Tyrosinase Model Research Articles

Raspberry ketone glucoside suppresses melanin synthesis through IL6/JAK1/STAT3 signal pathway.

This study aimed to evaluate the anti-melanogenic activity of raspberry ketone glucoside (RKG) and further explore the specific molecular mechanisms by which RKG affects melanogenesis. The B16F10 cells model, the mushroom tyrosinase model and the zebrafish model were used to assess the whitening activity of RKG. We subsequently identified possible pathways related to RKG inhibition of melanogenesis by RNA-seq analysis and qRT-PCR on the zebrafish model, and further explored the effects of key genes on the pathway on the melanogenic effect of RKG by using related pathway inhibitors and Tg [mpeg: EGFP] transgenic zebrafish line. RKG could noticeably inhibit melanogenesis in B16F10 cells in vitro and on zebrafish in vivo. The RNA-Seq analysis and the qRT-PCR in zebrafish embryos indicated that the inhibition of melanogenesis by RKG could be achieved by activating JAK1/STAT3 signal pathway and inhibiting the expression levels of the MITFa, TYR, TYRP1a genes directly associated with melanogenesis. The inhibitor tests revealed that the inhibitory effect of the RKG on melanogenesis was restored by the IL6, JAK1/2, and STAT3 inhibitors, specifically STAT3 inhibitor. We further examine the relationship between the JAK1/STAT3 signal pathway and the MITFa. The achieved results indicate that the RKG could activate the zebrafish macrophages via the JAK1, but the inhibition of macrophage activation by loganin did not affect the anti-pigmentation effect of the RKG. RKG showed remarkable whitening activity on both B16F10 cells in vitro and zebrafish model in vivo. Furthermore, RKG could inhibit melanogenesis by activating the IL6/JAK1/STAT3 pathway, inhibiting the transcriptional activity of MITFa, and its downstream expression levels of the TYR and TYRP1a genes.

Pharmacological activities and effective substances of the component-based Chinese medicine of Ginkgo biloba leaves based on serum pharmacochemistry, metabonomics and network pharmacology.

As a potential drug candidate for the treatment of hypertension and complications, it is speculated that the component-based Chinese medicine of Ginkgo biloba leaves (GBCCM) which mainly composed of flavonoid aglycones (FAs) and terpene lactones (TLs) may have different pharmacological effects at different doses or ratios. Taking the normal mice as the study object, metabonomics was conducted by giving different doses of GBCCM. Based on the components of GBCCM absorbed into the blood, the network pharmacological prediction was carried out. By integrating the results of metabonomics and network pharmacology, predict the possible pharmacological effects of GBCCM and conduct experimental verification. It was found that eight of the 19 compounds in GBCCM could be absorbed into the blood. GBCCM mainly affected the signal pathways of unsaturated fatty acid, pyruvate, bile acid, melanin and stem cells. It was speculated that GBCCM might have activities such as lowering blood pressure, regulating stem cell proliferation and melanogenesis. By establishing the models of mushroom tyrosinase, rat bone marrow mesenchymal stem cells (BMSCs) and spontaneously hypertensive rats (SHRs), we found that FAs and TLs showed synergistic effect in hypertension and tyrosinase models, and the optimal ratio was 3:2 (4.4mg/kg) and 1:1 (0.4mg/ml), respectively. As effective substances, FAs significantly promoted the proliferation of rat BMSCs on the third and fifth days at the concentration of 0.2μg/ml (p < 0.05). GBCCM showed a variety of pharmacological effects at different doses and ratios, which provided an important reference for the druggability of GBCCM.

The bridge towards a more stable and active side-on-peroxido (Cu2II(μ-η2:η2-O2)) complex as a tyrosinase model system.

A novel dinucleating bis(pyrazolyl)methane ligand was developed for tyrosinase model systems. After ligand synthesis, the corresponding Cu(I) complex was synthesized and upon oxygenation, formation of a μ-η2:η2 peroxido complex could be observed and monitored using UV/Vis-spectroscopy. Due to the high stability of this species even at room temperature, a molecular structure of the complex could be characterized via single-crystal XRD. Additional to its promising stability, the peroxido complex showed catalytic tyrosinase activity which was investigated via UV/Vis-spectroscopy. Products of the catalytic conversion could be isolated and characterized and the ligand could be successfully recycled after catalysis experiments. Furthermore, the peroxido complex was reduced by reductants with different reduction potentials. The characteristics of the electron transfer reactions were investigated with the help of the Marcus relation. The combination of the high stability and catalytic activity of the peroxido complex with the new dinucleating ligand, enables the shift of oxygenation reactions for selected substrates towards green chemistry, which is furthered by the efficient ligand recycling capability.

Comparative Study of High-throughput Screening Models for Anti-hyperpigmentation Compounds

HIGHLIGHTS It is the first time to compare mushroom tyrosinase model, B16 mouse melanoma cell model and zebrafish model. A high-throughput method for the quantification of melanin in zebrafish skin based on the Phyton for the first time.

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.

Frontispiece: Exceptional Substrate Diversity in Oxygenation Reactions Catalyzed by a Bis(μ‐oxo) Copper Complex

The tyrosinase-like activity of a new bioinspired bis(μ-oxo) dicopper(III) catalyst was investigated herein. The hybrid guanidine-stabilized catalyst revealed a remarkable oxygenation activity on diverse phenolic substrate classes, products of which were consistently predicted by DFT calculations. The selective C−H activation of mono- and bicyclic substrates provides a new benchmark to future developments on tyrosinase model systems on the route to atom-economic oxygen-transfer reactions. More information can be found in the Communication by S. Herres-Pawlis et al. on page 7556 ff.

Exceptional Substrate Diversity in Oxygenation Reactions Catalyzed by a Bis(μ-oxo) Copper Complex.

The enzyme tyrosinase contains a reactive side‐on peroxo dicopper(II) center as catalytically active species in C−H oxygenation reactions. The tyrosinase activity of the isomeric bis(μ‐oxo) dicopper(III) form has been discussed controversially. The synthesis of bis(μ‐oxo) dicopper(III) species [Cu2(μ‐O)2(L1)2](X)2 ([O1](X)2, X=PF6 − , BF4 −, OTf−, ClO4 −), stabilized by the new hybrid guanidine ligand 2‐{2‐((dimethylamino)methyl)phenyl}‐1,1,3,3‐tetramethylguanidine (L1), and its characterization by UV/Vis, Raman, and XAS spectroscopy, as well as cryo‐UHR‐ESI mass spectrometry, is described. We highlight selective oxygenation of a plethora of phenolic substrates mediated by [O1](PF6)2, which results in mono‐ and bicyclic quinones and provides an attractive strategy for designing new phenazines. The selectivity is predicted by using the Fukui function, which is hereby introduced into tyrosinase model chemistry. Our bioinspired catalysis harnesses molecular dioxygen for organic transformations and achieves a substrate diversity reaching far beyond the scope of the enzyme.

In-silico analysis of deleterious missense SNPs of human TYR gene associated with oculocutaneous albinism type 1 (OCA1)

Single Nucleotide Polymorphisms (SNPs) are among the major genetic anomalies behind genetically rare disorders. The human TYR gene encodes a key regulatory enzyme, tyrosinase for melanin biosynthesis. Missense mutations in the TYR gene may disrupt the normal function of tyrosinase and cause oculocutaneous Albinism type 1 (OCA1). The present study involves the use of multiple computational tools based on different algorithms for the identification of potential deleterious missense SNPs from the TYR gene. In this study, we identified ten deleterious missense mutations which may be contributing factors for OCA1. The 3D modeling analysis of mutant models of tyrosinase also strengthen the hypothesis that these mutations may induce significant structural and conformational changes on the native tyrosinase enzyme.

A Stereoselective Tyrosinase Model Compound Derived from an m-Xylyl-l-histidine Ligand.

The aim of mimicking enzyme activity represents an important motivation for the development of new catalysts. A challenging objective is the development of chiral complexes for bioinspired enantioselective oxidation reactions. Herein, we report a new chiral dinuclear copper(II) complex based on a m-xylyl-bis(histidine) ligand (mXHI) as a biomimetic catalyst for tyrosinase and catechol oxidase. The new ligand improves a previous system also containing two tridentate N3 units derived from l-histidine that were connected by a short, rigid ethanediamine bridge. In mXHI the bridge is provided by the more extended m-xylyl moiety. The dicopper(II) complex [Cu2(mXHI)]4+ was studied as a catalyst for stereoselective oxidations of enantiomeric couples of chiral catechols of biological interest (L/D-dopa, L/D-dopa methyl ester, and ( R/ S)-norepinephrine), showing excellent discrimination capability, particularly for the methyl esters of dopa enantiomers. The catechol oxidation was studied in acetate buffer as slightly acidic medium, and a role of acetate as bridging ligand between the two coppers, preorganizing the dinuclear center in a more catalytic efficient structure, could be established. The oxidation of β-naphthol and 3,5-ditertbutylphenol was studied as a model monophenolase reaction. The oxidation proceeds stoichiometrically, and the partial incorporation of 18O into β-naphthol when the reaction was performed using 18O2 suggests the existence of two competitive reaction pathways, a genuine monooxygenase mechanism and a radical pathway. However, the more challenging reaction on derivatives of l-/d-tyrosine did not lead to the desired monooxygenase product but only to products of radical oxidation. Complex [Cu2(mXHI)]4+ was also used for the catalytic sulfoxidation of thioanisole in the presence of hydroxylamine as cosubstrate, in a preliminary attempt to model the reaction of external monooxygenases. The reaction proceeds with 25 turnovers, but the enantiomeric excess of sulfoxide was modest.

Employing Linear Tridentate Ligands with Pyrazole End Groups in Catalytic Tyrosinase Model Chemistry: Does Hemilability Matter?

Several copper(I) and copper(II) complexes supported by hemilabile bis(pyrazolylmethyl)amine (pzma) ligands are synthesized and structurally characterized. The copper(I) complexes with hexafluoridophosphate or perchlorate anions are employed as catalysts for the tyrosinase‐like oxygenation of 2,4‐di‐tert‐butylphenol (DTBP‐H). Their activities are comparable to that of [Cu(MeCN)2PMP]PF6 (PMP= pyrazolylmethylpyridine) investigated earlier. In contrast to the copper(I) pzma complexes, their congeners supported by non‐hemilabile bis(pyrazolylethyl)amine (pzea) ligands are found to be catalytically inactive.

Tyrosinase Model Systems Supported by Pyrazolylmethylpyridine Ligands: Electronic and Steric Factors Influencing the Catalytic Activity and Impact of Complex Equilibria in Solution

Two new copper(I) complexes supported by pyrazolylmethylpyridine (PMP) ligands are synthesized and investigated regarding their ability to catalyze the oxygenation of several monophenolic substrates. The PMP ligands containing a pyridine and a pyrazole donor group represent hybrids between dipyridylmethane (DPM) and bis(pyrazolyl)methane (BPM) ligands. The catalytic activity of the two new [Cu(MeCN)2PMP]PF6 complexes is found to be intermediate between that of catalytically inactive [Cu(MeCN)2DPM]PF6 and highly active [Cu(MeCN)2BPM]PF6, suggesting that the electronic properties of a multidentate ligand can be designed in a modular fashion. DFT calculations are used to explore the differences in reactivity between the two systems. Regarding the behavior of these complexes in solution, evidence for an equilibrium between homoleptic and heteroleptic forms is presented. The crystal structure of a dinuclear complex exhibiting two homoleptic CuII units bridged by a fluorido ligand is obtained, which might represent one of the decay products of PMP‐type catalysts after prolonged reaction times.

Record Broken: A Copper Peroxide Complex with Enhanced Stability and Faster Hydroxylation Catalysis

Tyrosinase model systems pinpoint pathways to translating Nature's synthetic abilities for useful synthetic catalysts. Mostly, they use N-donor ligands which mimic the histidine residues coordinating the two copper centres. Copper complexes with bis(pyrazolyl)methanes with pyridinyl or imidazolyl moieties are already reported as excellent tyrosinase models. Substitution of the pyridinyl donor results in the new ligand HC(3-tBuPz)2 (4-CO2 MePy) which stabilises a room-temperature stable μ-η2 :η2 -peroxide dicopper(II) species upon oxygenation. It reveals highly efficient catalytic activity as it hydroxylates 8-hydroxyquinoline in high yields (TONs of up to 20) and much faster than all other model systems (max. conversion within 7.5 min). Stoichiometric reactions with para-substituted sodium phenolates show saturation kinetics which are nearly linear for electron-rich substrates. The resulting Hammett correlation proves the electrophilic aromatic substitution mechanism. Furthermore, density functional theory (DFT) calculations elucidate the influence of the substituent at the pyridinyl donor: the carboxymethyl group adjusts the basicity and nucleophilicity without additional steric demand. This substitution opens up new pathways in reactivity tuning.

Melanogenesis Inhibitors: Strategies for Searching for and Evaluation of Active Compounds.

Hyperpigmentation disorders constitute important medical and aesthetical conditions. Dark areas or dark spots on the skin result from inappropriate amount and/or deposition of skin pigments - melanins. Several depigmenting agents, such as kojic acid, arbutin, aloesin, ellagic acid, resveratrol, azelaic acid, niacinamide, tretinoin, glycolic acid, lactic acid, and citric acid, have already been identified and are used in topical drugs or cosmetic formulations for the treatment of hyperpigmentations. However, these compounds are characterized by insufficient effectiveness and multiple adverse effects. As a result, there is still a need for searching for new active substances. The current paper summarizes strategies for searching for novel melanogenesis inhibitors. In the review, they are divided according to approach, into in silico, in vitro and in vivo experiments. In silico research includes computational studies with models of tyrosinase and di-copper complexes. The in vitro approach is based on tests using tyrosinase (a key enzyme in melanin biosynthesis), cell cultures, cell co-cultures, pigmented human skin equivalents or Streptomyces bikiniensis model. In vivo studies involve zebrafish, rodents or humans. Examples of protocols and laboratory procedures are presented, with a focus on utilization of various models for evaluation of mechanisms of action of tested compounds. The potential limitations of the methods are also discussed, together with future perspectives in the field of searching for melanogenesis inhibitors.

Recombinant Tyrosinase from Polyporus arcularius: Overproduction in Escherichia coli, Characterization, and Use in a Study of Aurones as Tyrosinase Effectors.

Tyrosinases act in the development of organoleptic properties of tea, raisins, etc., but also cause unwanted browning of fruits, vegetables, and mushrooms. The tyrosinase from Agaricus bisporus has been used as a model to study tyrosinase inhibitors, which are also indispensable in the treatment of skin pigmentation disorders. However, this model has disadvantages such as side enzyme activities and the presence of multiple isoenzymes. Therefore, we aimed to introduce a new tyrosinase model. The pro-tyrosinase from Polyporus arcularius was overproduced in Escherichia coli. Trypsin digestion led to a cleavage after R388 and hence enzyme activation. The tyrosinase was a homodimer and transformed L-DOPA and tert-butylcatechol preferentially. Various aurons were examined as effectors of this enzyme. 2'- and 3'-hydroxyaurones acted as its activators and 2',4'-dihydroxyaurone as an inhibitor, whereas 4'-hydroxyaurones were its substrates. The enzyme is a promising model for tyrosinase effector studies, being a single isoenzyme and void of side enzyme activities.

Donor-driven conformational flexibility in a real-life catalytic dicopper(ii) peroxo complex.

The conformers of the real-life tyrosinase model [Cu2O2{HC(3-tBuPz)2(Py)}2](2+) which displays catalytic hydroxylation reactivity were investigated by density functional theory (DFT) studies including second-order perturbation theory and charge decomposition analysis (CDA). We elucidated the donor competition between pyrazolyl and pyridinyl moieties and found that pyrazolyl units are the stronger donors in bis(pyrazolyl)pyridinylmethane copper complexes. Geometry optimisations and TD-DFT calculations on all conformers proved to be robust in the prediction of the experimental data: the XAS distances and both charge-transfer bands are well reproduced. The CDA analyses gave insights into the electronic structure of the real-life peroxo dicopper species. The donor interplay as well as the multitude of interactions within two prototypical conformers have now been dissected in detail for the first time for a catalytic real-life system without simplifications. We find that the N donor interactions to the core are extremely stabilising and that in the conformer with both pyrazolyl units in equatorial position, these interactions are more stabilising than the axial ones. In the conformer with pyridinyl/pyrazolyl equator, the picture is more mixed but the general trend keeps consistent. We relate the extraordinary catalytic activity of the [Cu2O2{HC(3-tBuPz)2(Py)}2](2+) system to the subtle interplay of the different donor moieties.

Catalytic Models of Tyrosinase: Reactivity Differences between Systems Based on Mono‐ and Binucleating Ligands

AbstractA new tyrosinase model based on the binucleating ligand Lpy2 is synthesized and characterized. The ligand Lpy2 contains a combination of an imine and a pyridine function in the sidearms, which are bridged by a flexible alkyl spacer. As shown by UV/Vis and NMR spectroscopy, the Cu2Lpy2 complex catalyzed the conversion of the monophenol 2,4‐di‐tert‐butylphenol (DTBP‐H) into the o‐quinone 3,5‐di‐tert‐butylquinone (DTBQ) with a turnover number (TON) of 18. The dicopper complex of Lpy2 thus shows monophenolase activity that is comparable to that of the recently developed Lpy1 model of tyrosinase, which is based on a known mononucleating ligand (M. Rolff, J. Schottenheim, G. Peters, F. Tuczek, Angew. Chem. Int. Ed. 2010, 122, 6583). The electron‐poor substrate 4‐hydroxybenzoic acid methyl ester (MeBA‐OH), in contrast, is converted by Cu2Lpy2 into the semiquinone. For both substrates, the oxygenation reactions were also conducted in a stoichiometric fashion to obtain information on the intermediates involved. For the substrate MeBA‐OH, we detected a binuclear μ‐catecholato copper(II) complex by high‐resolution ESI mass spectrometry. These studies were complemented by investigations of deactivation mechanisms that could be invoked to explain the limitation of the TON. To this end, a bis‐μ‐hydroxido Lpy2 dicopper(II) complex as well as a semiquinone Lpy2 complex were prepared. Both complexes may represent decay products of the catalyst.

Biomimetic Hydroxylation Catalysis Through Self‐Assembly of a Bis(pyrazolyl)methane Copper–Peroxo Complex

AbstractWe synthesised and characterised four copper complexes (with copper in the oxidation states I and II) with the bis(pyrazolyl)methane ligands HC(3‐tBuPz)2(Py) and HC(3‐tBuPz)2(Qu). With the quinolinyl ligand (2‐quinolinyl)bis(3‐tert‐butylpyrazolyl)methane [HC(3‐tBuPz)2(Qu)] we obtained the tetrahedral monofacial complex [CuCl{HC(3‐tBuPz)2(Qu)}] (C1) and with the pyridinyl ligand (2‐pyridinyl)bis(3‐tert‐butylpyrazolyl)methane [HC(3‐tBuPz)2(Py)] we obtained the three complexes [CuCl{HC(3‐tBuPz)2(Py)}] (C2), [CuBr2{HC(3‐tBuPz)2(Py)}] (C3) and [CuCl2{HC(3‐tBuPz)2(Py)}] (C4), which are also monofacially coordinated. The molecular structures were analysed and compared with density functional theory calculations, which included natural bond orbital (NBO) analysis. C1 can, when generated in situ, serve as part of a precursor, used for the activation of oxygen as tyrosinase model. We observe the self‐assembly of a peroxo–dicopper complex P with the HC(3‐tBuPz)2(Qu) ligand, which is able to perform catalytic hydroxylation catalysis with phenols. DFT calculations were also carried out to understand the electronic transitions responsible for the UV/Vis bands in the corresponding spectra of the peroxo species.

Modeling the Met Form of Human Tyrosinase: A Refined and Hydrated Pocket for Antagonist Design

Tyrosinases are type 3 copper proteins involved in melanin biosynthesis, responsible for skin and hair color in mammals. To steer tyrosinase inhibitor discovery for therapeutic and cosmetic purposes, structural information about human tyrosinase is necessary. As this protein has never been crystallized so far, we derived a robust homology model built using structural information from Streptomyces castaneoglobisporus and Ipomea batata catecholoxidase enzymes. The active site containing two copper atoms in co-ordination with six histidine residues was refined through an optimization protocol based on molecular mechanics parameters for copper co-ordination and charges calculated by quantum mechanics methods. Five structural water molecules and a hydroxyl ion were found to be essential for optimization. The superimposition of the human homology model on crystallographic structures of tyrosinases from other species revealed similar overall backbone topologies, active site conformations, and conserved water molecules. Phenylthiourea (PTU), the tyrosinase inhibitor of reference, was then docked into the solvated human active pocket. A binding mode consistent with crystallographic information was obtained. Taken together, these findings demonstrated that the human tyrosinase model, deposited in the Protein Model Database, is a reliable structure for future rational inhibitor design projects.

In Silico Prediction of Tyrosinase and Adenylyl Cyclase Inhibitors from Natural Compounds

Although many herbal medicines are effective in the treatment of hyperpigmentation, the potency of different constituents remains unknown. In this work, more than 20,000 herbal ingredients from 453 herbs were docked into the crystal structures of adenylyl cyclase and a human homology tyrosinase model using Surflex-Dock. These two enzymes are responsible for melanin production and inhibition of them may attain a skin-whitening effect superior to currently available agents. The essential drug properties for topical formulation of the herbal ingredients, including skin permeability, sensitization, irritation, corrosive and carcinogenic properties were predicted by Dermwin, Skin Sensitization Alerts (SSA), Skin Irritation Corrosion Rules Estimation Tool (SICRET) and Benigni/Bossa rulebase module of Toxtree. Moreover, similarity ensemble and pharmacophore mapping approaches were used to forecast other potential targets for these herbal compounds by the software, SEArch and PharmMapper. Overall, this study predicted seven compounds to have advanced drug-like properties over the well-known effective tyrosinase inhibitors, arbutin and kojic acid. These seven compounds have the highest potential for further in vitro and in vivo investigation with the aim of developing safe and high-efficacy skin-whitening agents.

New catalytic model systems of tyrosinase: fine tuning of the reactivity with pyrazole-based N-donor ligands

Two new Cu(i) complexes have been synthesized and investigated as model systems of the enzyme tyrosinase. The corresponding ligands are based on a combination of an imine function with two different pyrazole groups. The reactivity of the prepared systems with respect to the conversion of monophenols to the corresponding ortho-quinones is investigated. The resulting data are compared to results obtained for other catalytic model systems of tyrosinase.