Article pubs.acs.org/JAFC
Discovery of Potent Cysteine-Containing Dipeptide Inhibitors against Tyrosinase: A Comprehensive Investigation of 20 × 20 Dipeptides in Inhibiting Dopachrome Formation Tien-Sheng Tseng,†,∥ Keng-Chang Tsai,‡,∥ Wang-Chuan Chen,§,#,∥ Yeng-Tseng Wang,⊥ Yu-Ching Lee,Δ,⊗ Chung-Kuang Lu,‡ Ming-Jaw Don,‡ Chang-Yu Chang,Π Ching-Hsiao Lee,Π Hui-Hsiung Lin,†,‡ Hung-Ju Hsu,‡ and Nai-Wan Hsiao*,† †
Institute of Biotechnology, National Changhua University of Education, Changhua, Taiwan National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan § The School of Chinese Medicine for Post Baccalaureate, I-Shou University, Kaohsiung, Taiwan # Department of Chinese Medicine, E-Da Hospital, Kaohsiung, Taiwan ⊥ Department of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Δ The Center of Translational Medicine, Taipei Medical University, Taipei, Taiwan ⊗ Ph.D. Program for Biotechnology in Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan Π Department of Medical Technology, Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli, Taiwan ‡
S Supporting Information *
ABSTRACT: Tyrosinase is an essential copper-containing enzyme required for melanin synthesis. The overproduction and abnormal accumulation of melanin cause hyperpigmentation and neurodegenerative diseases. Thus, tyrosinase is promising for use in medicine and cosmetics. Our previous study identified a natural product, A5, resembling the structure of the dipeptide WY and apparently inhibiting tyrosinase. Here, we comprehensively estimated the inhibitory capability of 20 × 20 dipeptides against mushroom tyrosinase. We found that cysteine-containing dipeptides, directly blocking the active site of tyrosinase, are highly potent in inhibition; in particular, N-terminal cysteine-containing dipeptides markedly outperform the C-terminal-containing ones. The cysteine-containing dipeptides, CE, CS, CY, and CW, show comparative bioactivities, and tyrosine-containing dipeptides are substrate-like inhibitors. The dipeptide PD attenuates 16.5% melanin content without any significant cytotoxicity. This study reveals the functional role of cysteine residue positional preference and the selectivity of specific amino acids in cysteine-containing dipeptides against tyrosinase, aiding in developing skin-whitening products. KEYWORDS: tyrosinase, peptide, melanin, molecular docking, eumelanin formation, dopachrome
■
INTRODUCTION Melanin, which is produced within melanosomes, is the most essential determinant of hair, eye, and skin color in mammals.1 In general, melanin protects the skin from ultraviolet radiation2 and inhibits photocarcinogenesis. Hyperpigmentation, which is caused by melanin overproduction, typically presents as freckles, age spots, uneven coloring, and sometimes melasma and has become a public concern.3 Medicines or medical cosmetics that contain depigments or skin-whitening components are used for treating hyperpigmentation. However, only a few natural and synthetic depigments are used as therapeutic agents because of safety concerns and low whitening bioactivity.4,5 Melanin synthesis in melanocytes, in which tyrosinase plays a critical role, is mediated by several enzymes.6 The pathway of melanin biosynthesis can be divided into two main parts: the production of eumelanin (eumelanogenesis) and the production of pheomelanin (pheomelanogenesis) (Figure 1).7 These two pigments mix to form mixed melanin, the main pigment affecting skin and hair color. During eumelanogenesis, Ltyrosine is converted to 3,4-dihydroxyphenylalanine (DOPA) by © 2015 American Chemical Society
tyrosinase, which then catalyzes the conversion of DOPA to dopaquinone. Dopaquinone, the main determinant in the formation of various melanin pigments, reacts with glutathione (GSH) or cysteine to form glutathionyldopa (GSH-dopa) or cysteineyldopa and eventually forms yellow-red pheomelanin. In the pathway of eumelanin formation, dopaquinone is converted to 5,6-dihydroxyindole-2-carboxylic acid and 5,6-dihydroxyindole intermediates, subsequently causing the formation of brown-black eumelanin. Tyrosinase is the rate-limiting enzyme for melanin synthesis; therefore, we describe the structure of tyrosinase and the details of the mechanism underlying reactions involving tyrosinase. Tyrosinase (monophenol or o-diphenol oxygen oxidoreductase), a copper-containing enzyme widely distributed in microorganisms, plants, and animals,8 catalyzes the hydroxylaReceived: Revised: Accepted: Published: 6181
February 26, 2015 June 12, 2015 June 17, 2015 June 17, 2015 DOI: 10.1021/acs.jafc.5b01026 J. Agric. Food Chem. 2015, 63, 6181−6188
Article
Journal of Agricultural and Food Chemistry
Figure 1. Biosynthetic pathway of melanin involving mushroom tyrosinase. Melanin biosynthesis includes eumelanogenesis and pheomelanogenesis that generate eumelanin and pheomelanin, respectively.
product, A5,36 that exhibits an apparent tyrosinase inhibitory ability. Moreover, the structure of A5 resembled that of the dipeptide WY. Therefore, in this study, we synthesized 20 × 20 dipeptides to comprehensively investigate their tyrosinase inhibitory ability. We found that cysteine-containing dipeptides exhibit an outstanding inhibitory potency against tyrosinase among all others. The tyrosine moiety of tyrosine-containing dipeptides competes with the substrate binding site of tyrosinase to reduce tyrosinase activity. Dipeptides (CA, YC, PD, and DY) exhibiting divergent inhibition against tyrosinase were subjected to a cell viability assay and melanin content assessment to study their effects on cytotoxicity and depigmentation. In addition, to identify dipeptides with the strongest tyrosinase inhibitory potency, the IC50 values of cysteine-containing dipeptides, which are superior to those of other dipeptides, were calculated. This study reveals the functional roles of the cysteine residue positional preference and the selectivity of specific amino acids of dipeptides for inhibiting tyrosinase. The results can be applied in developing inhibitors for interrupting melanin biosynthesis.
tion of tyrosine to DOPA and the oxidation of DOPA to dopaquinone during melanin formation from tyrosine.9 In plants, tyrosinase is responsible for browning, which occurs after the long-term storage of fruits and vegetables.10 In animals, tyrosinase is responsible for skin and hair pigmentation and skin anomalies, namely, hypo- (vitiligo) or hyper- (freckles or flecks) pigmentation.11,12 Moreover, tyrosinase may play a role in neurodegenerative diseases (e.g., Parkinson’s disease) and cancer.13,14 Therefore, tyrosinase is a crucial topic in the fields of medicine, agriculture, and cosmetics, and considerable attention has been paid to developing and screening for tyrosinase inhibitors.15 Some tyrosinase inhibitors, such as hydroquinone,16,17 arbutin,18 kojic acid,19,20 azelaic acid,21,22 and electron-rich phenols,23 have been used in cosmetics. However, only a few such inhibitors are sufficiently potent for practical use and are in compliance with safety regulations. Therefore, continual efforts have been exerted to obtain tyrosinase inhibitors synthetically (in laboratories) 24−26 and naturally (through plant extractions).27−29 Recently, we used phage-displayed tetrapeptide inhibitors30 and a T1 compound from Gastrodia elata,31 and other researchers have employed peptide-derived compounds for tyrosinase inhibition.4,32,33 Certain short-sequence oligopeptides, P3 and P4, inhibit mushroom tyrosinase with no cytotoxicity to human melanocyte.32 The tyrosinase inhibitory potency of kojic acid−tripeptide is superior to that of kojic acid.34 In addition, a mimosine tetrapeptide, mimosine−FFY, exhibits a marked tyrosinase inhibitory ability when the half-maximal inhibitory concentration (IC50) is 18.3 μM.33 Peptides are reported to bind with exact specificity to their in vivo targets, resulting in an exceptionally high potency of action and relatively few off-target side effects.35 Thus, tyrosinase inhibitors have great potential for use as skin-whitening components in medicine and cosmetics. In a previous study, we established a reasonable and reliable pharmacophore model for screening potent natural products for mushroom tyrosinase inhibition. We identified a potent natural
■
MATERIALS AND METHODS
Materials. Molecular docking studies were conducted using Genetic Optimization for Ligand Docking (GOLD; GlaxoSmithKline PLC, Astex Technology Ltd., Cambridge Crystallographic Data Centre). All calculations were performed at the National Center for HighPerformance Computing in Taiwan. Mushroom tyrosinase, L-tyrosine, and a potassium phosphate buffer (PBS, NaH2PO4−Na2HPO4, 67 mM, pH 6.8) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All 400 peptides (purity = 0.95%) used in this study were customsynthesized by Kelowna International Scientific, Inc. (New Taipei City, Taiwan). Methods. Examination of the Inhibitory Potency of Dipeptides against Tyrosinase. A tyrosinase inhibition assay was performed, according to a previously reported method,36 on the synthesized 20 × 20 dipeptides to investigate their effects on tyrosinase activity.37 In brief, 25 μL of a 5 mM dipeptide solution, 125 μL of a 5 mM L-tyrosine, and 80 μL of a 67 mM PBS were mixed and added to each well of a 96-well enzymelinked immunosorbent assay plate, which was incubated at 25 °C for 5 min. Subsequently, 20 μL of a 1250 U/mL mushroom tyrosinase 6182
DOI: 10.1021/acs.jafc.5b01026 J. Agric. Food Chem. 2015, 63, 6181−6188
Article
Journal of Agricultural and Food Chemistry
Figure 2. 20 × 20 dipeptide-based tyrosinase inhibition matrix. The comprehensive pre-estimation of the inhibitory ability of 20 × 20 dipeptides against mushroom tyrosinase is summarized in the matrix. The Y-axis represents the amino acids that constitute the N terminus of the dipeptides, and the X-axis represents the 20 amino acids that constitute the C terminus of the dipeptides. According to the chemical properties of the side chains, 20 amino acids are divided into eight categories: (1) G; (2) nonpolar aliphatic amino acids, A, V, L, I, and P; (3) nonpolar aromatic amino acids, F, W, and Y; (4) polar hydroxyl amino acids, S and T; (5) sulfur amino acids, C and M; (6) amidic amino acids, N and Q; (7) polar negative amino acids, D and E; and (8) polar positive amino acids, R, H, and K. The numbers in each small square with different colors are inhibition indices, which represent the distinct levels of tyrosinase activities. The scale of inhibition indices is from 0 (complete abolished tyrosinase activity; red) to 1 (full tyrosinase activity; blue). An asterisk denotes that the dipeptides are not completely dissolved in ddH2O. The dipeptides that change the color of the reaction solution and therefore might interfere with the results are marked with a dotted pattern. solution was added to each well; thus, the final volume in each well was 250 μL. The plate was incubated again at 25 °C for 5 min. In addition, the same concentrations of kojic acid and β-arbutin under the same conditions as those in the wells were used as controls. The amount of dopachrome produced was determined against a blank (solution without tyrosinase) and recorded using a spectrophotometer (Varian Cary 50 Bio UV−visible spectrophotometer) at 475 nm every 10 s for 10 min. The amount of dopachrome produced in the reaction solution was determined using a previously described method.37 The tyrosinase activity was calculated using the equation tyrosinase activity % =
(OD405) of supernatant in each cell of the plate was measured using a microplate reader (Molecular Devices Spectra Max M2). Molecular Modeling. Models of the dipeptides in complex with tyrosinase were built using molecular docking analysis. GOLD (version 5.2) featuring the Goldscore scoring function was used to predict the positions of dipeptides at the active sites. All cofactors and water molecules were removed before docking. Three-dimensional (3D) structures of the desired dipeptides were generated and optimized through energy minimization by using Discovery Studio version 4.0 (Accelrys Software, Inc., San Diego, CA, USA) and further docked into the active sites of mushroom tyrosinase (PDB ID 2Y9X) by turning on the flexible docking option. Initially, 100 genetic algorithm cycles of computation were performed with ligand torsion angles varying between −180° and 180°. The search efficiency was set at 200% to explore the docking conformational space. All other parameters were applied as the default settings. Finally, from the 100 docking conformations of each dipeptide, the conformation with the highest GOLD fitness score was examined to investigate its binding with the mushroom tyrosinase active site by using Goldscore. The molecular models of the dipeptide−tyrosinase complexes were presented using PyMOL software (http://www.pymol.org).
⎛S − B⎞ ⎜ ⎟ × 100 ⎝ C ⎠
where S is the OD475 absorbance of the test samples, B is the OD475 absorbance of the blank, and C is the OD475 absorbance of controls. The IC50 of cysteine-containing dipeptides was determined by performing dose-dependent inhibition experiments in triplicate. Cell Viability and Melanin Quantification Assay. Normal human epidermal melanocytes were purchased from Cascade Biologics (Portland, OR, USA) and cultured in Medium 254 supplemented with human melanocyte growth supplement (Cascade Biologics). During the experiment, confluent cells were trypsinized and suspended in melanocyte growth medium M2 containing 2 × 105 cells/mL. The cells were then placed in 96-well plates (2 × 104 cells/well) for the cell viability assay and in 24-well plates (1 × 105 cells/well) for the melanin content assay. After 24 h, the cells were treated with arbutin (50 μM, 2.5 mM), kojic acid (50 μM, 500 μM), and 1, 10, and 100 μM CA, YC, PD, and DY. The cells were then incubated at 37 °C for 72 h. Cell viability was determined using the MTT assay.38,39 For measuring the melanin content, the melanocyte pellets were dissolved in 1 N hot NaOH (70 °C) for 1 h and centrifuged for 10 min at 10000g. The optical density
■
RESULTS Comprehensive Investigation of Inhibitory Abilities of 20 × 20 Dipeptides against Tyrosinase. In our previous study, we found a natural product, A5,36 that exhibits an apparent tyrosinase inhibitory ability. Moreover, the structure of A5 resembles that of dipeptide WY. To investigate the inhibitory potency of dipeptides against mushroom tyrosinase, 400 dipeptides were constructed and subjected to the tyrosinase inhibition assay. Their inhibitory abilities were pre-estimated and 6183
DOI: 10.1021/acs.jafc.5b01026 J. Agric. Food Chem. 2015, 63, 6181−6188
Article
Journal of Agricultural and Food Chemistry summarized in a 20 × 20 dipeptide matrix (Figure 2). The result showed that 5 mM cysteine-containing dipeptides completely ceased the activity of tyrosinase (normalized percentage inhibition index is 0.0−0.3; Figure 2). In addition, 5 mM PD, DY, and YK reduced the activity of tyrosinase by 60−70%. However, dipeptides with a tyrosine residue located at the C or N terminus exerted moderate effects on tyrosinase inhibitory formation efficiency; their normalized inhibition index was approximately 0.5−0.7. The remaining dipeptides showed little or no inhibition against tyrosinase, and some did not even completely dissolve in double-distilled (dd) H2O (46 peptides labeled with asterisks). Twenty-seven dipeptides (the color of the reaction solution changed; therefore, these might have interfered with the result) are denoted with a dotted pattern (Figure 2). Cell Viability and Melanin Quantification Assay. According to the 20 × 20 dipeptide matrix for the inhibition of tyrosinase (Figure 2), we randomly selected four dipeptides, two cysteine-containing dipeptides (CA and YC) and two noncysteine-containing dipeptides (PD and DY), to estimate their effects on cell viability and melanogenesis. The cell viability of human normal melanocytes treated with 1.0, 10, and 100 μM dipeptides (CA, YC, PD, and DY) is shown in Figure 3; no
PD attenuated 5.6 and 16.5% melanin content, respectively, at 100 μM; notably, PD could attenuate 20−30% melanin content at 400 μM. However, YC and DY showed little or no antimelanogenesis effects. Determination of Inhibitory Potency (IC50) against Tyrosinase. The pre-estimation of the effects of dipeptides on tyrosinase inhibition demonstrated that cysteine-containing dipeptides markedly reduced the activity of tyrosinase. We further determined the IC50 to precisely investigate and compare the functional variations. The IC50 values of N-terminal cysteinecontaining dipeptides are shown in Table 2. According to the table, most of the values ranged from 2.0 to 22.6 μM, showing the effective inhibitory potency, except for CP (IC50 = 55.8 μM). In addition, CS, CY, CF, CC, CI, CM, and CQ considerably inhibited tyrosinase; the IC50 was 6-fold) compared with EC, SC, YC, AC, and WC. To elucidate the functional deviations of these cysteinecontaining dipeptides, we further analyzed the structural interactions of these dipeptides with mushroom tyrosinase by using molecular modeling. Dipeptides CE, CS, CY, CA, and CW and their reverse forms (EC, SC, YC, AC, and WC) were docked into the active sites of mushroom tyrosinase (PDB ID 2Y9X), and their detailed molecular interactions were analyzed (Supporting Information Table S1). The result showed that the cysteine residues of all dipeptides most likely contacted closely with the binuclear copper ions at the active sites of tyrosinase, regardless of their location (N or C terminus; Supporting Information Figure S1). In addition, CE, with the most profound tyrosinase inhibitory ability, showed extensive strong molecular interactions with tyrosinase (Supporting Information Figure S2 and Table S1). The electrostatic and cation−π interactions supported by residues His244, Asn260, Glu256, and Phe264 were commonly observed within the docked CE, CD, CS, CY, CA, and CW. In addition, hydrogen bonds and hydrophobic interactions were observed among CE, CS, CY, and CW active-site residues Arg268, Gly281, Ser282, and Val283 (Supporting Information Table S1). Molecular modeling of CD was performed to clarify its functional divergence with CE. The result showed that only four of six molecular interactions were observed between tyrosinase and CD. The molecular interactions of EC, SC, YC, AC, and WC with tyrosinase are summarized in Table S1. All N-terminal cysteine-containing dipeptides exhibited electrostatic attractions with Glu256, hydrogen-bond interactions with Asn260, and hydrophobic interactions with Val283. In addition, the molecular models of tyrosine-containing dipeptides, YK and DY, in complex with mushroom tyrosinase were observed (Figure 4). The tyrosine moiety of YK and DY went deep into the substrate binding pocket, and π−π interactions were observed between the phenol ring of tyrosine and the imidazole ring of His263. The lysine moiety of YK interacted with Phe264 and Arg268 through cation−π interactions and electrostatic interactions, respectively (Figure 4D). The dipeptide DY interacted with H85, Ser282, and
Figure 3. Effects of arbutin, kojic acid, and dipeptides CA, YC, PD, and DY on cell viability.
significant cell death was observed under these conditions compared with the controls. The depigmenting effects of these dipeptides were estimated under the same conditions as those of the cell viability assay (Table 1). The results revealed that CA and Table 1. Effects of Dipeptides CA, YC, PD, and DY on Melanogenesis in Normal Human Epidermal Melanocytes melanin content (%) peptide ID
1 μM
CA 94.5 ± 1.8 YC 85.9 ± 3.3 PD 88.6 ± 1.1 DY 91.1 ± 3.4 kojic acid, 50 μM kojic acid, 500 μM β-arbutin, 50 μM β-arbutin, 2.5 mM
10 μM
100 μM
93.4 ± 2.9 90.2 ± 5.7 87.0 ± 2.1 92.8 ± 2.4 97.7 ± 1.8 91.6 ± 1.0 102.1 ± 4.6 90.6 ± 1.8
94.4 ± 1.8 109.7 ± 1.8 83.5 ± 1.2 102.9 ± 1.3
400 μM
71.5 ± 1.5
6184
DOI: 10.1021/acs.jafc.5b01026 J. Agric. Food Chem. 2015, 63, 6181−6188
Article
Journal of Agricultural and Food Chemistry
Table 2. Comparison of Tyrosinase Inhibitory Ability (IC50) among N-Terminal Cysteine-Containing Dipeptides (Cx) and CTerminal Cysteine-Containing Dipeptides (xC) in ddH2O for 10 and 30 mina
a
dipeptide
IC50
SEM
xC/Cx
dipeptide
IC50
SEM
xC/Cx
CE EC* CS SC CY YC* CF FC CC CC CI IC CM MC CQ QC CK KC CV VC
2.0 140.1 4.5 28.3 3.1 131.6 2.7 7.9 3.2 3.2 4.0 4.5 4.9 10.7 3.5 5.9 5.9 39.9 8.2 8.3
0.3 16.7 2.8 5.4 0.7 26.4 0.2 1.3 0.4 0.4 0.9 0.5 0.8 1.6 1.0 0.3 0.5 2.4 2.1 1.5
70.7
CG GC CR RC CW WC CL LC CA AC CT TC* CH HC* CN NC CD DC* CP* PC
5.9 24.6 8.0 32.3 5.4 47.3 8.0 14.5 9.6 62.2 8.2 44.0 10.7 20.0 22.6 5.3 14.1 56.1 55.8 20.3
2.5 8.5 3.0 12.9 1.5 2.5 2.4 2.1 5.7 17.1 2.1 15.3 2.1 6.9 8.8 0.8 4.6 8.9 4.6 1.0
4.1
6.3 42.4 3.0 1.0 1.1 2.2 1.7 6.8 1.0
4.0 8.8 1.8 6.5 5.3 1.9 0.2 4.0 0.4
SEM denotes the standard error of mean. An asterisk represents the dipeptides that did not completely dissolve in ddH2O.
The functional variations resulting from the positional preference of the cysteine residue and the selectivity of specific amino acids among N- and C-terminal cysteine-containing dipeptides were further analyzed using molecular modeling. According to the models, the cysteine residue most likely coordinates with the binuclear copper ions through the sulfur atom of the cysteine residue, regardless of its location (N or C terminus; Figure S1). In addition, one anion−π interaction, one hydrogen bond, and two electrostatic forces were frequently observed in all N-terminal cysteine-containing dipeptides for tyrosinase interactions (Table S1). However, only one hydrogen bond, one hydrophobic bond, and one electrostatic interaction were frequently observed in all C-terminal cysteine-containing dipeptides for tyrosinase interaction. Thus, these variations may mainly cause a functional divergence between N- and C-terminal cysteine-containing dipeptides in the inhibition of tyrosinase. The different inhibitory efficacies of the N-terminal cysteinecontaining dipeptides against tyrosinase can be explained by their corresponding interactions with tyrosinase. For example, CE and CY display a maximum of six molecular interactions with tyrosinase; therefore, they show the strongest inhibitory ability (Figure S1 and Table S1), and CA, featuring the fewest molecular interactions with tyrosinase, exhibits the weakest inhibitory potency (Table S1). Similar explanations can be applied to interpret the diverse inhibitory efficiency of C-terminal cysteinecontaining dipeptides, except for EC and YC; their inferior inhibitory ability compared with that of SC and AC may result from incomplete dissolution in ddH2O. Moreover, the glutamic acid residue of CE, with a longer carboxyl side chain than that of aspartic acid, results in four extra molecular interactions with the active-site residues of tyrosinase (Figure S1A and Table S1), causing a stronger inhibitory ability than that of CD. Furthermore, the tryptophan residue of CW additionally contacts tyrosinase (one electrostatic interaction with His224, one hydrogen bond with the main chain of Gly281, and an extra hydrophobic interaction with Phe264), rendering it more efficient than WC in the inhibition of tyrosinase.
Val283 through electrostatic attractions, hydrogen bonding, and hydrophobic interactions, respectively (Figure 4C).
■
DISCUSSION According to our previous finding that a natural compound, A5,36 which possesses potent tyrosinase inhibitory ability, resembles the structure of dipeptide WY, dipeptides have great potential for tyrosinase inhibitor development. In this study, we comprehensively estimated 20 × 20 dipeptides for mushroom tyrosinase inhibition, and the results revealed that cysteine-containing dipeptides significantly inhibit tyrosinase. The IC50 of cysteinecontaining dipeptides was determined for investigating the positional preference of the cysteine residue and the selectivity of functional amino acids against tyrosinase. The results revealed that N-terminal cysteine-containing dipeptides outperformed the C-terminal cysteine-containing dipeptides in inhibiting tyrosinase, and CE exhibited the strongest inhibitory potency (IC50 = 2.0 μM) (Table 2). In addition, most of the cysteinecontaining dipeptides exhibited modest divergence in their inhibitory abilities when the cysteine residue was reversed at the terminus. By contrast, the inhibitory abilities of CE, CS, CY, CA, and CW decreased >6-fold when the cysteine residues shifted from the N terminus to the C terminus. Furthermore, an extensively attenuated inhibition of tyrosinase was observed; the inhibitory potencies of CY−YC and CE−EC were reduced 42.4and 70.7-fold, respectively. These results indicate that the glutamic acid residue (E) is more functional at the C terminus than at the N terminus of dipeptides for inhibition, and the hydroxyl groups of serine and tyrosine residues at the C terminus, not the N terminus, considerably contribute to the inhibitory ability. Moreover, the bulk indole ring of the tryptophan residue located at the C terminus of dipeptides is more bioactive in inhibiting tyrosinase than that located at the N terminus. This study not only demonstrates the importance of the positional preference of the cysteine residue but also reveals the selectivity of the functional amino acids of cysteinecontaining dipeptides for inhibiting tyrosinase. 6185
DOI: 10.1021/acs.jafc.5b01026 J. Agric. Food Chem. 2015, 63, 6181−6188
Article
Journal of Agricultural and Food Chemistry
Figure 4. Molecular models of dipeptides DY and YK in complex with mushroom tyrosinase. (A) Docking model of the dipeptide DY (cyan) at the active sites of mushroom tyrosinase. (B) Docking model of the dipeptide YK (yellow) in complex with mushroom tyrosinase. The residues interacting with dipeptides DY and YK are denoted by white and green (six histidine residues), respectively. Green, magenta, and cyan dashed lines represent the Hbonding, electrostatic, and anion−π interactions, respectively, and yellow lines represent the hydrophobic interactions. (C) 2D schematic of the interactions between the dipeptide DY and active-site residues of mushroom tyrosinase. (D) 2D schematic of the interactions between the dipeptide YK and active-site residues of mushroom tyrosinase. In 2D schemes, green and magenta dashed lines denote H-bonding and electrostatic interactions, respectively; circles in distinct colors represent the relative hydrophobic and/or π−π interactions among the dipeptide and active-site residues of mushroom tyrosinase, respectively.
In this study, the inhibitory abilities of dipeptides were investigated in the presence of tyrosine as the tyrosinase substrate, and dipeptide activity was evaluated for dopachrome formation. Therefore, the inhibition of tyrosinase activity could be accomplished by one of the following: (a) reducing agents, leading to the reduction of dopaquinone; (b) o-dopaquinone scavengers, such as thiol-containing compounds; (c) alternative enzyme substrates−phenolic compounds; (d) nonspecific enzyme inactivators, such as acids or bases; or (e) specific tyrosinase inactivator−mechanism-based inhibitors, also called suicide substrates.7 Cysteine-containing dipeptides are similar to o-dopaquinone scavengers, which react with dopaquinone, causing the formation of thiol−dopa conjugates. In vivo, cysteine and GSH are natural thiol compounds that form pheomelanin instead of eumelanin.40 Similarly, cysteine-containing dipeptides have the potential to form dopaquinone conjugates, thus attenuating dopachrome formation, abolishing eumelanin synthesis, and increasing the pheomelanin amount (Figure 1). The structural components of CE, the most potent dipeptide in the inhibition of dopachrome formation, exhibit similarities with those of GSH (Figure 1). Thus, we proposed that cysteine-
containing dipeptides can either interact with the active sites of tyrosinase or act as the o-dopaquinone scavenger to inhibit dopachrome formation. However, this was disproved by our HPLC and mass spectrometry analysis results (Figure S3, Table S2, and Figure S4). To clarify and identify the possible inhibition mechanism of cysteine-containing dipeptides against tyrosinase, we performed HPLC to purify the products of both N-terminal and C-terminal cysteine-containing dipeptides that reacted with tyrosinase. In addition, mass spectrometry was employed to determine the precise molecular weight of the purified samples. The results showed that none of the peaks corresponding to cysteinyldopa-peptidesthe products resulting from the reactions of cysteine-containing dipeptides interacting with dopaquinonewas observed in the HPLC profile, and no molecular weight equilibrant to cysteinyldopa-peptide was identified using mass spectrometry (Figure S4). These results reveal that cysteine-containing dipeptides most likely bind and inhibit tyrosinase directly, instead of interacting with dopaquinone to form cysteinyldopa-peptide. However, the tyrosine-containing dipeptides possibly belong to the alternative enzyme substrate, where the tyrosine entity can 6186
DOI: 10.1021/acs.jafc.5b01026 J. Agric. Food Chem. 2015, 63, 6181−6188
Journal of Agricultural and Food Chemistry
■
compete with the tyrosine substrate at the active site of tyrosinase. To elucidate the inhibition mechanism of tyrosinecontaining dipeptides against tyrosinase, CY, DY, YC, and YK were directly reacted with tyrosinase in the absence of a substrate (tyrosine); the absorbance at 400 and 475 nm of the products was then evaluated. The result showed that the products obtained from the reactions of CY and YC interacting with tyrosinase exhibited no absorption at 400 and 475 nm (Figure S5A), respectively, indicating that none of the products belongs to a dopachrome-containing and/or dopaquinone-containing peptide. This proved that cysteine-containing dipeptides are inhibitors that use the cysteine moiety mainly to interact with the active site of tyrosinase instead of using the tyrosine. The products from DY and YK showed weak absorption at 400 nm but not at 475 nm (Figure S5B), indicating that the tyrosinecontaining dipeptides are substrate-like inhibitors subject to further catalysis by tyrosinase to form dopaquinone-containing dipeptide. These results clarify that the tyrosine-containing dipeptides act as substrate binding at the active site of tyrosinase, thus exhibiting varying inhibitory efficacies against tyrosinase. When these tyrosine-containing dipeptides exhibit a favorable affinity with tyrosinase, dopachrome formation is attenuated. In addition, approximately 27 tyrosine-containing dipeptides that changed the color of the tyrosinase reaction solutions were observed. Therefore, free tyrosine can react with tyrosinase and form dopachrome. However, tyrosine in a dipeptide can be oxidized by tyrosinase, thus inhibiting dopachrome formation. Conventional pharmaceutical skin-whitening drugs, such as hydroquinone,16,17 arbutin,18 and kojic acid,19,20 are insufficient because they are reported to be severely carcinogenic and damage the structural architecture of several tissues.41−43 Moreover, an intriguing recent story was published on a skinwhitening rhododendrol, which has been withdrawn from the cosmetic market because of its toxicity.44 Recently, several studies have discovered novel proteins and peptides from natural resources, such as the housefly,45 silk,46 honey,47 wheat,48 and milk,49 for tyrosinase inhibition. In particular, cyclic peptides,50 short-sequence oligopeptides,32 kojic acid tripeptide,34 mimosine tetrapeptides,33 and octameric peptides15 have been thoroughly investigated to determine their tyrosinase inhibitory ability. The current study is the first on the systematical annotation of critical amino acids that constitute potent dipeptides for tyrosinase inhibition in dopachrome formation. The cytotoxicity and depigmenting effects of cysteine-containing YC and CA, as well as the less potent noncysteine-containing PD and DY, were examined using cell viability and melanin quantification assays. No significant cytotoxicity was observed in these dipeptide-treated melanocytes, and CA and PD attenuated 5.6 and 16.5% melanin content, respectively, at 100 μM. In addition, the cells treated with dipeptide PD (400 μM) could reduce 20−30% of melanin content. This indicates that the inhibitory effects of CA and PD on melanin production are not attributable to their cytotoxicity, and CA and PD can be safely used in skin-whitening products without influencing melanocyte growth. In conclusion, this study provides insight into the functional role of the cysteine residue positional preference and the selectivity of specific amino acids of cysteine-containing dipeptides for the inhibition of tyrosinase. Therefore, the findings can be applied in developing new inhibitors against tyrosinase.
Article
ASSOCIATED CONTENT
S Supporting Information *
Five figures (S1, S2, S3, S4, and S5) and two tables (S1 and S2). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b01026.
■
AUTHOR INFORMATION
Corresponding Author
*(N.-W.H.) Phone: +886-4-7232105, ext. 3421; Fax: +886-47211156. E-mail:
[email protected]. Author Contributions ∥
T.-S.T., K.-C.T., and W.-C.C. contributed equally to this work.
Funding
This study was supported by the National Research Institute of Chinese Medicine, Ministry of Health and Welfare (MM102110153 and MM10401-0394) and the Ministry of Science and Technology (MOST 103-2320-B-077-001-MY3 to K.-C.T.), Taipei, Taiwan. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We thank Chia-Ying Wu, Jin-Yu Shiu, and Chia-Chia Chang (Institute of Biotechnology, National Changhua University of Education, Changhua, Taiwan) for their input and assistance. In addition, we are grateful to the National Center for HighPerformance Computing for use of computers and facilities. The computational work of GOLD and Discovery Studio was conducted at the National Center for High-Performance Computing, Taiwan.
■
REFERENCES
(1) Lee, H. E.; Kim, E. H.; Choi, H. R.; Sohn, U. D.; Yun, H. Y.; Baek, K. J.; Kwon, N. S.; Park, K. C.; Kim, D. S. Dipeptides Inhibit Melanin Synthesis in Mel-Ab Cells through Down-Regulation of Tyrosinase. Korean J. Physiol. Pharmacol. 2012, 16, 287−291. (2) Brenner, M.; Hearing, V. J. The protective role of melanin against UV damage in human skin. Photochem. Photobiol. 2008, 84, 539−549. (3) Schallreuter, K.; Slominski, A.; Pawelek, J. M.; Jimbow, K.; Gilchrest, B. A. What controls melanogenesis? Exp. Dermatol. 1998, 7, 143−150. (4) Wang, H. M.; Chen, C. Y.; Wen, Z. H. Identifying melanogenesis inhibitors from Cinnamomum subavenium with in vitro and in vivo screening systems by targeting the human tyrosinase. Exp. Dermatol. 2011, 20, 242−248. (5) Lo, C. Y.; Liu, P. L.; Lin, L. C.; Chen, Y. T.; Hseu, Y. C.; Wen, Z. H.; Wang, H. M. Antimelanoma and antityrosinase from Alpinia galangal constituents. Sci. World J. 2013, 2013, No. 186505. (6) 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. (7) Chang, T. S. An updated review of tyrosinase inhibitors. Int. J. Mol. Sci. 2009, 10, 2440−2475. (8) Seo, S. Y.; Sharma, V. K.; Sharma, N. Mushroom tyrosinase: recent prospects. J. Agric. Food Chem. 2003, 51, 2837−2853. (9) Passi, S.; Nazzaro-Porro, M. Molecular basis of substrate and inhibitory specificity of tyrosinase: phenolic compounds. Br. J. Dermatol. 1981, 104, 659−665. (10) Marusek, C. M.; Trobaugh, N. M.; Flurkey, W. H.; Inlow, J. K. Comparative analysis of polyphenol oxidase from plant and fungal species. J. Inorg. Biochem. 2006, 100, 108−123. (11) Katsambas, A. D.; Stratigos, A. J. Depigmenting and bleaching agents: coping with hyperpigmentation. Clin Dermatol 2001, 19, 483− 488.
6187
DOI: 10.1021/acs.jafc.5b01026 J. Agric. Food Chem. 2015, 63, 6181−6188
Article
Journal of Agricultural and Food Chemistry
inhibitory activities on neuraminidase and tyrosinase. J. Agric. Food Chem. 2011, 59, 12858−12863. (34) Noh, J. M.; Kwak, S. Y.; Kim, D. H.; Lee, Y. S. Kojic acid-tripeptide amide as a new tyrosinase inhibitor. Biopolymers 2007, 88, 300−307. (35) Craik, D. J.; Fairlie, D. P.; Liras, S.; Price, D. The future of peptidebased drugs. Chem. Biol. Drug Des. 2013, 81, 136−147. (36) Hsiao, N. W.; Tseng, T. S.; Lee, Y. C.; Chen, W. C.; Lin, H. H.; Chen, Y. R.; Wang, Y. T.; Hsu, H. J.; Tsai, K. C. Serendipitous discovery of short peptides from natural products as tyrosinase inhibitors. J. Chem. Inf. Model. 2014, 54, 3099−3111. (37) Takahashi, M.; Takara, K.; Toyozato, T.; Wada, K. A novel bioactive chalcone of Morus australis inhibits tyrosinase activity and melanin biosynthesis in B16 melanoma cells. J. Oleo Sci. 2012, 61, 585− 592. (38) Kumar, K. J.; Yang, J. C.; Chu, F. H.; Chang, S. T.; Wang, S. Y. Lucidone, a novel melanin inhibitor from the fruit of Lindera erythrocarpa Makino. Phytother. Res. 2010, 24, 1158−1165. (39) Wang, K. H.; Lin, R. D.; Hsu, F. L.; Huang, Y. H.; Chang, H. C.; Huang, C. Y.; Lee, M. H. Cosmetic applications of selected traditional Chinese herbal medicines. J. Ethnopharmacol. 2006, 106, 353−359. (40) del Marmol, V.; Ito, S.; Bouchard, B.; Libert, A.; Wakamatsu, K.; Ghanem, G.; Solano, F. Cysteine deprivation promotes eumelanogenesis in human melanoma cells. J. Invest. Dermatol. 1996, 107, 698− 702. (41) McGregor, D. Hydroquinone: an evaluation of the human risks from its carcinogenic and mutagenic properties. Crit. Rev. Toxicol. 2007, 37, 887−914. (42) Philips, N.; Burchill, D.; O’Donoghue, D.; Keller, T.; Gonzalez, S. Identification of benzene metabolites in dermal fibroblasts as nonphenolic: regulation of cell viability, apoptosis, lipid peroxidation and expression of matrix metalloproteinase 1 and elastin by benzene metabolites. Skin Pharmacol. Physiol. 2004, 17, 147−152. (43) Charlin, R.; Barcaui, C. B.; Kac, B. K.; Soares, D. B.; RabelloFonseca, R.; Azulay-Abulafia, L. Hydroquinone-induced exogenous ochronosis: a report of four cases and usefulness of dermoscopy. Int. J. Dermatol. 2008, 47, 19−23. (44) Ito, S.; Ojika, M.; Yamashita, T.; Wakamatsu, K. Tyrosinasecatalyzed oxidation of rhododendrol produces 2-methylchromane-6,7dione, the putative ultimate toxic metabolite: implications for melanocyte toxicity. Pigm. Cell Melanoma Res. 2014, 27, 744−753. (45) Daquinag, A. C.; Sato, T.; Koda, H.; Takao, T.; Fukuda, M.; Shimonishi, Y.; Tsukamoto, T. A novel endogenous inhibitor of phenoloxidase from Musca domestica has a cystine motif commonly found in snail and spider toxins. Biochemistry 1999, 38, 2179−2188. (46) Kato, N.; Sato, S.; Yamanaka, A.; Yamada, H.; Fuwa, N.; Nomura, M. Silk protein, sericin, inhibits lipid peroxidation and tyrosinase activity. Biosci., Biotechnol., Biochem. 1998, 62, 145−147. (47) Ates, S.; P, S.; Cokmus, C. Partial characterization of a peptide from honey that inhibits mushroom polyphenol oxidase. J. Food Biochem. 2001, 25, 127−137. (48) Okot-Kotber, M.; L, A.; Yong, K. J.; Bagorogoza, K. Activity and inhibition of polyphenol oxidase in extracts of bran and other milling fractions from a variety of wheat cultivars. Cereal Chem. 2001, 78, 514− 520. (49) Chen, M. J.; L, J.; Sheu, J. F.; Lin, C. W.; Chuang, C. L. Study on skin care properties of milk kefir whey. Asian-Australas. J. Anim. Sci. 2006, 19, 905−908. (50) Morita, H.; Kayashita, T.; Kobata, H.; Gonda, A.; Takeya, K.; Itokawa, H. Pseudostellarins D−F, new tyrosinase inhibitory cyclic peptides from Pseudostellaria heterophylla. Tetrahedron 1994, 50, 9975− 9982.
(12) Solano, F.; Briganti, S.; Picardo, M.; Ghanem, G. Hypopigmenting agents: an updated review on biological, chemical and clinical aspects. Pigm. Cell Res. 2006, 19, 550−571. (13) Asanuma, M.; Miyazaki, I.; Ogawa, N. Dopamine- or L-DOPAinduced neurotoxicity: the role of dopamine quinone formation and tyrosinase in a model of Parkinson’s disease. Neurotoxic. Res. 2003, 5, 165−176. (14) Pan, T.; Li, X.; Jankovic, J. The association between Parkinson’s disease and melanoma. Int. J. Cancer 2011, 128, 2251−2260. (15) Schurink, M.; van Berkel, W. J.; Wichers, H. J.; Boeriu, C. G. Novel peptides with tyrosinase inhibitory activity. Peptides 2007, 28, 485−495. (16) Arndt, K. A.; Fitzpatrick, T. B. Topical use of hydroquinine as a depigmenting agent. J. Am. Med. Assoc. 1965, 194, 117−119. (17) Fitzpatrick, T. B.; Arndt, K. A.; el-Mofty, A. M.; Pathak, M. A. Hydroquinone and psoralens in the therapy of hypermelanosis and vitiligo. Arch. Dermatol. 1966, 93, 589−600. (18) Germanas, J. P.; Wang, S.; Miner, A.; Hao, W.; Ready, J. M. Discovery of small-molecule inhibitors of tyrosinase. Bioorg. Med. Chem. Lett. 2007, 17, 6871−6875. (19) Ahn, S. M.; Rho, H. S.; Baek, H. S.; Joo, Y. H.; Hong, Y. D.; Shin, S. S.; Park, Y. H.; Park, S. N. Inhibitory activity of novel kojic acid derivative containing trolox moiety on melanogenesis. Bioorg. Med. Chem. Lett. 2011, 21, 7466−7469. (20) Elsner, P., Maibach, H. I., Eds. Cosmeceuticals: Drugs vs. Cosmetics; CRC Press: Boca Raton, FL, USA, 2000. (21) Breathnach, A. C.; Nazzaro-Porro, M.; Passi, S.; Zina, G. Azelaic acid therapy in disorders of pigmentation. Clin. Dermatol 1989, 7, 106− 119. (22) Rigoni, C.; Toffolo, P.; Serri, R.; Caputo, R. [Use of a cream based on 20% azelaic acid in the treatment of melasma]. G. Ital. Dermatol. Venereol. 1989, 124, I−VI. (23) Jimbow, K. N-Acetyl-4-S-cysteaminylphenol as a new type of depigmenting agent for the melanoderma of patients with melasma. Arch. Dermatol. 1991, 127, 1528−1534. (24) Hamidian, H.; Tagizadeh, R.; Fozooni, S.; Abbasalipour, V.; Taheri, A.; Namjou, M. Synthesis of novel azo compounds containing 5(4H)-oxazolone ring as potent tyrosinase inhibitors. Bioorg. Med. Chem. 2013, 21, 2088−2092. (25) Hamidian, H. Synthesis of novel compounds as new potent tyrosinase inhibitors. BioMed Res. Int. 2013, 2013, No. 207181. (26) Zhu, T. H.; Cao, S. W.; Yu, Y. Y. Synthesis, characterization and biological evaluation of paeonol thiosemicarbazone analogues as mushroom tyrosinase inhibitors. Int. J. Biol. Macromol. 2013, 62, 589− 595. (27) Liang, C.; Lim, J. H.; Kim, S. H.; Kim, D. S. Dioscin: a synergistic tyrosinase inhibitor from the roots of Smilax china. Food Chem. 2012, 134, 1146−1148. (28) Zheng, Z. P.; Tan, H. Y.; Wang, M. Tyrosinase inhibition constituents from the roots of Morus australis. Fitoterapia 2012, 83, 1008−1013. (29) Sarkhail, P.; Sarkheil, P.; Khalighi-Sigaroodi, F.; Shafiee, A.; Ostad, N. Tyrosinase inhibitor and radical scavenger fractions and isolated compounds from aerial parts of Peucedanum knappii Bornm. Nat. Prod. Res. 2013, 27, 896−899. (30) Lee, Y. C.; Hsiao, N. W.; Tseng, T. S.; Chen, W. C.; Lin, H. H.; Leu, S. J.; Yang, E. W.; Tsai, K. C. Phage display-mediated discovery of novel tyrosinase-targeting tetrapeptide inhibitors reveals the significance of N-terminal preference of cysteine residues and their functional sulfur atom. Mol. Pharmacol. 2015, 87, 218−230. (31) Chen, W. C.; Tseng, T. S.; Hsiao, N. W.; Lin, Y. L.; Wen, Z. H.; Tsai, C. C.; Lee, Y. C.; Lin, H. H.; Tsai, K. C. Discovery of highly potent tyrosinase inhibitor, T1, with significant anti-melanogenesis ability by zebrafish in vivo assay and computational molecular modeling. Sci. Rep. 2015, 5, No. 7995. (32) Abu Ubeid, A.; Zhao, L.; Wang, Y.; Hantash, B. M. Shortsequence oligopeptides with inhibitory activity against mushroom and human tyrosinase. J. Invest. Dermatol. 2009, 129, 2242−2249. (33) Upadhyay, A.; Chompoo, J.; Taira, N.; Fukuta, M.; Gima, S.; Tawata, S. Solid-phase synthesis of mimosine tetrapeptides and their 6188
DOI: 10.1021/acs.jafc.5b01026 J. Agric. Food Chem. 2015, 63, 6181−6188
