21倍(P<0.05)。这些结果表明,DKK3可以通过Wnt/β-catenin信号通路介导MITF下调羊驼黑色素细胞中黑色素的生成。     

lpa-miR-nov-66通过调控cAMP路径抑制α-MSH介导的羊驼黑色素生成

α-黑素细胞刺激素(α-MSH)和lpa-miR-nov-66在羊驼黑色素细胞产生黑色素过程中均起重要的调控作用,但二者之间的关系尚未报道.本研究在体外培养的羊驼黑色素细胞中通过转染lpamiR-nov-66和添加α-MSH处理,用实时定量PCR和Western印迹检测黑色素细胞内基因表达水平,ELISA法检测c AMP和cGMP的产量,RTCA实时无标记细胞功能分析黑色素细胞增殖以及紫外分光光度法检测黑色素产量,证实二者在调控羊驼黑色素细胞产生黑色素颗粒过程中的关系.结果显示,与单纯α-MSH处理相比,lpa-miR-nov-66转染结合α-MSH处理组中,小眼转录因子(MITF)和酪氨酸酶(TYR)在转录水平和翻译水平的表达均降低,而酪氨酸酶相关蛋白2(TYRP2)在转录和翻译水平的表达均升高;cGMP的产量升高,cAMP的产量下降;黑色素细胞增殖没有显著变化;黑色素细胞内黑色素产量下降.与单纯转染lpa-miR-nov-66相比,lpa-miR-nov-66转染结合α-MSH处理组中,MITF、TYR和TYRP2在转录水平和翻译水平的表达均升高;cGMP的产量下降,cAMP的产量升高;黑色素细胞增殖没有显著变化;黑色素细胞内黑色素产量升高.上述结果证明,lpa-miR-nov-66通过调控羊驼黑色素细胞中毛色形成的c AMP路径,抑制α-MSH对黑色素细胞产生黑色素的促进作用.