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Tyrosinase (TYR) is a multifunctional metalloenzyme present in various species including fungi, plants, insects, bacteria, animals and human, that plays a crucial role in the biosynthesis of natural pigment molecules called melanins. Structural and activation characteristics of tyrosinase differ from species to species, nevertheless, they all possess an active site containing two copper ions CuA and CuB each coordinated with 3 histidine residues . Human tyrosinase (h-TYR) is a monomeric 529-amino acid membrane bound glycoprotein , produced only by melanocytes which are mainly expressed in the basal layer of the dermis.
Melanocytes are characterized by their unique subcellular lysosome-like organelles called melanosomes, responsible for the synthesis, storage and transport of melanins. H-tyrosinase is mainly sorted in the melanosome membrane and subsequently in melanosome’s cytoplasm, perinuclear region, lysosome and Golgi-associated vesicles . However, other TYR tissue localizations include the epidermal melanocyte, oral mucosa, retinal pigment epithelium cells, hair, bone marrow plasma cell and fetal cells .
Despite the fact that tyrosinase family is also comprised of other important members (TRP-1, TRP-2) it is only tyrosinase the necessary enzyme for the complex biochemical-catalyzed reactions to occur during melanogenesis.
That is because it catalyzes the first and rate-limiting step of the overall cascade  which is the oxidation of L-tyrosine and/or L-dihydroxyphenylalanine (L-DOPA) to Dopaquinone (DQ). The latter acts as the substrate for three distinct pathways which can then spontaneously proceed in the physiological pH environment to originate the polymer of pheomelanine (red-yellow pigment) and two eumelanin polymers that is 5,6-dihydroxy-2-indolylcarboxylic acid (DHICA melanin, brown pigment) and 5,6-dihydroxyindole (DHI melanin, black pigment), a step which is also possible to be catalyzed by tyrosinase .
It is apparent from the above, that TYR expression not only determines the variation in skin, eye and hair pigmentation amongst population , but also controls the process of melanogenesis which makes it a key molecular target in order to treat abnormal melanin synthesis. In addition to melanogenesis, there are studies confirming that tyrosinase is also involved in the development of neuromelanin in brain  and in the oxidative homeostatic process that protects human skin from the UV radiations.
The concentration of epidermal TYR is subject to geographical stratification. By increasing distance from the equator, human populations tend to express more the TYR allele rs2733832  which is associated with lighter pigmentation, whereas populations with European ancestry present the highest variation in eye and hair color compared with other population of the world.
Gene expression of TYR, as well as TRP-1 and TRP-2, is controlled by a series of intracellular signaling pathways associated with the melanocyte inducing transcription factor MITF, which is the master regulator of melanogenesis (Figure 1). The core regulatory pathways include cAMP/ PKA and PI3K/Akt signaling both induced by melanocortin 1 receptor bound with melanocyte-stimulating hormone (MC1R/a-MSH), Wingless Integrated (Wnt)/β-catenin and SCF/c-kit signaling pathways. The latter, together with the endothelin-1 (EDN-1) pathway trigger both the mitogen-activated protein kinase (MAPK) signaling cascade. In addition, nitric oxide (NO), cytokines (IL-13) as well as mechanisms involving autophagy (toll-like receptor TLR9) are also modulating melanogenesis and the expression of TYR. The initiation and propagation of the signaling pathways is influenced by extrinsic factors like chemical drugs and UV radiation and by various intrinsic factors such as pregnancy and diabetes .
To date, 127 identified genes are known to regulate pigmentation . Mutations of TYR gene leads results pathological conditions such as several types of albinism in which tyrosinase’s partially or complete deletion leads to a dysregulation of melanin synthesis and thus to its absence in skin, hair, and eyes. On the other hand, excessive production of melanin causes hyperpigmentation of the skin with the most common dermatological manifestations to be freckles, age spots, melasma and cancer. As already mentioned, tyrosinase also contributes in the brain neuromelanin development in mice and human via synthesis of substantia nigra. However, an excessive formation of dopaquinone derived by its main catalytic activity results in neuronal damage and cell death. Hence, there are evidence which link tyrosinase to neurodegenerative disorders (Parkinson’s, Alzheimer’s and Huntington’s diseases) . Some genome-wide association studies also suggest that tyrosinase may be involved in vitiligo acting as an important autoantigen . Finally, there are several other diseases characterized by hyperpigmentation manifestations which often contribute to their diagnosis, such as Addison’s disease  and Cushing’s syndrome .
Hyperpigmentation, is a dermatological disorder where skin assumes darker coloration due to increased amounts of melanin produced by the melanocytes, and less commonly, to an increased number of the latter . In the first case, clinical manifestations include epidermal melanosis, freckles and melasma while the second is related with epidermal melanocytosis and lentigines. Abnormal distribution of melanin may also account for skin darkening. The most widely documented triggering factors include skin inflammation (sun burns, injuries, acne, eczema), post-inflammation conditions evoked by previously affected dermal situs (former acne scars) and laser treatment . Extend exposure to sun is deleterious for the development of hyperpigmentation as UV radiation stimulates pigmentation in various pathways such as by increasing the expression of αMSH, endothelin1, interleukin-1, SCF, NGF, keratinocyte’s p53 and fibroblast’s growth factors as HGF and bFGF . Other incriminated parameters include genetic predisposition, allergic reaction to medications and cosmetics, contraceptives, hormone therapies, phototoxic drugs (phenothiazines, tricyclic antidepressants and anticonvulsants, antimalarials) .
Particular subpopulations are more prone to manifest certain hyperpigmentation conditions. In this respect, melasma is less common in men (10% of the cases)  when women, especially those in light-brown color skin and under hormone therapy (HRT) or contraceptive medications are in higher risk . A survey in 2009, reported that 48% of the women cases had a positive family history in melasma  underlying the significance of genetic predisposition in women. Pregnant women  and patients suffering from thyroid disease are also more susceptible to melasma , while people from the age of 40 are prone to aging spots . Finally, people with skin of color (SOC) mainly of African, Asian, and Mediterranean ancestry are more likely to manifest post-inflammatory hyperpigmentation .
As SOC represent the majority of today’s world population and their number is estimated to increase further by the year 2050 , the incidence of the disease as well as it’s variability would also increase. This necessitates the discovery of efficient anti-hyperpigmentation drugs which would also prevent future skin cancers. Treating hyperpigmentation would also alleviate the psychological impact of patients who often suffer from anxiety and depression having consequences in their self-esteem, social interactions and employability .
Tyrosinase can provide a useful target for treating hyperpigmentation by modulating its catalytic center, maturation and migration to melanosomes and the transcription of its mRNA  with all the involving signaling pathways to be previously described.
In order to treat hyperpigmentation, sun avoidance is crucial, as recurrence is very common during the summer. Current treatments rely on the use of topical phenolic bleaching compounds (hydroquinone HQ, monobenzyl hydroquinone, 4-methoxyphenol, isopropylcatechol) followed up by non-phenolic compounds (azealic acid, kojic acid, ascorbic acid and corticosteroids)  which can sufficiently address to epidermal hyperpigmentation whereas dermal pigmentation is less responsive and may additionally require laser treatment. For some dermatological cases like erythromelanosis follicularis faciei, hydroqionone is being used in combination with keratinolytics (urea, ammonium lactate), while for cases for which no effective therapy has been found such as for erythema dyschromicum perstans, UVB phototherapy and medications as dapsone, clofazimine have been used .
Unfortunately, existing therapies have several drawbacks regarding their efficacy and side effects hence the few commercially agents (kojic acid and arbutin). In detail, HQ has been associated with erythema, dermatitis, burning and hypochromia  and the EU Cosmetic Regulation has banned its use together with corticosteroids, monobenzyl hydroquinone and tretinoin. Other causes for their limited use are their low stability (ascorbic acid, kojic acid), low bioavailability (ellagic acid) and week activity in h-TYR .
Along with tyrosinase inhibitors, some of the most promising novel approaches include targeting the protease activate receptor 2 (PAR-2) which regulates the phagocytosis of melanosome from the keratinocytes as well as targeting the autophagy mechanism which can lead to degradation of already existing melanin . Regulation of melanogenesis can also achieved by adding or depleting glucose or galactose, modulating intramelanosomal pH and extracellular pH  and by inactivating protein phosphatase 2 via heat treatment . A-2 adrenergic receptor antagonist (yohimbine) , antituberculotic and antithyroid drugs have also been studied. Oral treatment with procyanidin combined with vitamins A, C, E has shown great promise  while there is also a tendency in exploiting natural resources, compounds with coexisting antioxidative activity and hybrid compounds with two pharmacophores . By producing recombinant h-TYR, the next generation drugs are expected to have greater biodisponibility and stability, lower side effects, they may combine antioxidant properties and treat already formed pigmentated skin rather than preventing from new damage.
Due to lack of purified human tyrosinase, most of the inhibitory studies until today have been performed with mushroom tyrosinase the crystallographic structure of which has been recently resolved . In this respect, a proposed simplified process of drug design would implicate molecular modelling, docking and simulation studies (MD) that could predict the 3D h-TYT structure and thus, progress with the best human inhibitor compounds economizing time and resources. This may include the use of MODELLER 9v8 tool, Ramachandran graph and Molprobity server . A good lead compound would present higher binding affinity for human tyrosinase compared with standard inhibitors such as kojic acid and arbutin, by forming strong bonds with adjacent residues of the binding pocket. In addition, tyrosinase inhibition essays would reveal the IC50 of the compound that could be followed by determination of the copper chelation via UV/Vis spectra. Animal studies would determine the viability of mice melanoma cells (B16F10) via colorimetric essay  and thus, provide valuable information regarding SAR and kinetics compared with already known m-TYR inhibitors. Penetration essays are useful to determine the transdermal bioavailability of the lead compound while the antioxidant activity, if present, could be evaluated by radical scavenging capacity assays . Overall, a hypothetical candidate should present lower discrepancy in activity between m-TYR and h-TYR and improved biostability and bioavailability properties than the already existing. However, the discovery of safer anti-pigmentation drugs remains the priority since current agents are related with several side effects.
Exploiting tyrosinase inhibitors is a validate approach to treat hyperpigmentation nevertheless could not be without drawbacks. This relies on the difficulty by which purified human tyrosinase can be obtained and the requirement of human clinical trials that often need the cooperation of cosmetic and biotechnology companies.
TYR inhibition has recently been approached by different aspects as via regulating the quality control of tyrosinase which has direct impact on pigmentation . Furthermore, zebra fish has proved to be a useful model in the inhibitory studies whereas the isolation of natural compounds provide a variety of chemical scaffolds to be exploited from the medicinal chemistry . In 2006, e scientific group successfully yielded h-TYR using recombinant baculovirus expression in insect cell line (High Five) providing a new insight in deducting both crystallization and high-throughput screening essays .
In recent years more and more people invest in whitening agents and it is estimated that by the year 2020, the global market would reach U.S. $23 billion . Thus, discovery of new tyrosinase inhibitors is of a primary importance. Furthermore, tyrosinase inhibitors are also important in preventing melanin-related disorders (Parkinson’s, Alzheimer’s, Huntington’s disease), they are implicated in pathologies characterized by hyperpigmentation (Addison’s, Cushing’s diseases) and could offer an interesting approach to treat diseases involving metalloproteinases like heart diseases, AIDS and cancer . For all the above reasons, together with several other applications of TYR inhibitors including dye production, biopolymers, bioremediation, food-processing industries  and pesticide controlling, investing in tyrosinase inhibitors is highly likely to be rewarded.
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