Goenka Shilpi, Simon Sanford R
Department of Biomedical Engineering, Stony Brook University, Stony Brook, NY 11794-5281, USA.
Department of Biochemistry and Cellular Biology, Stony Brook University, Stony Brook, NY 11794-5281, USA.
Biomedicines. 2020 Oct 13;8(10):411. doi: 10.3390/biomedicines8100411.
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.
CMT - 308是一种非抗菌性化学修饰四环素（CMT），我们之前已表明它具有抗真菌活性和多效抗炎活性，包括抑制基质金属蛋白酶（MMPs）的酶活性。基于其化学结构，我们推测CMT - 308可能抑制黑色素生成，并且可能是治疗因黑色素生物合成和/或转运失调而发生的皮肤色素沉着紊乱的候选药物。首先使用纯化的蘑菇酪氨酸酶制剂在体外研究CMT - 308对酪氨酸酶活性的任何影响；通过Lineweaver - Burk和Dixon图以及非线性最小二乘法拟合评估对可溶性真菌酶的抑制模式。接下来，在使用B16F10小鼠黑色素瘤细胞的哺乳动物细胞培养物中测试CMT - 308的效果，并在深色色素沉着的人黑素细胞（HEMn - DP）中进一步验证。我们的结果表明，微摩尔浓度的CMT - 308使用黑色素生成途径中的前两种底物（L - 酪氨酸和L - 3,4 - 二羟基苯丙氨酸（L - DOPA））抑制蘑菇酪氨酸酶的酶活性；CMT - 308主要通过混合抑制模式抑制蘑菇酪氨酸酶，其中竞争模式起主要作用。在B16F10细胞培养物中，CMT - 308（10 μM）在基础和激素刺激条件下均显著降低总黑色素水平，选择性降低细胞外黑色素水平，在72小时内无任何细胞毒性。对B16F10细胞中黑色素生成抑制潜在机制的研究表明，在哺乳动物细胞中，CMT - 308不抑制细胞内酪氨酸酶活性或α - 葡萄糖苷酶的活性，α - 葡萄糖苷酶是一种调节酪氨酸酶成熟的酶。然而，CMT - 308抑制B16F10细胞中MITF蛋白表达，并在无细胞系统中显示出铜螯合活性和抗氧化活性。在含有10 μM时获得的显著较低的细胞外黑色素水平表明，CMT - 308的抗黑色素生成作用可能归因于对黑素小体输出的选择性抑制以及黑素小体在核周聚集，而不是对黑色素生物合成中酪氨酸酶催化步骤的直接影响。这些结果在HEMn - DP细胞中得到验证，其中CMT - 308以完全可逆的方式抑制树突状形态，而不影响细胞内黑色素合成。此外，在HEMn - DP和HaCaT的共培养物中保留了CMT - 308抑制黑素小体输出的能力。总之，我们的结果为使用低微摩尔浓度的CMT - 308对抗色素沉着影响的治疗策略带来了希望。
