Inhibitors of Melanogenesis: An Updated Review - Journal of

Perspective Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX

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Inhibitors of Melanogenesis: An Updated Review Thanigaimalai Pillaiyar,*,† Vigneshwaran Namasivayam,† Manoj Manickam,‡ and Sang-Hun Jung‡ †

PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany ‡ College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon 34134, Korea S Supporting Information *

ABSTRACT: Melanins are pigment molecules that determine the skin, eye, and hair color of the human subject to its amount, quality, and distribution. Melanocytes synthesize melanin and provide epidermal protection from various stimuli, such as harmful ultraviolet radiation, through the complex process called melanogenesis. However, serious dermatological problems occur when there is excessive production of melanin in different parts of the human body. These include freckles, melasma, senile lentigo, pigmented acne scars, and cancer. Therefore, controlling the production of melanin is an important approach for the treatment of pigmentation related disorderes. In this Perspective, we focus on the inhibitors of melanogenesis that directly/indirectly target a key enzyme tyrosinase as well as its associated signaling pathways.

1. INTRODUCTION

2. SYNTHESIS OF MELANIN AND ITS REGULATION Melanocytes produce two kinds of melanin pigments called eumelanin (brown-black or dark insoluble polymer) and pheomelanin (red-yellow soluble polymer). Complex enzymatic and biochemical-catalyzed reactions are involved in melanin synthesis. Enzymatically, tyrosinase (TYR), tyrosinaserelated protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2, also called dopachrome tautomerase) are three key regulators of the melanogenesis. Particularly, TYR is exclusively important for the melanin synthesis. Synthesis of melanin begins by the oxidation of L-tyrosine and/or L-dihydroxyphenylalanine (L-DOPA) to dopaquinone (DQ), which serves as a substrate for the synthesis of eumelanin and pheomelanin (Figure 1). As an important enzyme, TYR performs ratelimiting activity in the oxidation reaction because all other reaction sequences can proceed in the presence of physiological pH condition. In pheomelanin synthesis pathway, the formation of DQ reacts with cysteine to obtain 3- or 5cysteinyl DOPAs, which are further oxidized and polymerized to form red-yellow soluble pheomelanin pigments. In eumelanin synthesis pathway, DQ cyclizes to form DOPAchrome, which then decarboxylates spontaneously to generate 5,6-dihydroxyindole (DHI) and then continues to form DHI-2-carboxylic acid (DHICA), as a minor product in the presence of TRP-2. Finally, these DHI and DHICA rapidly oxidize and polymerize to produce eumelanin. In this process, TRP-1 was reported to catalyze the oxidation of DHICA.22 On the other hand, the recent X-ray crystallographic structure of human TRP-1 showed that it does not act as a DHICA

It is estimated that approximately 15% of the populaton in the world invests for skin whitening.1 Worldwide the market for skin whitening agents is expected to reach nearly U.S. $23 billion by 2020.1 Inhibition or reduction of melanin synthesis by overactive melanocytes is the common mechanism for most of the whitening agents available in the market. Melanin protects human skin against a harmful ultraviolet radiation (UVR) and stress from the different source of environmental pollutants,2 toxic drugs, and chemicals. Melanins, a cluster of natural pigments, which originate from the epidermis, where melanocytes synthesize melanin through melanogenesis.3,4 Melanocytes are derived from melanoblasts,5,6 and each6 is enclosed by keratinocytes approximately in the ratio of 1:∼36 in the epidermis.7 During melanogenesis, melanin is deposited inside the melanocytes in melanosomes8,9 and transported to keratinocytes via dendrites.10,11Although melanin is crucial for protecting the skin, the abnormal production of melanin that leads to acute dermatological problems include melasma,12−15 postinflammatory melanoderma,16 solar lentigo, freckles, pigmented acne scars, and age spots. Continuous UV irradiation can result in an increased risk of skin damage and cancer. Moreover, studies reported that many melanogenesis disorders have been linked to the neurodegenerative diseases including Parkinson’s, Alzheimer’s, and Huntington’s diseases.17−21 In this Perspective, we emphasize the recently identified tyrosinase inhibitors that directly/indirectly target the catalytic activity of tyrosinase and melanogenesis signaling pathway inhibitors from all sources. © XXXX American Chemical Society

Received: June 30, 2017 Published: May 15, 2018 A

DOI: 10.1021/acs.jmedchem.7b00967 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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produced only by melanocyte cells. The production and subsequent glycosylation of TYR are further matured and trafficked to melanosomes, wherein the melanin synthesis occurred.31 The protein level of TYR is also regulated by two degradation systems, the proteasomal and endosomal/lysosomal systems, for proteolysis of misfolded or unfolded proteins during maturation processing.32−34 Since the first X-ray structure of tyrosinase was solved in 2006, further new crystal structures have been reported in order to elucidate the substrate binding, important amino acids in the active site, and the interaction between inhibitors and enzymes. The list of crystal structures from different species was reported in Table S1 in Supporting Information. In general, the structure of tyrosinase can be classified into three domains, namely, the central, the N-terminal, and the C-terminal domains (Figure 2).36 Among tyrosinases from different species, the central domain is the most conserved domain, which comprises six histidine residues and two copper ions (CuA and CuB). Structural investigations of the crystal structures of tyrosinases from different species include Bacillus megaterium, Streptomyces castaneoglobisporus from bacteria, Aspegilus oryzae, Agaricus bisporus from mushroom, and Juglans regia from the plant show the involvement of residues in the catalytic mechanism. In the active site, a thioether bond is formed between the cysteine and histidine residues, and the histidine coordinates one of the copper ions for the catalytic mechanism. On the oher hand, the thioether bond is formed to stabilize the histidine residue in the binding site. In N-terminal domain, a transit peptide regulates the final positioning of the enzyme and undergoes a proteolytic cleavage.37,38 In human and mushroom tyrosinases, this peptide is considered to be involved in melanosome transfer, while in plants, it regulates the chloroplast, whereas it is absent in the fungal tyrosinase. A latent precursor of tyrosinase called protyrosinase consists of the central and C-terminal domains that block the entrance to the active site through a “placeholder” residue. This residue enters the active site similar to the substrate or an inhibitor. 3.2. Catalytic Cycle of Tyrosinase. The active site of tyrosinase is highly conserved with two copper ions (CuA and

Figure 1. Melanogenesis or melanin synthesis27,28(Raper−Mason pathway).29,30

oxidase.23,24 This builds the question about the exact function of TRP-1 in melanin synthesis. Furthermore, the roles of TRP124 and TRP-2 were identified to stabilize and increase the activity of TYR. Melanogenesis can be regulated at three different levels: gene, cellular, and subcellular levels.25,26 During embryo development, melanocytes migrate to the epidermis and hair follicles, and these migration patterns are the first level in the melanogenesis regulation. Next, the melanogenesis is regulated by melanosomes based on its size, number, and densities. Finally, the gene expression determined by melanogenic enzymes (TYR, TRP-1, and TRP-2) controls melanogenesis at the subcellular level.

3. STRUCTURE AND FUNCTION OF TYROSINASE 3.1. Overall Structure and Function of Tyrosinase. Tyrosinase (EC 1.14.18.1), a dicopper oxidase, is a type 3 copper containing metalloenzyme that is largely distributed in bacteria, fungi, insects, plants, and animals, including humans to produce melanin pigments. It is a glycoprotein and exclusively

Figure 2. The crystallographic structure of tyrosinase from Agaricus bisporus in deoxy form (PDB code 2Y9X)35 is shown and represented in cartoon model with the N- and C-terminal domains highlighted in pink and blue, respectively. The catalytic site consisting of two copper ions (orange color) surrounded by six conserved histidine moieties and the placeholder residue (red color) is shown. B



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Figure 3. Catalytic cycles of tyrosinase: monophenolase and diphenolase catalytic cycles. Three types of tyrosinase are Eoxy, Emet, and Edeoxy, respectively. EoxyT, EoxyD, and EmetD are Eoxy-tyrosine, Eoxy-dopamine, and Emet-dopamine complexes, respectively. Sphere shape represents copper ions with different oxidation states.

CuB) complexed with six histidine residues.39 The catalytic mechanism of tyrosinase involves three different states, namely, oxy-, met-, and deoxy-tyrosinases, with differences in the oxidation state of copper ions (Figure 3). In the monophenolase activity (oxidation of L-tyrosine to L-DOPA), deoxytyrosinase (Edeoxy) binds to oxygen and form oxy-tyrosinase (Eoxy) and then to L-tyrosine, which is further catalyzed to LDOPA. In this process, the enzyme is finally recycled as Edeoxy for further dioxygen binding. In the diphenolase activity (oxidation of L-DOPA to DQ), the regeneration of oxytyrosinase drops only one oxygen atom to form met-tyrosinase (Emet), where the two close copper centers (dCU‑CU ≈ 2.9−4.9 Å) are bridged by an aqua (hydroxo) ligand. Tyrosinase occurs mainly as the Emet form, which cannot oxidize phenols, e.g., tyrosine, and needs to be reduced to Edeoxy by an L-DOPA before the tyrosine oxidation initiated. This explains the higher affinity of Emet to L-DOPA as compared to other forms, and thus Emet is considered as an important target for the discovery of tyrosinase inhibitors.40 After catalyzing L-DOPA, Emet loses the oxygen atom to regenerate Edeoxy form. Catechol oxidation is based on the phenolic oxidative mechanism that results in a reduction of copper to Cu° and tyrosinase deactivation.41−44

studies confirmed that tyrosinase is not only involved in the synthesis of melanin of peripheral tissues but also involved in the substantia nigra (SN) of mice and humans contributing an important role in the brain neuromelanin development. Excessive formation of dopaquinone results in neuronal damage and cell death. Evidence has linked tyrosinase to neurodegenerative disorders including Parkinson’s and Huntington’s diseases.17−21 Moreover, tyrosinase is an evolving target in the food industry, as tyrosinase inhibitors are being utilized to avoid enzymatic browning of fruits and vegetables. In plants, sponges, and many other invertebrates, tyrosinases are essential for wound healing and primary immune responses. They also play a role in sclerotization in arthropods and in bacteria, and tyrosinases protect DNA from UV damage.43,45−47 On the basis of the ability to oxidize phenolic molecules including tyrosine in peptides and proteins, the applications of tyrosinase have been extended to biotechnologies (bioremediation, dye production, and biopolymer cross-linking).46 Because TYR catalyzes rate-limiting steps of overall melanogenesis, it has been recognized as a therapeutic target for controlling abnormal melanin synthesis. Many approaches were reported to control melanogenesis targeting the key tyrosinase, and these include the modulation of TYR expression, maturation, degradation, and direct inhibition of a catalytic activity. The most common commercially available cosmetics or skin whitening agents are inhibitors of tyrosinase enzyme. They

4. IMPORTANCE OF TARGETING TYROSINASE Tyrosinase is important in oxidative homeostasis process and protects the human skin from ionizing radiations. Recent C



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Figure 4. Chemical structures of resveratrol derivatives and analogs.

target tyrosinase and possibly inhibit the melanogenesis without any side effects. Although a huge number of tyrosinase inhibitors were identified so far, relatively few of them have been reached to clinical applications as skin-whitening agents due to safety concerns and weak whitening effects. The skin whitening agents include azelaic acid,48,49 magnesium Lascorbyl-2-phosphate, phenols,50 hydroxyanisole, corticosteroids,51 N-acetyl-4-S-cysteaminylphenol, resinoids52,53 arbutin (hydroquinone-β-D-glucopyranoside), salicylhydroxamic acid, kojic acid,54−56 hydroquinone (HQ),57−60 monobenzyl hydroquinone, tretinoin, and mercury salts and are commonly used in the cosmetics industry. They are recommended worldwide, although associated with certain drawbacks and side effects. HQ is toxic to mammalian cells and associated with a series of side effects that include contact dermatitis, irritation, burning, hypochromia, ochronosis, and chestnut spots on the nails.61−64 The EU Cosmetic Regulation bans the use of corticosteroids, HQ, monobenzyl hydroquinone, tretinoin, and mercury salts as whitening agents. The usage of kojic acid has

been restricted due to its carcinogenicity and instability problems. L-Ascorbic acid degrades easily,65 and the bioavailability of ellagic acid is poor.66 For the tranexamic acid, the exact molecular target is yet to be identified.67 In in vitro assays, a large number of inhibitors targeting tyrosinase were proved to be effective, but only a few of them were possibly induce the same effects in clinical trials. Thus, there has been increasing need for novel tyrosinase inhibitors with drug-like properties.

5. TYROSINASE INHIBITORS In most cases, mushroom tyrosinase (mTYR) from Agaricus bisporus has been applied as a model of hTYR for screening TYR inhibitors, since mTYR is commercially available in a purified form.68,69 For screening, kojic acid, HQ, or arbutin has been used as references. L-Tyrosine or L-DOPA has been utilized to identify the monophenolase or diphenolase activity. In general, inhibitors of tyrosinase can be classified into four categories: competitive, noncompetitive, uncompetitive, and mixed type.70−74 A competitive inhibitor binds to the free D



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potent inhibitors of mTYR. Among them, (E)-4-((4-hydroxyphenylimino)methyl)benzene-1,2-diol (11) was identified as the most potent (IC50 = 17.22 μM) with a noncompetitive inhibition of mTYR, and it was a more potent inhibitor than the kojic acid (IC50 = 51.11 μM).81 In another study, a series of (E)-2-((substituted phenyl)diazenyl)phenyl-4-methylbenzenesulfonate and (E)-2-((substituted phenyl)diazenyl)phenol derivatives (12−14, Figure 4) were found to strongly inhibit the tyrosinase in a dose-dependent manner. In particular, (E)-2((2,4-dihydroxyphenyl)diazenyl)phenyl-4-methylbenzenesulfonate (13) was identified as a highly potent tyrosinase inhibitor (IC50 = 17.85 μM) with a competitive inhibition mechanism. Moreover, in murine B16F10 melanoma cells 13 inhibited cellular tyrosinase activity and melanin formation. Aza-resveratrols show potent inhibitory activity against mTYR.82 For example, compounds 15 and 16 resulted in high inhibitory activity of 56.25% (IC50 = 50.20 μM and 72.75% (IC50 = 36.28 μM) at 50 μM, respectively, in comparison to the kojic acid (IC50 = 51.11 μM).82 The results show that aza-resveratrol with high log P value could be better in comparison to resveratrol in the identification of effective skin whitening agents. 5.2. Peptides or Peptidomimetics. In recent times, peptides emerge as effective cosmetic agents as several peptides including dipeptides,83 cyclic peptides,84 oligopeptides85 and kojic acid peptides86 have been investigated as potent tyrosinase inhibitors. In particular, oligopeptides were confirmed as promising inhibitors for tyrosinase. For example, an octapeptide P3 (Arg-Ala-Asp-Ser-Arg-Ala-Asp-Cys) and a decapeptide P4 (Tyr-Arg-Ser-Arg-Lys-Tyr-Ser-Ser-Trp-Tyr) were found to be potent against mTYR and human tyrosinase (hTYR) without inducing melanocyte cytotoxicity.87 The promising clinical output of a decapeptide P4 (also called decapeptide-12) led to commercialization as the key active component in a skin-whitening product.88 This makes scientists search for new and potent tyrosinase inhibitors without cytotoxicity to the melanocytes. Kojic acid is an important antimelanogenic agent, although the usage has been restricted due to its carcinogenicity and stability issue on storage.89,90 These limitations led scientists to synthesize and evaluate active kojic acid peptides. Li et al. showed a series of hydroxypyridinone-L-phenylalanine conjugates (17 and 18, Figure 5) with moderate potencies compared to kojic acid (IC50 = 26.8 and 20 μM for monophenolase and diphenolase activity) in inhibiting tyrosinase.91 Structure−activity relationship studies (SARs)

enzyme and prevents substrate binding, while uncompetitive inhibitor binds to enzyme−substrate complex. Mixed type inhibitor binds to both free enzyme and enzyme−substrate complex, while noncompetitive inhibitor binds with a free enzyme and enzyme−substrate complex with the same equilibrium constant. The strength of inhibition was expressed in IC50 value. The Ki value specifies the binding affinity of the ligand toward the enzyme: if the value of Ki is lower, it means that binding affinity is higher; if the value of Ki is higher, it means that binding affinity is lower. For noncompetitive inhibitors, Ki value has the same numerical value as IC50 of the inhibitors, whereas for competitive inhibitors, the Ki is reduced to one-half that of the numerical values of IC50. Melanoma B16 cells were often employed for the in vitro model, which shares similar melanogenesis mechanism of normal human melanocytes. 5.1. Resveratrol Derivatives and Analogs. Resveratrol (trans-3,4′,5-trihydroxystilbene, 1, Figure 4) is a natural stilbenoid available in skins of grape and particularly in wine.75 It inhibited the tyrosinase activity through the suicide substrate type (Kcat) inhibition.76 In B16 cells, 1 inhibited the α-MSH stimulated melanin synthesis via reducing TYR, TRP-1, TRP-2, and MITF expressions,77 without causing any cytotoxicity effects up to 200 μM.76 In an in vivo study using the resveratrol treated with the UVB-irradiated dorsal skin of guinea pigs visually decreased the hyperpigmentation. However, when the drug was administered orally, it was having a low in vivo bioavailability. Rigon et al. prepared resveratrol-loaded solid lipid nanoparticles (SLNs, diameter of 1000 μM). SARs suggested meta-position of phenyl ring and carboxylic acid on the benzene ring (3-PBA)

Figure 8. Indole-derived tyrosinase inhibitors. G



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Figure 9. Docked pose of 41 in the binding pocket of tyrosinase obtained from Agaricus bisporus in deoxy form (PDB code 2Y9X). (A) Compound 41 (orange color) shown in stick model in the binding pocket of tyrosinase represented in the surface model. (B) The important amino acids in the binding pocket are represented in stick model (white) and the copper ions in spheres (marine blue).

Figure 10. Thiourea-derived tyrosinase inhibitors.

H



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Figure 11. Thiosemicarbazone and hydroxyl cinnamic acid derivatives as tyrosinase inhibitors.

fluorobenzyl)piperidine moiety of compound 41 was extended outside from the cavity forming interactions with His263 (π−π interaction), His244 (cation−π interaction), and Val283 (van der Waals interaction). Additionally, the indole core of compound 41 occupied the location near the cavity entrance formed by catalytic copper ions. 5.5. Thiourea and Thiosemicarbazone Derivatives. Phenylthiourea (PTU, 42, Figure 10) is an important tyrosinase inhibitor, and its inhibition mode is based on the chelation of sulfur atom with active site copper ions.103−105 Jung and coworkers explored a wide variety of PTU derivatives as tyrosinase inhibitors; see for some examples 43−45 in Figure 10.106,107 The summary of SARs provided crucial insights into structural requirements for the tyrosinase inhibitory activity: (i) sulfur atom was necessary for the chelating ability; (ii) direct linking of π-planar to thiourea unit was necessary; (iii) hydrophobic substituent at para- or meta-position (43 and 44) on the aryl ring was tolerated, while ortho substitution and the replacement of 3-aminohydrogens (45) by any substituent abolished the activity. This suggested that C2-substituted phenyl or 3-amino substitution might prevent the formation of thiourea in complex with copper ions at the binding site of tyrosinase. Repurposing of existing drugs is a smart approach in drug discovery program since it has numerous advantages that include time-savings, availability of drugs, reduced cost, and safety/tolerability. Choi et al. screened a drug library contained chemical scaffolds similar to PTU and identified ethionamide (46, Figure 10) and its analogs (47-49, Figure 10) including prothionamide (47) as tyrosinase inhibitors.108−110 Compounds 46 and 47 are antituberculosis drugs, and 47 was tested for the use in the treatment of leprosy. Instead, isoniazid (49), a first-line antituberculosis drug, displayed a very weak tyrosinase inhibitory activity. This showed that thiocarbamide unit was crucial for interacting with the copper ions at the tyrosinase active site. On the other hand, pyridine-2carbothioamide (48) and thiobenzamide (50) significantly decreased the melanin production in B16 cells to the values of 44% and 37%, respectively. In another study, antithyroid drugs such as methimazole 51,111 carbimazole 52,111 thiouracil 53,111

methylthiouracil 54,111 and propylthiouracil 55 (Figure 10)112 were reported as mTYR inhibitors.113,114,111 From the SARs, it was concluded that thiourea was a crucial moiety for the tyrosinase inhibitory activity and they showed a noncompetitive mode of action. This was supported by another study where Naryl-N′-substituted phenylthiourea analogs were estimated for their diphenolase inhibitory activity of mTYR (56−60).115 SARs revealed the presence of 2-(1,3,4-thiadiazol-2-yl)thioacetic acid was more beneficial for the increase in the tyrosinase inhibitory activity. In general, thiosemicarbazones and PTU share a similar mechanism of inhibition as both have thiourea moiety, which is responsible for chelating copper ions at the tyrosinase active site and shows enzymatic inhibitory activity. Recently, a huge number of thiosemicarbazones have been reported as potent tyrosinase inhibitors.116−125 You et al. reported the inhibitory activity for 4- or 3-aminoacetophenones derived thiosemicarbazones against mTYR.126 It was found that acylamino compounds 61−63 (Figure 11) displayed potent tyrosinase inhibition in comparison to kojic acid (IC50 = 28.5 μM).126 Specifically, compound 63 was the most potent compound (IC50 = 0.291 μM). In another study, a set of new analogs from 4-alkoxy- and 4-acyloxy-phenylethylenethiosemicarbazone were reported.127 The results indicate that both 4-alkoxy- and 4acyloxy-phenylethylenethiosemicarbazones significantly inhibited the tyrosinase activity with an IC50 value lower than 1.0 μM (see for examples 64−66, Figure 11). The SARs indicated that the thiosemicarbazone moiety is essential for the tyrosinase inhibitory activity. 5.6. Hydroxycinnamic Acid Derivatives. Caffeic acid (CA, 67, Figure 11) is a common phenolic acid found in vegetables, fruits, grains, and seeds. It has many medical properties that include antioxidant, antitumor, anti-inflammatory, antimicrobial, and antidiabetic activity. Antioxidants play a significant role in reducing aging effects, and thus the antioxidant property in designing whitening agent is highly considered. In a study, Kwak et al.128 identified 67 as a potent antioxidant with tyrosinase inhibitions. In another study, Kwak et al.129 evaluated caffeoyl-amino acidyl-hydroxamic acids 68 and 69 as cosmetic agents by evaluating their anti-tyrosinase and antioxidant activities. The results indicated that the I



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Figure 12. Chemical structure of chalcones and flavanone tyrosinase inhibitors.

Bronaugh-type diffusion cells. However, the results found no traces of 67 even after 24 h exposure of the gel to the skin cells. Chlorogenic acid (70, Figure 11) is a natural hydroxycinnamic acid derivative and largely presented in coffee, pears, and apples. Several publications reported this compound with an anti-inflammatory, antidiabetic, antiviral, antioxidant activities. Very few studies reported the chlorogenic acid with the antityrosinase property.131 In B16 cells, compound 70 at 500 μM suppressed the melanin content by inhibiting intracellular tyrosinase activity. 5.7. Chalcones and Flavanone Analogs. Chalcones are the most common natural products and largely distributed in vegetables, fruits, spices, tea, soya-based food products. They show a wide variety of biological activity including potent

caffeoyl-prolyl-hydroxamic acid 68 (Figure 11) and caffeoylphenylalanyl-hydroxamic acid 69 (Figure 11) exhibited good antioxidant and anti-tyrosinase activities. In particular, 69 showed a good tyrosinase inhibition activity with an IC50 value of 4.9 μM. The skin permeation of the cosmetic ingredients is an important criterion to achieve a desired therapeutic benefit. The stratum corneum of the skin behaves as a barrier, which often limited the entry of some cosmetic ingredients or foreign materials. It has been reported that physicochemical parameters include the molecular weight and lipophilic or hydrophilic balance alter the permeability of compounds in cosmetic formulation through the skin.130 Recently Zilius et al. studied in vitro permeation studies of 67 along with other agents utilizing J



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Figure 13. Human tyrosinase inhibitors.

μM. SARs revealed ortho-methoxy with para-nitro substituents (ring B) in 77 or an electron donating para-dimethylamino ring (ring B) in 78 was responsible for potent tyrosinase inhibition. Both 77 and 78 inhibited the cellular melanin formation in αMSH induced B16 cells. Four new compounds isolated from Camylotropis hirtella (79−82, Figure 12) showed potent inhibitory activities against tyrosinase.135 Among them, neorauflavane (81) was identified as the most potent inhibitor (IC50 = 30 nM for monophenolase and IC50 = 500 nM for diphenolase activity of tyrosinase). The compound 81 was 400-fold potent in comparison to kojic acid (13.2 μM),135 against the monophenolase activity of tyrosinase. In α-MHS induced B16 cells, 81 efficiently decreased the melanin content, without influencing cell viability. Structurally, the reduction of geranyl side chain was important for improving the tyrosinase inhibitory activity. As shown in Figure 12, a flavone named morusone (83, IC50 = 290 μM) and 16 known compounds isolated from twigs of

antityrosinase activities. The natural chalcones 71−74 (Figure 12) isolated from Morus australis were found as potent inhibitors (71−74).132 In particular, compound 71 exhibited 700-fold potent inhibition compared to arbutin. SARs indicated that resorcinol construction at both ring A and ring B was important for inhibiting the tyrosinase. Azachalcones were recently investigated as tyrosinase inhibitors.133 Compounds 75 and 76 (Figure 12) were potent tyrosinase inhibitors in comparison to kojic acid (IC50 = 27.30 μM),107 with a competitive inhibitory mechanism (75, Ki = 2.62 μM; 76, Ki = 8.10 μM). SARs revealed that the presence of pyridine ring was important for tyrosinase inhibitory activity. In another study, chalcones with oxime functionality were reported as potent tyrosinase inhibitors.134 For example, compounds 77 (IC50 = 4.77 μM) and 78 (IC50 = 7.89 μM) were more potent tyrosinase inhibitors (Figure 12) than kojic acid (IC50 = 22.25 μM).134 Kinetic studies indicated a competitive type of inhibition with Ki values of 5.25 and 8.33 K



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inhibitor.146 It shows inhibition against TYR and TRP-1 in vitro as well as in B16 melanoma cells.147,148 More specifically, 93 exhibits much higher specificity for TRP-1 than TYR.149,150 Recently, the inhibition mechanism was studied by Lee et al.,150 and they found that 93 inhibited the tyrosinase and melanin synthesis more effectively in intact cells than in cell lysates. Moreover, in α-MSH induced B16 cells, 93 completely inhibited the cellular tyrosinase at 100 μM. Western blot and reverse transcription-PCR experiments identified 93 has reduced protein levels of tyrosinase, whereas there is no reduction in mRNA levels in B16 cells. An elucidation of the mode of inhibition revealed 93 inhibited melanogenesis by enhancing proteolytic degradation of tyrosinase as well as competitive binding to tyrosinase. As a potent inhibitor of hTYR 93 (IC50 = 21 μM) exhibited complete inhibition of tyrosinase at concentrations above 100 μM. The inhibitory potency of 93 was about 20-fold potent in comparison to kojic acid (IC50 = 500 μM). Other common whitening agents such as arbutin (IC50 = 6500 μM) and HQ (IC50 = 4400 μM) were poor inhibitors of hTYR. In melanoDerm skin model, 93 displayed a highly potent inhibition with an IC50 value of 13.5 μM. The potency of the compounds has been assigned in the following order: 93 (IC50 = 13.5 μM) > HQ (IC50 = 40 μM) > kojic acid (IC50 = 400 μM). From the clinical studies, the efficacy and safety of 93 have been proven in treating the patients with melasma. A formula containing 4-butylresorcinol (93), 4-hexylresorcinol (94, Figure 13), and 4-phenylethylresorcinol (95, Figure 13) treated patients with age spots. Among them, 93 showed promising result in tyrosinase inhibition and reduced the appearance of age spots within 8 weeks, while 94 and 95 showed significant effects only after 12 weeks. The clinical output of 93 on hyperpigmentation suggested that it could be a promising inhibitor in the treatment of pigmentation disorders. In fact, a topical cream containing 0.1−0.3% of 93 has been utilized in melasma treatment with rapid efficacy, safety, and tolerability.151,152 Linderanolide B (96) and subamolide A (97) are two natural products proved to inhibit mTYR activity (Figure 13).153,154 At a dosage level of 1 μM concentration, both 96 and 97 reduce 50% of human tyrosinase activities and effectively inhibited (40% reduction) the melanin formation in human epidermal melanocytes (HEM), neonatal, moderately pigmented donor (HEMn-MP). In zebrafish, these two compounds (at 10 μM) showed remarkable reduction about 30% and 25%, respectively, in pigmentation level and showed no toxicity. In 1930s, thujaplicins (isopropyl cycloheptatrienolones) and related chemical substances were isolated from Thuja plicata (Western redcedar tree).155 The three regioisomers αthujaplicin (98), β-thujaplicin (hinokitiol, 99), and γ-thujaplicin (100) exhibited potent antifungal, antibacterial,156 and antioxidant properties.157 Recently, thujaplicins (98−100, α, β, and γ isomers, Figure 13) were identified as potent hTYR and mTYR inhibitors.144 Specifically, β- and γ-thujaplicins (99 and 100) efficiently inhibited the hTYR activity with IC50 values of 8.98 and 1.15 μM, respectively, in comparison to kojic acid (IC50 = 571.17 μM).118 The rank order potency of thujaplicins was in the following order: γ > β > α-thujaplicin with the same order for mTYR inhibitory activity with IC50 values obtained for γ (IC50 = 0.07 μM) > β (IC50 = 0.09 μM) > α (IC50 = 9.53 μM). However, a huge difference in the inhibitory activities was observed for hTYR and mTYR. For example, the inhibitory activity of kojic acid was estimated to

Morus alba L. were examined for their inhibitory activities against tyrosinase.112 The results showed that compounds steppogenin (84, IC50 = 0.98 μM), 2,4,2′,4′-tetrahydroxychalcone (85, IC50 = 0.07 μM), morachalcone A (86, IC50 = 0.08 μM), and moracin M (87, IC50 = 8.00 μM) have significant tyrosinase inhibitory activities than the kojic acid (IC50 = 58.30 μM),97 except the new compound morusone that displayed a weak anti-tyrosinase activity. The prenylated compounds 88−91 (Figure 12) obtained from the Dalea pazensis Rusby roots were tested for their in vitro inhibition of mTYR and in relation to their effect on melanogenesis in B16 cells.136,137 The results showed that compound 91 (IC50 = 2.32 μM)138 was effective and 90 (IC50 = 49.89 μM) moderately inhibited the tyrosinase, while compounds 88 and 89 were inactive in comparison to kojic acid (IC50 = 4.93 μM).138 The SARs suggest the presence of 4substituted phloroglucinol in ring A and resorcinol moiety in ring B were key factors for the potent inhibitory activity.139,140 Next, compounds were evaluated on melanoma B16 cells. All compounds were shown to inhibit the content of melanin with cytotoxicity in a concentration-dependent manner. In that study, kojic acid was proved to be cytotoxic at 5000 μM. The inhibitory strength of compounds was arranged as followed 88 (0.75 μM) > 91 (1.0 μM) > 89 (5.0 μM) = 90 (5.0 μM) > kojic acid (2000 μM). In order to investigate the possible mechanism that is involved in reducing melanin content, compounds were examined in murine B16 melanoma cells for their intracellular inhibitory activity of tyrosinase. The result was very clear that compound 91 inhibits the intracellular tyrosinase activity because of the presence of two prenyl groups and hydroxyl groups on both ring A and ring B. Arung et al.141 suggested that both prenyl and OH groups and the type of substitution pattern in flavones are important for decreasing the melanin production in B16 cells, and their potency was related to the number of isoprenoid substitutions. Compounds 88−90 were inactive although they reduced extracellular melanin but in an independent tyrosinase manner and possibly involved in the different pathway for decreasing melanin content in B16 cells.

6. INHIBITORS OF HUMAN TYROSINASE As opposed to a tetrameric mTYR, the monomeric human tyrosinase (hTYR) is a glycosylated membrane-bound protein with 13% carbohydrate content.142,143 Glycosylation is required for tyrosinase activity, and the amino acid sequences between hTYR and mTYR show only 23% identity. In comparison to the mTYR, the hTYR was found to be 6-fold higher for LDOPA oxidation activity, and the Km value of human and mushroom TYR for L-DOPA is 0.31 mM and 1.88 mM, respectively.144 The inhibitory activity of the kojic acid showed 10-fold higher activity for mTYR (IC50 = 53.70 μM) than hTYR (IC50 = 571.17 μM).144 Indeed the binding study also supported that the kojic acid has a high binding affinity for mTYR (Ki = 4.3 μM) than the hTYR (Ki = 350 μM).145 In addition, other inhibitors such as phenylthiourea, L-mimosine, cinnamic acid, benzoic acid, and aesculetin showed significant differences in the inhibitory activities between mTYR and hTYR (see Table S2). Importantly, aesculetin is another potent mTYR inhibitor, but it showed no detectable inhibitory activity against hTYR. These comparisons show that hTYR is unique and the identification of the human tyrosinase inhibitors is of high importance. Since 1995, the resorcinol derivative 4-butylresorcinol (93, Figure 13) has been known as a potent melanogenesis L



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120 μM for 106. Authors suggest that the scarce ability of compounds 105−107 to cross MNT-1 cells is possibly due to the zwitterionic nature of the HOPNO group. However, 105 is the potent inhibitor of the human tyrosinase identified to date. It is noteworthy to mention that the reference inhibitor kojic acid was almost inactive on both lysate and whole cells. p-Coumaric acid (PCA, 108, Figure 13), a common secondary metabolite obtained from plants, is widely distributed in vegetables, fruits, and mushrooms. Compound 108 (PCA) and its analogs are associated with many biological properties including their depigmenting potential, antioxidant, anticollagenase, antimicrobial, and anti-inflammatory activities.162 The chemical structure of 108 has been of interest due to its close similarity to tyrosine, a natural substrate of tyrosinase. Indeed 108 has been reported as a potent mTYR inhibitor by competing for tyrosine at the active sites of tyrosinase.163,164 An et al. reported 108 as a potent inhibitor of melanogenesis in murine melanoma cells stimulated with αMSH.165 Compound 108 inhibited cellular melanin formation much stronger than the compounds that are structurally similar such as 3-(4-hydroxyphenyl)propionic acid, cinnamic acid, and caffeic acid (67). These results indicate that 108 is the suitable structure for the inhibition of cellular melanogenesis.165 In an advance study, Song et al. evaluated the melanin inhibitory activities of 108 and its methyl ester (MPA, 109, Figure 13) in HEM.166 The results showed that 108 was a potent tyrosinase inhibitor with an IC50 value of 3 μM, while 109 (MPA) was almost inactive (IC50 = 30 μM). Additionally, in order to prove the cosmeceutical potential of these two compounds 108 and 109, semisolid creams were prepared. For skin permeation studies of compounds, the porcine skin was used as a model and evaluated the transdermal bioavailability. Compound 108 crossed biological membranes; however, 109 could not achieve the same result. Topical application of 108 as a cream attenuated the UVB-induced erythema formation and pigmentation in mice models which is more effective in comparison to MPC cream. An et al. explored inhibitory activities of 108 on different TYRs from mushroom, murine, and human and compared them with the reference compounds arbutin and kojic acid.167 Compound 108 showed a weak inhibition against mTYR but stronger inhibition against human or murine TYR in comparison to kojic acid and arbutin. Enzyme kinetics analysis indicated that 108 showed a mixed (for tyrosine) or competitive (for DOPA) type of inhibitory action against the hTYR. The physiological relevance of hTYR inhibition by 108 was studied in HEM exposed to UVB. The results show that 108 inhibited melanogenesis and loss of viability in human melanocytes-induced by UVB irradiation if used before irradiation (pretreatment), whereas during the post-treatment it had inhibited only melanin synthesis. The results also indicated that 108 could be a potential candidate for the development of hypopigmenting agent as it inhibited both melanin synthesis and UVB-induced cytotoxicity. Seo et al. conducted an in vivo study for the use of 108 as a whitening agent in the human using as cream (CR90416F10LHJ, Snow White cream; Cosmax, Seoul, Korea) which contains 1.5% of 108.168 The results showed that 108 decrease UV-induced erythema formation and subsequent pigmentation in the human skin.

have ∼11-fold weaker inhibition for hTYR (IC50 = 571.17 μM) in comparison to mTYR (IC50 = 53.70 μM). Thujaplicins had approximately >105-fold (α -isomer), 100-fold (β-isomer), and 17-fold (γ-iosmer) weaker inhibition against hTYR when compared to mTYR. Aurones (Z-benzylidenebenzofuran-3(2H)-ones) are naturally occurring and structurally isomeric to flavones.158 In aurone, a chalcone-like group is closed into a five-membered ring and there are two isomers, E- and Z-configurations. Okombi et al. investigated naturally occurring aurones and analogs as hTYR inhibitors. Aurones with hydroxyl groups on ring A and with different substituents on ring B were examined for their diphenolase inhibitory activity of hTYR. SARs revealed the overt aurones were weak inhibitors of hTYR. Instead, the derivatives of aurones with two or three hydroxyl groups possibly at 4,6 and 4′-positions inhibit the human melanocytetyrosinase. A few potent inhibitors (101−103) were shown in Figure 13. Among them, the most potent aurone was a naturally occurring 4,6,4′-trihydroxyaurone (103), which induces 75% inhibition at 0.1 mM concentration, and it was more potent than kojic acid. Furthermore, the investigation of active compound 103 on melanocytes, obtained from three different individuals with white, dark, and black skin, exhibited no significant change in inhibitory activity. The efficacy and safety of 103 evaluated in animal model showed that it did not possess any toxicity after oral administration to rats at 5 g/kg dose. In rabbits, topical and eye application of 103 did not indicate any significant irritation. The results indicate that this potent hTYR inhibitor is possibly a suitable candidate molecule in the development of new skin whitening agent. 2-Hydroxypyridine-N-oxide (HOPNO, 104, Figure 13), a catechol mimic, was previously reported as a non-natural transition state mTYR inhibitor having a competitive inhibition constant (Kic) value of 1.8 μM.159 Embedding this HOPNO moiety into the aurone backbone, Haudecoeur and co-workers described a new series of aurones as potent hTYR inhibitors.160 They also evaluated the effects of aurones on the oxidation of LDOPA by the hTYR, which was obtained from insect cells.145 From the experiments, compounds were found to inhibit in the competitive mode of inhibition (105−107, Figure 13). In particular, compound 105 (Kic = 0.35 μM) was found to be the most active inhibitor in comparison to other analogs (compare 105 vs 106 (Kic = 1.02 μM) and 107 (Kic = 1.2 μM). SARs indicated that the hydroxyl group at position 4 of the aurone was a key factor. On the other hand, the HOPNO moiety was identified as a very weak inhibitor of the hTYR with a Ki value of 128 μM, which is approximately 350 times higher than the value measured for compound 105, a potent mTYR inhibitor with an IC50 value of 1.5 μM.161 These data emphasize the crucial role of the aurone backbone in enhancing the inhibitory activities of hTYR. With further investigation in a human integrated cellular model (human melanoma MNT-1 cells), the hybrid aurones were found to prevent melanogenesis in a human complex cytoplasmic environment with a similar inhibition potency obtained from the isolated tyrosinase inhibition assay. For example, compound 105 resulted in a lower IC50 value (IC50 = 16.6 μM) compared to compounds 106 (IC50 = 30 μM) and 107 (IC50 = 30 μM), respectively (see Figure 13). In MNT-1 whole cells, the melanogenesis inhibitory activities of compounds were found 3.5−5 times higher than those obtained from lysates. For example, compound 105 showed potent inhibition with an IC50 of 85.3 μM, 119 μM for 107, and M



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Figure 14. Core regulatory pathways of melanogenesis.27,28

7. CHALLENGES IN CRYSTALLIZATION OF HUMAN TYROSINASE Human TYR catalyzes the rate-determining steps in melanin synthesis. It shared an overall 40% sequence identity and 70% similarity with TRP-1 and TRP-2. Therefore, a crystal structure of the human TYR with high-resolution is absolutely necessary for understanding the catalytic mechanism of TYR, and also it is important to compare the mechanisms of TRP-1 and TRP-2 at the molecular level. Moreover, a high-resolution hTYR bound with substrate/inhibitor provides atomic insights into the residues and shape of the substrate-binding pocket that would be useful for screening compounds and efficient inhibitors design. In contrast to the terameric mTYR, the monomeric hTYR is in inactive glycosylated membrane-bound form. One of the major problems encountered in determining the crystal structure of hTYR is obtaining highly pure and active protein in an adequate quantity. Although several efforts have been made to recombinant expression protocols for obtaining TYR, only few research groups were successful in at least getting some primary results. Kong et al. were the first to report the expression of hTYR in a bacterial expression system. They obtained the full-length protein169 and the intramelanosomal domain170 with good tyrosinase activity. Later, Chen et al. expressed hTYR in the same system.171 However, this protocol was never reproduced by other research groups. Dolinska et al. solved the reproducibility and glycosylation problem by generating the intramelanosomal domain of TYR with a fused heterologous signal peptide in a fully active and glycosylated form produced in Trichoplusia ni larvae as an expression host.172 The solubility and overexpression yield of the protein were enhanced, but still the recombinant expression in insect larva was slow and required multistep purifications, which may

significantly reduce the yield for screening many crystallization conditions. Recently, Fogal et al. succeded in producing overexpressed active, full-length TYR with the α-helical transmembrane and the flexible C-terminal domains in Spodoptera f rugiperda (sf9) cells using a baculovirus expression system.145 However, both structures were likely detrimental for crystallization. More recently, Lai et al. set up a protocol using baculovirus expression vector system in High Five cells (derived from Trichopulsia ni cells) to yield active TYR, which requires only two purification steps for obtaining highly pure protein sample.173 The process obtained a high expression yield of 4−6 mg per liter of culture, which was suitable both for crystallization screening and for high-throughput screening of skin whitening agents. Moreover, they developed a deglycosylation protocol that was suitable for crystallization purposes and subsequently obtained good TYR crystals (3.5 Å), although this resolution in crystallization was not suitable for a structure determination.

8. SIGNALING PATHWAY INHIBITORS Melanogenesis is modualted by a series of intracellular signaling pathways, and these include cAMP/PKA-depended pathway via MC1R/α-MSH, PI3K/Akt signaling, Wnt/β-catenin signaling, SCF/c-kit mediated signaling, nitric oxide (NO) or cytokines, MAPK cascade, and autophagy-mediated associated mechanisms.174 There are several extrinsic (UVB radiation and chemical drugs) and intrinsic (molecules secreted by surrounding keratinocytes or melanocytes, fibroblasts, inflammatory, neural or endocrine) factors that influence the initiation and extension of melanogenesis signaling. Figure 14 shows the most common signaling pathways of melanogenesis. All signaling pathways are associated with a master regulator of melanogenesis MITF, which controls melanogenesis gene expression of TYR, TRP-1, and TRP-2. N



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Figure 15. α-MSH induced melanogenesis inhibitors.

8.1. Inhibitors That Act on Melanocortin 1 Receptor/ α-Melanocyte-Stimulating Hormone α-MSH (MC1R/αMSH). MC1R is one of the key receptors involved in regulating the melanin synthesis. It positively regulates the melanogenesis by binding to α-MSH while negatively regulates by binding to agouti signal protein (ASP). Binding to MC1R, α-MSH stimulates the production of intracellular concentration of cyclic adenosine monophosphate (cAMP) by activating adenylyl cyclase (AC). cAMP production protein kinase A (PKA) activation in turn activates MITF by phosphorylating cAMP response element (CREB) protein. Binding to the Mbox, MITF regulates the expression of TYR, TRP-1, and TRP-2 (Figure 14).175 Glyceollins I−III (110−112), a group of phytoalexins generated from soybeans, inhibit melanin formation in B16 cells.176 As a molecular mechanism, glyceollins inhibited the intracellular cAMP levels followed by MITF expression and a subsequent reduction of tyrosinase expression in mRNA and protein levels. Methyl and ethyl linoenates (113 and 114)

isolated from Oxalis triangularis inhibited melanogenesis through the downregulation of cAMP levels in B16 cells.177 Platycodin (115) is another natural triterpene saponin from Platycodon grandif lorum that inhibited melanin synthesis.178 In an advanced study, it was confirmed that the inhibitory activity of 115 was mediated through the downregulation of cAMP, thereby inhibiting the melanogenesis genes. Bisabolangelone (BISA, 116, Figure 15), 4-hydroxy-3-methoxycinnamaldehyde (4H3MC, 117, Figure 15)179 and diphenylmethylenehydrazine carbothiamide (QNT3−80,147 118, Figure 15)180,181,106 inhibited α-MSH-induced melanin production in B16 cells, and in particular, compound 116 showed 99% inhibition at 30 μM (IC50 = 9 μM).179 Further studies revealed that compounds inhibited the PKA, thereby leading to inhibition of melanin production.182 In the study of UVB-tanned guinea pigs, compounds 117 and 118 were promising in reducing the expression of MITF and TYR and subsequently suppressed the melanin formation. Chrysin (119), a 5,7-dihydroxyflavone demonstrated AC O



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Figure 16. Chemical structures of PI3K/Akt signaling pathway inhibitors.

reported that dopamine receptors (DRs) regulate the melanogenesis in human skin.193 Jung et al. recently found that dopamine D4 receptor (DRD4A) antagonist 124 (Figure 16) inhibited the melanin content in B16194 and Mel-Ab cells as well as in human melanocytes. However, there is no inhibitory effect on TYR catalytic activity. A further study reported that 124 suppressed melanogenesis through the downregulation of MITF via an activation of the ERK signaling pathway. Eupafolin (125, Figure 16) isolated from Artemisia princeps Pampanini reduced melanin content and melanogenesis enzymes in B16 cells195 and was found to inhibit melanogenesis via the Akt pathway. Recents studies reported that Akt signaling contributed to the negative regulation of melanogenesis, which means the activation of Akt signaling contributes to the inhibition of melanin synthesis. For example, curcumin (126, Figure 16), a polyphenol isolated from the rhizome of Curcuma longa, inhibited melanogenesis in mouse B16 cells196 and human melanocytes197 through Akt signaling activation. In addition, Omethyl-fructofuranose (127, Figure 16) isolated from the Schisandra chinensis fruit198 and haginin A (128, Figure 16) from Lespedeza cyrtobotrya199 were also found to activate Akt signaling for their melanogenesis inhibition. Therefore, the role of Akt in regulating melanogenesis is not determined yet. 8.3. SCF/c-Kit-MAP Kinase-Mediated Melanogenesis Inhibitors. Stem cell factor (SCF) plays an important role in regulation of the human melanocytes life cycle. Accumulating evidence suggests that SCF is involving in the regulation of human and mouse melanocytes differentiation and proliferation.200 The expression of SCF is regulated by many factors, which include microphage migration inhibitory factor (MIF), protease-activated receptor 2 (PAR-2), and endothelins (EDNs) (Figure 14). Microphage Migration Inhibitory Factor (MIF). MIF catalyzes the tautomerization of DQ to DHI (see Figure 1),201 and thus MIF possibly contributed to the regulation of neuromelanin synthesis.202 Moreover, MIF mediates the melanogenesis through the activation of PAR-2 and SCF. Protease-Activated Receptor 2 (PAR-2). PAR-2 is a Gprotein-coupled receptor, and it has been linked with various functions on skin pigmentation including melanosome transfer via keratinocytes.203 Recently, Kim et al. reported the

inhibitory activity, followed by downregulating the intracellular cAMP and consequently inhibited melanogenesis.183 Paeonol (120), isolated from Moutan Cortex, inhibits melanogenesis through the inhibition of tyrosinase and MITF in mRNA levels in B16 melanoma cells.184 More specifically, the treatment using paeonol inhibited the phosphorylation of CREB that leads to inactivation of the expressions of tyrosinase and MITF and subsequently inhibited melanin synthesis.185 Caffeic acid derivatives are well-known TYR inhibitors,186 and N-(4-methoxyphenyl)caffeamide (121, Figure 15) was evaluated for its melanogenesis inhibitory effect on B16 melanoma cell lines.187 The result indicated that 121 at 1000 μM concentration significantly reduced mTYR (36.8%) activity. It also inhibited the formation of melanin and cellular tyrosinase activity in B16 melanoma cells. Compound 121 was found to be nontoxic to B16 cells from 0.5 to 2.5 μM concentrations. Further studies elucidating the mechanism of action revealed that 121 inhibited melanogenesis through the activation of phosphorylation of protein kinase B (AKT), GSK3β and subsequent inhibition of MITF transcription activity. Recently, ginsenoside Rb1 (122, Figure 15) showed antimelanogenesis activity in B16 cells.188 Ginsenoside Rb1 122 significantly reduced the melanin formation and subsequently inhibited the cellular tyrosinase activity in a dose-dependent manner, without inducing cytotoxicity at the dosage between 15.63 and 500 μM. However, at high concentrations of 1000 μM, it displayed substantial value (P < 0.001) of cytotoxicity and reduced the cell viability value to 4.67%. 8.2. PI3K/Akt Signaling Pathway Inhibitors. In PI3K/ Akt pathway, the production of intracellular cAMP controls MITF expression by inhibiting the phosphatidylinositol 3kinase (PI3K) and by stimulating the glycogen synthase kinase 3β (GSKβ) activity. Furthermore, the phosphorylation of MITF by GSKβ enhanced the transcriptional activation of pigmentary-related genes and synthesis of melanin (Figure 14).189−191 Hesperidin (123, Figure 16), a flavonoid compound, displayed antimelanogenesis activity in normal human melanocytes and B16 cells.192 The study elucidating the molecular mechanism revealed that 123 inhibited the cellular TYR activity, protein levels of TYR, TRP-1, TRP-2, and MITF, through Erk1/2 and Akt signaling pathways. It has been P



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Figure 17. SCF/c-kit/EDNs and Wnt/β-catenin mediated signaling pathway melanogenesis inhibitors.

Nobiletin (132, Figure 17) inhibited the melanogenesis in normal human melanocytes through the modulation of EDN and SCF.208 As a molecular mechanism, 132 reduced tyrosinase activity through the reduction of endothelin (EDN), phosphorylation of SCF-induced Raf-1/MEK and ERK. Further study using a 3D human epidermal model, 132 effectively inhibited EDN and SCF induced melanin content and MITF/ tyrosinase expression. Withaferin A (133, Figure 17) is a naturally occurring compound from Withania somnifera extract (WSE) inhibited melanogenesis by interfering with both EDN-1 and SCFtriggered intracellular signaling cascades.209 Astaxanthin (134, Figure 16) is a keto-carotenoid that inhibits melanogenesis by interrupting the SCF-induced intracellular signaling. However, it was not able to interrupt the EDN-triggered intracellular cascade in the human epidermal equivalents (HEEs).209 Tocotrienols belong to the vitamin E family and there are four types (α-, β-, γ-, and δ-tocotrienols). δ-Tocotrienol (135, δT3, Figure 16) significantly inhibited the melanin production and the reactive oxygen species (ROS) in B16 cells without toxicity.210 Furthermore, the inhibitory mechanism revealed 135 reduced the expression of a pigmentary related protein via ERK signaling pathway. 8.4. Wnt/β-Catenin Signaling Pathway Inhibitors. Wnt signaling plays an important role in the melanocyte development, and β-catenin is the key controller. Wnt ligand binds to the receptor Frizzled and triggers a displacement of the glycogen synthase kinase-3 β (GSK3β), which phosphorylates

contribution of PAR-2 receptor and the expression of SCF, which binds to the c-kit receptor (tyrosine-protein kinase kit or CD117) and triggers melanogenesis through mitogen-activated protein kinase (MAPK) cascades. Endothelin-1 (EDN-1). It has been reported that endothelins have an indispensable role in pigmentary related disorders.182 Endothelins peptides EDN, EDN-1, EDN-2, and EDN-3 bind to its receptor (EDNRB), and among them, EDN-1 (vasoconstrictor peptide) is a significant member as it induces melanogenesis in the human melanocytes.204 Upon binding to EDNRB, EDN-1 activates melanogenesis through MAPK cascade; the activation PKC in this pathway phosphorylates Raf or Raf-1 which activates MAPK cascade via phosphorylation and further downstream pathways that regulate melanogenesis. In the MAPK cascade, ERK, JNK, or p38 plays a significant role in the regulation of melanogenesis. Phosphorylation of ERK or JNK initiates the expression of MITF and its degradation followed by the downregulation of melanin synthesis. On contrary, the phosphorylation of p38 activates MITF and stimulating melanin production.205 Recently, 3,3′-bisdemethylpinoresinol (129, Figure 17) and americanin A (130, Figure 17) from Morinda citrifolia206 and sulforaphane (131, Figure 17) from broccoli207 were reported as melanogenesis inhibitors by reducing TYR expression, which resulted from the downregulation of MITF. The detailed mechanistic study with the respect to the melanogenesis downregulation found that they were inhibiting p38 phosphorylation. Q



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β-catenin211 and then degrades through an ubiquitin-dependent mechanism. Besides, Wnt signaling negatively regulates GSK3β and leads to β-catenin accumulation. Stabilized β-catenin is displaced to the nucleus and enhances MITF gene expression and melanogenesis.212,213 Cardamonin (136, Figure 16), a chalcone isolated from Aplinia katsumadai, and fingolimod (FTY720, 137, Figure 16)214 were reported as melanogenesis inhibitors targeting Wnt signaling. Studies elucidated that compound 136 induced the intracellular β-catenin degradation and downregulated MITF and TYR expression, but 137 (FTY720) inhibited melanin synthesis through the downregulation of β-catenin expression in Mel-Ab cells. Bellei et al. reported the pyridinyl imidazoles as melanogenesis inhibitors through the inhibition of the canonical Wnt/β-catenin pathway activity in B16-F0 cells.215 In particular, compounds 138, 139, and 140 inhibited melanin synthesis with IC50 values of 30, 34, and 89 nM, respectively. Recently, labdane diterpenoid andrographolide (141, Figure 16) was found to have potent inhibitory activities of the melanin content and intracellular TYR activity in B16 cells.216 In an in vivo UVB-induced brown guinea pig model, compound 141 reduced melanin and TYR content. Further study revealed andrographolide induced the degradation of β-catenin through the ubiquitin-dependent mechanism.

In general, it is important to design cosmetic products with effective antioxidant activity, which plays an important role in reducing aging effects. Recently there has been a growing number of melanogenesis inhibitors that have been associated with antioxidant properties. However, further advanced studies of inhibitors from the human clinical perspective are required. In drug discovery program, the use of existing drugs is one of the safe and effective methods, since it has several advantages that include time-savings, drug availability, safety/tolerability, and cost-effectiveness. Several antituberculosis and antithyroids drugs were proven potent tyrosinase inhibitors without causing significant cytotoxicity to the cells. Therefore, further studies in advancing as whitening agents must be done in the future. In conclusion, we believe that this Perspective will be beneficial for updating the inhibitors of melanogenesis and their mechanism of action involved in melanogenesis signaling pathways.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00967. X-ray crystal structures of tyrosinase and comparison of inhibitors between hTYR and mTYR (PDF)

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9. CONCLUSIONS In recent years a large number of melanogenesis inhibitors have been reported. In most cases, these reagents are directly targeting the tyrosinase catalytic activity or its expression by various downstream signaling pathways. Tyrosinase has been the most important molecular target because it acts as a ratelimiting enzyme in melanin synthesis. More importantly, it is expressed only by melanocytes, and therefore targeting tyrosinase might inhibit melanogenesis in cells without any side effects. Numerous mushroom tyrosinase inhibitors from synthetic, semisynthetic, or natural sources have been identified, but only a few of them reached clinical applications as skin-whitening agents due to safety concerns and weak whitening effects. Especially in recent years, natural products bring much attention because of fewer side effects and effective skin-whitening properties. Nevertheless, the skin-whitening efficiency and viability need to be studied in the animal study or in suitable in vivo models. On the other hand, identification of human tyrosinase inhibitors is of great importance. From this updated literature survey, we have found that there has been a huge difference in inhibitory activities between the mTYR and the hTYR. In fact, many mTYR inhibitors eventually became approximately 10fold less potent or even inactive against hTYR. For example, the well-known kojic acid has a high binding affinity for mTYR (Ki = 4.3 μM) compared with the hTYR (Ki = 350 μM), while aesculetin identified as a potent mTYR inhibitor showed no detectable inhibitory activity of hTYR. Although it is very clear that mTYR is economical and commercially available for skin whitening agents screening, the efficiency should be validated against human tyrosinase before going for any in vivo or animal studies. Many efforts have been made to recombinant expression protocols for producing hTYR. More recently, Lai et al. succeeded in overproducing the active hTYR using baculovirus expression vector system in High Five cells in high yield and pure form, which would be useful both for crystallization screening and for high-throughput screening of skin whitening agents in the future.

AUTHOR INFORMATION

Corresponding Author

*Phone: +49-228732360. E-mail: [email protected]. ORCID

Thanigaimalai Pillaiyar: 0000-0001-5575-8896 Notes

The authors declare no competing financial interest. Biographies Thanigaimalai Pillaiyar received his Doctoral degree in Medicinal Chemistry in 2011 under the supervision of Prof. Dr. Sang-Hun Jung at Chungnam National University, South Korea. In 2011, he won a “Japanese Society for the Promotion of Science Postdoctoral Fellowship (JSPS)” for 2 years with Prof. Dr. Yoshio Hayashi at Tokyo University of Pharmacy and Life Sciences, Japan. He was awarded an Alexander von Humboldt postdoctoral fellowship (AvH) in 2013 for 2 years with Prof. Dr. Christa E. Müller at University of Bonn, Germany. Currently he is developing modulators/inhibitors for various G-protein-coupled receptors. Vigneshwaran Namasivayam is a Senior Research Scientist at Pharmaceutical Institute, University of Bonn, Germany (since 2010), and involved in the field of cheminformatics, computational chemistry, data analysis, and molecular modeling. He gained his Master of Technology in Bioinformatics from SASTRA University, India (2004), and Doctoral degree under the supervision of Prof. Dr. Hans-Jörg Hofmann from Leipzig University, Germany (2009). He carried out his postdoctoral research at the Technical University of Munich, Germany (2010). Prior to his doctoral studies in Germany, he worked as a Research Executive (2004−2006) at Orchid Chemical and Pharmaceutical Limited, Chennai, India. Manoj Manickam received his Ph.D. in 2010 from Bharathiar University under the supervision of Prof. Dr. K. J. Rajendra Prasad, Coimbatore, India. He continued to work as a Research Associate at Orchid Chemicals and Pharmaceuticals Ltd. Then he moved to Chungam National University, South Korea, to continue his research. Currently, he is a Senior Research Scientist at the Department of R



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(9) Ito, S.; Wakamatsu, K. Chemistry of mixed melanogenesis− pivotal roles of dopaquinone. Photochem. Photobiol. 2008, 84, 582− 592. (10) Coudrier, E. Myosins in melanocytes: to move or not to move? Pigm. Cell Res. 2007, 20, 153−160. (11) Wu, X.; Hammer, J. A., 3rd. Making sense of melanosome dynamics in mouse melanocytes. Pigm. Cell Res. 2000, 13, 241−247. (12) Ahn, S. J.; Koketsu, M.; Ishihara, H.; Lee, S. M.; Ha, S. K.; Lee, K. H.; Kang, T. H.; Kima, S. Y. Regulation of melanin synthesis by selenium-containing carbohydrates. Chem. Pharm. Bull. 2006, 54, 281− 286. (13) Iozumi, K.; Hoganson, G. E.; Pennella, R.; Everett, M. A.; Fuller, B. B. Role of tyrosinase as the determinant of pigmentation in cultured human melanocytes. J. Invest. Dermatol. 1993, 100, 806−811. (14) Li, G.; Ju, H. K.; Chang, H. W.; Jahng, Y.; Lee, S. H.; Son, J. K. Melanin biosynthesis inhibitors from the bark of Machilus thunbergii. Biol. Pharm. Bull. 2003, 26, 1039−1041. (15) Unver, N.; Freyschmidt-Paul, P.; Horster, S.; Wenck, H.; Stab, F.; Blatt, T.; Elsasser, H. P. Alterations in the epidermal-dermal melanin axis and factor XIIIa melanophages in senile lentigo and ageing skin. Br. J. Dermatol. 2006, 155, 119−128. (16) Nordlund, J. J.; Boissy, R. E.; Hearing, V. J.; King, R. A.; Oetting, W. S.; Ortonne, J.-P. The Pigmentary System: Physiology and Pathophysiology, 2nd ed.; Blackwell Publishing Ltd.; Malden, MA, 2006; pp 1163−1174. (17) Cavalieri, E. L.; Li, K. M.; Balu, N.; Saeed, M.; Devanesan, P.; Higginbotham, S.; Zhao, J.; Gross, M. L.; Rogan, E. G. Catechol orthoquinones: the electrophilic compounds that form depurinating DNA adducts and could initiate cancer and other diseases. Carcinogenesis 2002, 23, 1071−1077. (18) Hasegawa, T. Tyrosinase-expressing neuronal cell line as in vitro model of Parkinson’s disease. Int. J. Mol. Sci. 2010, 11, 1082−1089. (19) Tessari, I.; Bisaglia, M.; Valle, F.; Samori, B.; Bergantino, E.; Mammi, S.; Bubacco, L. The reaction of alpha-synuclein with tyrosinase: possible implications for Parkinson disease. J. Biol. Chem. 2008, 283, 16808−1681. (20) Vontzalidou, A.; Zoidis, G.; Chaita, E.; Makropoulou, M.; Aligiannis, N.; Lambrinidis, G.; Mikros, E.; Skaltsounis, A. L. Design, synthesis and molecular simulation studies of dihydrostilbene derivatives as potent tyrosinase inhibitors. Bioorg. Med. Chem. Lett. 2012, 22, 5523−5526. (21) Greggio, E.; Bergantino, E.; Carter, D.; Ahmad, R.; Costin, G. E.; Hearing, V. J.; Clarimon, J.; Singleton, A.; Eerola, J.; Hellstrom, O.; Tienari, P. J.; Miller, D. W.; Beilina, A.; Bubacco, L.; Cookson, M. R. Tyrosinase exacerbates dopamine toxicity but is not genetically associated with Parkinson’s disease. J. Neurochem. 2005, 93, 246−256. (22) Kobayashi, T.; Urabe, K.; Winder, A.; Jimenez-Cervantes, C.; Imokawa, G.; Brewington, T.; Solano, F.; Garcia-Borron, J. C.; Hearing, V. J. Tyrosinase related protein 1 (TRP1) functions as a DHICA oxidase in melanin biosynthesis. EMBO J. 1994, 13, 5818− 5825. (23) Lai, X.; Wichers, H. J.; Soler-Lopez, M.; Dijkstra, B. W. Structure of human tyrosinase related protein 1 reveals a binuclear zinc active site important for melanogenesis. Angew. Chem., Int. Ed. 2017, 56, 9812−9815. (24) Boissy, R. E.; Sakai, C.; Zhao, H.; Kobayashi, T.; Hearing, V. J. Human tyrosinase related protein-1 (TRP-1) does not function as a DHICA oxidase activity in contrast to murine TRP-1. Exp. Dermatol. 1998, 7, 198−204. (25) Yamaguchi, Y.; Brenner, M.; Hearing, V. J. The regulation of skin pigmentation. J. Biol. Chem. 2007, 282, 27557−27561. (26) Bennett, D. C.; Lamoreux, M. L. The color loci of mice−a genetic century. Pigm. Cell Res. 2003, 16, 333−344. (27) Pillaiyar, T.; Manickam, M.; Jung, S. H. Inhibitors of melanogenesis: a patent review (2009−2014). Expert Opin. Ther. Pat. 2015, 25, 775−788. (28) Pillaiyar, T.; Manickam, M.; Jung, S. H. Downregulation of melanogenesis: drug discovery and therapeutic options. Drug Discovery Today 2017, 22, 282−298.

Pharmacy and Institute of Drug Research and Development, Chungnam National University, working with Professor Sang-Hun Jung. Sang-Hun Jung received his MS degree from the College of Pharmacy of the Seoul National University in 1976. He received his Ph.D. from the Chemistry Department at the University of Houston, U.S., in 1984. He served as a Postdoctoral Fellow at the University of Pittsburgh until 1985 and as a Principle Investigator of LG Life Sciences from 1985 to 1989. He has been a professor at the College of Pharmacy, Chungnam National University, South Korea since 1989. He has served as a Department Chairman (1993−2000), Dean of the College of Pharmacy (2003−2004), and President of Institute of Drug Research and Development of Chungnam National University (2007− 2009). His research interests include antimicrotubule-based anticancer agents, novel inotropes with selective activation of cardiac myosin, and melanogenesis inhibitors.

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ACKNOWLEDGMENTS M.M. and S.-H.J. thank the Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant 2009-0093815).

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ABBREVIATIONS USED TYR, tyrosinase; TRP-1, tyrosinase-related protein-1; TRP-2, tyrosinase-related protein-2; DQ, dopaquinone; DCT, dopachrome tautomerase; UVR, ultraviolet radiation; CNS, central nervous system; α-MSH, α-melanocyte stimulating hormone; MC1R, melanocortin 1 receptor; ED, endothelin; EDBR, endothelin B receptor; SCF, stem cell factor; NO, nitric oxide; AC, adenylyl cyclase; cAMP, cyclic adenosine monophosphate; PAK, protein kinase A; CREB, cAMP response element binding protein; PI3K, phosphatidylinositol 3-kinase; MIF, microphage migration inhibitory factor; PAR-2, protease-activated receptor 2; IP3, inositol triphosphate; DAG, diacyglycerol; GC, guanylyl cyclase; cGMP, cyclic guanosine monophosphate; ROS, reactive oxygen species; CAGR, compound annual growth rate.

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