Introduction

Skin, eye, and hair color are largely the result of melanin synthesized by melanocytes within a specialized organelle termed the melanosome (Seiji et al., 1963; Jimbow et al., 1976; Shen et al., 2001; Hearing, 2005). Melanin plays an important role in the protection of the skin from the deleterious effects of UV radiation. Agents that promote pigmentation hold the potential to reduce UV-induced skin damage and carcinogenesis (Brown, 2001). These agents might also be useful in the treatment of hypopigmentation disorders such as certain forms of albinism. Conversely, a large number of skin diseases, which include acquired hyperpigmentation, such as melasma, postinflammatory melanoderma, and solar lentigines, are owing to increased production and accumulation of melanin (Cullen, 1998; Urabe et al., 1998). Lightening agents have become increasingly important in the cosmetic and medicinal products used for the treatment of hyperpigmentation. Over the years, many efforts have been devoted to screening for agents that regulate pigmentation.

Knowledge of melanocyte biology and the mechanism of melanin synthesis have progressed remarkably over recent years. However, the regulation of mammalian pigmentation remains incompletely understood because of its complexity. First, more than 100 genes are involved in the process of melanogenesis, encoding important structural, enzymatic, and regulatory proteins (Bennett and Lamoreux, 2003). Alterations in the transcription, translation, processing, or intracellular trafficking of any of these proteins might affect melanin synthesis. Second, melanocytes also respond to various factors produced by either the environment or by neighboring cells in the skin (Nordlund et al., 1998; Slominski et al., 2004). One of the best studied of such factors is melanocyte-stimulating hormone and its receptor the melanocortin-1 receptor (Böhm et al., 2006). The transfer of melanosomes from melanocytes to keratinocytes and their breakdown in keratinocytes are also processes that play important roles in the regulation of pigmentation in the skin and hair (van den Bossche et al., 2006; Goding, 2007).

Chemical genetics offers a powerful tool in the identification of therapeutic candidates and their targets. It has been successfully employed to identify novel small molecules that control various biological functions from compound collections or combinatorial libraries (Schreiber, 1998, 2003; Gray, 2001; Tan, 2002; Ward et al., 2002; Lokey, 2003; Burdine and Kodadek, 2004; Khersonsky and Chang, 2004; Smukste and Stockwell, 2005; Zon and Peterson, 2005). Here, we discuss our chemical genetic approach to identify novel compounds and their targets involved in the regulation of mammalian pigmentation.

Screening tagged-triazine libraries

Combinatorial techniques allow for the rapid screening of large numbers of small molecules that can be identified by the production of novel phenotypes in a cellular or embryonic system (Jung, 1999; Nicolaou et al., 2002). An affinity matrix made of the immobilized active compounds is often used to identify biological targets.

Novel tagged-triazine libraries were screened in immortalized murine melanocytes (melan-a), albino murine melanocytes (melan-p) or zebrafish to identify new pigmentation modulators (Figure 1). A triazine scaffold was chosen because of its ease of manipulation and structural similarity to purine and pyrimidine (Khersonsky et al., 2003; Mitsopoulos et al., 2004). Purine- and pyrimidine-based libraries have already proven useful in a variety of biological systems (Verdugo et al., 2001; Chang et al., 2002; Gangjee et al., 2003). Although chemically related to purine/pyrimidine, the triazine scaffold has threefold symmetry, and therefore positional modification is much more flexible than in a purine or pyrimidine. One advantage of the tagged-triazine libraries is that a triethyleneglycol (TG), meta-position amide substituted phenyl (MP), acetamide substituted phenyl (AP), or para-position amide substituted phenyl (PP) linker is included as an intrinsic component of the original molecules (Figure 1a). The linkers provide greater chemical diversity and most importantly can be used to directly attach each identified "hit" compound to an agarose support, which eliminates common time-consuming downstream steps in the isolation of cellular targets for the molecules of interest, such as the need for a traditional structure–activity relationship study to find the proper position to which to add the linker without loss of target-binding activity (Mitsopoulos et al., 2004).

Figure 1.

Novel tagged-triazine libraries and the screening procedure. (a) Structures of the triazine libraries with a TG, MP, AP, or PP linker. The four linkers are each shown in a different color. R1 and R2 groups vary among different compounds. (b) Flowchart of the screening procedure. Screening was performed with cultured melanocytes (in 24-well plates) or zebrafish eggs (in 96-well plates) followed by melanin assay (melanocytes) or microscopic imaging (zebrafish). Using the compound bound to agarose beads as an affinity matrix, the cellular binding target of each active compound was identified.

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We performed screenings of library compounds with cultured melanocytes (24-well plates) or zebrafish eggs (96-well plates) followed by melanin assay (melanocytes) or microscopic imaging (zebrafish). "Hit" compounds were further confirmed for their abilities to modulate pigmentation by retesting in triplicate at various concentrations. The cellular binding target of each active compound was identified by incubating total melanocyte lysates with each compound-conjugated affinity matrix. The proteins bound to the affinity matrix were fractionated by SDS–PAGE gel and revealed by silver staining or Western blotting. Multiple controls were performed to ensure that identified bands are bound specifically to the compound in question and not to structurally related but inactive compounds. The binding targets were confirmed by various approaches and the mechanism of action was further investigated. A flowchart of the screening procedure is shown in Figure 1b.

Cell-based screening identifies a pigmentation enhancer that binds prohibitin

The TG-tagged triazine library was screened for compounds that enhance pigmentation in cultured immortalized murine melanocytes (melan-a cells) (Snyder et al., 2005). Melanogenin (TGV28) was identified as a potent pigmentation enhancer with an EC₅₀ of 2.5 M (Figure 2a). Melanogenin induced melanin formation in a dose-dependent manner and was far more potent than the known enhancer isobutylmethylxanthine (Figure 2b). Melanogenin induced an upregulation in both levels and activity of tyrosinase (Tyr), the rate-limiting enzyme in melanogenesis, although having no effect on the level of Tyr-related protein 1. Prohibitin was identified as a melanogenin-binding target by affinity chromatography and it bound specifically to the melanogenin-conjugated matrix but not to the agarose beads alone nor to a chemically related but biologically inactive compound conjugated-matrix (negative control compound) (Figure 2c). The specificity of melanogenin binding to prohibitin was further verified by the fact that only free melanogenin effectively competed for the binding of prohibitin to the melanogenin matrix (Figure 2d).

Figure 2.

Identification of a pigmentation enhancer that binds prohibitin in a cell-based screening. (a) Chemical structure and EC₅₀ of pigmentation enhancer melanogenin (TGV28). (b) Melanogenin induces melanin synthesis in a dose-dependent manner in immortalized murine melanocytes (melan-a cells). Melanin content is normalized to the total amount of protein in the cell lysates. (c) Melanogenin binds prohibitin specifically. Silver-stained 5–15% gradient SDS–PAGE gel illustrates melanogenin-specific protein binding. A is the unconjugated agarose bead matrix, TGV28-A is the melanogenin-conjugated affinity matrix, and negative-A is the negative control compound conjugated affinity matrix. Arrow indicates the protein band that specifically binds to melanogenin-conjugated matrix. (d) Antiprohibitin Western blot from melanocyte lysate incubated with melanogenin-conjugated agarose beads (TGV28) or agarose beads alone (A). Melanogenin-prohibitin binding was abolished on preincubation of cell lysate with free melanogenin but not with inactive control compound (negative). (e) Prohibitin gene silencing effectively attenuates the cellular response to melanogenin. Melanin assay of B16-F10 melanoma cells transfected with two distinct prohibitin siRNA sequences (Phb1 and Phb2), negative control siRNA, or irrelevant Lamin A/C siRNA followed by no treatment (control), 5 M melanogenin, or 100 M isobutylmethylxanthine treatment.

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Prohibitin is a cellular protein that has been identified in mitochondria (Nijtmans et al., 2000), the nucleus (Fusaro et al., 2003), and on the plasma membrane (Sharma and Qadri, 2004). Prohibitin has been functionally linked to many important cellular processes such as cellular transcriptional control, cell-cycle progression, apoptosis, cellular senescence, and mitochondrial biogenesis (Mishra et al., 2005, 2006). Prohibitin appears to be a "scaffold protein", interacting with a variety of transcriptional factors including, for example, steroid hormone receptors. As no previous small molecule inhibitors or stimulators of prohibitin function were known, we confirmed a functional role for prohibitin in the pro-pigmentary effects of melanogenin by using siRNA technology. The cellular response to melanogenin was significantly attenuated when prohibitin levels in B16-F10 melanoma cells were depleted by transfection with prohibitin-specific siRNAs (Figure 2e). B16-F10 melanoma cells were used in this particular experiment because of the fact that without electroporation, melan-a cells are extremely difficult to transfect. Note that, in contrast to melan-a cells, in our hands, isobutylmethylxanthine at 100 M did not show a significant effect on the pigmentation of B16-F10 cells. Our study revealed a novel functional role for prohibitin in melanin induction and suggested that prohibitin might be a potential target for modulation of pigmentation. Immunofluorescence microscopy revealed a mitochondrial localization of prohibitin in melanocytes. No alteration in prohibitin localization was observed in melanocytes on binding of melanogenin. We proposed that the binding of melanogenin to prohibitin might affect interactions between prohibitin and one or more transcriptional factors. Treatment with melanogenin may result in the release of transcriptional factors bound to prohibitin, allowing them to traffic to the nucleus, thus altering the level of Tyr gene transcription (Figure 6).

Figure 6.

Cellular binding targets and proposed roles of the newly identified pigmentation modulators in the regulation of Tyr transcription, translation, processing, and trafficking in melanocytes. Melanogenin (TGV28) binds to prohibitin (Phb), a scaffold protein that interacts with various transcriptional factors (TF). Treatment with melanogenin may result in the release of transcriptional factors bound to prohibitin, allowing them to traffic to the nucleus, thus altering the level of Tyr gene transcription. TGH11, TGD10, TGD39, and TGJ29 are a novel class of potent Tyr inhibitors that most probably compete with L-3, 4-dihydroxyphenylalanine for the dihydroxyphenylalanine-specific site of Tyr. PPA, MPC11, MPD11, APC25, APC32, PPA01, and PPJ01 induce melanin synthesis by inhibiting mitochondrial F₁F₀-ATPase-mediated transport of H⁺ ions, resulting in the alkalinization of the cytosol.

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Identification of novel tyrosinase inhibitors by screening the TG-tagged triazine library in immortalized murine melanocytes

Tyr plays a central role in melanin biosynthesis (Korner and Pawelek, 1982; Hearing, 1987; Tripathi et al., 1992). Overproduction or dysregulation of melanin synthesis can cause skin hyperpigmentation disorders. The inhibition of Tyr activity has been intensively investigated and many depigmenting compounds used for the treatment of hyperpigmentation in both cosmetic and medical products are known to act as Tyr inhibitors (Briganti et al., 2003; Parvez et al., 2006). Most of the available depigmenting agents have only modest activities and some exhibit toxicities that lead to adverse side effects after long-term usage. Therefore, there is a recognized need for new depigmenting agents.

Four compounds, TGH11, TGD10, TGD39, and TGJ29, were identified as potent pigmentation inhibitors with IC₅₀ values in the range of 10 M after screening the TG-tagged triazine library in immortalized melan-a cells (Figure 3a and b) (Ni-Komatsu et al., 2005). The IC₅₀ value of the widely used depigmenting agent, hydroquinone, was reported to be approximately 75 M (Briganti et al., 2003). By comparison, in our system, the IC₅₀ values of two previously recognized Tyr inhibitors, phenylthiourea and hydroquinone were 29 and 36 M, respectively (Hall and Orlow, 2005). These newly identified pigmentation inhibitors directly inhibited Tyr activity in a dose-dependent manner with IC₅₀ values below 10 M. Further characterization of these novel pigmentation inhibitors suggests that they act through competitive inhibition of Tyr without affecting Tyr protein expression, processing, or trafficking. Kinetic data suggested that these inhibitors most probably compete with L-3, 4-dihydroxyphenylalanine for the dihydroxyphenylalanine-specific binding site on Tyr. Tyr was further confirmed as the cellular target of these novel pigmentation inhibitors by affinity chromatography (Figure 3c). Tyr was found to bind specifically to the TGD10-, TGD39-, and TGJ29-conjugated beads but neither to the agarose beads alone nor to the beads conjugated to structurally closely related compounds that do not inhibit pigmentation. Our data suggest that TGH11, TGD10, TGD39, and TGJ29 are a class of novel potent Tyr inhibitors that may prove to be beneficial in the treatment of hyperpigmentation (Figure 6).

Figure 3.

Identification of novel Tyr inhibitors by screening the TG-tagged triazine library in immortalized murine melanocytes. (a) Chemical structures and IC₅₀ of four novel pigmentation inhibitors, TGH11, TGD10, TGD39, TGJ29, and two known pigmentation inhibitors, hydroquinone and phenylthiourea. (b) Effects of novel triazine pigmentation inhibitors on melanin synthesis in melan-a cells. The final concentration of each compound was 10 M. Data are presented as the percentage of control. (c) Identification of Tyr as the cellular target of the novel pigmentation inhibitors by affinity chromatography. Eluted proteins were separated by 7.5% SDS–PAGE and subjected to Western blot analysis. The membrane was probed with Pep7, a rabbit antisera against mouse Tyr (Jimenez et al., 1991). D10, D39, and J29 are each individual compound-conjugated affinity matrix; lysate is the melan-a cell lysates.

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Identification of compounds that bind mitochondrial F₁F₀-ATPase for correction of albinism by cell-based screening

Multiple forms of oculocutaneous albinism (OCA) have been shown to be because of the inappropriate processing and trafficking of Tyr. The most common form of albinism, OCA2, is because of mutations in the human P gene, homologue of the murine pink-eyed dilution (p) gene (Rinchik et al., 1993). In melanocytes cultured from p-null mice (melan-p cells), Tyr is misprocessed and trapped in a perinuclear compartment rather than trafficking to melanosomes (Manga et al., 2001; Chen et al., 2002; Toyofuku et al., 2002). This misrouting can be corrected by transfection with an expression vector encoding wild-type p or by incubating melan-p cells with the vacuolar adenotriphosphatase (ATPase) inhibitor bafilomycin A1, the ionophore monensin, or ammonium chloride (Chen et al., 2004). The vacuolar ATPase inhibitors, ionophores or ammonium chloride may increase the pH of the endoplasmic reticulum–Golgi intermediate compartments, the early Golgi compartments, and/or the cytosol, thereby inducing the proper folding and release of Tyr from these compartments and enabling correct trafficking to the melanosomes (Chen et al., 2004).

The AP-, MP-, and PP-tagged triazine libraries were screened in OCA2 murine melanocytes (melan-p cells) to identify compounds that enhance pigmentation in these cells (Williams et al., 2004). Six compounds, MPC11, MPD11, APC25, APC32, PPA01, and PPJ01, were identified as potent pigmentation enhancers in melan-p cells (Figure 4a). Affinity chromatography using immobilized compounds allowed the identification of mitochondrial F₁F₀-ATPase as the cellular binding target for these novel albinism-correcting compounds. Moreover, two well-studied mitochondrial F₁F₀-ATPase inhibitors, oligomycin and aurovertin B, also enhanced melanin synthesis in melan-p cells and competed with these six compounds for their binding to mitochondrial F₁F₀-ATPase in albino melanocytes. Immunofluorescence analysis indicated that these six compounds as well as oligomycin and aurovertin B all induced pigmentation in melan-p cells by correcting Tyr and Tyr-related protein 1 trafficking (Figure 4b). Treating melan-p cells with these pigmentation enhancers produced colocalization of Tyr and Tyr-related protein 1 that resembled the distribution pattern observed in non-albino cells. A similar effect was also observed in a non-melanocytic system, where Tyr was stably expressed in a human epithelial 293 cell line (293/Tyr). In this system, Tyr was misprocessed and transfection of p gene product could partially correct it. The compounds that inhibit mitochondrial F₁F₀-ATPase could also induce the maturation of Tyr and melanin synthesis in this non-melanocytic system in the absence of p gene product (Ni-Komatsu and Orlow, 2006).

Figure 4.

Identification of compounds that bind mitochondrial F₁F₀-ATPase for correction of OCA2 albinism by cell-based screening. (a) Chemical structures and EC₅₀ of compounds that correct albinism. (b) Immunomicroscopic analysis of the effect of compounds that correct albinism on the distribution of Tyr and Tyr-related protein 1 in OCA2 murine melanocytes (melan-p cells).

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The mitochondrial F₁F₀-ATPase had not been considered previously as a potential therapeutic target for restoring pigmentation in albino melanocytes. We proposed that this group of compounds induces melanin synthesis in melan-p cells by inhibiting mitochondrial F₁F₀-ATPase-mediated transport of H⁺ ions, resulting in the alkalinization of the cytosol, which is similar to the effects of compounds like bafilomycin A1 (Figure 6). Alkalinization of the cytosol facilitates the proper folding and release of Tyr from endoplasmic reticulum–Golgi intermediate compartments, the early Golgi compartments, and further trafficking of Tyr to the melanosomes. This hypothesis is supported by the fact that these novel pigmentation enhancers influenced mitochondrial membrane potential in a manner similar to that exhibited by oligomycin (Williams et al., 2004).

Identification of a pigmentation enhancer that binds mitochondrial F₁F₀-ATPase by screening the PP-tagged triazine library in zebrafish

The zebrafish has become an important research model because of the availability of relevant genomic resources, the ability to obtain large batches of transparent embryos, its similarity to mammals, and the ease with which large, phenotype-based screenings can be performed. Zebrafish are increasingly used at various stages of the drug discovery process including target identification, disease modeling, and toxicology (Zon and Peterson, 2005).

The PP-tagged triazine library was screened in zebrafish to identify compounds that enhance pigmentation (Jung et al., 2005). One compound, PPA, was found to produce a marked increase in pigmentation throughout the embryo (Figure 5a and b). PPA also enhanced pigmentation in cultured melan-a (wild-type), melan-p (OCA2), and mouse B16-F10 melanoma cells (Figure 5c). However, the concentration of PPA needed to enhance pigmentation in albino melan-p cells (20–50 M) was far higher than the concentrations required for the effects of those compounds shown in Figure 4a, which were originally identified by their ability to correct melan-p pigmentation. The cellular binding target of PPA was identified as the mitochondrial F₁F₀-ATPase by affinity chromatography and was further confirmed by the ability of oligomycin to compete with PPA for its cellular target in melanocytes. Perhaps, not surprisingly, given its ubiquitous cellular target, PPA also causes a number of morphologic features in the fish embryos in addition to just affecting pigmentation.

Figure 5.

Identification of a pigmentation enhancer that binds mitochondrial F₁F₀-ATPase by screening the PP-tagged triazine library in zebrafish. (a) Chemical structures and EC₅₀ of a pigmentation enhancer (PPA) identified by screening in zebrafish. (b) Effects of PPA on pigmentation in zebrafish embryo. Treatment with PPA induced a dose-dependent increase in pigmentation throughout the embryo. Increased pigmentation was most notable in the body of the fish embryo just caudal to the developing head (see boxes). (c) PPA increases melanin synthesis in melan-a melanocytes, albino melan-p melanocytes, and B16-F10 melanoma cells.

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This study attests to the power of chemical genetic screening in zebrafish as a means of identifying mammalian targets and once again suggests the mitochondrial F₁F₀-ATPase as a potential target for modulating pigmentation in both melanocytes and melanoma cells (Figure 6). However, the potential toxicity of known mitochondrial F₁F₀-ATPase inhibitors, which are broadly active, makes it difficult for these novel pigmentation enhancers to be considered as therapeutic agents.

Conclusion and Perspectives

We have successfully demonstrated the power of chemical genetic screening in shedding new light on pathways influencing mammalian pigmentation. By screening tagged triazine-based combinatorial libraries in immortalized murine melanocytes, albino melanocytes or zebrafish, 12 compounds were identified as either potent pigmentation enhancers or inhibitors in various systems and their mechanisms of action and cellular targets were further investigated (Figure 6). Novel pigmentation-enhancing compounds such as TGV28 and PPA could lead to new products that increase pigmentation in the skin, hair, and eyes for medical and/or cosmetic purposes without the need for UV exposure (a so-called "safe tan"). Compounds like MPC11, MPD11, APC25, APC32, PPA01, and PPJ01 may advance rational drug design for the treatment of OCA2. On the other hand, TGH11, TGD10, TGD39, and TGJ29 may have potential beneficial effects in the treatment of unwanted cutaneous hyperpigmentation.

Screening combinatorial compounds carrying a linker that can be easily attached to agarose beads greatly facilitates the identification of their cellular binding targets. The binding targets of each group of compounds in our studies were successfully identified by affinity chromatography. Functional roles of prohibitin and mitochondrial F₁F₀-ATPase in the regulation of mammalian pigmentation were revealed for the first time and may provide novel drug and cosmereutical targets for the treatment of various pigmentary disorders.

Zebrafish pigmentation research has focused on identifying and characterizing mutations that alter pigmentation or influence the development or subsequent migration of pigmentation precursor cells in the neural crest (Raible and Eisen, 1994; Maldonado et al., 2006). Attention has now turned to the use of zebrafish in chemical genetic screening. The applications of zebrafish as a powerful tool for identifying novel pigmentation modulators by chemical genetic screenings are further supported by our identification of PPA as a potent pigmentation enhancer in melanocytes, melanoma cells, and zebrafish. Zebrafish may represent a useful and cost-effective alternative animal model for further testing the pigmentation modulators identified in our cell-based screenings. "Skin equivalents" such as MelanoDerm^TM can also be used to address ability of these compounds to modulate pigmentation in a model more closely representative of human skin.

Conflict of Interest

The authors state no conflict of interest.

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Acknowledgments

We thank our many collaborators in the Orlow laboratory and the laboratory of Dr Young-Tae Chang (Department of Chemistry, New York University). We would like to thank Mr Edwin Staples, the medical illustrator at University of North Carolina, for his assistance with the figures. The work summarized herein was supported in part by the National Institutes of Health Grants AR 41880 and EY 10223 (S.J.O.).