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PIGMENT CELL & MELANOMA Research Inulavosin and its benzo-derivatives, melanogenesis inhibitors, target the copper loading mechanism to the active site of tyrosinase Hideaki Fujita, José C. J. M. D. S. Menezes, Sérgio M. Santos, Sadaki Yokota, Shrivallabh P. Kamat, José A. S. Cavaleiro, Tomonori Motokawa, Tomomi Kato, Mayu Mochizuki, Toshiyuki Fujiwara, Yuki Fujii and Yoshitaka Tanaka
DOI: 10.1111/pcmr.12225 Volume 27, Issue 3, Pages 376–386 If you wish to order reprints of this article, please see the guidelines here
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ORIGINAL ARTICLE
Pigment Cell Melanoma Res. 27; 376–386
Inulavosin and its benzo-derivatives, melanogenesis inhibitors, target the copper loading mechanism to the active site of tyrosinase C. J. M. D. S. Menezes4,5*, Se rgio M. Santos6, Sadaki Yokota1, Shrivallabh Hideaki Fujita1,2,3*, Jose 5 4 A. S. Cavaleiro , Tomonori Motokawa7, Tomomi Kato7, Mayu Mochizuki7, P. Kamat , Jose Toshiyuki Fujiwara1, Yuki Fujii1 and Yoshitaka Tanaka2,8 1 Section of Functional Morphology, Faculty of Pharmaceutical Sciences, Nagasaki International University, Nagasaki, Japan 2 Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan 3 Innovation Center for Medical Redox Navigation, Kyushu University, Fukuoka, Japan 4 Department of Chemistry and QOPNA, University of Aveiro, Aveiro, Portugal 5 Department of Chemistry, Goa University, Taleigao, Goa, India 6 Department of Chemistry and CICECO, University of Aveiro, Aveiro, Portugal 7 Skin Research Department, POLA Chemical Industries, Inc., Totsuka-ku, Yokohama, Japan 8 Organelle Homeostasis Research Center, Kyushu University, Fukuoka, Japan
KEYWORDS B16 melanoma cells/copper chaperon/ lysosomes/melanogenesis/melanosomes/tyrosinase PUBLICATION DATA Received 30 September 2013, revised and accepted for publication 17 January 2014, published online 31 January 2014 doi: 10.1111/pcmr.12225
CORRESPONDENCE Hideaki Fujita and Yoshitaka Tanaka, e-mails: [email protected] and [email protected] *Authors who contributed equally to this study.
Summary Tyrosinase, a melanosomal membrane protein containing copper, is a key enzyme for melanin synthesis in melanocytes. Inulavosin inhibits melanogenesis by enhancing a degradation of tyrosinase in lysosomes. However, the mechanism by which inulavosin redirects tyrosinase to lysosomes is yet unknown. The analyses of structure–activity relationship of inulavosin and its benzo-derivatives reveal that the hydroxyl and the methyl groups play a critical role in their inhibitory activity. Intriguingly, the docking studies to tyrosinase suggest that the compounds showing inhibitory activity bind through hydrophobic interactions to the cavity of tyrosinase below which the copper-binding sites are located. This cavity is proposed to be required for the association with a chaperon that assists in copper loading to tyrosinase in Streptomyces antibioticus. Inulavosin and its benzoderivatives may compete with the copper chaperon and result in a lysosomal mistargeting of apo-tyrosinase that has a conformational defect.
Introduction Melanin is produced by melanocytes, which are localized in the basal layer of the skin (Costin and Hearing, 2007; Lin and Fisher, 2007; Yamaguchi and Hearing, 2009).
Tyrosinase and its two related proteins (Tyrosinaserelated proteins; Tyrp-1 and Tyrp-2) are involved in melanin synthesis (Jimbow et al., 2000; Orlow et al., 1993; Vijayasaradhi et al., 1995; Wang and Hebert, 2006). Within the cells, they are translated in the endoplasmic
Significance Many of the melanogenesis inhibitors prevent the access of tyrosine to the active site of tyrosinase where the histidine residues containing copper ions are located. However, the mechanism underlying inulavosin, a novel melanogenesis inhibitor, remains unknown. Our works show that inulavosin and its benzo-derivatives directly bind to the cavity of tyrosinase where unidentified copper chaperon(s) bind. They may inhibit the copper loading process in living melanocytes. The resultant apo-tyrosinase is delivered to lysosomes, instead of its bona fide destination, melanosomes. This type of melanogenesis inhibitors is quite unique. Our findings have significant implications for designing future novel melanogenesis inhibitors.
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ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Inulavosin inhibits a copper loading to tyrosinase
Inula nervosa as a racemic mixture, leads to hypopigmentation of melanoma cells without affecting both transcription and enzymatic activity of tyrosinase (Fujita et al., 2009). We found that racemic 1 specifically accelerates tyrosinase degradation in lysosomes. However, how 1 misleads tyrosinase to lysosomes is still an open question. Here, to understand the structure–activity relationship in the inhibitory effect of 1, we tested several racemic benzo-derivatives (2–5; see Figure 1A) (Menezes et al., 2011) which share the 2-phenyl chroman 6 structure with 1. Based on the results obtained through biochemical experiments on compounds 2–5 and representative molecular docking simulations of 1, 2, and 4 to tyrosinase, we propose a novel mechanism underlying the 1 and its derivatives-induced lysosomal degradation of tyrosinase in melanocyte.
reticulum as a type I membrane glycoprotein, trafficked through Golgi and delivered to melanosomes where melanin is synthesized and accumulated (Hearing, 2005; Marks and Seabra, 2001; Raposo and Marks, 2002). Among them, the structure and catalytic mechanism of tyrosinase has been extensively studied (Olivares and Solano, 2009). The active site of tyrosinase, containing two copper-binding domains named CuA and CuB, is highly conserved among different species (Furumura et al., 1998; Schweikardt et al., 2007; Spritz et al., 1997). As many membrane proteins, a proper folding and N-glycan processing of tyrosinase in the endoplasmic reticulum is crucial for its catalytic activity (Choi et al., 2007). Furthermore, copper loading to tyrosinase, which takes place in the trans-Golgi network (TGN) area, is also an important step to acquire the enzymatic activity (Petris et al., 2000; Setty et al., 2008). In the recent cosmetic market, the identification and development of safe and natural skin-lightening substances has attracted much attention (Gillbro and Olsson, 2011; Solano et al., 2006). Large numbers of tyrosinase inhibitors have been reported. Nevertheless, while the crystal structure of tyrosinase from mushroom and several species of fungus has been solved (Decker et al., 2006; Matoba et al., 2006), there is limited information on the intermolecular interactions between inhibitor and tyrosinase (Khan, 2012). Previously, we have reported that inulavosin (henceforth denoted 1), a melanogenesis inhibitor isolated from A
Results Inulavosin derivatives inhibit cell pigmentation To determine the minimum 1 type structure required for this activity, we tested five compounds (2–6) that share a common 2-phenyl chroman core (shown in red; Figure 1A). We applied them to the culture medium and observed their effects on cell pigmentation using B16 melanoma cells, as described previously (Fujita et al., 2009). The two structurally isomeric compounds, 2-(2,4,4trimethyl-3,4-dihydro-2H-benzo[h]chromen-2-yl)-1-naphthol
B Control (DMSO)
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Figure 1. Inulavosin 1 and its derivatives (2–5) inhibit melanogenesis in B16 melanoma cells. (A) Structures of compounds used in this study. The structure of 1 and that conserved in 2–6 are shown by red. (B) The cells treated with 15 lM of each compounds for 72 h were collected. (C) The cells treated with the indicated concentration of each compounds for 72 h were collected. (D) B16 melanoma cells were treated with 15 lM of each compounds for 72 h. Differential interference contrast microscopic images of the cells were shown. The representative localization of heavily pigmented melanosomes to the tip of the cells was denoted by red dotted circles in control cells. Scale bar, 50 lm.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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* :P < 0.0001 ** :P < 0.005 *** :P < 0.05
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Figure 2. Late stages of melanosomes decrease in B16 melanoma cells treated with 1, compounds 2–5, except in case of 6. (A) The cells treated with 15 lM of each compounds for 72 h were processed for electron microscopic analysis. Scale bars, 1 lm. Red rectangle areas were enlarged and shown below each. Red arrows indicated the accumulated stage II melanosomes. Scale bars, 0.5 lm. (B) Melanosomes are morphologically classified into Stage I–IV based on the following definitions. Stage I: multivesicular structures containing no visible pigment. Stage II: oval-shaped structures sometime with an ordering of internal filaments into parallel arrays. Stage III: accumulation of melanin along with the internal filaments. Stage IV: melanosomes are completely filled with melanin. The representative morphological structures of all stages of melanosomes were shown in the right. The numbers of each stage of melanosomes (Stages I–IV) were counted (10–20 images/compound). The percentages of each stage were shown as averages standard deviation (SD). Data were analyzed for statistical significance using Student’s t test. P values of < 0.05 were considered significant.
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2 and 3-(2,4,4-trimethyl-3,4-dihydro-2H-benzo[g]chromen2-yl)-2-naphthol 4, and their corresponding acetates (3 and 5, respectively), (Menezes et al., 2011) effectively inhibited hyperpigmentation of the cells to the same extent as 1 (Figure 1B). However, the 2-phenyl chroman 6 showed no inhibitory effects. Their effects were dose-dependent (Figure 1C). Concentrations over 50 lM of all compounds indicated toxicity to the cells and therefore we decided to use 15 lM throughout all of the experiments. The effects of these compounds were also confirmed by differential interference contrast (DIC) images (Figure 1D). The B16 melanoma cells treated with DMSO and 6 showed the accumulation of melanin deposited in heavily pigmented granules, melanosomes. However, the cells treated with compounds 2–5 lost the melanosomes to the same extent as 1. Inulavosin derivatives decrease ratio of late stages of melanosomes Electron microscopic (EM) analyses revealed that while control cells were predominantly filled with the late stages of melanosomes (Stages III and IV), the cells treated with 1 and compounds 2–5, contained less number of the late stage melanosomes except in case of compound 6 (Figure 2A). Instead, the accumulations of stage II melanosomes were observed. We counted the number of each stage of melanosomes from the EM pictures. We found that compounds 2–5 significantly reduced the ratio of late stages of melanosomes (Stages III and IV) and increased early ones (Stages I and II), to the same extent as 1 did (Figure 2B). Intriguingly, as observed by enlarged images, the formation of Stage II melanosomes, oval-shaped structures sometime with an ordering of internal filaments into parallel arrays, was not affected by compounds 1–5 (red arrows in Figure 2). These results suggest that 1 and its benzo-derivatives (2–5) specifically reduce the melanin accumulation to the melanosomes without affecting on their morphology and formation of early stages of melanosomes. These compounds exhibited no specific effects on the morphology of mitochondria and the appearance of cytosol. Our previous works demonstrated that inulavosin 1 has no effect on the cell viability at the concentration we used (15 lM) (Fujita et al., 2009). We examined the cell toxicity of inulavosin 1 and compound 2 by WST assay and revealed that the IC50 values of these two compounds to cell viability were above 40 lM (data not shown); thus, we concluded that there is no significant damages to the cells at the concentration we used in this study. Inulavosin derivatives lead to tyrosinase degradation in lysosomes We next examined the expression and intracellular localization of tyrosinase by confocal microscopic analyses (Figure 3). In control cells, tyrosinase localized to the small granules that concentrated near the tips and peripheral region of the cells (Figure 3A, B, green arrows). In the ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
presence of inulavosin 1 or compound 2 (the compound possibly showing the highest inhibitory effect, see Table 1), the tyrosinase signals were significantly reduced and only found in the small vesicles near the TGN. A small fraction of these vesicles were colocalized with TGN38 (Figure 3A) and syntaxin-6 (Figure 3B), respectively (denoted by arrowheads in each color). Importantly, the morphology of TGN was not affected by these compounds. Tyrp-1, another melanosomal membrane protein, colocalized well with LAMP-1, a marker of late endosomes and lysosome, near the tips and peripheral region of the cells (indicated by arrows in each color). Neither inulavosin nor compound 2 affected on the expression and distribution of them, suggesting that these compounds specifically targeted to tyrosinase. Western blotting analyses revealed that compounds 2–5 specifically reduced the expression of tyrosinase, but not Tyrp-1, as 1 did (Figure 4A). Compound 6, however, continued to show no effect. Lysosomal protease inhibitors effectively restored the expression of tyrosinase (Figure 4B), suggesting that compounds 2–5, but not 6, accelerated tyrosinase degradation in lysosomes similar to 1. Inulavosin and its derivatives dock to a cavity near the active site of tyrosinase To understand the probable mechanism of action, we performed molecular docking simulations to assess the affinity and binding modes of 1 and compounds 2, 4, and 6 to tyrosinase. Although a racemic mixture of these compounds was used in the biochemical tests, in the docking simulations, their R- and S-enantiomeric forms were studied individually to understand the possible influence of each isomer on the tyrosinase degradation mechanism. These simulations were performed on the only known di-copper loaded crystal structure of tyrosinase, solved from Streptomyces castaneoglobisporus (Matoba et al., 2006). The docking simulations showed that the S-enantiomers of 1, 2, and 4 appear to have significantly higher binding affinities for the enzyme than the R-isomers, although both enantiomeric forms fit inside the catalytic site (see Figure 5A for representative examples of 1-S, 2-S, and 4-S and Table 1 for the corresponding binding energies). From the set of studied inhibitors, compound 2-S exhibits the highest binding affinity (ΔGbinding,2-S = 17.53 kcal/mol). However, if one looks at 4-S, which is structurally isomeric with 2-S, then the binding affinity decreases considerably (ΔGbinding,4-S = 8.41 kcal/mol), which clearly reflects the better fit between the inhibitor 2 and the cavity near the catalytic center of the enzyme. Interestingly, the binding affinity value of 1 (ΔGbinding,1-S = 9.31 kcal/mol) and 4 is closer to each other than to that of 2, which is structurally isomeric with 4. Compound 6 (control) shows negligible affinity toward the enzyme (see Table 1). Interestingly, although the hydroxyl function present in 1, 2, and 4 provides the possibility for formation of hydrogen bonds, these do not exist in any of the found conformations. In 379
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Figure 3. Inulavosin 1 and compound 2 specifically altered the intracellular localization of tyrosinase in B16 melanoma cells. The cells treated with DMSO or 15 lM of each compound for 72 h were processed for double-immunofluorescence analysis by confocal microscopy. (A, B) The fixed cells were labeled with antibodies to tyrosinase (green) and TGN38 (red in (A)) or syntaxin-6 (red in (B)]. Tyrosinase, found near the tips and peripheral region of the cells, was indicated by green arrows. In the cells treated with inulavosin 1 or compound 2, the colocalizations of tyrosinase with TGN38 or syntaxin-6 were denoted by arrowheads. (C) The cells were labeled with antibodies to Tyrp-1 (green) and mouse LAMP-1 (red). Tyrp-1 well colocalized with LAMP-1 near the tips and peripheral region of the cells (indicated by arrows in each color). Neither inulavosin 1 nor compound 2 affected on the expression and distribution of them. Scale bars, 20 lm.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Inulavosin inhibits a copper loading to tyrosinase
C
LAMP1
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Figure 3. (continued)
Table 1. Relative binding free energies (ΔGbinding, relatively to 6-S) of inulavosin and its derivatives to tyrosinase
1 (Inulavosin) Enantiomer ΔGbinding/kcal/mol
S
2 R
9.31
3.40
4
S
R 17.53
fact, and apart from the hydroxyl group, the major difference between 6 and the remaining compounds (1, 2, and 4) resides in the presence of methyl and benzene groups which are both significantly hydrophobic. These results, along with a better fit of 2 to the enzyme, suggest that the nature of the interaction between 1 and its derivatives (2 and 4) and the enzyme is mainly hydrophobic. We tested whether the hypopigmentation efficacy depends on the docking efficacy by measuring the cellular melanin contents using racemic 1, 2, and 4. The data clearly showed that they all had significant inhibitory effect on cell pigmentation (Figure 5B). Importantly, the racemic 2 showed the highest inhibitory activity as was predicted by the docking study. While the efficacy difference between compounds 1 and 4 was not significant. The docking results also show that the inhibitors do not interact directly with the copper catalytic core of the enzyme. In fact, the shortest distance observed ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
6
S 2.53
8.41
R
S
R
4.79
0.00
2.22
between any atom of the studied inhibitors and the copper cations is approximately 4.6 A, which is clearly too long to suggest direct interaction. As this cavity is proposed to be necessary for the association with the copper chaperons, we predict that the inhibitors affect the process of loading of the copper cations to tyrosinase, but cannot simply block access of the substrates to the catalytic center of the enzyme. To test this assumption, their direct inhibitory effect on tyrosinase activity was examined by in vitro assay of tyrosinase activity using the cell lysate prepared from the B16 cells cultured without any drugs (Figure 5C, D). While 4-n-butylresorcinol, one of the highly effective tyrosinase inhibitors (Kim et al., 2005), strongly reduced both tyrosine hydroxylation and dopa oxidation activities, neither compound 1 nor compound 2 showed any inhibitory effect on these tyrosinases activities, up to 150 lM, the concentration 10 times higher than that we used in cell-based assay.
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Discussion Analyses of a structure–activity relationship in the inhibitory effect of 1 suggest that the hydroxyl group existing in 1, 2, and 4, or any of the methyl groups in 1 and 2–5, may play a major role in their inhibitory activity. In the case of the acetates 3 and 5, we have hypothesized the possibility that they may be hydrolyzed by esterase(s) existing in B16 melanoma cells. The reliability of our docking simulation was partially proved by measuring melanin contents in the cells treated with compounds 1, 2, and 4, the racemic 2 showed the highest inhibitory activity (Figure 5B). The docking experiments further indicate that the S-isomers of compounds 1, 2, and 4 possess higher inhibitory effects over tyrosinase activity than the control compound 6 (see Table 1). The preferential association of the S-isomers seems to arise from the specific conformation adopted by the central triply methylated 6-membered pyran ring, which allows the two bulky geminal methyl groups to be directed toward outside the binding cavity. This arrangement permits the aromatic moieties of the inhibitors to better fit into the enzyme’s binding pocket. The bulkiness of these two geminal methyls appears to impede the adequate fit of the R-isomers to the cavity because of stereochemical hinderance. Hence, and given that the key functional groups directly involved in the inhibitor–enzyme interaction are either aromatic or aliphatic, the hydrophobic nature of interaction of these compounds with tyrosinase is further strengthened, as the large concave like cavity above the dicopper center which acts like putative substrate-binding pocket is formed by the hydrophobic
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Compound 5
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Figure 4. Inulavosin 1 and its derivatives (2–5) accelerated a lysosomal degradation of tyrosinase in B16 melanoma cells. (A) Cell lysates prepared from the cells treated with each compound (15 lM) for 72 h were subjected to Western blotting with antibodies to tyrosinase, Tyrp-1, and b-actin, respectively. (B) Cell lysates prepared from the cells treated as in (a) either in the absence ( ) or presence (+) of lysosomal protease inhibitors (100 lM of leupeptin, pepstatin-A, and 20 lg/ml of E64d) were subjected to Western blotting with antibodies to tyrosinase and b-actin, respectively.
residues (Matoba et al., 2006). Nevertheless, this structure–binding-affinity relationship observed in compounds 2 and 4 can also be partially explained on the basis of the inherent perpendicular orientation between the naphthopyran ring containing the three methyl groups and the naphthalene ring carrying the hydroxyl group (Srinivasan et al., 2012). This perpendicular disposition of the two parts of the molecule, which also exists in 1 (since they all have similar structural core; see Figure 1A), may preferentially direct the S-enantiomers of them in the right orientation toward the cavity above the catalytic center. Furthermore, the higher binding affinity observed in 2 can be associated with the inherent structural angular juxtaposition of the benzene ring in comparison with the linear one in 4. It can be argued that the angular or C-type ring structure existing in the naphthopyran part of 2 fits well into this cavity, and the additional benzene ring confers slightly better hydrophobic interactions in comparison with 1 and 4. The relative closeness between the calculated binding affinity values for 1 and 4 in comparison with 2 can also be rationalized on the basis of less hydrophobic nature of methyl group in 1 and the linear juxtaposition of the benzene ring in 4 over the angular structure in 2. As also observed in the docking experiments, the hydroxyl function present in 1, 2, and 4 does not provide the possibility for formation of hydrogen bonds under the studied conditions, which further supported the interaction between inhibitor and enzyme being mainly hydrophobic. Nonetheless, the influence of hydroxyl group (in case of acetates 3 and 5 which are hypothesized to be hydrolyzed to the active hydroxyl by esterases) cannot be ruled out, as various factors like hydrogen bonding to the
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Inulavosin inhibits a copper loading to tyrosinase
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Figure 5. Inulavosin 1 and its derivatives bind to the cavity of tyrosinase, but do not inhibit its activity. (A) Representative binding configurations of the S-enantiomers of inulavosin (1, top), 2 (middle), and 4 (bottom) to tyrosinase were obtained from docking simulations. The compounds (shown as spheres) fit into the cavity of the catalytic site (right column), without directly interacting with the copper cations burried deep inside the catalytic core (green spheres; left column) or the sulfur atoms of methionine residue (yellow spheres; right column). (B) The inhibitory effects of inulavosin 1 and compound 2 on melanin production in B16 cells were measured. Results are the averages of five experiments SD. Data were analyzed for statistical significance using Student’s t test. P values of
PIGMENT CELL & MELANOMA Research Inulavosin and its benzo-derivatives, melanogenesis inhibitors, target the copper loading mechanism to the active site of tyrosinase Hideaki Fujita, José C. J. M. D. S. Menezes, Sérgio M. Santos, Sadaki Yokota, Shrivallabh P. Kamat, José A. S. Cavaleiro, Tomonori Motokawa, Tomomi Kato, Mayu Mochizuki, Toshiyuki Fujiwara, Yuki Fujii and Yoshitaka Tanaka
DOI: 10.1111/pcmr.12225 Volume 27, Issue 3, Pages 376–386 If you wish to order reprints of this article, please see the guidelines here
EMAIL ALERTS Receive free email alerts and stay up-to-date on what is published in Pigment Cell & Melanoma Research – click here
Submit your next paper to PCMR online at http://mc.manuscriptcentral.com/pcmr
Subscribe to PCMR and stay up-to-date with the only journal committed to publishing basic research in melanoma and pigment cell biology As a member of the IFPCS or the SMR you automatically get online access to PCMR. Sign up as a member today at www.ifpcs.org or at www.societymelanomaresarch.org
To take out a personal subscription, please click here More information about Pigment Cell & Melanoma Research at www.pigment.org
ORIGINAL ARTICLE
Pigment Cell Melanoma Res. 27; 376–386
Inulavosin and its benzo-derivatives, melanogenesis inhibitors, target the copper loading mechanism to the active site of tyrosinase C. J. M. D. S. Menezes4,5*, Se rgio M. Santos6, Sadaki Yokota1, Shrivallabh Hideaki Fujita1,2,3*, Jose 5 4 A. S. Cavaleiro , Tomonori Motokawa7, Tomomi Kato7, Mayu Mochizuki7, P. Kamat , Jose Toshiyuki Fujiwara1, Yuki Fujii1 and Yoshitaka Tanaka2,8 1 Section of Functional Morphology, Faculty of Pharmaceutical Sciences, Nagasaki International University, Nagasaki, Japan 2 Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan 3 Innovation Center for Medical Redox Navigation, Kyushu University, Fukuoka, Japan 4 Department of Chemistry and QOPNA, University of Aveiro, Aveiro, Portugal 5 Department of Chemistry, Goa University, Taleigao, Goa, India 6 Department of Chemistry and CICECO, University of Aveiro, Aveiro, Portugal 7 Skin Research Department, POLA Chemical Industries, Inc., Totsuka-ku, Yokohama, Japan 8 Organelle Homeostasis Research Center, Kyushu University, Fukuoka, Japan
KEYWORDS B16 melanoma cells/copper chaperon/ lysosomes/melanogenesis/melanosomes/tyrosinase PUBLICATION DATA Received 30 September 2013, revised and accepted for publication 17 January 2014, published online 31 January 2014 doi: 10.1111/pcmr.12225
CORRESPONDENCE Hideaki Fujita and Yoshitaka Tanaka, e-mails: [email protected] and [email protected] *Authors who contributed equally to this study.
Summary Tyrosinase, a melanosomal membrane protein containing copper, is a key enzyme for melanin synthesis in melanocytes. Inulavosin inhibits melanogenesis by enhancing a degradation of tyrosinase in lysosomes. However, the mechanism by which inulavosin redirects tyrosinase to lysosomes is yet unknown. The analyses of structure–activity relationship of inulavosin and its benzo-derivatives reveal that the hydroxyl and the methyl groups play a critical role in their inhibitory activity. Intriguingly, the docking studies to tyrosinase suggest that the compounds showing inhibitory activity bind through hydrophobic interactions to the cavity of tyrosinase below which the copper-binding sites are located. This cavity is proposed to be required for the association with a chaperon that assists in copper loading to tyrosinase in Streptomyces antibioticus. Inulavosin and its benzoderivatives may compete with the copper chaperon and result in a lysosomal mistargeting of apo-tyrosinase that has a conformational defect.
Introduction Melanin is produced by melanocytes, which are localized in the basal layer of the skin (Costin and Hearing, 2007; Lin and Fisher, 2007; Yamaguchi and Hearing, 2009).
Tyrosinase and its two related proteins (Tyrosinaserelated proteins; Tyrp-1 and Tyrp-2) are involved in melanin synthesis (Jimbow et al., 2000; Orlow et al., 1993; Vijayasaradhi et al., 1995; Wang and Hebert, 2006). Within the cells, they are translated in the endoplasmic
Significance Many of the melanogenesis inhibitors prevent the access of tyrosine to the active site of tyrosinase where the histidine residues containing copper ions are located. However, the mechanism underlying inulavosin, a novel melanogenesis inhibitor, remains unknown. Our works show that inulavosin and its benzo-derivatives directly bind to the cavity of tyrosinase where unidentified copper chaperon(s) bind. They may inhibit the copper loading process in living melanocytes. The resultant apo-tyrosinase is delivered to lysosomes, instead of its bona fide destination, melanosomes. This type of melanogenesis inhibitors is quite unique. Our findings have significant implications for designing future novel melanogenesis inhibitors.
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ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Inulavosin inhibits a copper loading to tyrosinase
Inula nervosa as a racemic mixture, leads to hypopigmentation of melanoma cells without affecting both transcription and enzymatic activity of tyrosinase (Fujita et al., 2009). We found that racemic 1 specifically accelerates tyrosinase degradation in lysosomes. However, how 1 misleads tyrosinase to lysosomes is still an open question. Here, to understand the structure–activity relationship in the inhibitory effect of 1, we tested several racemic benzo-derivatives (2–5; see Figure 1A) (Menezes et al., 2011) which share the 2-phenyl chroman 6 structure with 1. Based on the results obtained through biochemical experiments on compounds 2–5 and representative molecular docking simulations of 1, 2, and 4 to tyrosinase, we propose a novel mechanism underlying the 1 and its derivatives-induced lysosomal degradation of tyrosinase in melanocyte.
reticulum as a type I membrane glycoprotein, trafficked through Golgi and delivered to melanosomes where melanin is synthesized and accumulated (Hearing, 2005; Marks and Seabra, 2001; Raposo and Marks, 2002). Among them, the structure and catalytic mechanism of tyrosinase has been extensively studied (Olivares and Solano, 2009). The active site of tyrosinase, containing two copper-binding domains named CuA and CuB, is highly conserved among different species (Furumura et al., 1998; Schweikardt et al., 2007; Spritz et al., 1997). As many membrane proteins, a proper folding and N-glycan processing of tyrosinase in the endoplasmic reticulum is crucial for its catalytic activity (Choi et al., 2007). Furthermore, copper loading to tyrosinase, which takes place in the trans-Golgi network (TGN) area, is also an important step to acquire the enzymatic activity (Petris et al., 2000; Setty et al., 2008). In the recent cosmetic market, the identification and development of safe and natural skin-lightening substances has attracted much attention (Gillbro and Olsson, 2011; Solano et al., 2006). Large numbers of tyrosinase inhibitors have been reported. Nevertheless, while the crystal structure of tyrosinase from mushroom and several species of fungus has been solved (Decker et al., 2006; Matoba et al., 2006), there is limited information on the intermolecular interactions between inhibitor and tyrosinase (Khan, 2012). Previously, we have reported that inulavosin (henceforth denoted 1), a melanogenesis inhibitor isolated from A
Results Inulavosin derivatives inhibit cell pigmentation To determine the minimum 1 type structure required for this activity, we tested five compounds (2–6) that share a common 2-phenyl chroman core (shown in red; Figure 1A). We applied them to the culture medium and observed their effects on cell pigmentation using B16 melanoma cells, as described previously (Fujita et al., 2009). The two structurally isomeric compounds, 2-(2,4,4trimethyl-3,4-dihydro-2H-benzo[h]chromen-2-yl)-1-naphthol
B Control (DMSO)
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Figure 1. Inulavosin 1 and its derivatives (2–5) inhibit melanogenesis in B16 melanoma cells. (A) Structures of compounds used in this study. The structure of 1 and that conserved in 2–6 are shown by red. (B) The cells treated with 15 lM of each compounds for 72 h were collected. (C) The cells treated with the indicated concentration of each compounds for 72 h were collected. (D) B16 melanoma cells were treated with 15 lM of each compounds for 72 h. Differential interference contrast microscopic images of the cells were shown. The representative localization of heavily pigmented melanosomes to the tip of the cells was denoted by red dotted circles in control cells. Scale bar, 50 lm.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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60
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* :P < 0.0001 ** :P < 0.005 *** :P < 0.05
n.s. : not significant
Figure 2. Late stages of melanosomes decrease in B16 melanoma cells treated with 1, compounds 2–5, except in case of 6. (A) The cells treated with 15 lM of each compounds for 72 h were processed for electron microscopic analysis. Scale bars, 1 lm. Red rectangle areas were enlarged and shown below each. Red arrows indicated the accumulated stage II melanosomes. Scale bars, 0.5 lm. (B) Melanosomes are morphologically classified into Stage I–IV based on the following definitions. Stage I: multivesicular structures containing no visible pigment. Stage II: oval-shaped structures sometime with an ordering of internal filaments into parallel arrays. Stage III: accumulation of melanin along with the internal filaments. Stage IV: melanosomes are completely filled with melanin. The representative morphological structures of all stages of melanosomes were shown in the right. The numbers of each stage of melanosomes (Stages I–IV) were counted (10–20 images/compound). The percentages of each stage were shown as averages standard deviation (SD). Data were analyzed for statistical significance using Student’s t test. P values of < 0.05 were considered significant.
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ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Inulavosin inhibits a copper loading to tyrosinase
2 and 3-(2,4,4-trimethyl-3,4-dihydro-2H-benzo[g]chromen2-yl)-2-naphthol 4, and their corresponding acetates (3 and 5, respectively), (Menezes et al., 2011) effectively inhibited hyperpigmentation of the cells to the same extent as 1 (Figure 1B). However, the 2-phenyl chroman 6 showed no inhibitory effects. Their effects were dose-dependent (Figure 1C). Concentrations over 50 lM of all compounds indicated toxicity to the cells and therefore we decided to use 15 lM throughout all of the experiments. The effects of these compounds were also confirmed by differential interference contrast (DIC) images (Figure 1D). The B16 melanoma cells treated with DMSO and 6 showed the accumulation of melanin deposited in heavily pigmented granules, melanosomes. However, the cells treated with compounds 2–5 lost the melanosomes to the same extent as 1. Inulavosin derivatives decrease ratio of late stages of melanosomes Electron microscopic (EM) analyses revealed that while control cells were predominantly filled with the late stages of melanosomes (Stages III and IV), the cells treated with 1 and compounds 2–5, contained less number of the late stage melanosomes except in case of compound 6 (Figure 2A). Instead, the accumulations of stage II melanosomes were observed. We counted the number of each stage of melanosomes from the EM pictures. We found that compounds 2–5 significantly reduced the ratio of late stages of melanosomes (Stages III and IV) and increased early ones (Stages I and II), to the same extent as 1 did (Figure 2B). Intriguingly, as observed by enlarged images, the formation of Stage II melanosomes, oval-shaped structures sometime with an ordering of internal filaments into parallel arrays, was not affected by compounds 1–5 (red arrows in Figure 2). These results suggest that 1 and its benzo-derivatives (2–5) specifically reduce the melanin accumulation to the melanosomes without affecting on their morphology and formation of early stages of melanosomes. These compounds exhibited no specific effects on the morphology of mitochondria and the appearance of cytosol. Our previous works demonstrated that inulavosin 1 has no effect on the cell viability at the concentration we used (15 lM) (Fujita et al., 2009). We examined the cell toxicity of inulavosin 1 and compound 2 by WST assay and revealed that the IC50 values of these two compounds to cell viability were above 40 lM (data not shown); thus, we concluded that there is no significant damages to the cells at the concentration we used in this study. Inulavosin derivatives lead to tyrosinase degradation in lysosomes We next examined the expression and intracellular localization of tyrosinase by confocal microscopic analyses (Figure 3). In control cells, tyrosinase localized to the small granules that concentrated near the tips and peripheral region of the cells (Figure 3A, B, green arrows). In the ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
presence of inulavosin 1 or compound 2 (the compound possibly showing the highest inhibitory effect, see Table 1), the tyrosinase signals were significantly reduced and only found in the small vesicles near the TGN. A small fraction of these vesicles were colocalized with TGN38 (Figure 3A) and syntaxin-6 (Figure 3B), respectively (denoted by arrowheads in each color). Importantly, the morphology of TGN was not affected by these compounds. Tyrp-1, another melanosomal membrane protein, colocalized well with LAMP-1, a marker of late endosomes and lysosome, near the tips and peripheral region of the cells (indicated by arrows in each color). Neither inulavosin nor compound 2 affected on the expression and distribution of them, suggesting that these compounds specifically targeted to tyrosinase. Western blotting analyses revealed that compounds 2–5 specifically reduced the expression of tyrosinase, but not Tyrp-1, as 1 did (Figure 4A). Compound 6, however, continued to show no effect. Lysosomal protease inhibitors effectively restored the expression of tyrosinase (Figure 4B), suggesting that compounds 2–5, but not 6, accelerated tyrosinase degradation in lysosomes similar to 1. Inulavosin and its derivatives dock to a cavity near the active site of tyrosinase To understand the probable mechanism of action, we performed molecular docking simulations to assess the affinity and binding modes of 1 and compounds 2, 4, and 6 to tyrosinase. Although a racemic mixture of these compounds was used in the biochemical tests, in the docking simulations, their R- and S-enantiomeric forms were studied individually to understand the possible influence of each isomer on the tyrosinase degradation mechanism. These simulations were performed on the only known di-copper loaded crystal structure of tyrosinase, solved from Streptomyces castaneoglobisporus (Matoba et al., 2006). The docking simulations showed that the S-enantiomers of 1, 2, and 4 appear to have significantly higher binding affinities for the enzyme than the R-isomers, although both enantiomeric forms fit inside the catalytic site (see Figure 5A for representative examples of 1-S, 2-S, and 4-S and Table 1 for the corresponding binding energies). From the set of studied inhibitors, compound 2-S exhibits the highest binding affinity (ΔGbinding,2-S = 17.53 kcal/mol). However, if one looks at 4-S, which is structurally isomeric with 2-S, then the binding affinity decreases considerably (ΔGbinding,4-S = 8.41 kcal/mol), which clearly reflects the better fit between the inhibitor 2 and the cavity near the catalytic center of the enzyme. Interestingly, the binding affinity value of 1 (ΔGbinding,1-S = 9.31 kcal/mol) and 4 is closer to each other than to that of 2, which is structurally isomeric with 4. Compound 6 (control) shows negligible affinity toward the enzyme (see Table 1). Interestingly, although the hydroxyl function present in 1, 2, and 4 provides the possibility for formation of hydrogen bonds, these do not exist in any of the found conformations. In 379
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Figure 3. Inulavosin 1 and compound 2 specifically altered the intracellular localization of tyrosinase in B16 melanoma cells. The cells treated with DMSO or 15 lM of each compound for 72 h were processed for double-immunofluorescence analysis by confocal microscopy. (A, B) The fixed cells were labeled with antibodies to tyrosinase (green) and TGN38 (red in (A)) or syntaxin-6 (red in (B)]. Tyrosinase, found near the tips and peripheral region of the cells, was indicated by green arrows. In the cells treated with inulavosin 1 or compound 2, the colocalizations of tyrosinase with TGN38 or syntaxin-6 were denoted by arrowheads. (C) The cells were labeled with antibodies to Tyrp-1 (green) and mouse LAMP-1 (red). Tyrp-1 well colocalized with LAMP-1 near the tips and peripheral region of the cells (indicated by arrows in each color). Neither inulavosin 1 nor compound 2 affected on the expression and distribution of them. Scale bars, 20 lm.
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Inulavosin inhibits a copper loading to tyrosinase
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Figure 3. (continued)
Table 1. Relative binding free energies (ΔGbinding, relatively to 6-S) of inulavosin and its derivatives to tyrosinase
1 (Inulavosin) Enantiomer ΔGbinding/kcal/mol
S
2 R
9.31
3.40
4
S
R 17.53
fact, and apart from the hydroxyl group, the major difference between 6 and the remaining compounds (1, 2, and 4) resides in the presence of methyl and benzene groups which are both significantly hydrophobic. These results, along with a better fit of 2 to the enzyme, suggest that the nature of the interaction between 1 and its derivatives (2 and 4) and the enzyme is mainly hydrophobic. We tested whether the hypopigmentation efficacy depends on the docking efficacy by measuring the cellular melanin contents using racemic 1, 2, and 4. The data clearly showed that they all had significant inhibitory effect on cell pigmentation (Figure 5B). Importantly, the racemic 2 showed the highest inhibitory activity as was predicted by the docking study. While the efficacy difference between compounds 1 and 4 was not significant. The docking results also show that the inhibitors do not interact directly with the copper catalytic core of the enzyme. In fact, the shortest distance observed ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
6
S 2.53
8.41
R
S
R
4.79
0.00
2.22
between any atom of the studied inhibitors and the copper cations is approximately 4.6 A, which is clearly too long to suggest direct interaction. As this cavity is proposed to be necessary for the association with the copper chaperons, we predict that the inhibitors affect the process of loading of the copper cations to tyrosinase, but cannot simply block access of the substrates to the catalytic center of the enzyme. To test this assumption, their direct inhibitory effect on tyrosinase activity was examined by in vitro assay of tyrosinase activity using the cell lysate prepared from the B16 cells cultured without any drugs (Figure 5C, D). While 4-n-butylresorcinol, one of the highly effective tyrosinase inhibitors (Kim et al., 2005), strongly reduced both tyrosine hydroxylation and dopa oxidation activities, neither compound 1 nor compound 2 showed any inhibitory effect on these tyrosinases activities, up to 150 lM, the concentration 10 times higher than that we used in cell-based assay.
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Discussion Analyses of a structure–activity relationship in the inhibitory effect of 1 suggest that the hydroxyl group existing in 1, 2, and 4, or any of the methyl groups in 1 and 2–5, may play a major role in their inhibitory activity. In the case of the acetates 3 and 5, we have hypothesized the possibility that they may be hydrolyzed by esterase(s) existing in B16 melanoma cells. The reliability of our docking simulation was partially proved by measuring melanin contents in the cells treated with compounds 1, 2, and 4, the racemic 2 showed the highest inhibitory activity (Figure 5B). The docking experiments further indicate that the S-isomers of compounds 1, 2, and 4 possess higher inhibitory effects over tyrosinase activity than the control compound 6 (see Table 1). The preferential association of the S-isomers seems to arise from the specific conformation adopted by the central triply methylated 6-membered pyran ring, which allows the two bulky geminal methyl groups to be directed toward outside the binding cavity. This arrangement permits the aromatic moieties of the inhibitors to better fit into the enzyme’s binding pocket. The bulkiness of these two geminal methyls appears to impede the adequate fit of the R-isomers to the cavity because of stereochemical hinderance. Hence, and given that the key functional groups directly involved in the inhibitor–enzyme interaction are either aromatic or aliphatic, the hydrophobic nature of interaction of these compounds with tyrosinase is further strengthened, as the large concave like cavity above the dicopper center which acts like putative substrate-binding pocket is formed by the hydrophobic
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Compound 5
–
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Figure 4. Inulavosin 1 and its derivatives (2–5) accelerated a lysosomal degradation of tyrosinase in B16 melanoma cells. (A) Cell lysates prepared from the cells treated with each compound (15 lM) for 72 h were subjected to Western blotting with antibodies to tyrosinase, Tyrp-1, and b-actin, respectively. (B) Cell lysates prepared from the cells treated as in (a) either in the absence ( ) or presence (+) of lysosomal protease inhibitors (100 lM of leupeptin, pepstatin-A, and 20 lg/ml of E64d) were subjected to Western blotting with antibodies to tyrosinase and b-actin, respectively.
residues (Matoba et al., 2006). Nevertheless, this structure–binding-affinity relationship observed in compounds 2 and 4 can also be partially explained on the basis of the inherent perpendicular orientation between the naphthopyran ring containing the three methyl groups and the naphthalene ring carrying the hydroxyl group (Srinivasan et al., 2012). This perpendicular disposition of the two parts of the molecule, which also exists in 1 (since they all have similar structural core; see Figure 1A), may preferentially direct the S-enantiomers of them in the right orientation toward the cavity above the catalytic center. Furthermore, the higher binding affinity observed in 2 can be associated with the inherent structural angular juxtaposition of the benzene ring in comparison with the linear one in 4. It can be argued that the angular or C-type ring structure existing in the naphthopyran part of 2 fits well into this cavity, and the additional benzene ring confers slightly better hydrophobic interactions in comparison with 1 and 4. The relative closeness between the calculated binding affinity values for 1 and 4 in comparison with 2 can also be rationalized on the basis of less hydrophobic nature of methyl group in 1 and the linear juxtaposition of the benzene ring in 4 over the angular structure in 2. As also observed in the docking experiments, the hydroxyl function present in 1, 2, and 4 does not provide the possibility for formation of hydrogen bonds under the studied conditions, which further supported the interaction between inhibitor and enzyme being mainly hydrophobic. Nonetheless, the influence of hydroxyl group (in case of acetates 3 and 5 which are hypothesized to be hydrolyzed to the active hydroxyl by esterases) cannot be ruled out, as various factors like hydrogen bonding to the
ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Inulavosin inhibits a copper loading to tyrosinase
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Figure 5. Inulavosin 1 and its derivatives bind to the cavity of tyrosinase, but do not inhibit its activity. (A) Representative binding configurations of the S-enantiomers of inulavosin (1, top), 2 (middle), and 4 (bottom) to tyrosinase were obtained from docking simulations. The compounds (shown as spheres) fit into the cavity of the catalytic site (right column), without directly interacting with the copper cations burried deep inside the catalytic core (green spheres; left column) or the sulfur atoms of methionine residue (yellow spheres; right column). (B) The inhibitory effects of inulavosin 1 and compound 2 on melanin production in B16 cells were measured. Results are the averages of five experiments SD. Data were analyzed for statistical significance using Student’s t test. P values of
