Small molecule reagents that upregulate melanin biosynthesis in melanocytes hold the potential to reduce UV-radiation induced skin damage and the onset of melanoma. In addition, such reagents may find use in the treatment of hypopigmentation disorders such as albinism. Conversely, a significant number of skin diseases such as melasma, postinflamitory melanoderma, and solar lentigines are the result of increased melanin biosynthesis.^[4] Small molecule reagents that increase or decrease melanin biosynthesis in melanocytes can be used as tools to examine the cellular mechanisms underlying melanogenesis, and potentially reveal previously unknown therapeutic targets for melanin-related diseases.

Currently, only a small number of molecules are known to alter the cellular level of melanin in melanocytes. The naturally occurring molecule forskolin^[5] (Figure 1), and synthetic reagents such as isobutylmethylxanthine^[6] (IBMX, Figure 1) are known to regulate adenylyl cyclase and phosphodiesterases, respectively, resulting in an increase in melanin biosynthesis in melanocytes. When applied topically to mice, forskolin provides protection against UV-induced carcinogenesis of skin.^[7] Recently, a synthetic reagent dubbed “melanogenin” was identified from small molecule screening, that increases melanin production in melanocytes (Figure 1).^[8] Unlike forskolin and IBMX, which act on known members of the canonical melanogenesis pathway, melanogenin was found to bind prohibitin and mitochondrial F₁F₀-ATPase resulting in re-trafficking of tyrosinase and tyrosinase-related protein 1. This example,^[8] and others,^[9] highlight the utility of small molecules with unique biological function to examine complex cellular processes, such as melanogenesis, and identify potential therapeutics and novel therapeutic targets.

As part of a research program designed to identify additional synthetic molecules capable of altering the cellular level of melanin in melanocytes, we synthesized and screened a 75-member targeted combinatorial library of functionally diverse amides. Library members were synthesized from carboxylic acid (A–E) and amine (1–15) building blocks in a two-step process to generate functionally diverse amides from inexpensive starting materials (Figure 2). All library members were characterized by mass spectrometry, and purity was determined by high performance liquid chromatography (HPLC). All compounds were determined to be ≥90% pure, and stored as 25 mM solutions in DMSO.

Screening the library for molecules that alter melanin levels in melanocytes was performed in triplicate in an immortalized murine melanocyte cell line (melan-a). Briefly, 1×10⁵ melan-a cells were plated in a 24-well tissue culture plate and allowed to stably adhere in 1 mL of RPMI 1640/10% FBS/0.2 μM TPA (referred to herein as growth media) for 18 hours at 37 °C under 5% CO₂. After such time, media was removed and replaced with growth media containing either 2.5 μM forskolin, which serves as a positive control for increased melanin levels, or 2.5 μM library member. As a negative control, cells were treated with growth media containing 0.1% DMSO (referred to herein as vehicle control).

After 72 hours, cells were trypsinized from the tissue culture multi-well plate, individually placed in a 1.5 mL plastic tube, pelleted, resuspended, and washed twice with phosphate buffered saline (PBS, pH 7.2). Following resuspension in 1 mL of PBS, a small aliquot of each cell mixture was removed, and cells were counted by hemocytometry. Cell count was used to measure the cytotoxicity of each library member, as well as to normalize the cellular level of melanin per cell in different samples.

After counting, the cell samples were repelleted, and lysed in 800 μL of 1 M NaOH/PBS for 2 hours on ice. Cell lysate was homogenized by pipetting, and melanin levels were measured by Abs₄₉₀. The cellular level of melanin in each sample was determined by reference to a A₄₉₀ standard curve using purified melanin (Sigma Aldrich) and normalized to cell count. From this screen, four compounds were identified that increase the cellular level of melanin by ≥ 1.5-fold in comparison to treatment with vehicle control. The most active compounds identified in our screen share structural features. 2-ethyl-quinoline and furan moieties represent the carboxylic acid building block, and three of the four compounds have either ortho- or para-fluorinated aryl groups as the amine building block. Comparison of these four identified compound A7 as the most effective activator of melanogenesis. Treating melan-a melanocytes with 2.5 μM A7 resulted in a 1.8 ± 0.3-fold increase in the cellular level of melanin, in comparison to treatment with vehicle control (Figure 3).

Treatment of melan-a cells with 0.5, 1.0, 2.5, or 5.0 μM A7 resulted in a dose-dependent increase in the cellular level of melanin, with a maximum effect observed at a dose ≥ 2.5 μM. In contrast, compound A7 did not have a significant effect on melan-a proliferation under the concentrations tested (Figure 4). In addition to these data, an A7-dependent increase in the cellular level of melanin was confirmed by microscopy. Melan-a melanocytes were grown on glass slides and treated with growth media containing either vehicle control or 2.5 μM A7 and incubated at 37 °C under 5% CO₂ for 72 hours. Cells were then washed three times with PBS and imaged. Unlike cells treated with vehicle control, which were absent of dark features, cells treated with 2.5 μM A7 were observed to have significant pigmentation. Closer analysis of cells treated with 2.5 μM A7 revealed a large number of dark punctate foci within each cell, consistent with high levels of melanin present in melanosomes (Figure 5).^[10] Taken together, these data are consistent with a model in which the cellular level of melanin is significantly increased by treatment with compound A7.

In addition to an increase in cell pigmentation, we observed significant changes in the morphology of melan-a cells following treatment with 2.5 μM A7 (Figure 5). This observation furthersupports an A7-dependent increase in melanogenesis. Previous reports have shown a strong correlation between changes in melanocyte morphology, such as dendricity, and the cellular level of melanin.^[11] In order to measure the observed change in morphology, as well as obtain additional data on the response of melanocytes to compound A7, we measured the dendricity of melan-a cells 72 hours after treatment with vehicle control or 2.5 μM A7. 1×10⁴ melan-a cells were plated on a Matrigel^™-coated glass slide and allowed to stably adhere for 18 hours. Cells were then treated in triplicate with growth media containing either vehicle control or 2.5 μM A7 and cultured for 72 hours at 37 °C under 5% CO₂. Cells were washed three times with PBS and fixed with 4% formaldehyde/PBS. The cell nucleus was stained with hematoxylin, and cells were imaged by microscopy. The number of dendrites per cell were counted manually. On average, 36% ± 3.1% of cells treated with growth media containing vehicle control had greater than 2 dendrites per cell. In comparison, 69% ± 5.2% of cells treated with 2.5 μM A7 had greater than 2 dendrites per cell (Figure 6). These data confirm our initial observations, and support A7-dependent induction of melanogenesis in melan-a cells.

We next sought to identify cellular targets of A7. Tyrosinase is a well characterized enzyme that catalyzes the oxidation of phenols (such as tyrosine) and is known to play a critical role in the canonical melanin biosynthetic pathway.^[12] In this pathway, tyrosinase catalyzes the hydroxylation of tyrosine to generate L-DOPA. An increase in the activity or expression of tyrosinase therefore results in an increase of melanin in melanocytes.^[13]

The direct correlation between the catalytic activity of tyrosinase or tyrosinase overexpression, and melanin production, makes tyrosinase a good starting point to examine potential cellular targets of compound A7 that result in an increased cellular level of melanin. In addition, because the role of tyrosinase in the canonical melanogenesis pathway is well established, the effect compound A7 has on tyrosinase activity and expression provides a valuable starting point for future efforts to further elucidate the mechanism of action of compound A7.

Tyrosinase activity was measured using a previously reported technique.^[14] The cellular level of tritiated water, which is formed as a byproduct of tyrosinase catalyzed hydroxylation of L-tyrosine-3,5-[³H] was measured by scintillation (Figure 7). An increase in ³H₂O levels would support an increase in tyrosinase activity, whereas a decrease in ³H₂O levels supports a decrease in tyrosinase activity.

Briefly, 1×10⁵ melan-a cells were plated in a 24-well tissue culture plate and allowed to stably adhere for 18 hours. Cells were then treated in quadrupet with growth media containing either vehicle control or 2.5 μM A7 and cultured for 72 hours. Melan-a cells were trypsinized from the tissue culture plate, pelleted, washed three times in PBS, and repelleted. Cells were then lysed in 75 μL of 80 mM K₂PO₄, 1% CHAPS, 2 mM PMSF, containing PICO2 protease inhibitor cocktail (CalBioChem) for 2 hours on ice. Total protein levels in each cell lysate sample were determined by Bradford Assay. 5.0 μg aliquots of total protein were incubated in 250 μL of 80 mM K₂PO₄ containing 250 nM L-tyrosine, 25 μM L-DOPA and 0.7 μCi of L-tyrosine-3,5-[³H] for 60 minutes at 37 °C. Total protein was then precipitated with 375 μL of 0.2% bovine serum albumin and 375 μL of 10% trichloroacetic acid, and pelletted. The resulting supernatant was added to 500 μL of a washed charcoal slurry to remove particulate. Charcoal was then pelletted, and the level of tritiated water in the supernatant was analyzed by a scintillation counter. Supernatant from melan-a cells incubated in growth media containing either 2.5 μM forskolin or vehicle control was used as a positive and negative control, respectively. Supernatant from melan-a cells that were untreated, and boiled after lysis, to denature total cellular protein, was used to determine background count level. Background was subtracted from the value obtained for each sample to provide an absolute measurement of tyrosinase activity.

On average, cells treated with vehicle control had 73,422 ±781 scintillation counts per minute. In comparison, cells treated with 2.5 μM A7 or 2.5 μM forskolin had 99,567 ±10,390 scintillation counts per minute and 121,588 ±5,016 scintillation counts per minute, respectively (Figure 8A). On average, this correlates to a 35% increase in tyrosinase activity in cells treated with compound A7 and a 65% increase in tyrosinase activity in cells treated with forskolin. These data suggest that treating melan-a cells with compound A7 results in a statistically significant increase in the activity of tyrosinase, a known participant in the canonical melanin biosynthetic pathway.

At least two models explain an A7-dependent increase in tyrosinase activity. In one model, treatment with compound A7 has no effect on the cellular level of tyrosinase, but does result in increased catalytic activity of tyrosinase. For example, a direct or allosteric interaction between compound A7 and tyrosinase, as well as an interaction between compound A7 and a member of the canonical melanin biosynthetic pathway upstream of tyrosinase, could influence the catalytic activity of tyrosinase. In another model, treatment with compound A7 does not influence the catalytic activity of tyrosinase, but does result in the overexpression of tyrosinase. Overexpression of tyrosinase would result in an increase of tyrosinase-dependent oxidation of tyrosine, resulting in higher cellular levels of melanin.

In an effort to provide support for one of these models, the cellular level of tyrosinase was measured in melan-a melanocytes treated with growth media containing either vehicle control or 2.5 μM A7. 1×10⁵ melan-a melanocytes were plated in a 12-well tissue culture plate and allowed to stably adhere for 18 hours at 37 °C under 5% CO₂. Cells were then treated with growth media containing either vehicle control or 2.5 μM A7, and cultured for 72 hours. After such time, growth media was removed and cells were washed three times with PBS. Cells were then lysed in RIPA buffer containing protease inhibitor cocktail (CalBioChem) and tyrosinase levels were measured by Western blot and normalized to β-actin. Cells treated with vehicle control or A7 were found to have similar cellular levels of tyrosinase (Figure 8 B and C). These data are consistent with a model in which treatment with compound A7 in melanocytes has no effect on tyrosinase expression, but does increase the activity of tyrosinase through mechanisms that are currently unknown.

The production of melanin in melanocytes is a critical defense mechanism to combat genome instability and disease, as a result of chronic exposure to UV-radiation.^[2] Small molecules that increase the cellular level of melanin in melanocytes therefore have significant therapeutic potential, and may be used to treat diseases such as albinism. In addition, a number of diseases are the result of hyper-melanogenesis.^[4] Small molecules that upregulate melanin production can be used to study melanogenesis, and have the potential to reveal new therapeutic targets for melanin-related diseases.

In this communication, we described the synthesis of a targeted combinatorial library of functionally diverse amides, and the screening of this library for molecules that alter the cellular level of melanin in melanocytes. From this screen, four compounds were identified that resulted in a ≥ 1.5-fold increase in melanin production in melan-a cells, in comparison to cells treated with vehicle control. Treating melan-a melanocytes with the most active molecule identified in our screen, A7, resulted in a 1.8 ± 0.3-fold increase in the cellular level of melanin, in comparison to cells treated with the vehicle control. An A7 dose-dependent increase in the cellular level of melanin was observed in melan-a cells treated with 0.5 μM – 5.0 μM A7, further supporting A7-dependent melanogenesis. In addition to an increase in pigmentation, an increase in the dendricity of melanocytes has been shown to correlate well with melanogenesis.^[11] On average, 69% of melan-a cells treated with 2.5 μM A7 had > 2 dendrites per cell, whereas 36% of melan-a cells treated with vehicle control had > 2 dendrites per cell. Tyrosinase-catalyzed oxidation of L-tyrosine to L-DOPA is a well studied, and critical step in melanin biosynthesis. As a starting point to examine the mechanism of A7-dependent melanogenesis, we examined the effect compound A7 has on the catalytic activity of tyrosinase, as well as cellular levels of tyrosinase. On average, treating melan-a cells with 2.5 μM A7 increased tyrosinase-catalyzed oxidation of L-tyrosine-3,5-[³H] by 35%, in comparison to vehicle control. Conversely, treating melan-a cells with 2.5 μM A7 did not result in a significant change in the cellular level of tyrosinase.

Taken together, our data show that compound A7 is a novel and potent activator of melanogenesis in melan-a melanocytes, and supports a model in which compound A7 acts in a way that increases the tyrosinase-dependent oxidation of tyrosine, resulting in higher cellular levels of melanin. A7 is simply prepared in a two-step synthesis, and has significant potential as a tool to study melanogenesis, as well as mechanisms by which tyrosinase activity can be increased in a small molecule-dependent manner. Future studies examining the role A7 plays in melanogenesis and tyrosinase activity, as well as structure activity relationships centered on A7 are currently underway, and will be reported in due course.

Supporting Information