Abstract
CMT-308 is a nonantimicrobial chemically-modified tetracycline (CMT), which we have previously shown exhibits antifungal activity and pleiotropic anti-inflammatory activities, including inhibition of the enzymatic activity of matrix metalloproteinases (MMPs). Based on its chemical structure, we hypothesized that CMT-308 could inhibit melanogenesis and might be a candidate for the treatment of skin hyperpigmentation disorders which occur due to unregulated melanin biosynthesis and/or transport. CMT-308 was first studied for any effects on activity of the enzyme tyrosinase in vitro using a purified preparation of mushroom tyrosinase; the mode of inhibition of the soluble fungal enzyme was evaluated by Lineweaver-Burk and Dixon plots as well as by non-linear least squares fitting. Next, the effects of CMT-308 were tested in mammalian cell cultures using B16F10 mouse melanoma cells and further validated in darkly-pigmented human melanocytes (HEMn-DP). Our results showed that micromolar concentrations of CMT-308 inhibited mushroom tyrosinase enzyme activity, using the first two substrates in the melanogenesis pathway (l-tyrosine and l-3,4-dihydroxyphenylalanine (l-DOPA)); CMT-308 inhibited mushroom tyrosinase primarily via a mixed mode of inhibition, with the major contribution from a competitive mode. In B16F10 cell cultures, CMT-308 (10 µM) significantly diminished total melanin levels with a selective reduction of extracellular melanin levels, under both basal and hormone-stimulated conditions without any cytotoxicity over a duration of 72 h. Studies of potential mechanisms of inhibition of melanogenesis in B16F10 cells showed that, in mammalian cells, CMT-308 did not inhibit intracellular tyrosinase activity or the activity of α-glucosidase, an enzyme that regulates maturation of tyrosinase. However, CMT-308 suppressed MITF protein expression in B16F10 cells and showed copper chelating activity and antioxidant activity in a cell-free system. The significantly lower extracellular melanin levels obtained at 10 µM indicate that CMT-308's anti-melanogenic action may be attributed to a selective inhibition of melanosome export with the perinuclear aggregation of melanosomes, rather than a direct effect on the tyrosinase-catalyzed steps in melanin biosynthesis. These results were validated in HEMn-DP cells where CMT-308 suppressed dendricity in a fully reversible manner without affecting intracellular melanin synthesis. Furthermore, the capacity of CMT-308 to inhibit melanosome export was retained in cocultures of HEMn-DP and HaCaT. In summary, our results offer promise for therapeutic strategies to combat the effects of hyperpigmentation by use of CMT-308 at low micromolar concentrations.