Docking Studies of Some Novel Kojic acid Derivatives as Possible Tyrosinase Inhibitors

Tyrosinase (E.C, 1.14.18.1) catalyzes two key reactions in mammalian melanogenesis. Hyperpigmentation caused by the overproduction of melanin in the skin. Enzymatic browning of fruits and vegetables and derived products is caused by tyrosinase. Therefore, tyrosinase inhibitors have potential applications in medicine, cosmetics and agriculture. The aim of this research is the bioinformatical study of tyrosinase inhibition by some Kojic acid Derivatives. In order to investigating the mode of interaction of the compounds with tyrosinase active site, Chemical structures of all compounds were designed using ChemDraw program, then were transferred into Hyperchem software and energy minimized. docking study was performed by AutoDock 4.2 program and The resulting docking poses were analyzed in AutoDock Tools, DS Visualizer and Ligplot softwares. Among the all studied compounds, Ligands 12, 20, 21 and 23 displayed good docking results, In fact, these compounds have the most negative ΔGbind that indicated favorable interactions with the key amino acid residues at active site of tyrosinase. The docked conformation revealed that these compounds could form metal-ligand interaction with The Cu2+ ions in the active site. These insilico results can thus serve as a template for further studies invitro and invivo.


INTRODUCTION
Tyrosinase is copper-dependent enzyme that catalyze the oxidations of both monophenols and o-diphenols into reactive o-quinones [1][2][3][4] .Melanin synthesis is initiated from tyrosine by tyrosinase.Although melanin is essential for protecting skin against UV irradiation damage, abnormal melanin production can lead to hyperpigmentation disorders such as melasma, lentigines, and post-inflammatory hyper pigmentation [5][6][7] .Enzymatic browning of fruits and vegetables takes place in the presence of oxygen when tyrosinase and its polyphenolic substrates are mixed after brushing and crushing operations, which leads to the rupture of cell structure 8,9 .Browning in crops is unfavorable and decreases the commercial value of the products 2,10 .Furthermore, tyrosinase inhibitors have been used as insecticides and insect control agents for years, because this enzyme is associated with the host defense, wound healing, molting and sclerotisation of insects [11][12][13][14] .Therefore, tyrosinase inhibition has become increasingly important for scientists in many branches of life science research.
The most intensively studied inhibitor of tyrosinase, 5-hydroxy-2-(hydroxymethyl)-4-pyrone, also termed as kojic acid (KA), was discovered in Japan by Saito in 1907 and is produced by various fungies 15 .KA chelates transition metal ions such as Cu 2+ and Fe 3+ and is an effective free radicals scavenger.This inhibitor is currently applied as a food additive to prevent enzymatic browning of fruits.KA is also added to cosmeceuticals such as skinlightening agents.It inhibits both the cresolase and catecholase activity of mushroom tyrosinase.However, its use has been limited, for its cytotoxicity, the skin irritation caused by its use in cosmetics and also its instability during storage 16,17 .Therefore, the development of novel, potent, non-toxic and stable kojic acid derivatives as tyrosinase inhibitors is of great importance.
Determination of the binding mode and affinity between the ligand and receptor is an essential issue in understanding the interaction mechanisms and designing therapeutic interventions in molecular recognition.The main aim of molecular docking is to accurately predict the conformation of a ligand locked up to the receptor binding site and to correctly estimate the strength of binding.Docking technique is a structure-based virtual screening that predicts the preferred binding orientation of the ligand to the desired receptor in a stable complex with respect to experiment 18 .This technique searches over many possible interactions in order to identify a set of ligand poses that represent local minimum-energy positions of the ligand.It calculates a binding energy that can be used to accurately rank-order different ligands relative to experimentally measured binding affinities 19 .this method is very informative to represent key structural characteristics and interactions to provide helpful data for proposing effective receptor inhibitors.
In the present study, twenty four derivatives of 3-hydroxypyridine-4-one scaffold were subjected to molecular docking studies.The preparation of some of these compounds is reported previously by this group 20,21 .These compounds are very close analogues of kojic acid having structural elements which provides them the ability to chelate copper ions and set up interactions with key residues located in the active site.Based on the in vitro evaluations some of these compounds had tyrosinase inhibition activity and were considered in the design of the study compounds 21 .

MATERIALS AND METHODS
For the protein-ligand docking simulation, we used AutoDock4.2software package 22 .Lamarckian Genetic Algorithm of the AutoDock 4.2 program was used to perform the flexible-ligand docking studies.An Intel-based core i5 personal computer with Linux (redhat 6) operating system was used for docking procedure.The crystal structure of tyrosinase from Agaricus bisporus (AbTYR; PDB code, 2Y9X) was chosen as the protein model for the present study.

Ligand Structure Preparation
General structure and the structural detailes of the compounds subjected to molecular docking simulations are provided in Table 1.All 2D structures of compounds were built using ChemDraw program (ChemDraw Ultra 10.0, Cambridge soft.), then were transferred into Hyperchem 8.0 software (HyperChem, Release 8.0 for Windows, Molecular Modeling System: HyperCube, 2007) and energy minimized with MM + force field using Polak-Ribiere conjugate gradient algorithm until the root mean square gradient of 0.01 Kcal/µ.mol.These optimized structures were used as inputs of the AutoDock tools.Then the partial charges of atoms were calculated using the Gasteiger-Marsili procedure implemented in the AutoDock tools package.Non-polar hydrogens of the compounds were merged and then rotatable bonds were assigned.

Protein Structure Preparation
A tyrosinase from the mushroom Agaricus bisporus has been successfully crystallized by Wangsa Ismaya and its structure has been elucidated by means of X-ray crystallography.The enzyme is commonly found as a H 2 L 2 tetramer.The H subunit is the tyrosinase domain with a molecular mass of 43 kDa.This subunit contains two copper atoms coordinated by six histidine residues, His85, His61, His94, His259, His263 and His296.The L subunit is 14 kDa with a lectin-like fold and the function of this subunit is unknown.This tetramer is stabilized by two holmium ions in the H_H interface 8 .
Co-crystallized ligands (tropolone), all chains except chain A, two holmium ions and water molecules of crystallization were removed from the complex using Discovery StudioVisualizer 23 .All missing hydrogens were added and after determining the Kolman united atom charges, nonpolar hydrogens were merged to their corresponding carbons using Autodock tools 24 .

Docking Procedure
The AutoGrid program performs precalculations for the docking of a ligand to a set of grids that describe the effect that the protein has on point charges.The effect of these forces on the ligand is then analyzed by the AutoDock program.Using Autogrid as a part of the Auto dock package, desolvation parameters and electrostatic interactions were assigned to each protein atom.The grid points were set as 40 × 40 × 40 with the spacing valued at 0.375(a quarter of the C-C bond) to the catalytic site of the tyrosinase while the grid center was placed between the two metal ions located in the active site.The Lamarckian Genetic Algorithm (LGA) approach was selected as the search algorithm for the global optimum binding position search among the three different search algorithms offered by AutoDock 4.2.This algorithm has been determined superior compared with simulated annealing and genetic algorithm.100 independent docking runs were carried out for each ligand.At the end of docking, the resulting docking poses were analyzed in AutoDockTools, DS Visualizer 3.5 and Ligplot softwares 25

RESULTS
Tropolone was redocked into the active site of tyrosinase for the method validation in the first step of docking procedure.The binding energy of the best bound conformation of tropolone was -3.11 kcal/mol.The molecular interactions of Tropolone with tyrosinase are illustrated in figure 1 and 2 .Tropolone interacts with HIS263 through αβ and cation-α interactions (orange lines) and its   OH group forms a hydrogen-bond with Asn260 (black dashed line) (Figure 1).This Compound interacts hydrophobically with Gly281, His259, Val283, Ser282, His61, His263, Ala286 and Met280.
After validation of the docking protocol, the 3D structures of the study compounds were docked into the tyrosinase active site.Docking analysis revealed that all active compounds, occupied the same space as tropolone with a similar binding mode.The estimated free binding energy values (ΔG bind ) of the docked positions, intermolecular energy, electrostatic energy, total internal energy and torsional energy of these inhibitors into the active site are listed in Table 2. ΔG bind is the sum of the intermolecular energy and the torsional freeenergy penalty and indicates favorable interactions and tight binding with key amino acid residues at the active site.Intermolecular energy is the sum of van der Waals, hydrogen binding, desolvation and electrostatic-energies 18,19 .The favorable interactions with the key amino acid residues at the active site of the enzyme are presented Tables 3.

DISSCUSSION
The binding mode of the study compounds against tyrosinase was investigated by performing molecular docking smulations.There is no X-ray crystallography data available for the three-dimensional structure of human tyrosinase 26 .Thus, the 3D structure of Agaricus bisporus mushroom tyrosinase was used for the docking study.Possible H-bonding, ð-sigma, cation-ð and metal interactions assessed using the Discovery StudioVisualizer program.Ligplot software Based on the structural diversity of the compounds, they are divided into three different groups.Interactions of the most active compounds in each group are provided in related sections.

Group I: Compounds 1-12
Compound 12 was the most active compound in this group.This compound interacts through H-bonding with The His61, His259, His296, His244, Asn260 and Glu256 residues of tyrosinase active site.The ÄG bind for this molecule was -13.24kcal/mol.Compound 12 interacts hydrophobically with Gly281, His94, Val283, Ser282, His61, His263, Asn260, His259, Phe264, Phe90, Phe292 and Ala286.The docking results for the most active compound in this group, are illustrated in Figure 2. The docked conformation revealed that the NO 2 group of compound 12, which resided 2.25-2.27Åadjacent to the di-copper nucleus, could form metal-ligand interaction.

Group II: Compounds 13-19
Molecular docking study revealed that compounds 13-19 may attach to tyrosinase active site via H-bond and hydrophobic interactions as other compounds.Members of this group exhibited weaker affinity to tyrosinase compared with the previous group.Compound 17 in this group exhibited the weakest affinity amongst the studiy compounds.None of the members in this group had interaction with the di-copper ion in the active site.The ÄG bind of the most active compound,  3.

Group III: Compounds 20-24
Compounds 22 and 24 exhibited the lowest interactions with tyrosinase in this group.They both did not interact with the di-copper ion in the active site.They lacked a free diketo acid group and due to the improper orientation of the esterified diketo acid moiety, were not able to have any interaction with the metal ions.Ligands 20, 21 and 23 displayed good docking results, with 20 as the most potent member of the group (-12.23 kcal/mol).In the case of compound 20, the carboxylic acid group substituted on C2 and the αhydroxy ketone moiety on the 3-hydroxypyridin 4-one ring together may form metal-ligand interactions with two metal ions as enolyzed diketo may in compounds 21 and 23.Two carbonyl moieties of compound 20 formed two hydrogen bonds with His85 and Ser282 and interacted again with the imidazole ring of His263 by a sigma-ð interaction.These observations are illustrated in Figure 4.This compound makes hydrophobic interactions with Asn260, Val283, Ser282, His61,His259, Phe292 and Ala286.In compounds 21 and 23 the diketo acid moiety may interact with both metal ions in the active site.It is demonstrated with molecular docking results (Table 3).

CONCLUSION
In agreement with binding energies, Ligands 12, 20, 21, and 23 displayed good docking results.The docking results for this compound are in accordance with the docking results reported by others in terms of the amino residues involved in interaction with the inhibitor molecule 1,[26][27][28][29] .
All of these four compounds have an efficient metal-ligand interaction with the Cu 2+ ions.In tyrosinase enzyme, each copper ion is coordinated by three histidine residues 4 .Among these six histidines, His61, His296 and His259 interact with compound 12, His58 interact with compound 20, His61, His296 and His259 interact with compound 21 and His296 and His259 interact with compound 23.Among the all studied compounds, the best docking result was obtained for compound 12.In fact, this compound had the most negative ÄG bind (-13.24Kcal/mol) that indicated favorable interactions and tight binding with the key amino acid residues at active site of tyrosinase.These insilico results can thus serve as a template for further studies invitro and invivo.

Fig. 1 :Fig. 2 :Fig. 3 :Fig. 4 :
Fig. 1: Redocking results of tropolone in the active site of tyrosinase.This figure was prepared using the Accelrys discovery studio visualizer program