Synthesis of a targeted 75 member combinatorial library from functionally diverse carboxylic acid and amine building blocks. Carboxylic acids (A-E) are converted to acid chloride by treatment with oxalyl chloride and catalytic DMF, followed by reaction with an amine building block (1–15) to generate mature library members. All compounds used in screening were ≥90% pure by HPLC, and stored as 25 mM solutions in DMSO

Concentration-dependent increase in A7-dependent melanin content. Fold change in melanin was determined by (A₄₉₀/10⁶ cells) 72 hours after treatment with 0.5, 1.0, 2.5, or 5.0 μM A7and is represented by dashed bars. Fold change in cytotoxicity was measured by hemocytometry 72 hours after treatment with 0.5, 1.0, 2.5, or 5.0 μM A7, and is represented as solid bars. Values are normalized to treatment with a vehicle control (0.1% DMSO). An A7 concentration-dependent change in melanin is observed. In contrast, cytotoxicity was uniformly low in the concentrations tested. Values and error bars represent the mean and SD of three independent experiments.

A7-dependent changes in the dendricity of melan-a cells. (A) Melan-a melanocytes imaged 72 hours after treatment with either vehicle control (0.1% DMSO) or 2.5 μM A7. The nucleus of each cell is stained with hematoxylin, a representative sample of cells are shown. Scale bar = 50 μm. (B) Percentage of cells with > 2 dendrites 72 hours after treatment with vehicle control (0.1% DMSO) or 2.5 μM A7. Treatment with 2.5 μM A7 results in ~ 2-fold more cells with > 2 dendrites. Values and error bars represent the mean and ± SD of three independent experiments.

(A) Tyrosinase activity in melan-a cells treated with vehicle control (0.1% DMSO), 2.5 μM A7, or 2.5 μM forskolin for 72 hours. Absolute tyrosinase activity levels is plotted. In comparison to treatment with DMSO, treatment with A7 and forskolin result in a 35% and 65% increase in tyrosinase activity, respectively. (B) Densitometry analysis of cellular level of tyrosinase protein in melan-a cells treated with 2.5 μM A7 or vehicle control (0.1% DMSO). (C) Representative Western blot of melan-a cells treated with either 2.5 μM A7 or vehicle control (0.1% DMSO). In (A) and (B), values and error bars represent the mean and ± SD of four and three independent experiments, respectively.

Forskolin, IBMX, and melanogenin: Compounds known to increase the cellular level of melanin in melanocytes.

Fold change in melanin and viable cell count in melan-a cells 72 hours after treatment with vehicle control (0.1% DMSO), 2.5 μM forskolin, or 2.5 μM of library member. Cellular level of melanin was determined by (Abs₄₉₀/10⁶ cells) and is represented by dashed bars. Cytotoxicity was determined by hemocytometry and is shown by solid bars. Values are normalized to treatment with vehicle control (0.1% DMSO). In each sample, cells demonstrated very low levels of small molecule-dependent cytoxicity; however, cells treated with the positive control (forskolin) and library members A5, A7, C1, and C5 resulted in a ≥ 1.5-fold increase in melanin. Values and error bars represent the mean and ± SD of three independent experiments. The structures of the most active compounds identified in the screen are shown (above).

Brightfield microscopy images of melan-a cells 72 hours after treatment with either vehicle control (0.1% DMSO) or 2.5 μM A7. Cells treated with vehicle control are non-pigmented whereas cells treated with A7 are pigmented, and contain a large number of dark punctate foci, consistent with high levels of melanin in melanosomes. Exposure time = 10 msec. Scale bar = 100 μm.

Tyrosinase catalyzed oxidation of L-tyrosine-3,5-[³H] to generate L-DOPA and ³H₂0.