Journal of Investigative Dermatology. Short-sequence oligopeptides with inhibitory activity against mushroom and human tyrosinase

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1 Short-sequence oligopeptides with inhibitory activity against mushroom and human tyrosinase Journal: Journal of Investigative Dermatology Manuscript ID: draft Manuscript Type: Original Article Date Submitted by the Author: Complete List of Authors: Abu Ubeid, Anan; elixir institute of regenerative medicine, Translational research Zhao, Longmei; elixir institute of regenerative medicine, Translational research Wang, Ying; elixir institute of regenerative medicine, Translational research Hantash, Basil; Stanford University, Surgery Key Words: Tyrosinase, hyperpigmentation, inhibition, melanocytes, oligopeptide

2 Page of 0 Journal of Investigative Dermatology Title: Short-sequence oligopeptides with inhibitory activity against mushroom and human tyrosinase Short title: Oligopeptide tyrosinase inhibitors Authors: Anan Abu Ubeid, BS,, Longmei Zhao, MD, PhD,, Ying Wang, PhD,, Basil M. Hantash, MD, PhD Elixir Institute of Regenerative Medicine, San Jose, CA These authors contributed equally to this manuscript Corresponding Author: Basil M. Hantash, MD, PhD Contact Information: Elixir Institute of Regenerative Medicine Optical Court San Jose, CA Phone No.: () 0-0 Fax No.: () basil@elixirinstitute.org Funding sources: None. Disclosure statement: None. Prior Presentation: None. Word Count:. Abbreviations: [E] = enzyme concentration; HQ = hydroquinone; [I] = inhibitor; PIH = postinflammatory hyperpigmentation; [S] = substrate; UV = ultraviolet.

3 Page of Abstract Cutaneous hyperpigmentation is a common disorder due to excess melanin production by the enzyme tyrosinase. The gold standard for treatment is hydroquinone (HQ), which reduces pigmentation through its toxicity to melanocytes rather tyrosinase inhibition. We screened an internal library for oligopeptides that inhibited both mushroom and human tyrosinase but demonstrated no cytotoxicity to human melanocytes. We identified two highly active inhibitory sequences, P and P, of and 0 amino acid length, respectively. Mushroom tyrosinase inhibition was dose-dependent with IC0 values of and 0 µm, respectively, compared to 0 µm for HQ. Other oligopeptides demonstrated weaker or no inhibitory activity. Kinetic studies revealed that P and P are competitive inhibitors of mushroom tyrosinase. At 00 µm, P and P inhibited human tyrosinase by to %,. This inhibition partially depended on whether L-dopa or L-tyrosine was the substrate, suggesting that tyrosinase may contain contains two distinct catalytic sites. Treatment of melanocytes with 00 µm P or P for days led to a % or % reduction in melanin content, respectively. This inhibition was independent of cell proliferation and cytotoxic effects. Our data suggest that peptide-mediated inhibition of melanogenesis is due to reduction of tyrosinase activity. Keywords Tyrosinase, hyperpigmentation, inhibition, melanocytes, oligopeptide, skin-whitening agent.

4 Page of 0 Journal of Investigative Dermatology Introduction Melanin, the end product of melanogenesis, determines the color of human skin, hair, and eyes and is synthesized within unique membrane bound organelles called melanosomes. The type and amount of melanin and its distribution pattern in the surrounding keratinocytes determine the actual color of skin (Parvez, Kang et al. 00). Melanogenesis is regulated by a variety of environmental, hormonal, and genetic factors, such as ultraviolet (UV) exposure, α-melanocytestimulating hormone, melanocortin receptor, and agouti-related protein (Petit and Pierard 00);(Kadekaro, Kanto et al. 00). The functions of melanin include protection of skin from UV radiation, the inhibition of photocarcinogenesis, and the synthesis of vitamin D (Lindquist ). Hyperpigmentation is a very common and usually harmless disorder in which patches of skin become darker in color than the normal surrounding skin. This darkening occurs when an excess of melanin is formed and deposited in the skin. The most common pigmentation disorders for which patients seek treatment are melasma and post-inflammatory hyperpigmentation (PIH) (Lynde, Kraft et al. 00). These conditions may have a major impact on a person s psychological and social well-being, contributing to lower productivity, social functioning, and self-esteem (Finlay ). Melasma is an acquired pigmentary disorder that occurs in women of all racial and ethnic groups (Rendon, Berneburg et al. 00). While the cause of melasma is unknown, factors include: genetic predisposition, UV light exposure, and estrogen (Johnston, Sviland et al. ). PIH represents a pathophysiologic response to cutaneous inflammation, such as acne, atopic dermatitis, lichen planus, chronic dermatitis, and photo toxic eruptions (allergic reactions to drugs coupled with exposure to sunlight) (Lacz, Vafaie et al. 00). Tyrosinase is a multifunctional copper containing oxidase, and is considered the key enzyme orchestrating mammalian melanogenesis (Sanchez-Ferrer, Rodriguez-Lopez et al. ). This key enzyme

5 Page of catalyzes two distinct reactions in the biosynthesis pathway of melanin: the hydroxylation of a monophenol (tyrosine) and the conversion of an o-diphenol (dopa) to the corresponding o- quinone (dopaquinone). A multitude of different tyrosinase inhibitors are used today to treat epidermal hyperpigmentation conditions. Skin-whitening agents in the cosmetics industry include hydroquinone (Jimbow, Obata et al. ), arbutin (Maeda and Fukuda ), kojic acid (Cabanes, Chazarra et al. ), vitamin C (Kojima, Yamaguchi et al. ), retinol (Pathak, Fitzpatrick et al. ), azelaic acid (Schallreuter and Wood 0), and other botanicals. The most popular depigmenting agent, hydroquinone (dihydroxybenzene; HQ), has been in use since. HQ, with formulations of % %, has been widely used for melasma treatment and reports of its clinical efficacy have been well documented (Ennes, Paschoalick et al. 000). HQ inhibits the conversion of dopa to melanin by inhibiting the activity of tyrosinase (Palumbo, d'ischia et al. ). In addition to tyrosinase inhibition, Penney et al. suggested that HQ exerts its effect on melanogenesis through degradation of melanosomes, and destruction of melanocytes (Penney, Smith et al. ). It was also found that alteration of melanosome functions, depletion of glutathione, generation of reactive oxygen species and subsequent oxidative damage of membrane lipids and proteins may play a role in the depigmenting effect of HQ (Briganti, Camera et al. 00). HQ is considered to be cytotoxic to melanocytes, potentially mutagenic to mammalian cells (Curto, Kwong et al. ), and causes skin irritation (Parvez, Kang et al. 00). Due to the long-term risk of mutagenesis, (DeCaprio ), the use of HQ in cosmetics has been banned by the European Committee (th Dir. 000//EC) and formulations of % or greater are available only by prescription in selected other countries (Solano, Briganti et al. 00). This has led to the use of alternative agents such as kojic acid and arbutin. However, these agents failed to demonstrate efficacy in vivo, as clinical trials have been disappointing to date (Curto,

6 Page of 0 Journal of Investigative Dermatology Kwong et al. ). The effectiveness of these products appears to be limited due to their adverse effects, poor skin penetrations, and low formulation stabilities (Hermanns, Pierard- Franchimont et al. 000). More recently, investigators have turned their attention to shortsequence peptides as potential therapeutic candidates for the treatment of skin disorders. The most widely studied and popular signal peptide is the sequence lysine-threonine-threonine-lysineserine (KTTKS) found on type-i procollagen (Katayama, Armendariz-Borunda et al. ). This pentapeptide has been demonstrated to stimulate feedback regulation of new collagen synthesis and to result in an increased production of extracellular matrix proteins such as types I and II collagen and fibronectin, significantly improving photoaged human facial skin (Robinson, Fitzgerald et al. 00; Lupo and Cole 00). In the present study, we screened an internal oligopeptide library for inhibitory activity against tyrosinase and absence of melanocyte toxicity. We discovered several short-sequence peptides which met both these criteria.

7 Page of Results Inhibition of mushroom tyrosinase by oligopeptides As the rate-limiting enzyme in melanin production, tyrosinase was targeted during the screening of seven potential hypopigmenting agents (table ). Of these oligopeptides, two showed substantial inhibitory effects on mushroom and human tyrosinase activity, whereas the other peptides had either poor or no inhibitory activity on tyrosinase. Dose-response curves were obtained after an incubation time of 0 min (Fig. ). Peptide P had no effect on tyrosinase activity, whereas peptides P, P, P and P showed an inhibitory effect at high concentrations. On the other hand, peptides P and P showed very impressive inhibition at relatively low concentrations. The IC0 for P and P was µm and 0 µm respectively, compared to 0 µm for HQ. Figure also illustrates that the inhibitory activity of the peptides was dosedependent, with P showing inhibitory activity at concentrations as low as 0 µm. Based on the above results, we focused our attention on peptides P and P. Figure shows the time response curves and indicates that P and P exhibited similar inhibitory activity as HQ. Moreover, peptide P showed a more potent inhibitory effect at concentrations as low as 0 µm. Kinetics of mushroom tyrosinase inhibition by oligopeptides We next determined the kinetics of the inhibition for P, P, and HQ using a standard assay (Pomerantz ). The inhibition mechanisms of the two peptides P and P, as well as that of HQ, are all competitive as demonstrated by Lineweaver-Burke plots in figure-. The double reciprocal plots illustrate that Vmax was independent of substrate concentration for all three inhibitors, whereas the Km was altered. The inhibition constant (K i ) for HQ, P and P was determined to be 0.0, 0.0 and 0.00 mm, respectively.

8 Page of 0 Journal of Investigative Dermatology Inhibition of human tyrosinase by oligopeptides We also examined the inhibitory activity of P and P relative to HQ against human tyrosinase under two settings: the first using L-dopa as the substrate, and the second using L-tyrosine as the substrate with L-dopa as co-factor. In the latter, L-dopa acts as a hydrogen donor helping eliminate the lag period observed during tyrosine hydroxylation (Pomerantz and Warner ). Figure shows dose-dependent inhibition for P, P and HQ. It can be seen that P and P are more potent than HQ at concentrations as low as 0 µm. At 00 µm, human tyrosinase activity was reduced by % for P and % for P. With L-dopa as the substrate, P was more potent than P at inhibiting the enzyme. On the other hand, when L-tyrosine was used as the substrate, P showed stronger inhibition than P. These data support the hypothesis that tyrosinase has two distinct catalytic sites for L-dopa and L-tyrosine (Olivares, Jimenez-Cervantes et al. 00). Effects of HQ, P and P on melanin content of melanocytes To determine the effect of the oligopeptides on melanogenesis, we incubated melanocytes with HQ, P, or P for week and then measured melanin content. At a concentration of 0 µm, HQ, P, and P reduced melanin content by ± %, ± % and 0 ± %, respectively (Fig. ). At 00 µm, P and P reduced melanin content by ± % and ± %, respectively, while HQ was found to be 00% toxic to melanocytes. Cytotoxic and proliferative effects of HQ, P and P on melanocytes To exclude the possibility that P and P were reducing melanin content by inhibiting cell proliferation or inducing cytotoxicity, melanocytes were incubated in the presence or absence of various concentrations of HQ, P, or P, and proliferation assessed at various time points using the WST- cell proliferation kit. Figure shows that treatment with 00 µm or greater of HQ completely abolished WST- activity (from 0. ± 0.0 at day down to 0.0 at day ). These data are consistent with our above findings and imply that HQ is cytotoxic to melanocytes at these

9 Page of concentrations. Unlike HQ, 0 µm, 00 µm, or mm P did not alter melanocyte proliferation rates relative to the control group. Although no effect on melanocytes proliferation was observed for P at 0 and 00 µm after days of incubation, proliferation was inhibited by 0 ± % at a concentration of mm. Table further supports our findings regarding the toxicity of HQ on melanocytes compared to peptides P and P. After a hr incubation of melanocytes with 0 µm HQ, viability staining with trypan blue indicated that % of cells were dead. This was one order of magnitude greater than that for P. The toxic effects of HQ were dose-dependent and at concentrations of 0 and 00 µm, and % of melanocytes were no longer viable, respectively. The highest rate of cell death for P was % after incubating the melanocytes with mm of P for six days. At concentrations greater than 00 µm for HQ there was 00% cell death starting within hrs of exposure. P was found to be considerably less toxic to melanocytes even at concentrations as high as mm (% cell death after six days).

10 Page of 0 Journal of Investigative Dermatology Discussion In this study, we screened an internal library for oligopeptides with inhibitory activity against mushroom and human tyrosinase. Even though the amino acid sequence similarity between mushroom and human tyrosinase is only %, our initial experiments were performed using mushroom tyrosinase since previous studies suggested a strong correlation of efficacy with agents tested against the human form (Sugimoto, Nishimura et al. 00). The dose and time response curves (Figs. & ) of these peptides showed that P and P were - and -fold more potent inhibitors of mushroom tyrosinase than HQ (IC0 values presented in table ). Based on this initial screen, we decided to further evaluate and characterize the effects of P and P. To determine the mode of inhibition, we performed Lineweaver Burk plots of P and P and compared the kinetics to those of HQ. These studies revealed that similar to HQ, P and P were competitive inhibitors of tyrosinase. Peptides P and P were also tested against fresh extracts of human tyrosinase. At a concentration of 00 µm, the peptides were able to inhibit human tyrosinase activity by % to %. Although the potency against human tyrosinase appeared reduced relative to that observed with mushroom tyrosinase, several experimental conditions may account for this discrepancy. First, the assay was performed on extracts from lysed melanocytes and not purified human tyrosinase. Although protease inhibitors were included to reduce degradation of human tyrosinase, we did not optimize these conditions. Second, the catalytic site of the extracted human tyrosinase can exist in different redox states than the purified form. We observed a lengthy lag period for tyrosine oxidation. The oxidation of L-tyrosine by tyrosinase has previously been distinguished by a lag period that is affected by enzyme and substrate concentration, ph, and hydrogen donors such as L-dopa (Pomerantz and Warner ). Therefore, when L-tyrosine was the substrate, L-dopa was added as a co-factor at low

11 Page 0 of micromolar levels to eliminate this lag period. The ability to act as substrate requires a free electron donor group, while competitive inhibition, on the contrary, requires the presence of powerful electron acceptor groups (Passi and Nazzaro-Porro ). These experiments also unexpectedly revealed that P inhibited human tyrosinase more potently in the presence of L- dopa than L-tyrosine, whereas P showed greater inhibition in the presence of L-tyrosine as the substrate. Hearing et al proposed that there are two catalytic sites on tyrosinase, one for tyrosine hydroxylase and another site specific for dopa oxidase activity (Hearing and Ekel ). Our findings with P and P further support the hypothesis of separate catalytic sites for L-dopa and L-tyrosine (Schallreuter, Kothari et al. 00). Despite the fact that little is known about the effect of short sequence peptides on melanogenesis and tyrosinase inhibition, there is great potential for these agents to replace more toxic drugs such as HQ, since they are comprised of naturally occurring amino acids that are easily metabolized in skin. Therefore, risks of systemic absorption and liver related mutagenesis may be avoided during the chronic treatment of hyperpigmentation disorders. Some amino acids such L-alanine, glycine, L-isoleucine and L-leucine have previously been shown to possess beneficial effects towards disruption of melanogenesis (Ishikawa, Kawase et al. 00). Ishikawa et al reported that a combination of these four amino acids had an additive effect on hypopigmentation similar to that of kojic acid. Schurink et al proposed that the peptide should contain one or more arginine residues for strong tyrosinase inhibitory and binding activity.(schurink, van Berkel et al. 00). Moreover, their studies indicated that tyrosinase inhibition is optimal when arginine and/or phenylalanine is/are combined with hydrophobic aliphatic residues such as valine, alanine or leucine. Besides hydrophobic residues, peptides containing the polar, uncharged residues cysteine and serine also demonstrated significant inhibitory activity. 0

12 Page of 0 Journal of Investigative Dermatology The amino acid tyrosine contains a phenol group, which makes it unique in that it can serve as a substrate or a competitive inhibitor. Oligopeptide P contains the combination Arg-Ala in positions / and / as well as the amino acids serine and cysteine at positions and respectively. The presence of these key residues may explain the strong inhibitory activity observed for P. For P, the presence of tyrosine at positions and 0, arginine at positions and, and serine at, and may explain the robust inhibition observed for this oligopeptide. One of the main advantages of oligopeptides P and P is the relative absence of melanocyte cytotoxicity (table ). While HQ was 00% toxic after a -hour treatment at 00 µm, P showed no toxic effects after six days of treatment. P also showed minimal (%; table ) toxicity after the same incubation period. Treatment with mm P resulted in a % toxicity rate (table ) and no effect on melanocyte proliferation (Fig. ). At a concentration -fold higher than its IC0 value, P began to show mild (%; table ) melanocyte toxicity after a prolonged incubation of days. We also confirmed these findings using proliferation assays which revealed that HQ was toxic to melanocytes at concentrations as low as 0 µm (data now shown), whereas mild reduction of proliferation was first noted only after days of incubation with mm P (Fig. ). Lower concentrations did not show any effect on melanocyte proliferation even after prolonged incubation for days, whereas HQ dramatically reduced melanocyte proliferation within hours at concentrations as low as 0 (data not shown) and 00 µm (Fig. ). In conclusion, our findings suggest that oligopeptides P and P may be ideal tyrosinase inhibitors due to their potent enzymatic inhibitory activity, relative absence of melanocyte toxicity, and minimal effects on melanocyte proliferation rates. Further studies are underway to optimize cell penetration and oligopeptide stability for transdermal applications. We believe our novel oligopeptides may hold significant therapeutic potential for the treatment of skin hyperpigmentation disorders. Since both oligopeptides demonstrated substantially improved

13 Page of potency against tyrosinase with minimal melanocyte toxicity, clinical use of these agents may obviate the need for HQ which derives its primary skin lightening capability via its cytotoxic activity. Materials and Methods Materials Mushroom tyrosinase, L-tyrosine, HQ and L-dopa were purchased from Sigma Aldrich. The peptides (table ) were synthesized by NeoMPS, Inc (San Diego, California, USA) using solidphase FMOC chemistry. Enzymatic assay of mushroom tyrosinase Tyrosinase inhibition activity was determined in vitro using L-tyrosine as the substrate using a modified method from Piao et al (Piao, Park et al. 00). The concentration of enzyme, substrate and inhibitor was denoted as [E], [S] and [I], respectively. The experiment was conducted in a - well flat bottom plate. Each well contained 0 µl of 0.0 M potassium phosphate buffer (ph.), 0 µl of mg/ml L-tyrosine dissolved in 0.0 M potassium phosphate buffer (ph.), 0 µl of the different concentrations of inhibitor in % DMSO solution, and 0 µl of 0 units/ml mushroom tyrosinase solution. The final volume of each well was 00 µl, containing mg/ml [S], units/ml [E] and varying concentrations of [I], ranging from µm to 0 mm. For the negative control wells, the inhibitors were substituted with % DMSO solution and adjusted to the total volume of 00 µl. HQ served as the positive control in our experiments. HQ was tested in parallel with the oligopeptides at the same concentrations and same conditions. The assay

14 Page of 0 Journal of Investigative Dermatology mixture was incubated at C. The optical density of the reaction mixtures, which correlates with the amount of dopachrome produced, was measured using a spectrophotometer (Varioskan,Thermo Electron Corporation) at nm and at different time periods. Kinetic studies The Lineweaver-Burk plot method was used to determine the reaction kinetics (Burk, Lineweaver et al. ). Experiments were conducted using the same protocol used for measuring mushroom tyrosinase activity except for changes in concentration of [E], [S] and [I]. [E] was units/ml. The series of [S] were 0.0 (or 0.0 for peptide inhibitors), 0., 0. and 0. mg/ml. The series of [I] were 0, 0.0 and 0.0 mm, respectively. The results were analyzed according to the Lineweaver-Burk plot method that facilitated the determination of the Michaelis constant (Km) and maximum velocity (Vm). Melanin content measurement Melanin content of the melanocytes was measured according to the method of Naeyaert et al (Naeyaert, Eller et al. ). Human melanocytes were cultured in -well plates and treated with individual test samples for days. P, P, and HQ were tested at 0 and 00 µm each, all in triplicate. Media was changed every other day and fresh oligopeptide or HQ was added. After seven days, the media was discarded and the wells were washed with PBS. Cells were detached by short incubation in trypsin/edta (0.%/0. % in PBS). An aliquot was used for cell counting and the remaining cells were sonicated and incubated overnight in the dark in 00 µl M NaOH at C. Melanin concentrations were calculated by comparison of the OD at nm of unknown samples with a standard curve obtained with synthetic melanin (Sigma Aldrich, St Louis, MO, USA). Melanin content was expressed as pg/cell.

15 Page of Assay of human tyrosinase activity The following methods are modifications of protocols adopted from Nagata et al, (Nagata, Takekoshi et al. 00) and Cheng et al (Cheng, Hsu et al. 00). Human melanocytes were cultured in 00 mm dishes using medium (Invitrogen, Carlsbad, CA), incubated at C in a humidified % CO chamber. At 0 to 0% confluence, cells were harvested and lysed using % Triton X-00 in 0.0 M potassium phosphate buffer (ph.). Cells were then sonicated twice at 0% intensity for 0 sec (on ice). Lysates were clarified by centrifugation at 0,000 g for 0 min. Protein content was determined using a Bio-Rad protein assay kit (Richmond, CA). Lysates were added to a well flat bottom plate containing equal amounts of protein (0µg), and adjusted with lysis buffer to reach 0 µl in each well. L-dopa was dissolved in lysis buffer and added to each well at a final concentration of. mm. Each triplicate of wells received different concentrations (0 to mm) of oligopeptide or HQ. The plate was incubated at C for 0 min, and spectrophotometric nm (Varioskan, Thermo Electron Corporation) were taken at 0 min intervals. In certain experiments, L-tyrosine was substituted for L-dopa, and used at a final concentration mm in lysis buffer. L-dopa was also added at a final concentration of 0.00% to serve as a cofactor. Several control wells were used in the experiment. Certain wells served as controls and contained 0 µg of protein extract in lysis buffer, and substrate at varying compositions but without peptide inhibitors. Other control wells contained lysis buffer, substrate, peptide inhibitors but no protein extract (no enzyme). WST assay for Melanocyte proliferation Melanocyte proliferation was determined using the WST- Cell Proliferation Kit (Roche, Palo Alto, CA). Cells were plated at. 0 /well in -well plates in a humidified atmosphere with

16 Page of 0 Journal of Investigative Dermatology % CO at ºC. Twenty-four hours after plating, test samples were added and cultures were incubated for an additional,, or days. Fresh medium and test samples were replaced every other day. At the end of the treatment period, 0 µl WST- mixture was added to each well containing 00 µl fresh medium. The plates were placed at ºC for h in the dark and the absorbance at 0 nm was read using the HTS 000 Plus Bio Assay reader (Perkim Elmer, Waltham, MA). Three replicate wells were measured for each group to be tested. Wells containing medium but no cells served as background controls. Evaluation of the cytotoxic effect of P, P and HQ on melanocytes Melanocytes were cultured in three -well plates at a density of x 0 cells/well for either,, or days in ml of medium containing different concentrations of peptides or HQ (0, 0, 0,00, 00, or 000 µm). The plates were incubated at C in a humidified % CO chamber. Media and inhibitors were changed every hours. After hours, the Plate labeled Day- was harvested and cells were counted in each well using a hemacytometer. The control well contained ml of media without any inhibitor. The toxicity of the test sample was measured according to the following formula: [- (# of cells in control well)- (# of live cells in test sample well) ] X 00% # of cells in control well The same procedure was repeated after hours of incubation for the plate labeled Day- and after hours for the Day- plate.

17 Page of Statistical analysis Each experiment was performed in triplicate and run a minimum of independent times. The results were averaged and standard error of the mean was calculated for all conditions using Microsoft Excel.

18 Page of 0 Journal of Investigative Dermatology Conflict of interest: There are no conflicts of interest.

19 Page of Acknowledgments: There are no acknowledgments.

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22 Page of 0 Journal of Investigative Dermatology Table. Oligopeptide inhibitory profile against mushroom tyrosinase Oligopeptide Sequence IC 0 [M] P SFLLRN mm P KFEKKFEK. mm P RADSRADC µm P YRSRKYSSWY 0 µm P PLG. mm P VLLK No effect P KFEFKFEF No effect IC0 [M] = concentration at 0% inhibition of mushroom tyrosinase in molar.

23 Page of Table. The toxic effect of HQ, P, and P. Concentration % Cell Death Day Day Day [µm] P P HQ P P HQ P P HQ Human melanocytes were incubated with HQ, P and P at the above concentrations for, and days. The assay was carried out in triplicates, and the percent cell death was calculated according to the formula discussed in the methods section.

24 Page of 0 Journal of Investigative Dermatology Figure Legends: Figure : Dose response curves for all seven peptides and HQ were taken at 0 min post-incubation with varying concentrations. The experiment was conducted in a -well flat bottom plate. Each well contained 0 µl of 0.0 M potassium phosphate buffer (ph.), 0 µl of mg/ml L- tyrosine dissolved in 0.0 M potassium phosphate buffer (ph.), 0 µl of the different concentrations of inhibitor in % DMSO solution, and 0 µl of 0 units/ml mushroom tyrosinase solution. The assay mixture was incubated at C and spectrophotometric nm were taken every 0 mins. (a) P, P, P, P and P. (b) HQ, P and P. Figure : Time response curves for peptides P and P along with the control HQ. The final volume of each well was 00 µl, containing mg/ml L-tyrosine, units/ml of mushroom tyrosinase and varying concentrations of inhibitors, ranging from 0 µm to 0 mm. The -well plate was C and the spectrophotometric readings taken every 0 mins. Each point on the graph is an average of five independent trials, displayed with error bars. (a) P. (b) P. (c) HQ. Figure : Lineweaver-Burk plots. (a) P (b) P and (c) HQ. The concentration of mushroom tyrosinase [E] was units/ml. The series of substrate [S] were 0.0 (or 0.0 for peptide inhibitors), 0., 0. and 0. mg/ml. The series of inhibitor [I] were 0, 0.0 and 0.0 mm, respectively. The results were analyzed according to the Lineweaver-Burk plot method that allows the determination of the Michaelis constant (Km) and maximum velocity (Vm). All inhibitors showed competitive inhibition pattern. Figure : Dose response curves with human tyrosinase. Melanocytes were lysed using % Triton X-00 in 0.0 M potassium phosphate buffer (ph.). Cells were then sonicated twice at 0% intensity for 0 sec. Lysates were clarified by centrifugation at 0,000 g for 0 min. after measuring protein content, an equal amount of 0µg was added to each well and adjusted with lysis buffer to reach 0 µl. Each triplicate of wells received different concentrations (0 to mm) of oligopeptide or HQ. (a) Using L-dopa as the substrate. L-dopa was dissolved in lysis buffer and added to each well at a final concentration of. mm. The plate was incubated at C for 0 min, and spectrophotometric nm were taken at 0 min intervals. (b) Using L-tyrosine as substrate with L-dopa at a final concentration of 0.00% as a co-factor.

25 Page of Figure : The effects of peptides and HQ on the melanin content of melanocytes, expressed in pg/cell. The tested concentrations were, 0 and 00µM. Melanocytes were incubated with test samples for a week. Cells were lysed and incubated O/N in M NaOH. Melanin content was determined by comparing the absorbance results with a standard curve generated from synthetic melanin Figure : Proliferation of human melanocytes treated with HQ, P, and P.,000 human melanocytes per well were incubated at ºC in the presence of 0, 00, 000 µm HQ, P and P in medium for,, days. After the incubation 0 µl WST- was added to each well and absorbance was read at 0 nm after four hour incubation at ºC.

26 Page of 0 Journal of Investigative Dermatology nm a Figure [Log] b [Log]

27 Page of a b c Figure

28 Page of 0 Journal of Investigative Dermatology a /[V o ] b /[V o ] c /[V o ] /[S] /[S] /[S] Figure

29 Page of nm a Figure L-dopa b L-tyrosine [Log] [Log]

30 Page of 0 Journal of Investigative Dermatology Melanin content (pg/cell) Figure (µm) Ctrl [HQ] [P] [P]

31 Page 0 of nm a 0 b c 0 Day Day Day Ctrl HQ P P [µm] Figure