ABSTRACT 

Melanogenesis is a process to synthesize melanin, which is primarily responsible for the pigmentation of human skin, eyes, and hair. Although numerous enzymatic catalyzed and chemical reactions are involved in the melanogenesis process, the enzymes such as tyrosinase and tyrosinase-related protein-1 (TRP-1) and TRP-2 played a major role in melanin synthesis. Specifically, tyrosinase is a key enzyme, which catalyzes a rate-limiting step of melanin synthesis, and the downregulation of tyrosinase is the most prominent approach for the development of melanogenesis inhibitors. Therefore, numerous inhibitors that target tyrosinase have been developed in recent years. The review focuses on the recent discovery of tyrosinase inhibitors that are directly involved in the inhibition of tyrosinase catalytic activity and functionality from all sources, including laboratory synthetic methods, natural products, virtual screening, and structure-based molecular docking studies.

According to relevant studies,cistanche is a common herb that is known as "the miracle herb that prolongs life". Its main component is cistanoside, which has various effects such as antioxidant, anti-inflammatory, and immune function promotion. The mechanism between cistanche and skin whitening lies in the antioxidant effect of cistanche glycosides. Melanin in human skin is produced by the oxidation of tyrosine catalyzed by tyrosinase, and the oxidation reaction requires the participation of oxygen, so the oxygen-free radicals in the body become an important factor affecting melanin production. Cistanche contains cistanoside, which is an antioxidant and can reduce the generation of free radicals in the body, thus inhibiting melanin production.

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KEYWORDS 

human tyrosinase; inhibitors; Parkinson’s disease; structure-activity relationships; skin whitening agents

Introduction

It is estimated approximately 15% of the world population invests in skin whitening agents with Asia being dominated1. Global industry analysts (GIA) have predicted that the universal market for skin lighteners will reach $23 billion by 2020, driven by new markets in Asia, particularly India, Japan, and China2. According to the SIRONA biochem report1, approximately $13 billion was spent on skin care products and cosmetics in Asia Pacific. In India alone, it is estimated that $432 million was spent in 2010 on skin-lightening creams and skin care agents. A recent survey showed that 80% of Indian men use fairness creams and the number of consumers is growing by 18% annually1. The molecular mechanism of these skin-lightening agents is to reduce melanin, which is the main source of skin color.

Melanin is primarily responsible for the pigmentation of human skin, eyes, and skin, which is produced from epidermis melanocytes in an approximate ratio of 1:36 with basal keratinocytes3. In response to ultraviolet B (UVB)-irradiation, melanocyte synthesizes melanin through the process called melanogenesis. The synthesized melanin in melanosomes is transported to neighboring keratinocytes in the epidermis4. Under normal physiological conditions, pigmentation has a beneficial effect on the photo-protection of human skin against harmful UV injury and plays an important evolutionary role in camouflage and animal mimicry5. However, excessive production of melanin causes dermatological problems such as freckles, solar lentigo (age spots), and melasma6–9, as well as cancer10 and postinflammatory melanoderma11. In addition, continuous UV-irradiation can result in DNA damage, gene mutation, cancer development, and impairment of the immune system or photoaging12.

Regulation of melanogenesis

Melanogenesis is a complex pathway involving a combination of enzymatic and chemical-catalyzed reactions. Melanocytes produce two types of melanin: eumelanin (brown-black) and pheomelanin (red-yellow) formed by the conjugation of cysteine or glutathione (Figure 1) 13–15.

The melanogenesis process is initiated with the oxidation of L-tyrosine to dopaquinone (DQ) by the key enzyme, tyrosinase (TYR). The resulting quinone will serve as a substrate for the synthesis of eumelanin and pheomelanin16,17. The formation of DQ is a rate-limiting step in melanin synthesis because the remainder of the reaction sequence can proceed spontaneously at a physiological pH value of 17. After DQ formation, it undergoes intramolecular cyclization to produce indoline, and leukodopachrome (cyclo-dopa). The redox exchange between leukodopachrome and DQ gives rise to dopachrome and L-3,4-dihydroxyphenylalanine (L-DOPA), which is also a substrate for TYR and oxidized to DQ again by the enzyme. Dopachrome gradually decomposes to give dihydroxy indole (DHI) and dihydroxy indole-2-carboxylic acid (DHICA). The latter process is catalyzed by TRP-2, now known as dopachrome tautomerase (DCT). Ultimately, these dihydroxy indoles (DHI and DHICA) are oxidized to eumelanin. TRP-1 is believed to catalyze the oxidation of DHICA to eumelanin. Alongside, DQ is converted to 5-S-cysteinyldopa or glutothionyldopa in the presence of cysteine or glutathione. Subsequent oxidation gives benzothiazine intermediates and finally produces pheomelanin. Although three enzymes, TYR, TRP-1, and TRP-2 are involved in the melanogenesis pathway, tyrosinase is exclusively necessary for melanogenesis.

Tyrosinase and its key role in melanin synthesis

Tyrosinase (monophenol or o-diphenol, oxygen oxidoreductase, EC 1.14.18.1, syn. polyphenol oxidase), a multifunctional membrane-bound type-3 copper-containing glycoprotein is located in the membrane of the melanosome18. Tyrosinase is produced only by melanocyte cells. Following its production and consequent processing in the endoplasmic reticulum and Golgi, it is trafficked to melanosomes, wherein the pigment melanin is synthesized. From the structural point of view, two copper ions are surrounded by three histidine residues that are responsible for the catalytic activity of tyrosinase (Figure 2) 19. The active site has three states; oxy, met, and deoxy forms in the formation of pigmentation. More specifically, at the active site, two copper ions interact with dioxygen to form a highly reactive chemical intermediate that directly participates in the hydroxylation of monophenols to diphenols (mycophenolate activity) and in the oxidation of o-diphenols to o-quinones (diphenols activity)20,21. Tyrosinase is also catalyzing the process of neuromelanin production in which the oxidation of dopamine produces dopaquinones. However, excessive production of dopaquinones results in neuronal damage and cell death. This suggests that tyrosinase might play a significant role in neuromelanin formation in the human brain and is responsible for the neurodegeneration associated with Parkinson’s disease and Huntington’s diseases22–26. Tyrosinase has also been linked to the browning of vegetables and fruits during the postharvest and handling process, leading to quick degradation27–29. The application of tyrosinase was further extended in the molting process of insects30. Thus, the regulation of melanin synthesis by inhibiting the tyrosinase enzyme is a major motivation for researchers in the context of preventing hyperpigmentation.

Tyrosinase inhibitors

Since tyrosinase is a crucial enzyme in synthesizing melanin through melanogenesis, it becomes the most prominent and successful target for melanogenesis inhibitors that directly inhibit the tyrosinase catalytic activity. Most cosmetics or skin-lightening agents commercially available are tyrosinase inhibitors. The fact that the inhibitors target tyrosinase may specifically inhibit melanogenesis in cells without side effects, as tyrosinase is exclusively produced only by melanocytes. Many tyrosinase inhibitors such as hydroquinone (HQ)31–34, arbutin, kojic acid35–37, azelaic acid38,39, L-ascorbic acid40–42, ellagic acid43–45, tranexamic acid46–48 have been used as skin-whitening agents, with certain drawbacks (Figure 3).

HQ is potentially mutagenic to mammalian cells and linked to several adverse reactions including contact dermatitis, irritation, transient erythema, burning, prickling sensation, leukoderma, chestnut spots on the nails, hypochromic and ochronosis49–52. Arbutin, a prodrug of hydroquinone, is a natural product and reduces or inhibits melanin synthesis by inhibiting tyrosinase. However, natural forms of arbutin are chemically unstable and can release hydroquinone which is catabolized to benzene metabolites with the potential toxicity for bone marrow53. Kojic acid use in cosmetics has been limited, due to its carcinogenicity and its instability during storage54. L-Ascorbic acid is sensitive to heat and degrades easily 55. Ellagic acid is insoluble and thus poorly bioavailable56, and for the tranexamic acid, the melanogenic pathway remains undetermined 47. Thus, it is in great need of developing new tyrosinase inhibitors with drug-like properties.

Mushroom tyrosinase inhibitors

Chalcones and flavanone inhibitors

In another study, chalcones 2a–2d isolated from Morus australis were evaluated for their inhibitory activity on mushroom tyrosinase using L-tyrosine as the substrate 58. The results showed that all four chalcone derivatives were extremely potent inhibitors for tyrosinase in comparison to the standard compound arbutin (Figure 5(a), 2a–2d). Especially, compound 2a was 700-fold potent inhibition in comparison to arbutin. The structure–activity relationship (SAR) analysis showed that resorcinol construction at both ring A and ring B could be the reason for strong tyrosinase inhibition. In addition, the substitution at the 30 positions of ring A plays an important role in enhancing inhibitory potency. For example, the steric bulky substituent at ring A on 2c reduced the potency compared to the unsubstituted 2a, this is due to the chelating ability towards the copper ions. Interestingly, compound 2d exhibited stronger inhibitory activity than 2a and 2c, indicating that the 4- hydroxy-1-pentene group at the 30 -position of 2d is responsible for the enhancement. Further, the effects of chalcones on melanin synthesis were tested in melanin-producing B16 murine melanoma cells, and compounds 2a, 2b, and 2d were strongly inhibited with little or no cytotoxicity.

Recently, Radhakrishnan et al., reported a library of aza chalcones and59 the inhibitory effects on mushroom tyrosinase using L-DOPA as substrate was investigated. Among the investigated compounds in the study, two compounds 3a and 3b, congeners (carbonyl reduction) of the pyridyl azachalcones were found to be more potent enzyme inhibitors than kojic acid (IC50¼27.30 lM) (Figure 5(a), 3a–3b). Furthermore, the kinetic analysis identified both 3a and 3b were competitive inhibitors with Ki values of 2.62 and 8.10 lM, respectively. The structure–function analysis showed that the nitrogen atom in the pyridine skeleton of the inhibitors could be complex with the copper ions present in the tyrosinase active site. The same research group has reported another chalcone series with oxime functionality as an inhibitor of tyrosinase and melanin formation in melanoma B16 cells.60 Two of the compounds (4a: IC50¼4.77 lM and 4b: IC5057.89 lM) exhibited potent tyrosinase inhibitory activities (Figure 5(a), 4a–4b) than the kojic acid (IC50522.25 lM). Kinetic studies revealed competitive inhibitors with Ki values of 5.25 and 8.33 lM. In a-melanocyte-stimulating hormone (a-MSH) induced B16 melanoma cells, these two compounds 4a and 4b inhibited melanin formation and tyrosinase without cytotoxicity. In terms of SAR analysis, it was identified that the presence of ortho-methoxy with para-nitro substituents (ring B) was responsible for potent tyrosinase inhibition (4a). In addition, an electron-donating para-dimethyl amino ring (ring B) exhibited the second most potent inhibition (4b).

In another study, a novel series of 2,3-dihydro-1H-inden-1-one chalcone-like derivatives were reported.61 Two of them, 5a and 5b, were identified as potent inhibitors of diphenolase activity of tyrosinase with IC50 values of 12.3 and 8.2 lM (Figure 5(a)). Further exploration of the mechanism, both the inhibitors 5a and 5b were found to be reversible and competitive.

Wang et al. isolated dihydrochalcones (Figure 5(a), 6a–6c) and flavanones (Figure 5(b), 7a–7c) from Flemingia philippinensis and investigated for their inhibitory activities on tyrosinase62. The results showed that they inhibit the mycophenolate (IC50¼1.01 to 18.4 lM) and diphenols (IC50¼5.22 to 84.1 lM) actions of tyrosinase. In particular, dihydrochalcone (6c) effectively inhibited both mycophenolate and diphenols activities of tyrosinase with IC50 values of 1.28 and 5.22 lM, respectively. The SAR analysis is very interesting because the pharmacophore is not associated with tyrosinase inhibition and it lacks the a,b-unsaturated ketone motif which is present in most of the inhibitors. In the case of flavanones, compounds containing resorcinol group (7a) were competitive and significantly inhibited mycophenolate (IC50¼1.79 lM) and diphenols (IC50¼7.48 lM) of tyrosinase.

In the search for new tyrosinase inhibitors, it was found that the extracts of Camylotropis hirtella show tyrosinase inhibition63. After the successful purification and isolation of fourteen compounds, four compounds (Figure 5(b), 8a–8d) showed potent inhibitory activities against tyrosinase. The most potent compound was found to be neorauflavane 8c exhibiting IC50 values of 30 and 500 nM against monophenolase and diphenolase activity of tyrosinase. Furthermore, compared with kojic acid (13.2 lM), 8c was 400-fold more potent against the mycophenolate activity of tyrosinase. The second most potent compound was geranylated isoflavone 8a inhibited mycophenolate and diphenols with IC50 values 2.9 and 128.2 lM, respectively, and was identified as a competitive and reversible inhibitor. In addition, compounds 8a and 8c efficiently reduced the melanin content in a-MHS-induced B16 melanoma cells, without influencing cell viability. From the structural point of view, the reduction of the geranyl side chain improves the tyrosinase inhibitory activity.

Resveratrol analogs

Resveratrol (3,5,40 -trihydroxy-trans-stilbene, 9) a widely distributed stilbenoid in nature such as in grapes, exhibited the inhibitory activity against mushroom tyrosinase through the mechanism of Kcat (suicide substrate) type inhibition64. In vitro analysis in a-MSH-stimulated B16 murine melanoma cells, resveratrol inhibited the cellular melanin production via suppression of melanogenesis-related proteins such as tyrosinase, TRP-1, TRP-2, and microphthalmia-associated transcription factor (MITF) expression65 without any cytotoxicity up to 200 lM.64 The inhibitory effects of resveratrol have been confirmed in an in vivo model using UVB-irradiated brownish guinea pigs. In this study, treatment of resveratrol with UVB-irradiated dorsal skin of guinea pigs visually decreased the hyperpigmentation.

Recently, a series of azo-resveratrol (13a–13e and 13 g) and azo-oxyresveratrol (13f) were reported (Figure 6) 69. Among these compounds, 13a and 13b exhibited high tyrosinase inhibitory activity of 56.25% and 72.75% at 50 lM, respectively 69. The 4-hydroxyphenyl moiety was found to be essential for higher inhibition and 3,5-dihydroxy phenyl or 3,5-dimethoxy phenyl derivatives showed better tyrosinase inhibition than 2,5-dimethoxy phenyl derivatives. Particularly, the introduction of hydroxyl or methoxy group into the 4-hydroxyphenyl moiety diminished or significantly reduced mushroom tyrosinase inhibition. Among the synthesized azo compounds, azo-resveratrol (13b) was the most potent mushroom tyrosinase inhibition with an IC50 value of 36.28 lM. The results indicate that azo-resveratrol with a high Log p-value might be superior to resveratrol for the development of whitening agents and pharmaceutical drugs in the treatment of hyperpigmentation.

Coumarin derivatives

Coumarins are a large family of benzopyrone compounds available from natural and synthetic origins with different pharmacological activities70. In recent studies, few coumarins proved to inhibit the mushroom tyrosinase, which includes esculetin and umbelliferone with stronger tyrosinase inhibitory activity71,72. In a continuous effort, Matos et al., have demonstrated a series of coumarin-resveratrol hybrid compounds, 3-phenyl coumarins with hydroxyl or alkoxy and bromo substituent at various positions in the scaffold73 (Figure 7). Among the series, a compound with a bromo atom and two hydroxyl groups in the 3-phenyl coumarin moiety (14), was identified as the best inhibitor with an IC50 value of 215 lM. This compound is a noncompetitive tyrosinase inhibitor with a Ki value of 0.189 mM.

In another study, a series of umbelliferone analogs were reported for their inhibitory effects on mushroom tyrosinase74. Specifically, compounds 15a and 15b possessing 3,4-dihydroxy and 3,4,5-dihydroxy phenyl scaffold showed more potent inhibitory activities against mushroom tyrosinase activity (Figure 7). Asthana et al. demonstrated a series of hydroxy coumarins (16a–16d) 75 (Figure 7). The SAR studies suggested that the position of hydroxyl substituent on the coumarin plays a role in enzymatic inhibition; the compounds with aromatic hydroxylated, 6-hydroxycoumarin (16c), and 7-hydroxycoumarin (16d), found to be weak substrates of the enzyme. Especially, 7-hydroxycoumarin strongly inhibited the dopachrome concentration in a range of 0.3 - 0.9 mM. At a maximum 7-hydroxycoumarin concentration, the inhibition reached 88%. The authors found the phenomenon was due to the specific inhibition of L-tyrosine conversion. On the other hand, the compounds with pyrone hydroxylated, 3-hydroxycoumarin (16a) and 4-hydroxycoumarin (16b) were not substrates of tyrosinase. 3-hydroxycoumarin (16a) was found to inhibit tyrosinase but not the compound with 4-hydroxycoumarin (16b), indicating the pyrone ring cannot be hydroxylated by tyrosinase.

Recently, a series of phosphonic acid diamides were screened for their tyrosinase inhibition activity76. The results showed that the substituent attached to C-5 and the stereochemistry of the two stereogenic centers (C-5 and phosphorus atom) were important for the tyrosinase inhibition (17a–17d). Diastereomers with unsubstituted phenyl did not show any inhibitory activity against tyrosinase (17a and 17a0 ). In contrast, compounds with a substituted phenyl showed various effects on tyrosinase activity, for example, compound 17b with p-chlorophenyl (80.65% tyrosinase inhibition) moderately inhibited the tyrosinase but its diastereomer 17b0 16.5% tyrosinase inhibition) was inactive. In another case, 17c (58.54% tyrosinase inhibition) and 17c0 containing p-methyl phenyl (61.80% tyrosinase inhibition) exhibited good tyrosinase inhibition. Compound 17d consisting of a 2-pyridinyl (97.40% tyrosinase inhibition) fragment was found to be the most potent tyrosinase inhibitor of the above study.

Inhibitors with b-phenyl-a,b-unsaturated carbonyl functionality

Recently, it was reported that benzylidene hydantoin 18a, benzylidene pyrrolidine dione 18b, and benzylidene thiazolidine-2,4-dione 18c (Figure 8(a)) derivatives as potential tyrosinase inhibitors and using in vivo studies the compounds were proved to be an effective skin whitening agents77–80. They exhibited strong inhibitory activities than kojic acid and arbutin. The compound 18a was designed by mimicking the chemical structure of the L-tyrosine and L-DOPA, tyrosinase substrates. The SAR studies revealed that the amide NH at the 1-position of hydantoin 18a can form hydrogen bonds with amino acids at the active site of tyrosinase. Furthermore, the imido group of 18a mimics the carboxylic acid group of the substrates. Based on this background, Kim et al. recently synthesized and evaluated a series of 5-(hydroxyl- or alkoxy substituted benzylidene)thiohydantoin analogs possessing b-phenyl-a,b-unsaturated carbonyl scaffolds.81 Among them, three compounds, 19a–19c, exhibited high inhibitory activities than kojic acid or resveratrol (Figure 8(a)). Especially, 2,4-dihydroxybenzylidene-2-thiohydantoin 19c (IC50¼1.07 lM) was found to be the best inhibitor of this study. In addition, 19c inhibited the cellular tyrosinase activity in B16 cells without any significant cytotoxicity.

In a continuation, (E)-2-benzoyl-3-(substituted phenyl)acrylonitriles (BPA analogs) with a linear b-phenyl-a,b-unsaturated carbonyl scaffold were synthesized and evaluated as potential tyrosinase inhibitors82. Among them, three compounds 20a–20c effectively inhibited the mushroom tyrosinase activity (Figure 8(a)). Especially, compound 20c significantly suppressed melanin biosynthesis and inhibited intracellular tyrosinase activity in B16 cells without influencing cell viability. The SAR analysis revealed that all active compounds have a 4-hydroxy group on the phenyl ring, and substitution of Br at 3-position or 3 and 5-position were found to be associated with potent tyrosinase inhibitory activity.

Recently, the same research group continued to explore the SAR of 3-(substituted phenyl)acrylonitriles. Accordingly, a series of (E)-2-cyano-3-(substituted phenyl)acrylamide derivatives possessing a linear b-phenyl-a,b-unsaturated carbonyl scaffold showed inhibitory activity against mushroom tyrosinase83. Among the compounds, 21a and 21b exerted inhibitory activity against mushroom tyrosinase (Figure 8(a)). Especially, compound 21a showed excellent inhibitory activity. In B16 cells, 21a significantly suppressed tyrosinase activity in a dose-dependent manner without any influence of cytotoxic effect. From the structural point of view, a “linear” b-phenyl-a,b-unsaturated carbonyl scaffold plays an essential role in showing an anti-melanogenic effect through the direct inhibition of tyrosinase enzyme.

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It has been a long time known that cinnamaldehyde was able to inhibit L-DOPA oxidation by mushroom tyrosinase84. Recently, Cui et al. reported a series of a-substituted derivatives of cinnamaldehyde derivatives85. The SAR studies showed that a-bromocinnamaldehyde 22a, a-chlorocinnamaldehyde 22b, and a-methyl cinnamaldehyde 22c compounds reduced both mycophenolate and diphenolase activity on tyrosinase (Figure 8(a)). The IC50 values of 22a–22c were 0.075, 0.140, and 0.440 mM on mycophenolate and 0.049, 0.110, and 0.450 mM on diphenols, respectively. Furthermore, it was suggested that the a-substituted cinnamaldehyde derivative was more potent compared to cinnamaldehyde.

Recently, thio/barbiturates have drawn attention in the field of tyrosinase inhibitors86, due to their attractive structural unit of b-phenyl-a,b-unsaturated carbonyl scaffold responsible for tyrosinase inhibitory function. In the literature, few 5-benzylidene (thio) barbiturates with hydroxyl substituent at 4-position of the phenyl ring had excellent inhibitory activities, for example, 23a and 23b inhibited with IC50 values of 13.98 and 14.49 lM, respectively87 (Figure 8(b)). Inspired by this work, Chen et al., recently explored the SAR of thio/barbiturates emphasizing the position and the number of hydroxyl substituents for the influence of tyrosinase inhibitory activity. Accordingly, a series of hydroxy- or methoxy-substituted 5-benzylidene(thio)barbiturates were reported for their inhibitory effects on the diphenols activity of mushroom tyrosinase88. The results show that compounds (23c–23 g) had potent tyrosinase inhibitory activities compared to kojic acid (IC50=18.25 lM). In particular, a compound with 3,4-dihydroxy substituents 23e was identified as the best inhibitor with an IC50 value of 1.52 lM. The SAR studies revealed that barbiturates were more potent than thiobarbiturates and 3,4-dihydroxyl groups on the phenyl ring improved the potency. Furthermore, these inhibitors were found to reversible type.

For more info: david.deng@wecistanche.com  WhatApp:86 13632399501