Inhibitors of Melanogenesis: An Updated Review - ACS Publications

May 15, 2018 - ABSTRACT: Melanins are pigment molecules that determine the skin, eye, and hair color of the human subject to its amount, quality, and ...
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Inhibitors of Melanogenesis: An Updated Review Thanigaimalai Pillaiyar, Vigneshwaran Namasivayam, Manoj Manickam, and Sang Hun Jung J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00967 • Publication Date (Web): 15 May 2018 Downloaded from http://pubs.acs.org on May 15, 2018

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Journal of Medicinal Chemistry

Inhibitors of Melanogenesis: An Updated Review

Thanigaimalai Pillaiyar,†,* Vigneshwaran Namasivayam,† Manoj Manickam, ∥ 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.

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, a serious dermatological problems occur when the 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.

Keywords: skin whitening agents, hyperpigmentation, melanogenesis, melasma, tyrosinase.

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1. Introduction It is estimated that approximately 15% of the papulaton in the world invests for skin whitening.1 Worldwide the market for skin whitening agents is expected to reach nearly US$ 23 billion by 2020.1 Inhibition or reduction of melanin synthesis by overactive melanocytes is the common mehcanism 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 are originated from the epidermis, where melanocytes synthsize 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 melanosomes,8,9 and transported to keratinocytes via dendrites.10,11Although melanin is crucial for protecting the skin, the abnormal production of melanin leads to acute dermatological problems include melasma,12-15 post-inflammatory melanoderma16 solar lentigo, freckles, pigmented acne scars and age spots. Continuous UVirradiation 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 emphasis the recently identified tyrosinase inhibitors that directly/indirectly target the catalytic activity of tyrosinase and melanogenesis signaling pathway inhibitors from all sources. 2. Synthesis of melanin and its regulation Melanocytes produce two kinds of melanin pigments called as eumelanin (brown-black or dark insoluble polymer) and pheomelanin (red-yellow soluble polymer). A complex enzymatic and biochemical-catalyzed reactions involve in melanin synthesis. Enzymatically, tyrosinase (TYR), 2 ACS Paragon Plus Environment

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tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2, also called as 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 (Fig. 1). As an important enzyme, TYR performs rate-limiting 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 5-cysteinyl DOPAs, which 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 continued 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 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 oxidase.23,24 This builds the question about the exact function of TRP-1 in melanin synthesis. Furthermore, the role of TRP-124 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 this 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) control melanogenesis at the subcellular level.

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

3. Structure and Function of Tyrosinase 3.1. Overall Structure and Function of Tyrosinase. Tyrosinase (EC 1.14.18.1), a di-copper 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 exlusuvely produced only by melanocyte cells. The production and subsequent glycosylation of tyrosinase are further matured and trafficked to melanosomes, wherein the melanin synthesis occurred.31 The protein level of tyrosinase is also regulated by two degradation systems, the proteasomal and endosomal/lysosomal systems for proteolysis of misfolded or unfolded proteins during maturation processing.32,33-34

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Figure 2. The crystallographic structure of tyrosinase from Agaricus bisporus in deoxy-form (PDB ID: 2Y9X)35 is shown and represented in cartoon model with the N- and C-terminal domains highlighted in pink and blue color, 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.

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 general, the structure of tyrosinase can be classified into three domains namely the central, the N-terminal and the C-terminal domains (Fig. 2).36 Among tyrosinases from different species, the central domain is the most conserved domain, 5 ACS Paragon Plus Environment

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which comprises of six histidine residues and two copper ions (CuA and CuB). Structural investigations of the crystal structure 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, the histidine coordinate 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 and whereas it is absent in the fungal tyrosinase. A latent precursor of tyrosinase called pro-tyrosinase 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 CuB) complexed with six histidine residues.39 The catalytic mechanism of tyrosinase involves in three different states namely oxy, met and deoxy-tyrosinases with differences in the oxidation state of copper ions (Fig. 3). In the monophenolase activity (oxidation of L-tyrosine to L-DOPA), deoxy-tyrosinase (Edeoxy) binds to oxygen and form oxytyrosinase (Eoxy) and then to L-tyrosine, which is further catalyzed to L-DOPA. 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 oxy-tyrosinase drops only one oxygen atom to form met-tyrosinase (Emet), where the two close copper centers (dCU-CU ≈ 2.9 to 4.9 Ǻ) bridged by an aqua (hydroxo) ligand. Tyrosinase occurs mainly as Emet form, which cannot oxidize phenols, e.g. tyrosine, and needs to be reduced to Edeoxy by an L-DOPA before the tyrosine oxidation 6 ACS Paragon Plus Environment

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Journal of Medicinal Chemistry

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 the 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 Cuo and tyrosinase deactivation.41-43,44

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

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4. The Importance of Targeting Tyrosinase Tyrosinase is important in oxidative homeostasis process and protects the human skin from ionizing radiations. Recent 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. In case of an excessive formation of dopaquinone results in a neuronal damage and cell death. Evidence has been 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 Based on 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 tyrosinase catalyzes rate-limiting steps of overall melanogenesis, it has been recognized as a therapeutic target for controlling abnormal melanin synthesis. Many approaches 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 target tyrosinase and possibly inhibit the melanogenesis without any side-effects. Although a huge number of tyrosinase inhibitors identified so far, relatively a 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-L-ascorbyl-2phosphate,

phenols,50

hydroxyanisole,

corticosteroids,51 8

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N-acetyl-4-S-cysteaminylphenol,

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resinoids52,53 arbutin (hydroquinone-β-D-glucopyranoside), salicylhydroxamic acid, kojic acid,5456

hydroquinone (HQ),57-60 monobenzyl hydroquinone, tretinoin and mercury salts are commonly

used in the cosmetic 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, and burning, hypochromia, ochronosis and chestnut spots on the nails.61-63,64 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. LAscorbic acid degrades easily65 and the bioavailability of Ellagic acid is poor.66 For the tranexamic acid, the exact molecular target 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 human tyrosinase for screening TYR inhibitors, since mTYR is commercially available in a purified form.68,69 For screening, kojic acid, HQ or arbutin have been used as references. LTyrosine 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 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 in the same equilibrium constant. The strength of inhibition was expressed in IC50 value. The Ki value 9 ACS Paragon Plus Environment

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specifies the binding affinity of the ligand towards the enzyme; if the value of Ki is lower means, binding affinity is higher and the value of Ki is higher means 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, Fig. 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 administered orally it was having a low in vivo bioavailability. Rigon et al. prepared resveratrol-loaded solid lipid nanoparticles (SLNs, diameter 1000 and >1000 µM). SARs suggested meta-position of phenyl ring and carboxylic acid on the benzene ring (3-PBA) resulted in a most potent inhibitory effect on mTYR. The docking studies supported that meta-carboxylic acid on the 3PBA was attributed to the strong binding interaction by coordinating to the active site copper ions. The importance of carboxylic acid was proved by evaluating its ester congener, as it was inactive against mTYR. The same research group extended the SARs by introducing 4’hydroxylation and 4’-methoxylation of PBA isomers (see for some examples 34-36 in Fig. 6).98 The results specify that the inhibitory activity of 3-PBA was slightly reduced by 4’-hydroxylation (35, IC50 5.0 and 10.59 µM) and further decreased by 4’-methoxylation (IC50 8.0 and 15.30 µM) against mTYR. It was surprising that 4’-hydroxylation (34, IC50 4.0 and 100.18 µM) appeared to be vital for the inhibitory activity in comparison to the methoxylation of 2-PBA. Instead, 4’hydroxylation (36, IC50 6.0 and 14.70 µM) of 4-PBA increased the inhibitory activity of mTYR. 5.4. Indole Derivatives. Ferro et al. evaluated indole derivatives on the diphenolase activity of mTYR using L-DOPA as substrate and the results were evaluated using the standard kojic acid (IC50 17.76 µM).99 Potent compounds 37-39 were shown in Figure 8. SARs suggested that the 4fluorobenzyl moiety substituted at the N1-position of indole influenced positively for the 17 ACS Paragon Plus Environment

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tyrosinase inhibitory activity, while the diketo group was not crucial. The kinetics of mTYR inhibition suggest that compounds 37 and 38 displayed mixed type inhibition and the compound possibly bind to free enzyme and enzyme-substrate complex.

Figure 8. Indole-derived tyrosinase inhibitors

In continuation, the same research group further identified 3-(4-benzylpiperidin-1-yl)-1-(1Hindol-3-yl)propan-1-one (40) as a potential candidate for mTYR inhibitor (IC50 252 µM, Fig. 8). SAR studies of 40 yielded 41 (Fig. 8) as the most potent inhibitor in the study.100 The docking study of 40 was performed with mTYR (Fig. 9), X-ray structure (PDB ID: 2Y9X ) resolved with the potent inhibitor tropolone in the H2L2 tetrameric complex.101,35,102The study predicted inhibitor 41 was overlapping with the orientation of inhibitor tropolone obtained from the crystal 18 ACS Paragon Plus Environment

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structure.35 The (4-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 to the cavity entrance formed by catalytic copper ions.

Figure 9. The docked pose of 41 in the binding pocket of tyrosinase obtained from Agaricus bisporus in deoxy-form (PDB ID: 2Y9X). (A) The 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).

5.5. Thiourea and Thiosemicarbazone Derivatives. Phenylthiourea (PTU, 42, Fig. 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 19 ACS Paragon Plus Environment

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Figure 10. Thiourea-derived tyrosinase inhibitors

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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 πplanar 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 3aminohydrogens (45) by any substituent abolished the activity. This suggested that C2substituted 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-saving, 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, Fig. 10) and its analogs (47-49, Fig. 10), including prothionamide (47) as tyrosinase inhibitors.108-110 Compounds 46 and 47 are anti-tuberculosis drugs and 47 were tested for the use in the treatment of leprosy. Instead, isoniazid (49), a first-line anti-tuberculosis 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-2-carbothioamide (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 54111 and propylthiouracil 55 (Fig. 10)112 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 non-competitive mode of action. This was supported by another study where N-aryl-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)thio acetic acid was more beneficial for the increase in the tyrosinase inhibitory activity. 21 ACS Paragon Plus Environment

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

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 acylamino compounds 61-63 ( Fig. 11) displayed potent tyrosinase inhibition in comparison to kojic acid (IC50 28.5 µM).126 Specifically, compound 63 was being 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 4-acyloxy-phenylethylenethiosemicarbazones significantly inhibited the tyrosinase activity with

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an IC50 value lower than 1.0 µM (see for examples 64-66, Fig. 11). The SARs indicated that the thiosemicarbazone moiety is essential for the tyrosinase inhibitory activity. 5.6. Hydroxycinnamic Acid Derivatives. Caffeic acid (CA, 67, Fig. 11) is a common phenolic acid found in vegetables, fruits, grains, and seeds. It has many medical properties that include anti-oxidant, anti-tumor, and anti-inflammatory, anti-microbial and anti-diabetic activity. Antioxidants play a significant role in reducing aging effects and thus the anti-oxidant property in designing whitening agent is of highly considered. In a study, Kwak et al.128 identified 67 as a potent anti-oxidant 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 antityrosinase and anti-oxidant activities. The results indicated that the caffeoyl-prolyl-hydroxamic acid 68 (Fig. 11) and caffeoyl-phenylalanyl-hydroxamic acid 69 (Fig. 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 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, Fig. 11) is a natural hydroxycinnamic acid derivative and largely presented in coffee, pears and apples. Several publications reported this compound with an antiinflammatory, anti-diabetic, anti-viral, anti-oxidant activities. Very few studies reported the 23 ACS Paragon Plus Environment

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chlorogenic acid with the antit-yrosinase property.131 In B16 cells, compound 70 at 500 µM suppressed the melanin content by inhibiting intracellular tyrosinase activity. HO

OH

HO

Tyrosinase inhibition IC50 ( M)a

A R OH O

OH

OH

B Melanin synthesis inhibition IC50 (( M)

N

N

OH

OH

76, IC50 2.30 M

75, IC50 1.70 M

71-74 71

R=H

0.21

5.0

72

R=

0.82

3.8 N

73

HO

R=

OH

NO2

OH O

N

OH

N

OH

78, IC50 7.89 M

77, IC50 4.77 M N.Db

4.62

HO

OH

O

HO

OH

O

OH

R HO

74

OH

OH

HO

R=

Arbutin a b

0.17

4.0

164

500

O

substrate: L-tyrosine N.D: Not detected

O

HO

O

OH O

HO O

HO

OH OH

HO

O A 6 OH O

88, IC50 >100 M

HO

O

OH

OH OH

HO

O HO

A OH O

4

OH

O

OH O

89, IC50 >100 M

90, IC50 49.89 M

Figure 12. Chemical structure of chalcones and flavanone tyrosinase inhibitors

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OH 87, Moracin M IC50 8.00 M

86, Morachalcone A IC50 0.08 M

B

O

HO

OH O

OH

O

OH B

HO

85, 2,4,2',4' -Tetrahydroxychalcone IC50 0.07 M

OH 2

O

OH

82 monophenolase: IC50 18.4 M diphenolase: IC50 144 M

OH

OH O

84, Steppogenin IC50 0.98 M

83, Morusone IC50 290 µM

OH

R

O

HO

OH O

O

O

HO

OH

OH

OH

81 (Neorauflavane) monophenolase: IC50 0.03 M diphenolase: IC50 0.5 M

OH

O

80 monophenolase: IC50 92.0 M diphenolase: IC50 >200 M

R=

O

O

HO

OH

OH

79 monophenolase: IC50 2.9 M diphenolase: IC50 128.2 M O

R

HO HO

4

OH

O

OH O 91, IC50 2.32 M

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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 antityrosinase activities. The natural chalcones 7174 (Fig. 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 (Fig. 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 the presence 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) displayed as potent tyrosinase inhibitors (Fig. 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 µM. SARs revealed ortho-methoxy with para-nitro substituents (ring B) in 77 or an electron donating para-dimethyl amino ring (ring B) in 78 was responsible for potent tyrosinase inhibition. Both 77 and 78 inhibited the cellular melanin formation in α-MSH induced B16 cells. Four new compounds isolated from Camylotropis hirtella (79-82, Fig. 12) showed potent inhibitory activities against tyrosinase.135 Among them, neorauflavane (81) identified as a 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

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decreased the melanin content, without influencing cell viability. Structurally, the reduction of geranyl side chain was important for improving the tyrosinase inhibitory activity. Recently, a flavone named morusone (83, IC50 290 µM) and sixteen known compounds isolated from twigs of Morus alba L. were examined for their inhibitory activities against tyrosinase.112 The results showed that compounds steppogenin (84, IC50 0.98 µM, Fig. 12), 2,4,2’,4’tetrahydroxychalcone (85, IC50 0.07 µM, Fig. 12), morachalcone A (86, IC50 0.08 µM, Fig. 12) and moracin M (87, IC50 8.00 µM, Fig. 12) 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 (Fig. 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 an 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 4-substituted 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 showed to inhibit the content of melanin with cytotoxicity in a concentrationdependent manner. In that study, kojic acid was proved 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 involves 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 26 ACS Paragon Plus Environment

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groups, 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.

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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 human and mushroom tyrosinase show only 23% identity. In comparison to the mTYR, the hTYR found 6-fold higher for L-DOPA oxidation activity, 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 hTYR is unique and the identification of the human tyrosinase inhibitors is of high importance. Since 1995, the resorcinol derivative 4-butylresorcinol (93, Fig. 13) has been known as a potent melanogenesis 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 found 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.

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Figure 13. The human tyrosinase inhibitors.

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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, Fig. 13) and 4-phenylethylresorcinol (95, Fig. 13) treated patients with age spots. Among them, 93 showed promising result in tyrosinase inhibition and reduced the appearance of age spots within eight 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% to 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 (Fig. 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. 30 ACS Paragon Plus Environment

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In 1930s, Thujaplicins (isopropyl cycloheptatrienolones) and related chemical substances were isolated from Thuja plicata (western redcedar tree).155 The three regioisomers; α-thujaplicin (98), β-thujaplicin (hinokitiol, 99), and γ-thujaplicin (100) exhibited potent anti-fungal, antibacterial156 and anti-oxidant properties.157 Recently, thujaplicins (98-100; α, β and γ isomers, Fig. 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 observed for hTYR and mTYR. For example, the inhibitory activity of kojic acid was estimated to have ~11 fold weaker inhibition for hTYR (IC50 571.17 µM) in comparison to mTYR (IC50 53.70 µM). Thujaplicins were approximately >105 (α -isomer)-, 100 (β -isomer)-, and 17 (γ-iosmer)-fold 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 31 ACS Paragon Plus Environment

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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 possibly a suitable candidate molecule in the development of new skin whitening agent. 2-Hydroxypyridine-N-oxide (HOPNO, 104, Fig. 13), a catechol mimic, was previously reported as a non-naturally 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 coworkers described a new series of aurones as potent hTYR inhibitors160 They also evaluated the effects of aurones on the oxidation of L-DOPA 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, Fig. 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. 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 32 ACS Paragon Plus Environment

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compounds 106 (IC50 30 µM) and 107 (IC50 30 µM), respectively (see Fig. 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 120 µM for 106. Authors suggest that the scarce ability of compounds 105-107 to cross MNT-1 cells 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, Fig. 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, anti-oxidant, anti-collagenase, anti-microbial and anti-inflammatory activities.162 The chemical structure of 108 has been attracted 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 α-MSH.165 Compound 108 inhibited cellular melanin formation much stronger than the compounds which are structurally similar such as 3-(4hydroxyphenyl)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, Fig. 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 33 ACS Paragon Plus Environment

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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 with the reference compounds arbutin and kojic acid.167 Compound 108 showed a weak inhibition against mTYR, while 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 because 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.

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 understanding the mechanisms of 34 ACS Paragon Plus Environment

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TRP-1 and TRP-2 at the molecular level. Moreover, a high-resolution hTYR bound with substrate/inhibitor provide 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 membranebound 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 one 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 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 trans-membrane and the flexible Cterminal domains in Spodoptera frugiperda (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 35 ACS Paragon Plus Environment

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per liter of culture, which was suitable for both crystallization screening and for high-throughput screening of skin whitening agents. Moreover, they developed a deglycosylation protocol which 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.

Figure 14. The core regulatory pathways of melanogenesis.27-28

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 autophagy36 ACS Paragon Plus Environment

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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 influence the initiation and extension of melanogenesis signaling. Figure 14 shows the most common signaling pathways of melanogenesis. All signaling pathways associated with a master regulator of melanogenesis MITF, which controls melanogenesis gene expression of TYR, TRP-1 and TRP-2. 8.1. Inhibitors that act on Melanocortin 1 receptor/ α-Melanocyte-stimulating hormone αMSH (MC1R/α-MSH). 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 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, which in turn activates MITF by phosphorylating cAMP response element (CREB) protein. Binding to the M-box, MITF regulates the expression of TYR, TRP-1, and TRP-2 (Fig. 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 grandiflorum inhibited melanin synthesis.178 In an advanced study, it was confirmed that the inhibitory activity of 115 was mediated through the down-regulation of cAMP, thereby inhibited the melanogenesis genes. Bisabolangelone (BISA, 116, Fig. 15), 4-Hydroxy-3-methoxycinnamaldehyde (4H3MC, 117, Fig. 15)179 and diphenylmethylenehydrazine carbothiamide (QNT3-80,147 118, Fig. 15)180-181,106 37 ACS Paragon Plus Environment

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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

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Figure 15. α-MSH induced melanogenesis inhibitors.

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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 inhibitory activity, followed by downregulating the intracellular cAMP and consequently inhibited melanogenesis.183 Paeonol (120), isolated from Moutan Cortex inhibit 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 inactivate the expressions of tyrosinase and MITF and subsequently inhibited melanin synthesis.185 Caffeic acid derivatives are well known TYR inhibitors186 and N-(4-methoxyphenyl) caffeamide (121, Fig. 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 found to be non-toxic 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, Fig. 15) showed anti-melanogenesis activity in B16 cells.188 Ginsenoside Rb1 122 significantly reduced the melanin formation and subsequently inhibited the cellular tyrosinase activity in a dosedependent 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 reducing 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 3-kinase (PI3K) and by stimulating the glycogen synthase kinase 3β (GSKβ) activity. Furthermore, the 40 ACS Paragon Plus Environment

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phosphorylation of MITF by GSKβ enhanced the transcriptional activation of pigmentary-related genes and synthesis of melanin (Fig. 14).189-191 Hesperidin (123, Fig. 16), a flavonoid compound displayed anti-melanogenesis 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 reported that dopamine receptors (DRs) regulate the melanogenesis in human skin.193 Jung et al. recently found that dopamine D4 receptor (DRD4A) antagonist 124 (Fig. 16) inhibited the melanin content in B16 194 and Mel-Ab cells as well as in human melanocytes. However, there is no inhibitory effect on TYR catalytic activity. A further study reported 124 suppressed melanogenesis through the down-regulation of MITF via an activation of the ERK signaling pathway. Eupafolin (125, Fig.16) isolated from Artemisia princeps Pampanini reduced melanin content and melanogenesis enzymes in B16 cells195 and found to inhibit melanogenesis via Akt pathway. Recents studies reported that Akt signaling is 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, Fig.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, O-methyl-fructofuranose (127, Fig.16) isolated from the Schisandra chinensis fruit198 and haginin A (128, Fig.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.

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Figure 16. Chemical structure of PI3K/Akt Signaling Pathway inhibitors

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) (Fig. 14). Microphage Migration Inhibitory Factor (MIF). MIF catalyzes the tautomerization of DQ to DHI (see Fig. 1),201 and thus MIF is 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 G protein-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 contribution of PAR-2 receptor and the 42 ACS Paragon Plus Environment

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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 play 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 contrarily, the phosphorylation of p38 activates MITF and stimulating melanin production.205 Recently, 3,3′-bisdemethylpinoresinol (129, Fig. 17) and americanin A (130, Fig. 17) from Morinda citrifolia206 and sulforaphane (131, Fig. 17) from broccoli207 were reported as melanogenesis inhibitors by reducing TYR expression, which was resulted from the downregulation of MITF. The detailed mechanistic study with the respect to the melanogenesis downregulation found that they were inhibiting p38 phosphorylation. Nobiletin (132, Fig. 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.

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Figure 17. SCF/c-kit/EDNs and Wnt/β-Catenin mediated Signaling Pathway melanogenesis inhibitors

Withaferin A (133, Fig. 17) is a naturally occurring compound from Withania somnifera extract (WSE) inhibited melanogenesis by interfering both EDN-1 and SCF-triggered intracellular signaling cascades.209 Astaxanthin (134, Fig.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 are belonging to the vitamin E family and there are four types (α, β, γ and δtocotrienols). δ-Tocotrienol (135, δT3, Fig. 16) significantly inhibited the melanin production and the reactive oxygen species (ROS) in B16 cells without a toxicity.210 Furthermore, the inhibitory 44 ACS Paragon Plus Environment

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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 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 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, Fig. 16), a chalcone isolated from Aplinia katsumadai, and fingolimod (FTY720, 137, Fig. 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 138-140 were inhibited melanin synthesis with IC50 values of 30, 34 and 89 nM, respectively. Recently, labdane diterpenoid andrographolide (141, Fig. 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.

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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 rate-limiting 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 source have been identified, but only a few of them reached to 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 10-fold 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) than 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 overproducing the active hTYR using baculovirus expression vector system in high five cells in high yield and pure form,

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which would be useful for both crystallization screening and for high-throughput screening of skin whitening agents in the future. In general, it is important that designing cosmetic products with effective anti-oxidant activity, which plays an important role in reducing aging effects. Recently there has been growing number of melanogenesis inhibitors that have been associated with anti-oxidant 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-saving, drug availability, safety/tolerability and cost-effective. Several anti-tuberculosis and anti-thyroids drugs were proven potent tyrosinase inhibitors without causing significant cytotoxicity to the cells. Therefore, further studies in advancing as whitening agents must be done on 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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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.

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

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.

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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 for continuing his research. Currently, he is a Senior Research Scientist at the Department of 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, USA, in 1984. He served as a postdoctoral fellow at the University of Pittsburg until 1985 and as a Principle investigator of LG life Science 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 (20032004) and President of Institute of Drug Research and Development of Chungnam National

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University (2007-2009). His research interests include antimicrotubule-based anticancer agents, novel inotropes with selective activation of cardiac myosin and melanogenesis inhibitors.

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; EDs, endothelins; 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; SCF, stem cell factor; PAR-2, protease-activated receptor 2; IP3, inositol triphosphate; DAG, diacyglycerol; NO, Nitric oxide; GC, guanylyl cyclase; cGMP, cyclic guanosine monophosphate, ROS, reactive oxygen species; CAGR, compound annual growth rate.

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Journal of Medicinal Chemistry

Table of Contents Graphic (TOC)

Tyrosinase inhibition EDN-1 mediated pathway

MAPK pathway

cAMP pathway Melanin Inhibition

Wnt/βcatenin pathway

PI3K/Akt pathway

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