The effect of fucoidan on tyrosinase: computational molecular dynamics integrating inhibition kinetics

Fucoidan is a complex sulfated polysaccharide extracted from brown seaweed and has a wide variety of biological activities. In this study, we investigated the inhibitory effect of fucoidan on tyrosinase via a combination of inhibition kinetics and computational simulations. Fucoidan reversibly inhibited tyrosinase in a mixed-type manner. Time-interval kinetics showed that the inhibition was processed as first order with biphasic processes. For further insight, we simulated dockings with various sizes of molecular models (monomer to decamer) of fucoidan and showed that the best binding energy change results were obtained from the pentamer (?1.89?kcal/mol) and the hexamer (?1.97?kcal/mol) models of AutoDock Vina. The molecular dynamics simulation confirmed the binding mechanisms between tyrosinase and fucoidan and suggested that fucoidan mostly interacts with several residues including copper ions located in the active site. Our study suggests that fucoidan might be a potential natural antipigment agent.     

Tyrosinase inhibition by isophthalic acid: kinetics and computational simulation

Using inhibition kinetics and computational simulation, we studied the reversible inhibition of tyrosinase by isophthalic acid (IPA). IPA inhibited tyrosinase in a complex manner with K(i)=17.8 ± 1.8mM. Measurements of intrinsic and ANS-binding fluorescence showed that IPA induced no changes in tertiary protein structure. For further insight, we predicted the 3D structure of tyrosinase and used a docking algorithm to simulate binding between tyrosinase and IPA. Simulation was successful (binding energies for Dock6.3: -25.19 kcal/mol and for AutoDock4.2: -4.28 kcal/mol), suggesting that IPA interacts with PRO175 or VAL190. This strategy of predicting tyrosinase inhibition based on hydroxyl group number and orientation may prove useful for the screening of potential tyrosinase inhibitors.     

The effect of histidine residue modification on tyrosinase activity and conformation: inhibition kinetics and computational prediction

We found that the histidine chemical modification of tyrosinase conspicuously inactivated enzyme activity. The substrate reactions with diethylpyridinecarbamate showed slow-binding inhibition kinetics (K(I) = 0.24 +/- 0.03 mM). Bromoacetate, as another histidine modifier, was also applied in order to study inhibition kinetics. The bromoacetate directly induced the exposures of hydrophobic surfaces following by complete inactivation via ligand binding. For further insights, we predicted the 3D structure of tyrosinase and simulated the docking between tyrosinase and diethylpyridinecarbamate. The docking simulation was shown to the significant binding energy scores (-3.77 kcal/mol by AutoDock4 and -25.26 kcal/mol by Dock6). The computational prediction was informative to elucidate the role of free histidine residues at the active site, which are related to substrate accessibility during tyrosinase catalysis.     

Relationship between the Circadian Variation of Uric Acid Level and Dietary Conditions

We studied the inhibitory effects of isorhamnetin on mushroom tyrosinase by inhibition kinetics and computational simulation. Isorhamnetin reversibly inhibited tyrosinase in a mixed-type manner at K _i=0.235 ± 0.013 mM. Measurements of intrinsic and 1-anilinonaphthalene-8-sulfonate(ANS)-binding fluorescence showed that isorhamnetin did not induce significant changes in the tertiary structure of tyrosinase. To gain insight into the inactivation process, the kinetics were computed via time-interval measurements and continuous substrate reactions. The results indicated that inactivation induced by isorhamnetin was a first-order reaction with biphasic processes. To gain further insight, we simulated docking between tyrosinase and isorhamnetin. Simulation was successful (binding energies for Dock6.3: ?32.58 kcal/mol, for AutoDock4.2: ?5.66 kcal/mol, and for Fred2.2: ?48.86 kcal/mol), suggesting that isorhamnetin interacts with several residues, such as HIS244 and MET280. This strategy of predicting tyrosinase interaction in combination with kinetics based on a flavanone compound might prove useful in screening for potential natural tyrosinase inhibitors.     

Effects of isorhamnetin on tyrosinase: inhibition kinetics and computational simulation

We studied the inhibitory effects of isorhamnetin on mushroom tyrosinase by inhibition kinetics and computational simulation. Isorhamnetin reversibly inhibited tyrosinase in a mixed-type manner at Ki=0.235±0.013 mM. Measurements of intrinsic and 1-anilinonaphthalene-8-sulfonate(ANS)-binding fluorescence showed that isorhamnetin did not induce significant changes in the tertiary structure of tyrosinase. To gain insight into the inactivation process, the kinetics were computed via time-interval measurements and continuous substrate reactions. The results indicated that inactivation induced by isorhamnetin was a first-order reaction with biphasic processes. To gain further insight, we simulated docking between tyrosinase and isorhamnetin. Simulation was successful (binding energies for Dock6.3: -32.58 kcal/mol, for AutoDock4.2: -5.66 kcal/mol, and for Fred2.2: -48.86 kcal/mol), suggesting that isorhamnetin interacts with several residues, such as HIS244 and MET280. This strategy of predicting tyrosinase interaction in combination with kinetics based on a flavanone compound might prove useful in screening for potential natural tyrosinase inhibitors.     

Effect of hesperetin on tyrosinase: inhibition kinetics integrated computational simulation study

Tyrosinase inhibitors have potential applications in medicine, cosmetics and agriculture to prevent hyperpigmentation or browning effects. Some of the flavonoids mostly found in herbal plants and fruits are revealed as tyrosinase inhibitors. We studied the inhibitory effects of one such flavonoid, hesperetin, on mushroom tyrosinase using inhibition kinetics and computational simulation. Hesperetin reversibly inhibited tyrosinase in a competitive manner with K_i = 4.03 ± 0.26 mM. Measurements of ANS-binding fluorescence showed that hesperetin induced the hydrophobic disruption of tyrosinase. For further insight, we used the docking algorithms to simulate binding between tyrosinase and hesperetin. Simulation was successful (binding energies for Dock6.3: −34.41 kcal/mol and for AutoDock4.2: −5.67 kcal/mol) and showed that a copper ion coordinating with 3 histidine residues (HIS61, HIS85, and HIS259) within the active site pocket was chelated via hesperetin binding. Our study provides insight into the inhibition of tyrosinase in response to flavonoids. A combination of inhibition kinetics and computational prediction may facilitate the identification of potential natural tyrosinase inhibitors such as flavonoids and the prediction of their inhibitory mechanisms.     

Mixed-Type Inhibition of Tyrosinase from Agaricus bisporus by Terephthalic Acid: Computational Simulations and Kinetics

Tyrosinase inhibition studies are needed due to the agricultural and medicinal applications. For probing effective inhibitors of tyrosinase, a combination of computational prediction and enzymatic assay via kinetics were important. We predicted the 3D structure of tyrosinase from Agaricus bisporus, used a docking algorithm to simulate binding between tyrosinase and terephthalic acid (TPA) and studied the reversible inhibition of tyrosinase by TPA. Simulation was successful (binding energies for Autodock4 = -1.54 and Fred2.0 = -3.19 kcal/mol), suggesting that TPA interacts with histidine residues that are known to bind with copper ions at the active site. TPA inhibited tyrosinase in a mixed-type manner with a K ( i ) = 11.01 ± 2.12 mM. Measurements of intrinsic and ANS-binding fluorescences showed that TPA induced no changes in tertiary structure. The present study suggested that the strategy of predicting tyrosinase inhibition based on hydroxyl groups and orientation may prove useful for screening of potential tyrosinase inhibitors.     

Kinetic, structural and molecular docking studies on the inhibition of tyrosinase induced by arabinose

Tyrosinase plays a central role in biological pigment formation, and hence knowledge of tyrosinase catalytic mechanisms and regulation may have medical, cosmetic, and agricultural applications. We found in this study that arabinose significantly inhibited tyrosinase, and this was accompanied by conformational changes in enzyme structure. Kinetic analysis showed that arabinose-mediated inactivation followed first-order kinetics, and single and multiple classes of rate constants were measured. Arabinose displayed a mixed-type inhibitory mechanism with K(i)=0.22±0.07 mM. Measurements of intrinsic and ANS-binding fluorescence showed that arabinose induced tyrosinase to unfold and expose inner hydrophobic regions. We simulated the docking between tyrosinase and arabinose (binding energies were -26.28 kcal/mol for Dock6.3 and -2.02 kcal/mol for AutoDock4.2) and results suggested that arabinose interacts mostly with His61, Asn260, and Met280. The present strategy of predicting tyrosinase inhibition by simulation of docking by hydroxyl groups may prove useful in screening for potential tyrosinase inhibitors, as shown here for arabinose.     

Inhibition of tyrosinase by gastrodin: An integrated kinetic-computational simulation analysis

We studied the inhibitory effect of gastrodin on tyrosinase using inhibition kinetics and computational simulation. Gastrodin reversibly inhibited tyrosinase in a mixed-type manner with K_i = 123.8 ± 20.2 mM. Time-interval kinetics revealed the inhibition to be a first-order process with mono- and bi-phasic components. Using AutoDock Vina, we calculated a binding energy of ?6.3 kcal/mol for gastrodin and tyrosinase, and we performed a molecular dynamics simulation of the tyrosinase–gastrodin interaction. The simulation results suggested that gastrodin interacts primarily with histidine residues in the active site. A 10-ns molecular dynamics simulation showed that one copper ion in the tyrosinase active site was responsible for the interaction with gastrodin. Our study provides insight into the inhibition of tyrosinase by the hydroxyl groups of gastrodin. A combination of inhibition kinetics and computational calculations may help to confirm the inhibitory action of gastrodin on tyrosinase and define the mechanisms of inhibition.     

Melanogenesis inhibitory effect of low molecular weight fucoidan from <Emphasis Type="Italic">Undaria pinnatifida</Emphasis>

In this study, fucoidans with different molecular weight that were isolated from the brown alga Undaria pinnatifida (Phaeophyceae, Laminariales) were investigated for their ability to inhibit melanogenesis and scavenge superoxide and hydroxyl radicals. Fucoidan samples with low molecular weights of 89, 35, 17, and 6 kDa were prepared by radiation-degradation of a 378 kDa fucoidan isolated from U. pinnatifida. The inhibitory activity of fucoidan against melanin biosynthesis in B16BL6 melanoma cells was enhanced for low molecular weight samples. To investigate the increase in melanogenesis inhibition exhibited by the low molecular weight fucoidan, tyrosinase inhibition activity and radical scavenging activities were measured. There was an increase in the tyrosinase inhibition activity for low molecular weight samples. Additionally, the radical scavenging activity was increased for lower molecular weight fucoidans. These results suggest that low molecular weight fucoidans from seaweeds may have beneficial biological properties.     

Fucoidan-dependent conformational changes in annexin II tetramer

Fucoidan, a sulfated fucopolysaccharide, mimics the fucosylated glycans of glycoproteins and has therefore been used as a probe for investigating the role of membrane polysaccharides in cell-cell adhesion. In the present report we have characterized the interaction of fucoidan with the Ca(2+)- and phospholipid-binding protein annexin II tetramer (AIIt). AIIt bound to fucoidan with an apparent K(d) of 1.24 +/- 0.69 nM (mean +/- SD, n = 3) with a stoichiometry of 0.010 +/- 0.001 mol of fucoidan/mol of AIIt (mean +/- SD, n = 3). The binding of fucoidan to AIIt was Ca(2+)-independent. Furthermore, in the presence but not the absence of Ca(2+), the binding of fucoidan to AIIt caused a decrease in the alpha-helical content from 32% to 7%. A peptide corresponding to a region of the p36 subunit of AIIt, F(306)-S(313), which contains a Cardin-Weintraub consensus sequence for heparin binding, was shown to undergo a conformational change upon fucoidan binding. This suggests that heparin and fucoidan bound to this region of AIIt. The binding of fucoidan but not heparin by AIIt also inhibited the ability of AIIt to bind to and aggregate phospholipid liposomes. These results suggest that the binding of AIIt to the carbohydrate conjugates of certain membrane glycoproteins may have profound effects on the structure and biological activity of AIIt.     

The tyrosinase inhibitory effects of isoxazolone derivatives with a (Z)-β-phenyl-α, β-unsaturated carbonyl scaffold

Thirteen (Z)-4-(substituted benzylidene)-3-phenylisoxazol-5(4H)-ones were designed to confirm the geometric effect of the double bond of the β-phenyl-α, β-unsaturated carbonyl scaffold on tyrosinase inhibitory activity. Compounds 1a–1m, which all possessed the (Z)-β-phenyl-α, β-unsaturated carbonyl scaffold, were synthesized using a tandem reaction consisting of an isoxazolone ring formation and a Knoevenagel condensation, and three starting materials, ethyl benzoylacetate, hydroxylamine and benzaldehydes. Some of the compounds showed inhibitory activity against mushroom tyrosinase as potent as compounds containing the “(E)”-β-phenyl-α, β-unsaturated carbonyl scaffold. Compounds 1c and 1m showed greater inhibitory activity than kojic acid: IC₅₀?=?32.08?±?2.25?μM for 1c; IC₅₀?=?14.62?±?1.38?μM for 1m; and IC₅₀?=?37.86?±?2.21?μM for kojic acid. A kinetic study indicated that 1m inhibited tyrosinase in a competitive manner and that it probably binds to the enzyme’s active site. In silico docking simulation supported binding of 1m (?7.6?kcal/mol) to the active site of tyrosinase with stronger affinity than kojic acid (?5.7?kcal/mol). Similar results were obtained using cell-based assays, and in B16F10 cells, compound 1m dose-dependently inhibited tyrosinase activity and melanogenesis. These results indicate the anti-melanogenic effect of compound 1m is due to the inhibition of tyrosinase and (Z)-isomer of the β-phenyl-α, β-unsaturated carbonyl scaffold can, like its congener the (E)-isomer, act as an excellent scaffold for tyrosinase inhibition.     

An Integrated Study of Tyrosinase Inhibition by Rutin: Progress using a Computational Simulation

Abstract

Tyrosinase inhibition studies have recently gained the attention of researchers due to their potential application values. We simulated docking (binding energies for AutoDock Vina: ?9.1 kcal/mol) and performed a molecular dynamics simulation to verify docking results between tyrosinase and rutin. The docking results suggest that rutin mostly interacts with histidine residues located in the active site. A 10 ns molecular dynamics simulation showed that one copper ion at the tyrosinase active site was responsible for the interaction with rutin. Kinetic analyses showed that rutin-mediated inactivation followed a first-order reaction and mono- and biphasic rate constants occurred with rutin. The inhibition was a typical competitive type with K_i = 1.10 ± 0.25 mM. Measurements of intrinsic and ANS-binding fluorescences showed that rutin showed a relatively strong binding affinity for tyrosinase and one possible binding site that could be a copper was detected accompanying with a hydrophobic exposure of tyrosinase. Cell viability testing with rutin in HaCaT keratinocytes showed that no toxic effects were produced. Taken together, rutin has the potential to be a potent antipigment agent. The strategy of predicting tyrosinase inhibition based on hydroxyl group number and computational simulation may prove useful for the screening of potential tyrosinase inhibitors.     

An integrated study of tyrosinase inhibition by rutin: progress using a computational simulation

Tyrosinase inhibition studies have recently gained the attention of researchers due to their potential application values. We simulated docking (binding energies for AutoDock Vina: -9.1 kcal/mol) and performed a molecular dynamics simulation to verify docking results between tyrosinase and rutin. The docking results suggest that rutin mostly interacts with histidine residues located in the active site. A 10 ns molecular dynamics simulation showed that one copper ion at the tyrosinase active site was responsible for the interaction with rutin. Kinetic analyses showed that rutin-mediated inactivation followed a first-order reaction and mono- and biphasic rate constants occurred with rutin. The inhibition was a typical competitive type with K(i) = 1.10±0.25 mM. Measurements of intrinsic and ANS-binding fluorescences showed that rutin showed a relatively strong binding affinity for tyrosinase and one possible binding site that could be a copper was detected accompanying with a hydrophobic exposure of tyrosinase. Cell viability testing with rutin in HaCaT keratinocytes showed that no toxic effects were produced. Taken together, rutin has the potential to be a potent anti-pigment agent. The strategy of predicting tyrosinase inhibition based on hydroxyl group number and computational simulation may prove useful for the screening of potential tyrosinase inhibitors.     

The effect of thiobarbituric acid on tyrosinase: inhibition kinetics and computational simulation

Tyrosinase plays various roles in organisms and much research has focused on the regulation of tyrosinase activity. We studied the inhibitory effect of thiobarbituric acid (TBA) on tyrosinase. Our kinetic study showed that TBA inhibited tyrosinase in a reversible noncompetitive manner (K(i) 5 14.0 ± 8.5 mM and IC?? 5 8.0 ± 1.0 mM). Intrinsic and ANS-binding fluorescences studies were also performed to gain more information regarding the binding mechanism. The results showed that no tertiary structural changes were obviously observed. For further insight, we predicted the 3D structure of tyrosinase and simulated the docking between tyrosinase and TBA. The docking simulation was successful with significant scores (binding energy for AutoDock4: -5.52 kcal/mol) and suggested that TBA was located in the active site. The 11 ns molecular dynamics simulation convinced that the four HIS residues (residue numbers: 57, 90, 250, and 282) were commonly responsible for the interaction with TBA. Our results provide a new inhibition strategy that works using an antioxidant rather than targeting the copper ions within the tyrosinase active site.     

2-Aminothiazole-Oxadiazole Bearing N-Arylated Butanamides: Convergent Synthesis,Tyrosinase Inhibition,Kinetics, Structure-Activity Relationship,and Binding Conformations

A multi-step synthesis of novel bi-heterocyclic N-arylated butanamides was consummated through a convergent strategy and the structures of these medicinal scaffolds, 7a–h , were corroborated using spectral techniques. The in vitro analysis of these hybrid molecules revealed their potent tyrosinase inhibition as compared to the standard used. The kinetics mechanism was investigated through Lineweaver-Burk plots which exposed that, 7f , inhibited tyrosinase enzyme non-competitively by forming the enzyme-inhibitor complex. The inhibition constants K_i calculated from Dixon plots for this compound was 0.025 μM. Their binding conformations were ascertained by in silico computational studies whereby these molecules disclosed good binding energy values (kcal/mol). So, it was anticipated from the current research that these bi-heterocyclic butanamides might be probed as imperative therapeutic agents for melanogenesis.     

Antithrombin activity of fucoidan. The interaction of fucoidan with heparin cofactor II, antithrombin III, and thrombin

Fucoidan, poly(L-fucopyranose) linked primarily alpha 1----2 with either a C3- or a C4-sulfate, is an effective anticoagulant in vitro and in vivo (Springer, G. F., Wurzel, H. A., McNeal, G. M., Jr., Ansell, N. J., and Doughty, M. F. (1957) Proc. Soc. Exp. Biol. Med. 94, 404-409). We have determined the antithrombin effects of fucoidan on the glycosaminoglycan-binding plasma proteinase inhibitors antithrombin III and heparin cofactor II. Fucoidan enhances the heparin cofactor II-thrombin reaction more than 3500-fold. The apparent second-order rate constant of thrombin inhibition by heparin cofactor II increases from 4 x 10(4) (in the absence of fucoidan) to 1.5 x 10(8) M-1 min-1 as the fucoidan concentration increases from 0.1 to 10 micrograms/ml and then decreases as fucoidan is increased above 10 micrograms/ml. The fucoidan reaction with heparin cofactor II-thrombin is kinetically equivalent to a "template model." Apparent fucoidan-heparin cofactor II and fucoidan-thrombin dissociation constants are 370 and 1 nM, respectively. The enhancement of thrombin inhibition by fucoidan, like heparin and dermatan sulfate, is eliminated by selective chemical modification of lysyl residues either of heparin cofactor II or of thrombin. The fucoidan-antithrombin III reactions with thrombin and factor Xa are accelerated maximally 285- and 35-fold at fucoidan concentrations of 30 and 500 micrograms/ml, respectively. Using human plasma and 125I-labeled thrombin in an ex vivo system, the heparin cofactor II-thrombin complex is formed preferentially over the antithrombin III-thrombin complex in the presence of 10 micrograms/ml fucoidan. Our results indicate that heparin cofactor II is activated by fucoidan in vitro and in an ex vivo plasma system and suggest that the major antithrombin activity of fucoidan in vivo is mediated by heparin cofactor II and not by antithrombin III.     

Interaction of fucoidan with the proteins of the complement classical pathway

Fucoidan inhibits complement by mechanisms that so far remain to be unraveled, and the objective of this work was to delineate the mode of inhibition by this sulfated polysaccharide. For that purpose, low molecular weight fractions of algal (Ascophyllum nodosum) fucoidan containing the disaccharide unit [-->3)-alpha-L-Fuc(2SO3(-))-(1-->4)-alpha-L-Fuc(2,3diSO3(-))-(1-->](n) have been studied. Gel co-affinity electrophoresis and a new affinity capillary electrophoresis (ACE) method have been implemented to characterize fucoidan-complement protein complexes. Fucoidan binds C1q, likely to its collagen-like region through interactions involving lysine residues, and then prevents the association of the C1r(2)-C1s(2) subunit, required to form the fully active C1. In addition to C1q, fucoidan forms a complex with the protein C4 as observed by ACE. The fucoidan inhibits the first steps of the classical pathway activation that is of relevance in view of the proinflammatory effects of the subsequent products of the cascade. This study shows that a high level of inhibitory activity can be achieved with low molecular weight carbohydrate molecules and that the potential applicability of fucoidan oligosaccharides for therapeutic complement inhibition is worthy of consideration.