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How do skincare companies determine whether a new compound is a tyrosinase inhibitor and assess the relative efficacies of compounds in the class? The most obvious answer would be to replicate human tyrosinase (HTyr) in vitro, but this has proven to be exceedingly difficult. Extraction of HTyr at any substantial level is impossible, and scientists have to fully model its crystalline structure. Given these difficulties, industry was forced to turn to a non-human model, with mushroom tyrosinase (MTyr) being the most widely adopted. Extracted from the common species Agaricus Bisporus, MTyr has three important characteristics for its use as a substitute for HTyr: it is plentiful, cheap, and its chemical and 3-D structures are thought to roughly resemble those of its human counterpart. In addition, compounds that inhibit mushroom tyrosinase could have utility both as a whitening agent for humans and to reduce browning due to the natural aging or large-scale handling of fruits and vegetables.

As one would expect, the use of MTyr has its limitations. Indeed, many compounds that show inhibitory activity against MTyr fail to reproduce similar efficacy in vivo. This could be related to properties of the enzymes, which are notably different. For example, the optimal temperature for MTyr activity is 40 degrees Celsius, while HTyr operates best at 50 degrees C, and MTyr operates most efficiently at a more acidic pH than HTyr. Scientists are turning to big data and bioinformatics to replace MTyr with more reliable and applicable HTyr models.

One of these is the tyrosinase in B. Megatarium. Using homologous modeling (predicting the 3-D structure of an unknown protein using known proteins that are assumed similar to the unknown protein) to predict the structure of HTyr, scientists found that this bacterial tyrosinase had a high similarity (33.5%) to the assumed HTyr. Using docking simulations and molecular dynamic calculations of various known tyrosinase inhibitors to MTyr, B. Megatarium tyrosinase, and HTyr, the histamine binding residues responsible for the enzyme/inhibitor interaction were completely different in all three. Thus, while the bacteria-derived tyrosinase exhibited higher similarity to HTyr, the modeling showed that it likely is not sufficient to replicate HTyr function.

A more compelling answer may involve the novel production of a truncated HTyr, where primarily the catalytic (active) domain is produced in vitro by HEK 293 cells, a cell line derived from human kidney epithelial cells. Instead of relying on mammalian or fungal models, now scientists have a true comparative to analyze current (assumed) tyrosinase inhibitors and screen for new ones. A recent study (Mann et al, 2019) utilized this truncated HTyr in a high-throughput screen utilizing 50,000 compounds, with the goal of understanding the “structural motifs in small-molecule compounds that efficiently inhibit HTyr.”

Included in this compound library were numerous well-known lightening compounds, such as hydroquinone, kojic acid, resorcinol derivatives and arbutin. The results are, well, enlightening. Comparing Ki values (Ki is the inhibitor constant, a measure of inhibition potency, or the concentration required to produce half-maximum inhibition), the three resorcinol derivatives had Ki values between 9.1 and 39; kojic acid’s value was 145; and hydroquinone’s value was not determined (ie, unmeasurable).

What does this mean? The authors postulate that perhaps hydroquinone’s efficacy as a lightening agent lies not in its effect as a tyrosinase inhibitor, but, given these Ki results, in a more nefarious effect on melanocytes. When melanocytes were cultured in the presence of hydroquinone, melanogenesis reduced by 85%. However, when hydroquinone was removed from the culture, this reduction persisted two weeks later, indicating an effect that surpasses enzyme inhibition and may creep into cytotoxicity.

A more humanized model for tyrosinase will likely reveal newer, and possibly safer, inhibitors for our patients, in a much more rapid development process. Dermatologists should keep a close eye on the literature and new product launches to ensure we are offering our patients the most advanced, and likely effective, lightening agents.

Drs. Farris and Lain are co-founders of the Science of Skincare Summit, to be held October 28-30 in Austin, TX. For information:

Seo SY, Sharma VK, Sharma N. Mushroom tyrosinase: recent prospects. J Agric Food Chem. 2003 May 7;51(10):2837-53.

Mann T, Gerwat W, Batzer J, Eggers K, Scherner C, Wenck H, Stäb F, Hearing VJ, Röhm KH, Kolbe L. Inhibition of Human Tyrosinase Requires Molecular Motifs Distinctively Different from Mushroom Tyrosinase. J Invest Dermatol. 2018 Jul;138(7):1601-1608.

Nokinsee D, Shank L, Lee VS, Nimmanpipug P. Estimation of Inhibitory Effect against Tyrosinase Activity through Homology Modeling and Molecular Docking. Enzyme Res. 2015;2015:262364.

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