Coumarin, a Lead Compound of Warfarin, Inhibits Melanogenesis via Blocking Adenylyl Cyclase

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2. Materials and Methods

2.1. Reagents

Coumarin, forskolin, α-MSH, MTT and other chemical reagents were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). [³H]cAMP was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA).

2.2. Molecular Docking

AutoDock 4.2 ^[20] and AutoDock Vina 1 ^[21] were used for the docking study of coumarin. The crystallographic structures of two catalytic subunits of mAC, VC1 and IIC2 as well as that of forskolin, a known ligand of mAC, were adopted from the PDB file 1U0H ^[22]. To prepare ligands and the receptor molecule for docking, ADT ^[23] was used to add hydrogens and calculate atomic charges. Coumarin has zero rotatable bonds. Ten amino acid residues at the docking site in the receptor molecule were assumed to be flexible; Leu A438, Asp A440, Thr A512, Asn A515, Lys B938, Ile B940, Ser B942, Asp B1018, Asn B1025, and Lys B1065. A search box was defined to contain both binding sites of forskolin and 2 '(3')-O-(N-methylanthraniloyl) (MANT)-GTP (37.5 × 24.0 × 27.0Å³). AutoDock Vina uses a grid spacing of 1 Å and Autodock 4.2 uses 0.375 Å by default. The docking computation was performed on a 64bit Linux PC with a 3GHz, 8 multi-threaded Intel CPU and 16 GB RAM. The result was analyzed and visualized by the software PyMOL ^[24].

2.3. Cell Culture

B16 cells were purchased from Korean Cell Line Bank (Seoul, Korea) were cultured in DMEM with 10% fetal bovine serum (FBS; Gibco, NY, U.S.A.) and penicillin/streptomycin (100 IU/50μg/ml) in a humidified atmosphere containing 5% CO₂ in air at 37°C. B16 cells were cultured in 24-well plates for each assay. All the experiments were performed in triplicate and repeated three times to ensure reproducibility.

2.4. Measurement of [³H] cAMP

Intracellular cyclic AMP generation was determined by [³H]-cyclic AMP competition assay in binding to cyclic AMP binding protein as described previously by ^[25] with some modification. To determine the cyclic AMP production induced by α-MSH and forskolin, the B16 cells were stimulated with agonists for 20min in the presence of the phosphodiesterase inhibitor Ro 20-1724 (5μM) and the reaction was quickly terminated by three repeated cycles of freezing and thawing. The samples were then centrifuged at 2500×g for 5min at 4°C. The cyclic AMP assay is based on the competition between [³H]-labelled cyclic AMP and unlabelled cyclic AMP present in the sample for binding to a crude cyclic AMP-binding protein prepared from bovine adrenal cortex according to the method of ^[26]. Each sample was incubated with 50μl of [³H]-labelled cyclic AMP (5mCi) and 100μl of binding protein for 2h at 4°C. Separation of the protein-bound cyclic AMP from the unbound cyclic AMP was achieved by the adsorption of the free cyclic AMP onto charcoal (100μl) followed by centrifugation at 12,000×g at 4°C. The 200μl of supernatant were then placed into an Eppendorf tube containing 1.2ml of scintillation cocktail to measure the radioactivity. The cyclic AMP concentration in the sample was determined based on a standard curve and expressed as pmol per number of cells.

2.5. Estimation of Melanogenesis in B16 Cells

Melanin content was used as an index of melanogenesis. Determination of melanin content was performed using a modified method of previous study ^[27]. In brief, B16 cells (5X10⁴) were plated on 24-well, multi-dishes and pretreated with coumarin at concentrations ranging from 0 to 300 μM. Then added and incubated with 100 nM of forskolin for 24hrs. After washing twice with PBS, samples were dissolved in 100mll of 1 N NaOH. The samples were incubated at 60°C for 1 hr and mixed to solubilize the melanin. Absorbance at 405nm was compared with a standard curve of synthetic melanin.

2.6. Determination of Chrysin for Cell Viability

The cell viability assay was performed as described in the previous study ^[28] using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Shortly thereafter, 5 X 10⁴ B16 cells/well were plated in a 24-well plate. After cells were exposed to coumarin at various time scales, MTT solutions were added and the insoluble derivative formed by cellular dehydrogenase was solubilized with EtOH–dimethyl sulfoxide (DMSO) (1:1 mixture solution); the absorbance of each well was read at 560nm using a microplate spectrophotometer.

2.7. Statistical Analysis

The values are expressed as mean ± standard error (n = 3) and the biological significance p < 0.05 was determined by the Student’s t-test.

3. Results

3.1. Molecular Docking of Coumarin

Coumarin is a small and planar compound with the molecular weight of 146.04. This suggests that the docking of coumarin to two relatively large binding clefts of mAC might be a hard optimization problem. The docking solutions obtained from initial trials of coumarin to mAC with AutoDock 4.2 showed poor separations among the clusters of docked conformations. In total, 41 distinct conformational clusters were found out of 256 lamarckian genetic algorithm docking runs using an RMSD tolerance of 2.0Å. The difference in mean binding energy between the top and the second clusters was 0.30kcal/mol. Therefore, care must be taken in adopting the top solution right away.

An additional independent docking calculation was performed with AutoDock Vina which adopted a stochastic global optimization algorithm in addition to a modified scoring function. It is shown that Vina has a much improved speed and accuracy in predictions when compared to AutoDock 4 ^[21]. As a control experiment, docking of forskolin, the known ligand of mAC was performed using Vina. In 10 out of 10 docking runs with a different random seed, the crystallographic binding mode was successfully found with an average RMSD of 0.6Å, which is significant improvement when compared with 1.1Å from the same experiment in our previous report using AutoDock 4 ^[29].

For the docking study of coumarin, 20 independent docking runs using Vina were performed with different random seeds. As with AutoDock 4, the separation of the top cluster from the second cluster was only 0.3kcal/mol in average, which again suggests the existence of local minima in the energy landscape and, thus, several possible binding modes. Interestingly, the best solutions of 20 runs are distributed in two distinct binding sites. In 16 out of 20 runs, the best docking cluster of coumarin was found at the forskolin binding site with the minimal binding energy of -6.6 kcal/mol and in the remaining four runs, the best docking cluster was found at the MANT-GTP binding site with the minimal binding energy of -6.4 kcal/mol (Figure 2. panels A). The existence of two binding modes was also observed with the docking runs using AutoDock 4. In this case, the best cluster composed of 19 runs out of 256 runs was found at the MANT-GTP binding pocket, while the second best cluster composed of 37 runs was found at the forskolin binding pocket.

In both possible binding modes, the benzopyran moiety of coumarin occupies the hydrophobic cleft at the interface of two subunits of mAC. The orientation of coumarin seems to be guided by hydrogen bonding between the carbonyl group of coumarin and the nearby hydrogen donor, which is Ser B942 or Asn B1025, respectively (Figure 2. panel B and C).

Figure 2. The predicted binding mode of coumarin to mAC. (A). Two best docking solutions of coumarin (stick, white colored) are visualized on the mAC structure (ribbon). For comparison, the crystallographic conformations of forskolin (FSK) and MANT-GTP are also displayed (lines). (B and C). Each docked coumarin molecule is visualized with the electrostatic potential mapped on the surface of mAC. The docked molecules are viewed from the center of the binding pocket space. The calculation was done by PyMOL using AMBER99 vacuum electrostatic potential.

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Figure 3. The inhibition of α-MSH- and Forskolin- induced cAMP production with the increase in the concentration of coumarin treatment. B16 cells were preincubated with various concentrations of coumarin for 5min, then cells were stimulated with α-MSH (100nM, white bar) and forskolin(100nM, closed bar), respectively. The data are the mean±S.E.M. of assay triplicates with at least six independent experiments. (**p < 0.05).

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3.2. Inhibitory Action of Coumarin on Adenylyl Cyclase-Induced cAMP Production.

To check whether coumarin really blocks adenylyl cyclase activity in living cellular system or not, we chose the B16 melanoma cell line and investigated the coumarin’s inhibitory effect. Both α-MSH and forskolin are known to increase intracellular cAMP concentration via activating adenylyl cyclase signaling pathways in B16 cells ^[30]. We preliminarily checked the cAMP production induced by α-MSH and forskolin in B16 cells (data not shown), and EC50 for both were 100nM. 5min pretreatment of coumarin showed strong inhibitory-effect on α-MSH- and forskolin-induced cAMP production in a concentration-dependent manner (Figure 3) suggesting that coumarin inhibits cAMP production signaling pathways via blocking adenylyl cyclase.

3.3. Inhibitory Action of Coumarin on Melanin Content in B16 Cells

In order to obtain evidence for the inhibitory effect of coumarin on forskolin-induced melanogenesis signaling pathway in B16 cells, melanin production in B16 cells was examined. As shown in Figure 4a, the melanin content of the cells that were pretreated with coumarin decreased in dose-dependent manners, showing 50.5% at 18M and 35.5% at 35μM compared to the forskolin alone treated control group.

Figure 4. (a) The melanin content reduction with the increase in the concentration coumarin treatment on the melanin biosynthesis. The results are expressed as percentages of control, and the data are the mean±S.E.M. of at least three determinations. (**p < 0.05) (b) The viability of B16 melanocytes during the coumarin treatments. Cells were treated over the indicated time (from 0hrs to 24hrs) with 18μM of coumarin. The viability was evaluated by MTT assay and represented as percentages of control. The data are the mean±S.E.M. of assay triplicates.

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