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Inhibition of Human Tyrosinase Requires Molecular Motifs Distinctively Different from Mushroom Tyrosinase

Open AccessPublished:February 07, 2018DOI:https://doi.org/10.1016/j.jid.2018.01.019
      Tyrosinase is the rate-limiting enzyme of melanin production and, accordingly, is the most prominent target for inhibiting hyperpigmentation. Numerous tyrosinase inhibitors have been identified, but most of those lack clinical efficacy because they were identified using mushroom tyrosinase as the target. Therefore, we used recombinant human tyrosinase to screen a library of 50,000 compounds and compared the active screening hits with well-known whitening ingredients. Hydroquinone and its derivative arbutin only weakly inhibited human tyrosinase with a half-maximal inhibitory concentration (IC50) in the millimolar range, and kojic acid showed a weak efficacy (IC50 > 500 μmol/L). The most potent inhibitors of human tyrosinase identified in this screen were resorcinyl-thiazole derivatives, especially the newly identified Thiamidol (Beiersdorf AG, Hamburg, Germany) (isobutylamido thiazolyl resorcinol), which had an IC50 of 1.1 μmol/L. In contrast, Thiamidol only weakly inhibited mushroom tyrosinase (IC50 = 108 μmol/L). In melanocyte cultures, Thiamidol strongly but reversibly inhibited melanin production (IC50 = 0.9 μmol/L), whereas hydroquinone irreversibly inhibited melanogenesis (IC50 = 16.3 μmol/L). Clinically, Thiamidol visibly reduced the appearance of age spots within 4 weeks, and after 12 weeks some age spots were indistinguishable from the normal adjacent skin. The full potential of Thiamidol to reduce hyperpigmentation of human skin needs to be explored in future studies.

      Abbreviations:

      HTS (high-throughput screen), hTyr (human tyrosinase), IC50 (half maximal inhibitory concentration), Ki (inhibitor constant), mTyr (mushroom (Agaricus bisporus) tyrosinase)

      Introduction

      Melasma, actinic and senile lentigines, and postinflammatory hyperpigmentation are major cosmetic problems for which many patients seek medical advice. Generally, those disorders affect populations with darker skin complexions with a greater frequency and severity (
      • Stratigos A.J.
      • Katsambas A.D.
      Optimal management of recalcitrant disorders of hyperpigmentation in dark-skinned patients.
      ). Many topical products are available to treat hyperpigmentary disorders, and they contain diverse active ingredients to reduce melanin production and/or distribution. Although skin hyperpigmentation can be reduced by various mechanisms (
      • Briganti S.
      • Camera E.
      • Picardo M.
      Chemical and instrumental approaches to treat hyperpigmentation.
      ), tyrosinase, the rate-limiting enzyme of melanin production, is the obvious target for inhibitors of hyperpigmentation (
      • Kanteev M.
      • Goldfeder M.
      • Fishman A.
      Structure-function correlations in tyrosinases.
      ,
      • Lee S.Y.
      • Baek N.
      • Nam T.G.
      Natural, semisynthetic and synthetic tyrosinase inhibitors.
      ,
      • Ramsden C.A.
      • Riley P.A.
      Tyrosinase: the four oxidation states of the active site and their relevance to enzymatic activation, oxidation and inactivation.
      ). Many substances have been described in the literature as inhibitors of tyrosinase, but most of them lack clinical efficacy, and only a few compounds are currently used in topical dermatological products (
      • Chang T.S.
      An updated review of tyrosinase inhibitors.
      ,
      • Kim Y.J.
      • Uyama H.
      Tyrosinase inhibitors from natural and synthetic sources, structure, inhibition mechanism and perspective for the future.
      ,
      • Rescigno A.
      • Sollai F.
      • Pisu B.
      • Rinaldi A.
      • Sanjust E.
      Tyrosinase inhibition, general and applied aspects.
      ). Among those, kojic acid, hydroquinone, and arbutin are the most common (
      • Solano F.
      • Briganti S.
      • Picardo M.
      • Ghanem G.
      Hypopigmenting agents: an updated review on biological, chemical and clinical aspects.
      ).
      The unsatisfactory clinical efficacy of currently used tyrosinase inhibitors is largely due to the fact that those compounds were tested using only tyrosinase isolated from the mushroom Agaricus bisporus (mTyr) (
      • Espin J.C.
      • Varon R.
      • Fenoll L.G.
      • Gilabert M.A.
      • Garcia-Ruiz P.A.
      • Tudela J.
      • et al.
      Kinetic characterization of the substrate specificity and mechanism of mushroom tyrosinase.
      ,
      • Garcia-Molina F.
      • Hiner A.N.
      • Fenoll L.G.
      • Rodriguez-Lopez J.N.
      • Garcia-Ruiz P.A.
      • Garcia-Canovas F.
      • et al.
      Mushroom tyrosinase: catalase activity, inhibition, and suicide inactivation.
      ), which is the only active tyrosinase readily commercially available. The catalytic activities and substrate specificities of mTyr have been shown to be significantly different from the mammalian enzyme (
      • Hearing Jr., V.J.
      • Ekel T.M.
      • Montague P.M.
      • Nicholson J.M.
      Mammalian tyrosinase. Stoichiometry and measurement of reaction products.
      ). The three-dimensional structures of several tyrosinases were recently solved, among them the structures of mTyr (
      • Ismaya W.T.
      • Rozeboom H.J.
      • Weijn A.
      • Mes J.J.
      • Fusetti F.
      • Wichers H.J.
      • Dijkstra B.W.
      Crystal structure of Agaricus bisporus mushroom tyrosinase: identity of the tetramer subunits and interaction with tropolone.
      ) and of two bacterial enzymes from Streptomyces castaneoglobisporus (
      • Matoba Y.
      • Kumagai T.
      • Yamamoto A.
      • Yoshitsu H.
      • Sugiyama M.
      Crystallographic evidence that the dinuclear copper center of tyrosinase is flexible during catalysis.
      ) and Bacillus megaterium (
      • Sendovski M.
      • Kanteev M.
      • Ben-Yosef V.S.
      • Adir N.
      • Fishman A.
      First structures of an active bacterial tyrosinase reveal copper plasticity.
      ). By contrast, very little kinetic or structural information is available for human tyrosinase (hTyr), mainly because of substantial difficulties in obtaining sufficient amounts of hTyr from natural sources or by heterologous expression. hTyr has been transiently expressed in various animal cell lines (
      • Olivares C.
      • Garcia-Borron J.C.
      • Solano F.
      Identification of active site residues involved in metal cofactor binding and stereospecific substrate recognition in mammalian tyrosinase. Implications to the catalytic cycle.
      ,
      • Schweikardt T.
      • Olivares C.
      • Solano F.
      • Jaenicke E.
      • Garcia-Borron J.C.
      • Decker H.
      A three-dimensional model of mammalian tyrosinase active site accounting for loss of function mutations.
      ,
      • Tripathi R.K.
      • Hearing V.J.
      • Urabe K.
      • Aroca P.
      • Spritz R.A.
      Mutational mapping of the catalytic activities of human tyrosinase.
      ,
      • Wendt M.
      Rationales design neuer tyrosinase-inhibitoren. PhD thesis.
      ), but yields were always too low for a detailed characterization of the resulting hTyr preparations. More recently, several groups have developed more efficient expression systems for hTyr (
      • Cordes P.
      • Sun W.
      • Wolber R.
      • Kolbe L.
      • Klebe G.
      • Röhm K.H.
      Expression in non-melanogenic systems and purification of soluble variants of human tyrosinase.
      ,
      • Fogal S.
      • Carotti M.
      • Giaretta L.
      • Lanciai F.
      • Nogara L.
      • Bubacco L.
      • Bergantino E.
      Human tyrosinase produced in insect cells: a landmark for the screening of new drugs addressing its activity.
      ,
      • Lai X.
      • Soler-Lopez M.
      • Wichers H.J.
      • Dijkstra B.W.
      Large-scale recombinant expression and purification of human tyrosinase suitable for structural studies.
      ), but data on the three-dimensional structure of hTyr or kinetic data of hTyr inhibitors were still missing.
      In this study, we used a recombinant hTyr construct (
      • Cordes P.
      • Sun W.
      • Wolber R.
      • Kolbe L.
      • Klebe G.
      • Röhm K.H.
      Expression in non-melanogenic systems and purification of soluble variants of human tyrosinase.
      ) to conduct a high-throughput screen (HTS) of a large compound library to identify structural motifs in small-molecule compounds that efficiently inhibit hTyr. We also evaluated the effects of well-known whitening compounds, such as hydroquinone, arbutin, kojic acid, rhododendrol and 4-butylresorcinol, on hTyr activity and compared their efficacies with the new compounds identified by the HTS screen. Among the screening hits, we identified Thiamidol (Beiersdorf AG, Hamburg, Germany) (International Nomenclature of Cosmetic Ingredients name, isobutylamido thiazolyl resorcinol; International Union of Pure and Applied Chemistry name, N-(4-(2,4-dihydroxyphenyl)thiazol-2-yl)isobutyramide) as an especially potent inhibitor of hTyr, and we show that it is an effective and safe inhibitor of human hyperpigmentation in vivo.

      Results

      Inhibition of hTyr

      A screen of 50,000 compounds in the library, which spans a wide chemical space, yielded several hit series of active and effective hTyr inhibitors. Among them, derivatives of thiazolyl-resorcinol were the most promising group. This lead compound was then optimized to develop derivatives with high activity and physico-chemical properties compatible with topical formulations. Thiamidol (isobutylamido thiazolyl resorcinol, compound 1) (Figure 1) was identified as one of the most potent derivatives. In addition to Thiamidol, 4-butylresorcinol (compound 2) and the classical tyrosinase inhibitors kojic acid (compound 5), hydroquinone (compound 6), and arbutin (compound 7), as well as rhododendrol (compound 9), were also tested as inhibitors of the diphenolase (l-dopa oxidase) activity of hTyr over a wide range of concentrations (up to 4 orders of magnitude). The results are summarized in Figure 2a and Table 1. Among these actives, Thiamidol was by far the most efficient inhibitor of hTyr, with a half-maximal inhibitory concentration (IC50) of 1.1 μmol/L, with almost complete enzyme inhibition of hTyr occurring at concentrations above 10 μmol/L. The resorcinol derivatives 4-butylresorcinol, 4-hexylresorcinol, and 4-phenylethylresorcinol had IC50 values of 21 μmol/L, 94 μmol/L, and 131 μmol/L, respectively (Table 1). With an IC50 of about 500 μmol/L, kojic acid was 500 times less potent than Thiamidol. Hydroquinone and arbutin were both very poor inhibitors of hTyr, with IC50 values in the millimolar range. Kojic acid, arbutin, and hydroquinone were not able to completely inhibit hTyr in the concentration range tested. Racemic rhododendrol was also rather ineffective as an inhibitor of l-dopa oxidation, with an IC50 >1,200 μmol/L (Figure 2a).
      Figure 1
      Figure 1Chemical structure of inhibitory compounds evaluated in this study. Compound 1, Thiamidol (Beiersdorf AG, Hamburg, Germany) (isobutylamido thiazolyl resorcinol); compound 2, 4-butylresorcinol; compound 3, 4-hexylresorcinol; compound 4, 4-phenylethylresorcinol; compound 5, kojic acid; compound 6, hydroquinone; compound 7, arbutin; compound 8, dimethoxytolylpropyl resorcinol; and compound 9, rhododendrol.
      Figure 2
      Figure 2Inhibition of melanin production by Thiamidol, 4-butylresorcinol, kojic acid, rhododendrol, hydroquinone, and arbutin. (a) In vitro assays using purified hTyr in 50 mmol/L sodium phosphate buffer, pH 7.0, at a substrate (l-dopa) concentration of 1 mmol/L and various concentrations of inhibitors as noted. Data represent mean ± standard deviation of three independent experiments. (b) Kinetics of inhibition of hTyr by Thiamidol (Beiersdorf AG, Hamburg, Germany) at the concentrations noted. The experiment was performed in triplicate at pH 7.0. The data are plotted according to Lineweaver-Burk. (c) Inhibition of melanin production in MelanoDerm (MatTek Corporation, Ashland, MA) skin models. The melanin content of each MelanoDerm skin model was determined after 13 days of cultivation in the presence of various inhibitors at the concentrations noted. Data represent mean ± standard deviation of five independent experiments. AU, arbitrary unit; hTyr, human tyrosinase; M, mol/L.
      Table 1Kinetic data for inhibitors of hTyr and/or mTyr and inhibition of melanin production
      NumberCompoundKi (μmol/L)IC50 (μmol/L)
      hTyrhTyrmTyrMelanoDerm

      Skin Model
      1Thiamidol0.251.11080.9
      24-Butylresorcinol9.1210.613.5
      34-Hexylresorcinol39941.2
      IC50 value from Chen et al., 2004.
      n.d.
      44-Phenylethylresorcinol241310.3
      IC50 value calculated from Vielhaber et al., 2007.
      n.d.
      5Kojic acid1455006.0
      IC50 value from Curto et al., 1999.
      400
      6Hydroquinonen.d.>4,0001.1
      IC50 value from Kang et al., 2003.
      16.3
      7Arbutinn.d.>4,00040
      IC50 value from Ying et al., 1999.
      >4,000
      8Dimethoxytolylpropyl resorcinoln.d.No inh.0.24
      IC50 value from Nesterov et al., 2008.
      n.d.
      Abbreviations: hTyr, human tyrosinase; IC50, half maximal inhibitory concentration; Ki, inhibitor constant; M, mol/L; mTyr, mushroom tyrosinase; n.d., not determined; no inh., no inhibition.
      1 IC50 value from
      • Chen Q.X.
      • Ke L.N.
      • Song K.K.
      • Huang H.
      • Liu X.D.
      Inhibitory effects of hexylresorcinol and dodecylresorcinol on mushroom (Agaricus bisporus) tyrosinase.
      .
      2 IC50 value calculated from
      • Vielhaber G.
      • Schmaus G.
      • Jacobs K.
      • Franke H.
      • Lange S.
      • Herrmann M.
      • et al.
      4-(1-phenylethyl)1,3-benzenediol: a new, highly efficient lightening agent.
      .
      3 IC50 value from
      • Curto E.V.
      • Kwong C.
      • Hermersdörfer H.
      • Glatt H.
      • Santis C.
      • Virador V.
      • et al.
      Inhibitors of mammalian melanocyte tyrosinase: in vitro comparisons of alkyl esters of gentisic acid with other putative inhibitors.
      .
      4 IC50 value from
      • Kang H.H.
      • Rho H.S.
      • Hwang J.S.
      • Oh S.G.
      Depigmenting activity and low cytotoxicity of alkoxy benzoates or alkoxy cinnamte in cultured melanocytes.
      .
      5 IC50 value from
      • Ying Y.H.
      • Lee S.J.
      • Chung M.H.
      • Ying H.J.
      • Suk J.L.
      • Myung H.C.
      • et al.
      Aloesin and arbutin inhibit tyrosinase activity in a synergistic manner via a different action mechanism.
      .
      6 IC50 value from
      • Nesterov A.
      • Zhao J.
      • Minter D.
      • Hertel C.
      • Ma W.
      • Abeysinghe P.
      • et al.
      1-(2,4-dihydroxyphenyl)-3-(2,4-dimethoxy-3-methylphenyl)propane, a novel tyrosinase inhibitor with strong depigmenting effects.
      .
      A detailed kinetic analysis of the inhibition of hTyr by Thiamidol yielded a strictly competitive type of inhibition with an inhibitor constant (Ki) of 0.25 μmol/L (Figure 2b, Table 1). This value is in agreement with the IC50 value estimated from dose-response curves (1.1 μmol/L) (cf. Figure 2a) which, for competitive inhibition, should be about 3 times higher than the Ki. The Ki values for 4-butylresorcinol (9 μmol/L), 4-hexylresorcinol (39 μmol/L), and 4-phenylethylresorcinol (24 μmol/L) were also markedly higher than the Ki value of Thiamidol (Table 1). These data illustrate that the thiazolylamide moiety of Thiamidol conveys a much better inhibition of hTyr than do the hydrocarbon side chains present in three other derivatives of resorcinol (4-butyl-, 4-hexyl-, and 4-phenyl ethylresorcinol). As noted, the efficacy is distinctively different in mTyr, where 4-butylresorcinol, 4-hexylresorcinol, and 4-phenylethylresorcinol, and even kojic acid, are superior to Thiamidol in inhibiting the enzyme (Table 1). Thus, Thiamidol would not have been identified as positive in a screening using mTyr, and the efficacy of 4-phenylethylresorcinol would have been grossly overestimated.
      • Garcia-Jimenez A.
      • Teruel-Puche J.A.
      • Berna J.
      • Rodriguez-Lopez J.N.
      • Tudela J.
      • Garcia-Ruiz P.A.
      • Garcia-Canovas F.
      Characterization of the action of tyrosinase on resorcinols.
      recently reported that mTyr slowly oxidizes certain resorcinols, provided that the prevailing met- form of the enzyme is previously converted to either the oxy- or deoxy- form by additives like H2O2 and ascorbate and that the reaction is sustained by o-diphenols. Therefore, we used quantitative high-performance liquid chromatography analysis (
      • Ito S.
      • Wakamatsu K.
      A convenient screening method to differentiate phenolic skin whitening tyrosinase inhibitors from leukoderma-inducing phenols.
      ) to ascertain whether Thiamidol might also be a substrate of hTyr. In our normal assay conditions (i.e., in the absence of the additives mentioned by
      • Garcia-Jimenez A.
      • Teruel-Puche J.A.
      • Berna J.
      • Rodriguez-Lopez J.N.
      • Tudela J.
      • Garcia-Ruiz P.A.
      • Garcia-Canovas F.
      Characterization of the action of tyrosinase on resorcinols.
      ), no detectable oxidation of Thiamidol took place within several hours of incubation with hTyr, whereas rhododendrol was readily oxidized within that time frame (see Supplementary Figure S1 online). Thus, we assume that the reaction described by Garcia-Jimenez et al. is not relevant for Thiamidol and hTyr in physiological conditions.

      Inhibition of melanin production

      We then tested the potential inhibitory effects of these compounds using a three-dimensional model for human skin. As observed with purified hTyr, arbutin showed only a negligible efficacy at inhibiting melanin production in MelanoDerm (MatTek Corporation, Ashland, MA) skin models (IC50 > 4,000 μmol/L) (Figure 2c). Kojic acid inhibited melanin production with an IC50 of ∼400 μmol/L, showing a surprisingly steep dose-response curve, with concentrations below 200 μmol/L only slightly inhibiting melanin production (i.e., by 5% at 150 μmol/L). Rhododendrol showed only marginal effects on melanogenesis, with an apparent IC50 for inhibition of ∼1,200 μmol/L. Hydroquinone inhibited melanin production in MelanoDerm skin models with an IC50 of 15 μmol/L, suggesting that it has a mechanism other than tyrosinase inhibition. 4-Butylresorcinol inhibited melanin synthesis with an IC50 of 13.5 μmol/L. Again, Thiamidol was, by far, the most potent inhibitor of melanin production in MelanoDerm skin models, with an IC50 of 0.9 μmol/L, and in monolayer cultures, Thiamidol visibly reduced melanin formation (Figure 3a).
      Figure 3
      Figure 3Characteristics of hTyr inhibition by Thiamidol or hydroquinone. (a) Melanocytes from African donors were cultivated for 2 weeks (left) with or (right) without 5 μmol/L Thiamidol (Beiersdorf AG, Hamburg, Germany) in microplate dishes; photographs were taken in bright field mode. Scale bars = 200 μm. (b) Melanocytes from Caucasian donors were cultivated with 1 μmol/L hydroquinone or 1 μmol/L Thiamidol for 2 weeks to reduce melanin production and then were cultured for 2 more weeks without those active compounds to monitor the recovery of melanin production. Melanin content was measured at the times noted using a microplate reader, and data are reported as % control. Mean ± standard error of the mean, n = 5. M, mol/L.
      Hydroquinone and Thiamidol were then tested in long-term melanocyte monolayer cultures to check the potential reversibility of inhibition. Although 1 μmol/L Thiamidol reduced melanin production to less than 60% after 2 weeks, 1 μmol/L hydroquinone reduced melanin production only to approximately 85% (Figure 3b). However, upon further cultivation without the active compounds, melanocytes that had been inhibited by Thiamidol rapidly restarted their melanin production, reaching pretreatment levels within 1 week. In contrast, hydroquinone-treated cells did not recover their full capacity for melanin production within the 2-week culture period, and melanin production continued at 85% of pretreatment levels.

      Molecular modeling

      Possible binding modes of Thiamidol to hTyr were examined by virtual docking studies. Figure 4a shows the active site of the homology model of hTyr in the met- form, with a docked Thiamidol ligand in a lowest-energy conformation. The di-copper center with the bridging oxygen is visible on the left. Only amino acid residues immediately adjacent to the bound inhibitor are shown. (Residue numbering includes the signal peptide). The inner surface of the binding pocket is colored according to hydrophobicity on a scale from blue for hydrophilic to brown for hydrophobic. Although the environment of the di-copper center is distinctly hydrophilic, a strongly hydrophobic subpocket is formed mainly by the side chains of I368, V377, and F347. In the spatial orientation shown, the 1-hydroxy group of the aromatic ring of the ligand makes extensive contacts with the di-copper center, and the 3-hydroxy group is involved in hydrogen bonds with the side chain of S380 and the backbone carbonyl of M374. The thiazolyl ring is held in place by hydrophobic interactions with the nonpolar pocket (Figure 4b), formed by side chains of amino acids, most of which differ between mTyr and hTyr (Figure 4c).
      Figure 4
      Figure 4Structural aspects of hTyr and mTyr. (a) Thiamidol (Beiersdorf AG, Hamburg, Germany) docked into a homology model of hTyr as detailed in the text. The inner surface of the substrate binding pocket is colored according to hydrophobicity: gray, hydrophobic; blue, hydrophilic. (b) Schematic view of interactions stabilizing the modeled enzyme-ligand complex. Hydrophobic interactions are depicted by yellow arcs, hydrogen bonding interactions by red and green arrows, and π-π bonding by a blue arrow. (c) Comparison of the amino acid sequences of hTyr with mTyr isoenzymes PPO3 and PPO4 in the CuB region. Cu-coordinating histidines are highlighted in red, residues probably interacting with Thiamidol are in blue. hTyr, human tyrosinase; mTyr, mushroom tyrosinase.
      Comparable results were obtained when Thiamidol was docked to the recently published x-ray structure of the structurally similar TRP1, a Zn2+-containing melanogenic enzyme of yet unknown function in humans (
      • Ghanem G.
      • Fabrice J.
      Tyrosinase related protein 1 (tyrp1/gp75) in human cutaneous melanoma.
      ,
      • Lai X.
      • Wichers H.J.
      • Soler-Lopez M.
      • Dijkstra B.W.
      Structure of human tyrosinase related protein 1 reveals a binuclear zinc active site important for melanogenesis.
      ), suggesting that the TYRP1 enzyme is inhibited by Thiamidol as well (see Supplementary Figure S2 online).

      Clinical studies

      The in vivo efficacy of Thiamidol was then examined in clinical studies where elderly subjects treated age spots on their skin twice daily with a formula containing 0.2% Thiamidol or with the vehicle only as a control. Already after 4 weeks of treatment, the treated age spots were significantly lighter than the untreated control age spots (Figure 5a). Improvement continued over the entire treatment period, and after 12 weeks some of the age spots were indistinguishable from the surrounding normally pigmented skin (Figure 5b). EpiFlash (Canfield Scientific Inc., Parsippany, NJ) photographs showed visible improvement in the appearance of age spots, and the untreated control age spots remained unchanged (not shown). A follow-up study showed that concentrations of Thiamidol as low as 0.1% effectively reduced the visibility of age spots (see Supplementary Figure S3 online).
      Figure 5
      Figure 5Effects of Thiamidol on age spots in clinical studies. (a) Age spots on the volar forearms of each subject were treated twice daily for 12 weeks with 0.2% Thiamidol (Beiersdorf AG, Hamburg, Germany) or with the vehicle only as a control using a spot applicator. Efficacy was evaluated after 4, 8, and 12 weeks. Data represent the mean ± standard error of the mean of 17 subjects. P < 0.05, ∗∗P < 0.01; statistically significant versus the control. (b) Visual monitoring of the lightening of age spots during treatment. Photographs were taken (top) before and (bottom) after 12 weeks of treatment. Images show three representative age spots. Scale bar = 5 mm. Pre, before treatment; Post, after 12 weeks of treatment.

      Discussion

      The safest and most effective way to treat cutaneous hyperpigmentation is to reduce melanin production by inhibiting tyrosinase activity. However, most tyrosinase inhibitors described in the literature lack clinical efficacy when incorporated into topical products. Almost all of them were tested only against mTyr (
      • Espin J.C.
      • Varon R.
      • Fenoll L.G.
      • Gilabert M.A.
      • Garcia-Ruiz P.A.
      • Tudela J.
      • et al.
      Kinetic characterization of the substrate specificity and mechanism of mushroom tyrosinase.
      ,
      • Garcia-Molina F.
      • Hiner A.N.
      • Fenoll L.G.
      • Rodriguez-Lopez J.N.
      • Garcia-Ruiz P.A.
      • Garcia-Canovas F.
      • et al.
      Mushroom tyrosinase: catalase activity, inhibition, and suicide inactivation.
      ) and thus, although being effective against mTyr, turned out to be poor inhibitors of hTyr. Commercially available mTyr is not a homogeneous preparation but rather is a mixture of several tyrosinase isoenzymes and small amounts of additional enzyme activities that may affect inhibition studies in unpredictable ways (
      • Pretzler M.
      • Bijelic A.
      • Rompel A.
      Heterologous expression and characterization of functional mushroom tyrosinase (AbPPO4).
      ). Isoenzymes AbPPO3 and AbPPO4, the main components of commercially available mTyr, have amino acid sequences in the region of the active site that significantly differ from hTyr (Figure 4c). Both mTyr isoenzymes contain extra loops between Asn371 (one of the glycosylation sites of hTyr) and Gly372. Several of the residues interacting with Thiamidol in hTyr (cf. Figure 4b) are not conserved in mTyr, for example, Ile368, Ser375, and Ser380. Phe207 is structurally conserved in mTyr, whereas Phe347 is not. Because even small changes in enzyme-ligand interactions may have dramatic effects on binding affinities, the diverse inhibition profiles of hTyr and mTyr (summarized in Table 1) did not come as a surprise.
      The main objective of this study was to compare the effects of arbutin, hydroquinone, and kojic acid with various resorcinol derivatives on the catalytic function of hTyr and on melanin production in vivo. Except for Thiamidol, all of the tested substances have been described as tyrosinase inhibitors (
      • Kim H.
      • Choi H.R.
      • Kim D.S.
      • Park K.C.
      Topical hypopigmenting agents for pigmentary disorders and their mechanisms of action.
      ); however, their reported inhibitory activities are extremely divergent. In the medical literature, hydroquinone is considered the criterion standard for the treatment of skin hyperpigmentation, although there are severe concerns regarding its safety. Hydroquinone is banned in the European Union from use in cosmetics, but it is still sold in the United States as an over-the-counter drug in formulations containing up to 2% hydroquinone. Recently, the

      US Food and Drug Administration, Department of Health and Human Services. Skin bleaching drug products for over-the-counter human use; proposed rule. 71 Federal Register 51146-5115521 (codified at 21 CFR Part 310); 2006.

      expressed concern about hydroquinone; however, a final ruling is still pending. The published IC50 values for hydroquinone inhibition of mTyr cover a range from 1.1 μmol/L (
      • Kang H.H.
      • Rho H.S.
      • Hwang J.S.
      • Oh S.G.
      Depigmenting activity and low cytotoxicity of alkoxy benzoates or alkoxy cinnamte in cultured melanocytes.
      ) to 680 μmol/L (
      • Abu Ubeid A.
      • Zhao L.
      • Wang Y.
      • Hantash B.M.
      Short-sequence oligopeptides with inhibitory activity against mushroom and human tyrosinase.
      ). In our analysis, hydroquinone was remarkably ineffective against hTyr, inhibiting it only slightly, reaching just 50% inhibition at approximately 4,000 μmol/L. Although hydroquinone has been considered as a tyrosinase inhibitor since the early 1990s (
      • Palumbo A.
      • d'Ischia M.
      • Misuraca G.
      • Prota G.
      Mechanism of inhibition of melanogenesis by hydroquinone.
      ), our results suggest that its cytotoxic properties are actually more important, not only for its adverse effects on melanocytes but also for its efficacy as an inhibitor of melanogenesis (
      • Jimbow K.
      • Obata H.
      • Pathak M.A.
      • Fitzpatrick T.B.
      Mechanism of depigmentation by hydroquinone.
      ,
      • Penney K.B.
      • Smith C.J.
      • Allen J.C.
      Depigmenting action of hydroquinone depends on disruption of fundamental cell processes.
      ,
      • Smith C.J.
      • O’Hare K.B.
      • Allen J.C.
      Selective cytotoxicity of hydroquinone for melanocyte-derived cells is mediated by tyrosinase activity but independent of melanin content.
      ). This view is substantiated not only by our results with hTyr and the fact that hydroquinone significantly reduced melanin production in skin models but also by our experiments with melanocyte cultures. Here, hydroquinone reduced melanin production, but the treated cells did not regain the full capacity to produce melanin after removal of the active.
      Although arbutin is generally considered an effective tyrosinase inhibitor, the published IC50 values of arbutin for mTyr range from 40 μmol/L (
      • Ying Y.H.
      • Lee S.J.
      • Chung M.H.
      • Ying H.J.
      • Suk J.L.
      • Myung H.C.
      • et al.
      Aloesin and arbutin inhibit tyrosinase activity in a synergistic manner via a different action mechanism.
      ) to more than 30,000 μmol/L (
      • Sugimoto K.
      • Nomura K.
      • Nishimura T.
      • Kiso T.
      • Sugimoto K.
      • Kuriki T.
      Syntheses of α-arbutin-α-glycosides and their inhibitory effects on human tyrosinase.
      ). In our test system, we found very high IC50 values (>4,000 μmol/L) for arbutin with both purified hTyr and the MelanoDerm skin model. Data on the efficacy of both α-arbutin and β-arbutin have been published (
      • Garcia-Jimenez A.
      • Teruel-Puche J.A.
      • Berna J.
      • Rodriguez-Lopez J.N.
      • Tudela J.
      • Garcia-Canovas F.
      Action of tyrosinase on alpha and beta-arbutin: A kinetic study.
      ). However, both compounds are actually hydroquinone pro-drugs, with their biological activity dependent on the release of hydroquinone from the molecule (
      • Briganti S.
      • Camera E.
      • Picardo M.
      Chemical and instrumental approaches to treat hyperpigmentation.
      ). The European Union

      Scientific Committee on Consumer Products. Opinion on β-arbutin, http://ec.europa.eu/health/archive/ph_risk/committees/04_sccp/docs/sccp_o_134.pdf; 2008 (accessed November 21, 2017).

      published a critical opinion on arbutin. In view of the release of hydroquinone from the molecule, it regards the use of arbutin in cosmetic products as unsafe.
      The published IC50 values for tyrosinase inhibition by kojic acid range from 6 μmol/L (
      • Curto E.V.
      • Kwong C.
      • Hermersdörfer H.
      • Glatt H.
      • Santis C.
      • Virador V.
      • et al.
      Inhibitors of mammalian melanocyte tyrosinase: in vitro comparisons of alkyl esters of gentisic acid with other putative inhibitors.
      ) to more than 100 μmol/L (
      • Jeon S.H.
      • Kim K.H.
      • Koh J.U.
      • Kong K.H.
      Inhibitory effects of l-Dopa oxidation of tyrosinase by skin whitening agents.
      ). As an inhibitor of hTyr, kojic acid is much less efficient, with an IC50 of about 500 μmol/L. Kojic acid exhibits a mixed type of inhibition, with a Ki of 145 μmol/L, indicating that it binds to the deoxy- form of tyrosinase (
      • Sun W.
      • Wendt M.
      • Klebe G.
      • Röhm K.H.
      On the interpretation of tyrosinase inhibition kinetics.
      ). When used to treat the MelanoDerm model, kojic acid shows an exceptionally steep dose-response curve, with relative inhibition increasing from 5% at 150 μmol/L to more than 75% inhibition at 900 μmol/L (see Figure 2c). This fact may be the main reason for the very limited efficacy of kojic acid in vivo. Concerning the safety of kojic acid, the European

      Scientific Committee on Consumer Safety. Opinion on kojic acid, http://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_o_098.pdf; 2012 (accessed November 21, 2017).

      now considers kojic acid at concentrations up to 1.0% to be safe for cosmetic products when applied to healthy skin, a view shared by the Cosmetic Ingredient Review Expert Panel (
      • Burnett C.L.
      • Bergfeld W.F.
      • Belsito D.V.
      • Hill R.A.
      • Klaassen C.D.
      • Liebler D.C.
      • et al.
      Final report of the safety assessment of Kojic acid as used in cosmetics.
      ).
      Rhododendrol was granted quasi-drug status in Japan in 2008 and was used as a whitening ingredient in cosmetic products. It was assumed to be a competitive inhibitor of tyrosinase. However, in 2013, rhododendrol-containing products were recalled in 10 Asian countries when close to 20,000 consumers developed leukoderma after using the products. It was shown that rhododendrol is not only an inhibitor but is also a substrate of both hTyr (
      • Ito S.
      • Gerwat W.
      • Kolbe L.
      • Yamashita T.
      • Ojika M.
      • Wakamatsu K.
      Human tyrosinase is able to oxidize both enantiomers of rhododendrol.
      ) and mTyr (
      • Ito S.
      • Ojika M.
      • Yamashita T.
      • Wakamatsu K.
      Tyrosinase-catalyzed oxidation of rhododendrol produces 2-methylchromane-6,7-dione, the putative ultimate toxic metabolite: implications for melanocyte toxicity.
      ). The tyrosinase-dependent accumulation of endoplasmic reticulum stress and/or activation of the apoptotic pathway may contribute to the melanocyte cytotoxicity of rhododendrol (
      • Sasaki M.
      • Kondo M.
      • Sato K.
      • Umeda M.
      • Kawabata K.
      • Takahashi Y.
      • et al.
      Rhododendrol, a depigmentation-inducing phenolic compound, exerts melanocyte cytotoxicity via a tyrosinase-dependent mechanism.
      ).
      The 4-substituted resorcinol motif has been known for some time as an efficient chemical moiety that inhibits tyrosinase (
      • Khatib S.
      • Nerya O.
      • Musa R.
      • Shmuel M.
      • Tamir S.
      • Vaya J.
      Chalcones as potent tyrosinase inhibitors: the importance of a 2,4-substituted resorcinol moiety.
      ). Many natural compounds that have been identified as whitening agents, mainly flavonoids, contain this motif (
      • Shimizu K.
      • Kondo R.
      • Sakai K.
      Inhibition of tyrosinase by flavonoids, stilbenes and related 4-substituted resorcinols: structure-activity investigations.
      ,
      • Shimizu M.M.
      • Melo G.A.
      • Brombini Dos Santos A.
      • Bottcher A.
      • Cesarino I.
      • Araújo P.
      • et al.
      Enzyme characterisation, isolation and cDNA cloning of polyphenol oxidase in the hearts of palm of three commercially important species.
      ). Because the bioavailability of flavonoids is generally low, our goal was to identify resorcinol derivatives with better effectiveness and bioavailability. 4-Butylresorcinol had already been identified as an inhibitor of mouse and human tyrosinase (
      • Kim D.S.
      • Kim S.Y.
      • Park S.H.
      • Choi Y.G.
      • Kwon S.B.
      • Kim M.K.
      • et al.
      Inhibitory effects of 4-n-butylresorcinol on tyrosinase activity and melanin synthesis.
      ,
      • Kolbe L.
      • Mann T.
      • Gerwat W.
      • Batzer J.
      • Ahlheit S.
      • Scherner C.
      • et al.
      4-n-butylresorcinol, a highly effective tyrosinase inhibitor for the topical treatment of hyperpigmentation.
      ) and of the dihydroxyindole carboxylic acid oxidase activity of mouse TYRP1 (
      • Katagiri T.
      • Okubo T.
      • Oyobikawa M.
      • Futaki K.
      • Shaku M.
      • Kawai M.
      • et al.
      Inhibitory action of 4-n-butylresorcinol on melanogenesis and its skin whitening effect.
      ), and it is commercially available for the medical and cosmetic treatment of hyperpigmentation (

      Bohnsack K, Koop U, Hiddemann S, Kolbe L, Rippke F. Pigmentation reducing efficacy and tolerability of six new face care formulations containing 4-n-butylresorcinol, poster no. P864. Poster presented at: 21st EADV Congress, September 27-30, 2012; Prague, Czech Republic.

      ,
      • Jimenez M.
      • Garcia-Carmona F.
      4-substituted resorcinols (sulfite alternatives) as slow-binding inhibitors of tyrosinase catecholase activity.
      ,
      • Kim D.S.
      • Kim S.Y.
      • Park S.H.
      • Choi Y.G.
      • Kwon S.B.
      • Kim M.K.
      • et al.
      Inhibitory effects of 4-n-butylresorcinol on tyrosinase activity and melanin synthesis.
      ). Nevertheless, detailed kinetic data of 4-butylresorcinol were still missing. In the hTyr assay, we found a strictly competitive type of inhibition by 4-butylresorcinol with a Ki of 9.1 μmol/L, which is in excellent agreement with the IC50 value determined (Table 1).
      In our in vitro experiments, Thiamidol, with an IC50 of 1.1 μmol/L in the hTyr enzyme assay and 0.9 μmol/L in the MelanoDerm skin model, was by far the most effective of all substances tested. Further experiments confirmed that Thiamidol is a strictly competitive inhibitor (Figure 2b) and not a substrate for tyrosinase (see Supplementary Figure S1), and, thus, Thiamidol is not converted to a toxic and potentially leukoderma-inducing quinone. Therefore, Thiamidol was selected for clinical studies to assess its efficacy in vivo. A study of Thiamidol using a spot applicator showed continuous improvement in the appearance of age spots over the entire 12-week treatment period, reaching statistical significance as early as 4 weeks. These results show a strong pigment-reducing efficacy of the Thiamidol test product and a clear clinical benefit in the management of skin hyperpigmentation.
      In conclusion, our study shows that the structural differences between hTyr and mTyr are reflected in the molecular features of effective inhibitors. Highly effective inhibitors of hTyr are distinctively different from inhibitors of mTyr and vice versa. Thiamidol (isobutylamido thiazolyl resorcinol) was identified as a potent inhibitor of hTyr with a remarkable efficacy in vitro and in vivo. However, the full potential of this compound needs to be explored in further studies.

      Materials and Methods

      Human tyrosinase

      A truncated, His-tagged form of hTyr (hTyr-DHis) comprising the catalytic domain of hTyr was expressed in HEK 293 cells and purified by metal affinity chromatography on Ni2+-Sepharose (GE Healthcare, Munich, Germany) as described elsewhere (
      • Cordes P.
      • Sun W.
      • Wolber R.
      • Kolbe L.
      • Klebe G.
      • Röhm K.H.
      Expression in non-melanogenic systems and purification of soluble variants of human tyrosinase.
      ). The resulting preparation had the same catalytic properties as wild-type hTyr.

      Sources of inhibitors

      From the Evotec compound library (Evotec, Hamburg, Germany), 50,000 compounds, covering a wide chemical space, were selected to conduct an HTS for hTyr inhibitors, assessed using the Tyr assay described in the next section. Derivatives of promising lead compounds were then synthesized for further optimization. The other inhibitors were purchased from various suppliers (see Supplementary Materials online for details).

      Tyrosine assay and HTS procedure

      Full details of the l-dopa oxidase activity and the HTS screening procedures used can be found in the Supplementary Materials.

      Molecular modeling

      In silico docking was based on a new homology model of hTyr, described elsewhere (
      • Mann T.
      • Gerwat W.
      • Wenck H.
      • Röhm K.H.
      • Kolbe L.
      Isobutylamido thiazolyl resorcinol a new powerful inhibitor of human tyrosinase.
      ). The simulations were performed using Molegro Virtual Docker (Molegro, Aarhus, Denmark). Discovery Studio Visualizer 4.0 (Accelrys, San Diego, CA) was used for visual data analysis and presentation. The sequences were taken from the UniProt database (
      UniProt Consortium
      UniProt: the universal protein knowledgebase.
      ).

      Skin model assays

      Full details of MelanoDerm tissues used as a skin model and quantitation of their melanin content can be found in the Supplementary Materials.

      Melanocyte cultures

      Full details of melanocyte cultures and quantitation of their melanin content can be found in the Supplementary Materials.

      Clinical studies

      Two randomized in vivo studies (blinded for the test products, open for the untreated control) were conducted. One study enrolled 18 female subjects (56–71 years of age), with 17 subjects completing the study. The second study was performed with 19 subjects (18 females, 1 male; 58–70 years of age), with all 19 subjects completing the study. Each subject applied two different formulations twice daily to age spots on their volar forearms using a spot applicator. The formulations differed only in the active ingredient: 0.2% Thiamidol versus vehicle in the first study, 0.1% Thiamidol versus vehicle in the second study. One age spot per subject was treated with a formula containing the active ingredient, and a control spot was treated with the vehicle only. Pigmentation of the age spots was analyzed as described in the Supplementary Materials. The in vivo studies were conducted according to the recommendations of the current version of the Declaration of Helsinki and the guidelines of the International Conference on Harmonization Good Clinical Practice. All participants in these studies provided written informed consent. In addition, the studies were approved and cleared by the institutional review board of Beiersdorf AG (Hamburg, Germany).

      Conflict of Interest

      TM, WG, JB, KE, CS, HW, FS, and LK are employees of Beiersdorf AG. Thiamidol is patented by Beiersdorf AG. None of the other authors has a conflict of interest to declare.

      Supplementary Material

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      Linked Article

      • Next Time, Save Mushrooms for the Pizza!
        Journal of Investigative DermatologyVol. 138Issue 7
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          Mann et al. (2018) use recombinant human tyrosinase to screen for novel inhibitors of pigmentation. They develop thiamidol, a new thiazolyl-resorcinol derivative, that is a submicromolar tyrosinase inhibitor and effective for treating solar lentigines. Thiamidol and established inhibitors of pigmentation exhibit substantially different activities on human and mushroom tyrosinase, supporting use of the human enzyme in high-throughput screens.
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