Virtual Screening of Selected Natural Products as Human Tyrosinase-Related Protein 1 Blockers

Many researchers have widely explored the need to replace the harmful compound hydroquinone in skin-lightening creams with more skin-friendly compounds that can give similar results. Some compounds from the plant kingdom have been shown to possess human tyrosinase inhibitory action with no adverse effect on the skin. In this study, the virtual screen of glabridin, kojic acid, arbutin, niacinamide, ascorbic acid, salicin, lactic acid, glutathione, azelaic acid, linoleic acid, glycolic acid, acclaimed to possess this activity as well as the synthetic compound hydroquinone, as human tyrosinase-related protein 1 inhibitor was investigated using computational methods. Site-directed docking was performed at the binding pocket on the enzyme carrying the cocrystallized ligand tropolone. The binding affinity of salicin (-6.7 kcal/mol), a-arbutin (-6.3 kcal/mol), glutathione (-6.2 kcal/mol), ascorbic acid (-5.7 kcal/mol), and niacinamide (-5.7 kcal/mol) were higher than that of the cocrystallized ligand tropolone (-5.5 kcal/mol) and the synthetic skin lightening compound hydroquinone (-4.8 kcal/mol). a-arbutin and glutathione also interacted with similar amino acids units as hydroquinone, suggesting that they followed the exact mechanism of action. These findings strongly corroborate the claim that these natural products could inhibit melanin production and may serve to replace hydroquinone in skin lightening creams.

The Oxidation of Equol by Tyrosinase Produces a Unique Di-ortho-Quinone: Possible Implications for Melanocyte Toxicity

Equol (7-hydroxy-3-(4′-hydroxyphenyl)-chroman, EQ), one of the major intestinally derived metabolites of daidzein, the principal isoflavane found in soybeans and most soy foods, has recently attracted increased interest as a health-beneficial compound for estrogen-dependent diseases. However, based on its structure with two p-substituted phenols, this study aimed to examine whether EQ is a substrate for tyrosinase and whether it produces o-quinone metabolites that are highly cytotoxic to melanocyte. First, the tyrosinase-catalyzed oxidation of EQ was performed, which yielded three EQ-quinones. They were identified after being reduced to their corresponding catechols with NaBH4 or L-ascorbic acid. The binding of the EQ-quinones to N-acetyl-L-cysteine (NAC), glutathione (GSH), and bovine serum albumin via their cysteine residues was then examined. NAC and GSH afforded two mono-adducts and one di-adduct, which were identified by NMR and MS analysis. It was also found that EQ was oxidized to EQ-di-quinone in cells expressing human tyrosinase. Finally, it was confirmed that the EQ-oligomer, the EQ oxidation product, exerted potent pro-oxidant activity by oxidizing GSH to the oxidized GSSG and concomitantly producing H2O2. These results suggest that EQ-quinones could be cytotoxic to melanocytes due to their binding to cellular proteins.

Novel Anti-Melanogenic Compounds, (Z)-5-(Substituted Benzylidene)-4-thioxothiazolidin-2-one Derivatives: In Vitro and In Silico Insights

To confirm that the β-phenyl-α,β-unsaturated thiocarbonyl (PUSTC) scaffold, similar to the β-phenyl-α,β-unsaturated carbonyl (PUSC) scaffold, acts as a core inhibitory structure for tyrosinase, twelve (Z)-5-(substituted benzylidene)-4-thioxothiazolidin-2-one ((Z)-BTTZ) derivatives were designed and synthesized. Seven of the twelve derivatives showed stronger inhibitory activity than kojic acid against mushroom tyrosinase. Compound 2b (IC50 = 0.47 ± 0.97 µM) exerted a 141-fold higher inhibitory potency than kojic acid. Kinetic studies’ results confirmed that compounds 2b and 2f are competitive tyrosinase inhibitors, which was supported by high binding affinities with the active site of tyrosinase by docking simulation. Docking results using a human tyrosinase homology model indicated that 2b and 2f might potently inhibit human tyrosinase. In vitro assays of 2b and 2f were conducted using B16F10 melanoma cells. Compounds 2b and 2f significantly and concentration-dependently inhibited intracellular melanin contents, and the anti-melanogenic effects of 2b at 10 µM and 2f at 25 µM were considerably greater than the inhibitory effect of kojic acid at 25 µM. Compounds 2b and 2f similarly inhibited cellular tyrosinase activity and melanin contents, indicating that the anti-melanogenic effects of both were due to tyrosinase inhibition. A strong binding affinity with the active site of tyrosinase and potent inhibitions of mushroom tyrosinase, cellular tyrosinase activity, and melanin generation in B16F10 cells indicates the PUSTC scaffold offers an attractive platform for the development of novel tyrosinase inhibitors.

Urolithin and Reduced Urolithin Derivatives as Potent Inhibitors of Tyrosinase and Melanogenesis: Importance of the 4-Substituted Resorcinol Moiety

We previously reported (E)-β-phenyl-α,β-unsaturated carbonyl scaffold ((E)-PUSC) played an important role in showing high tyrosinase inhibitory activity and that derivatives with a 4-substituted resorcinol moiety as the β-phenyl group of the scaffold resulted in the greatest tyrosinase inhibitory activity. To examine whether the 4-substituted resorcinol moiety could impart tyrosinase inhibitory activity in the absence of the α,β-unsaturated carbonyl moiety of the (E)-PUSC scaffold, 10 urolithin derivatives were synthesized. To obtain more candidate samples, the lactone ring in synthesized urolithins was reduced to produce nine reduced urolithins. Compounds 1c (IC50 = 18.09 ± 0.25 μM), 1h (IC50 = 4.14 ± 0.10 μM), and 2a (IC50 = 15.69 ± 0.40 μM) had greater mushroom tyrosinase-inhibitory activities than kojic acid (KA) (IC50 = 48.62 ± 3.38 μM). The SAR results suggest that the 4-substituted resorcinol motif makes an important contribution to tyrosinase inhibition. To investigate whether these compounds bind to human tyrosinase, a human tyrosinase homology model was developed. Docking simulations with mushroom and human tyrosinases showed that 1c, 1h, and 2a bind to the active site of both tyrosinases with higher binding affinities than KA. Pharmacophore analyses showed that two hydroxyl groups of the 4-substituted resorcinol entity act as hydrogen bond donors in both mushroom and human tyrosinases. Kinetic analyses indicated that these compounds were all competitive inhibitors. Compound 2a inhibited cellular tyrosinase activity and melanogenesis in α-MSH plus IBMX-stimulated B16F10 melanoma cells more strongly than KA. These results suggest that 2a is a promising candidate for the treatment of skin pigment disorders, and show the 4-substituted resorcinol entity importantly contributes to tyrosinase inhibition.

Methoxy-Substituted Tyramine Derivatives Synthesis, Computational Studies and Tyrosinase Inhibitory Kinetics

Targeting tyrosinase for melanogenesis disorders is an established strategy. Hydroxyl-substituted benzoic and cinnamic acid scaffolds were incorporated into new chemotypes that displayed in vitro inhibitory effects against mushroom and human tyrosinase for the purpose of identifying anti-melanogenic ingredients. The most active compound 2-((4-methoxyphenethyl)amino)-2-oxoethyl (E)-3-(2,4-dihydroxyphenyl) acrylate (Ph9), inhibited mushroom tyrosinase with an IC50 of 0.059 nM, while 2-((4-methoxyphenethyl)amino)-2-oxoethyl cinnamate (Ph6) had an IC50 of 2.1 nM compared to the positive control, kojic acid IC50 16700 nM. Results of human tyrosinase inhibitory activity in A375 human melanoma cells showed that compound (Ph9) and Ph6 exhibited 94.6% and 92.2% inhibitory activity respectively while the positive control kojic acid showed 72.9% inhibition. Enzyme kinetics reflected a mixed type of inhibition for inhibitor Ph9 (Ki 0.093 nM) and non-competitive inhibition for Ph6 (Ki 2.3 nM) revealed from Lineweaver–Burk plots. In silico docking studies with mushroom tyrosinase (PDB ID:2Y9X) predicted possible binding modes in the catalytic site for these active compounds. Ph9 displayed no PAINS (pan-assay interference compounds) alerts. Our results showed that compound Ph9 is a potential candidate for further development of tyrosinase inhibitors.

Biochemical Properties of Tyrosinase from Aspergillus terreus and Penicillium copticola; Undecanoic Acid from Aspergillus flavus, an Endophyte of Moringa oleifera, Is a Novel Potent Tyrosinase Inhibitor

Tyrosinase is a copper-containing monooxygenase catalyzing the O-hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine then to dopaquinone that is profoundly involved in melanin synthesis in eukaryotes. Overactivation of tyrosinase is correlated with hyperpigmentation that is metabolically correlated with severe pathological disorders, so, inhibition of this enzyme is the most effective approach in controlling the overproduction of melanin and its hazardous effects. Thus, searching for a powerful, selective inhibitor of human tyrosinase to limit the hyper-synthesis of melanin is a challenge. Unlike the difficulty of overexpression of human tyrosinase, using fungal tyrosinase as a model enzyme to the human one to evaluate the mechanistics of enzyme inhibition in response to various compounds is the most feasible strategy. Thus, the purification of highly catalytic-efficient fungal tyrosinase, exploring a novel inhibitor, and evaluating the mechanistics of enzyme inhibition are the main objectives of this work. Aspergillus terreus and Penicillium copticola were reported as the most potential tyrosinase producers. The biochemical properties suggest that this enzyme displays a higher structural and catalytic proximity to human tyrosinase. Upon nutritional bioprocessing by Plackett–Burman design, the yield of tyrosinase was increased by about 7.5-folds, compared to the control. The purified tyrosinase was strongly inhibited by kojic acid and A. flavus DCM extracts with IC50 values of 15.1 and 12.6 µg/mL, respectively. From the spectroscopic analysis, the main anti-tyrosinase compounds of A. flavus extract was resolved, and verified as undecanoic acid. Further studies are ongoing to unravel the in vivo effect and cytotoxicity of this compound in fungi and human, that could be a novel drug to various diseases associated with hyperpigmentation by melanin.

Identification of Tyrosinase Inhibitors and Their Structure-Activity Relationships via Evolutionary Chemical Binding Similarity and Structure-Based Methods

Tyrosinase is an enzyme that plays a crucial role in the melanogenesis of humans and the browning of food products. Thus, tyrosinase inhibitors that are useful to the cosmetic and food industries are required. In this study, we have used evolutionary chemical binding similarity (ECBS) to screen a virtual chemical database for human tyrosinase, which resulted in seven potential tyrosinase inhibitors confirmed through the tyrosinase inhibition assay. The tyrosinase inhibition percentage for three of the new actives was over 90% compared to 61.9% of kojic acid. From the structural analysis through pharmacophore modeling and molecular docking with the human tyrosinase model, the pi–pi interaction of tyrosinase inhibitors with conserved His367 and the polar interactions with Asn364, Glu345, and Glu203 were found to be essential for tyrosinase–ligand interactions. The pharmacophore features and the docking models showed high consistency, revealing the possible essential binding interactions of inhibitors to human tyrosinase. We have also presented the activity cliff analysis that successfully revealed the chemical features related to substantial activity changes found in the new tyrosinase inhibitors. The newly identified inhibitors and their structure–activity relationships presented here will help to identify or design new human tyrosinase inhibitors.

Tyrosinase Nanoparticles: Understanding the Melanogenesis Pathway by Isolating the Products of Tyrosinase Enzymatic Reaction

Human Tyrosinase (Tyr) is the rate-limiting enzyme of the melanogenesis pathway. Tyr catalyzes the oxidation of the substrate L-DOPA into dopachrome and melanin. Currently, the characterization of dopachrome-related products is difficult due to the absence of a simple way to partition dopachrome from protein fraction. Here, we immobilize catalytically pure recombinant human Tyr domain (residues 19–469) containing 6xHis tag to Ni-loaded magnetic beads (MB). Transmission electron microscopy revealed Tyr-MB were within limits of 168.2 ± 24.4 nm while the dark-brown melanin images showed single and polymerized melanin with a diameter of 121.4 ± 18.1 nm. Using Hill kinetics, we show that Tyr-MB has a catalytic activity similar to that of intact Tyr. The diphenol oxidase reactions of L-DOPA show an increase of dopachrome formation with the number of MB and with temperature. At 50 °C, Tyr-MB shows some residual catalytic activity suggesting that the immobilized Tyr has increased protein stability. In contrast, under 37 °C, the dopachrome product, which is isolated from Tyr-MB particles, shows that dopachrome has an orange-brown color that is different from the color of the mixture of L-DOPA, Tyr, and dopachrome. In the future, Tyr-MB could be used for large-scale productions of dopachrome and melanin-related products and finding a treatment for oculocutaneous albinism-inherited diseases.

Functional in silico analysis of human tyrosinase and OCA1 associated mutations

Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase gene. OCA1 exists in two forms: OCA1A and OCA1B. OCA1A is caused by a full loss of the human tyrosinase protein (Tyr), leading to an absence of pigment in skin, hair, and eyes, while OCA1B has reduced Tyr catalytic activity and pigment. The current understanding of the disease is hampered by the absence of information regarding the alterations of protein structure and the effects leading to either form of OCA1. Here, we used computational methods to find a general mechanism for establishing this link. Tyr and mutant variants were built through homology modeling, glycosylated in silico, minimized, and simulated using 100 ns molecular dynamics in water. For OCA1B mutants, cavity size is linked to DDG values for mutants, suggesting that partial loss of Tyr is associated with the destabilizing effect of the EGF-like domain movement. In OCA1A, active site mutation simulations indicate that the absence of O2 leads to protein instability. OCA1B mutants are described in severity by the size of the cavity within the EGF–Tyr interface, while active site OCA1A mutants are unable to fully coordinate copper, leading to an absence of O2 and Tyr instability. In patients with known genotypes, free energy changes may help identify the severity of the disease by assessing either the allosteric effect of the EGF-Tyr cavity in OCA1B or the active site instability in OCA1A.