The inhibitory effect of some new synthesized xanthates on mushroom tyrosinase activities

Three iso-alkyldithiocarbonates (xanthates), as sodium salts, C₃H₇OCS₂Na (I), C₄H₉OCS₂Na (II) and C₅H₁₁OCS₂Na (III), were synthesized, by the reaction between CS₂ with the corresponding iso-alcohol in the presence of NaOH, and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agricus bisporus. 4-[(4-methylbenzo)azo]-1,2-benzendiol (MeBACat) and 4-[(4-methylphenyl)azo]-phenol (MePAPh) were used as synthetic substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed and competitive inhibition for the three xanthates and also for cresolase and catecholase activities of MT. For cresolase activity, I and II showed a mixed inhibition pattern but III showed a competitive inhibition pattern. For catecholase activity, I showed mixed inhibition but II and III showed competitive inhibition. These new synthesized compounds are potent inhibitors of MT with K_i values of 9.8, 7.2 and 6.1 μM for cresolase inhibitory activity, and also 12.9, 21.8 and 42.2 μM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater inhibitory potency towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (α>1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds in both cresolase and catecholase activities. The cresolase inhibition is related to the chelating of the copper ions at the active site by a negative head group (S^? ) of the anion xanthate, which leads to similar values of K_i for all three xanthates. Different K_i values for catecholase inhibition are related to different interactions of the aliphatic chains of I, II and III with hydrophobic pockets in the active site of the enzyme.     

The inhibitory effect of some new synthesized xanthates on mushroom tyrosinase activities

Three iso-alkyldithiocarbonates (xanthates), as sodium salts, C3H7OCS2Na (I), C4H9OCS2Na (II) and C5H11OCS2Na (III), were synthesized, by the reaction between CS2 with the corresponding iso-alcohol in the presence of NaOH, and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agricus bisporus. 4-[(4-methylbenzo)azo]-1,2-benzendiol (MeBACat) and 4-[(4-methylphenyl)azo]-phenol (MePAPh) were used as synthetic substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed and competitive inhibition for the three xanthates and also for cresolase and catecholase activities of MT. For cresolase activity, I and II showed a mixed inhibition pattern but III showed a competitive inhibition pattern. For catecholase activity, I showed mixed inhibition but II and III showed competitive inhibition. These new synthesized compounds are potent inhibitors of MT with K(i) values of 9.8, 7.2 and 6.1 microM for cresolase inhibitory activity, and also 12.9, 21.8 and 42.2 microM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater inhibitory potency towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (alpha > 1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds in both cresolase and catecholase activities. The cresolase inhibition is related to the chelating of the copper ions at the active site by a negative head group (S-) of the anion xanthate, which leads to similar values of K(i) for all three xanthates. Different K(i) values for catecholase inhibition are related to different interactions of the aliphatic chains of I, II and III with hydrophobic pockets in the active site of the enzyme.     

The inhibition effect of some n-alkyl dithiocarbamates on mushroom tyrosinase

Three new n-alkyl dithiocarbamate compounds, as sodium salts, C4H9NHCS2Na (I), C6H13NHCS2Na (II) and C8H17NHCS2Na (III), were synthesized and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agaricus bisporus in 10 mM phosphate buffer pH 6.8, at 293K using UV spectrophotometry. Caffeic acid and p-coumaric acid were used as natural substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed and competitive inhibition for catecholase and cresolase reactions, respectively. These new synthetic compounds can be classified as potent inhibitors of MT due to Ki values of 0.8, 1.0 and 1.8 microM for cresolase inhibitory activity, and also 9.4, 14.5 and 28.1 microM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater potency in the inhibitory effect towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (alpha > 1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds. The inhibition mechanism is presumably related to the chelating of the binuclear coppers at the active site and the different Ki values may be related to different interaction of the aliphatic chains of I, II and III with the hydrophobic pocket in the active site of the enzyme.     

The role of alkyl chain length in the inhibitory effect n-alkyl xanthates on mushroom tyrosinase activities

Sodium salts of four n-alkyl xanthate compounds, C2H5OCS2Na (I), C3H7OCS2Na (II), C4H9OCS2Na (III), and C6H13OCS2Na (IV) were synthesized and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) in 10 mM sodium phosphate buffer, pH 6.8, at 293 K using UV spectrophotometry. 4-[(4-Methylbenzo)azo]-1,2-benzendiol (MeBACat) and 4-[(4-methylphenyl)azo]-phenol (MePAPh) were used as synthetic substrates for the enzyme for catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed, competitive or uncompetitive inhibition for the four xanthates. For the cresolase activity, I and II showed uncompetitive inhibition but III and IV showed competitive inhibition pattern. For the catecholase activity, I and II showed mixed inhibition but III and IV showed competitive inhibition. The synthesized compounds can be classified as potent inhibitors of MT due to their Ki values of 13.8, 11, 8 and 5 microM for the cresolase activity, and 1.4, 5, 13 and 25 microM for the catecholase activity for I, II, III and IV, respectively. For the catecholase activity both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (alpha > 1) and this negative cooperativity increases with increasing length of the aliphatic tail of these compounds. The length of the hydrophobic tail of the xanthates has a stronger effect on the Ki values for catecholase inhibition than for cresolase inhibition. Increasing the length of the hydrophobic tail leads to a decrease of the Ki values for cresolase inhibition and an increase of the Ki values for catecholase inhibition.     

The inhibitory effect of benzenethiol on the cresolase and catecholase activities of mushroom tyrosinase

The inhibitory effect of benzenethiol on the cresolase and catecholase activities of mushroom tyrosinase (MT) have been investigated at two temperatures of 20 and 30°C in 10 mM phosphate buffer solution, pHs 5.3 and 6.8. The results show that benzenethiol can inhibit both activities of mushroom tyrosinase competitively. The inhibitory effect of benzenethiol on the cresolase activity is more than the catecholase activity of MT. The inhibition constant (K_i) value at pH 5.3 is smaller than that at pH 6.8 for both enzyme activities. However, the K_i value increases in cresolase activity and decreases in catecholase activity due to the increase of temperature from 20 to 30°C at both pHs. Moreover, the effect of temperature on K_i value is more at pH 6.8 for both cresolase and catecholase activities. The type of binding process is different in the two types of MT activities. The binding process for catecholase inhibition is only entropy driven, which means that the predominant interaction in the active site of the enzyme is hydrophobic, meanwhile the electrostatic interaction can be important for cresolase inhibition due to the enthalpy driven binding process. Fluorescence and circular studies also show a minor change in the tertiary structure, without any change in the secondary structure, of the enzyme due to the electrostatic interaction in cresolase inhibition by benzenethiol at acidic pH.     

Dual effects of aliphatic carboxylic acids on cresolase and catecholase reactions of mushroom tyrosinase

Catecholase and cresolase activities of mushroom tyrosinase (MT) were studied in presence of some n-alkyl carboxylic acid derivatives. Catecholase activity of MT achieved its optimal activity in presence of 1.0, 1.25, 2.0, 2.2 and 3.2?mM of pyruvic acid, acrylic acid, propanoic acid, 2-oxo-butanoic acid, and 2-oxo-octanoic acid, respectively. Contrarily, the cresolase activity of MT was inhibited by all type of the above acids. Propanoic acid caused an uncompetitive mode of inhibition (K_i=0.14?mM), however, the pyruvic, acrylic, 2-oxo-butanoic and 2-oxo-octanoic acids showed a competitive manner of inhibition with the inhibition constants (K_i) of 0.36, 0.6, 3.6 and 4.5?mM, respectively. So, it seems that, there is a physical difference in the docking of mono- and o-diphenols to the tyrosinase active site. This difference could be an essential determinant for the course of the catalytic cycle. Monophenols are proposed to bind only the oxyform of the tyrosinase. It is likely that the binding of acids occurs through their carboxylate group with one copper ion of the binuclear site. Thus, they could completely block the cresolase reaction, by preventing monophenol binding to the enzyme. From an allosteric point of view, n-alkyl acids may be involved in activation of MT catecholase reactions.     

Potent inhibitory effects of benzyl and p-xylidine-bis dithiocarbamate sodium salts on activities of mushroom tyrosinase

A novel monofunctional benzyldithiocarbamate, C₆H₅CH₂NHCSSNa (I), and a bifunctional p-xylidine-bis(dithiocarbamate), NaSSCNHCH₂C₆H₄CH₂NHCSSNa (II), as sodium salts, were synthesized by reaction between p-xylylenediamine or benzylamine with CS₂ in the presence of NaOH. They were characterized by spectroscopic techniques such as ¹H NMR, IR, and elemental analysis. These water-soluble compounds were examined for their inhibition of both activities of mushroom tyrosinase (MT) from a commercial source of Agricus bisporus. l-3,4- Dihydroxyphenylalanine (L-DOPA) and l-tyrosine were used as natural substrates for the catecholase and cresolase enzyme reactions, respectively. Kinetic studies showed noncompetitive inhibition of I and mixed type inhibition of II on both activities of MT. The inhibition constant (K_I) of II was smaller than that of I. Raising the temperature from 27 to 37°C caused a decrease in K_I values of I and an increase in values of II. The binding process for inhibition of I was only entropy driven, which means that the predominant interaction in the active site of the enzyme is hydrophobic; meanwhile, the electrostatic interaction can be important for the inhibition of II due to the enthalpy driven binding process. Fluorescence studies showed a decrease of emission intensity without a shift of emission maximum in the presence of different concentrations of compounds. An extrinsic fluorescence study did not show any considerable change of the tertiary structure of MT. Probably, the conformation of inhibitor-bound MT is stable and inflexible compared with uninhibited MT.     

The inhibitory effect of benzenethiol on the cresolase and catecholase activities of mushroom tyrosinase

The inhibitory effect of benzenethiol on the cresolase and catecholase activities of mushroom tyrosinase (MT) have been investigated at two temperatures of 20 and 30 degrees C in 10 mM phosphate buffer solution, pHs 5.3 and 6.8. The results show that benzenethiol can inhibit both activities of mushroom tyrosinase competitively. The inhibitory effect of benzenethiol on the cresolase activity is more than the catecholase activity of MT. The inhibition constant (K(i)) value at pH 5.3 is smaller than that at pH 6.8 for both enzyme activities. However, the K(i) value increases in cresolase activity and decreases in catecholase activity due to the increase of temperature from 20 to 30 degrees C at both pHs. Moreover, the effect of temperature on K(i) value is more at pH 6.8 for both cresolase and catecholase activities. The type of binding process is different in the two types of MT activities. The binding process for catecholase inhibition is only entropy driven, which means that the predominant interaction in the active site of the enzyme is hydrophobic, meanwhile the electrostatic interaction can be important for cresolase inhibition due to the enthalpy driven binding process. Fluorescence and circular studies also show a minor change in the tertiary structure, without any change in the secondary structure, of the enzyme due to the electrostatic interaction in cresolase inhibition by benzenethiol at acidic pH.     

The inhibitory effect of ethylenediamine on mushroom tyrosinase

The inhibitory effect of ethylenediamine on both activities of mushroom tyrosinase (MT) at 20 °C in a 10 mM phosphate buffer solution (pH 6.8), was studied. L-DOPA and L-tyrosine were used as substrates of catecholase and cresolase activities, respectively. The results showed that ethylenediamine competitively inhibits both activities of the enzyme with inhibition constants (K(i)) of 0.18±0.05 and 0.14±0.01 μM for catecholase and cresolase respectively, which are lower than the reported values for other MT inhibitors. For further insight a docking study between tyrosinase and ethylenediamine was performed. The docking simulation showed that ethylenediamine binds in the active site of the enzyme near the Cu atoms and makes 3 hydrogen bonds with two histidine residues of active site.     

Carbonic anhydrase inhibitors: inhibition of human and bovine isoenzymes by benzenesulphonamides,cyclitols and phenolic compounds

Carbonic anhydrase inhibitors (CAIs) are a class of pharmaceuticals used as anti-glaucoma agents, diuretics and anti-epileptics. We report here the inhibitory capacities of benzenesulphonamides, cyclitols and phenolic compounds 1–11 against three human CA isozymes (hCA I, hCA II and hCA VI) and bovine skeletal muscle carbonic anhydrase III (bCA III). The four isozymes showed quite diverse inhibition profiles with K_i values ranging from low micromolar to millimolar concentrations against all isoenzymes. Compound 5 and 6 had more powerful inhibitory action against hCA I and very similar action against hCA II and hCA VI as compared with acetazolamide (AZA) and sulphapyridine (SPD), specific CAIs. Probably the inhibition mechanism of the tested compounds is distinct of the sulphonamides with RSO₂NH₂ groups and similar to that of the coumarins/lacosamide, i.e. binding to a distinct part of the active site than that where sulphonamides bind. These data may lead to drug design campaigns of effective CAIs possessing a diverse inhibition mechanism compared to other sulphonamide/sulphamate inhibitors.     

Synthesis and inhibitory properties of some carbamates on carbonic anhydrase and acetylcholine esterase

A series of carbamate derivatives were synthesized and their carbonic anhydrase I and II isoenzymes and acetylcholinesterase enzyme (AChE) inhibitory effects were investigated. All carbamates were synthesized from the corresponding carboxylic acids via the Curtius reactions of the acids with diphenyl phosphoryl azide followed by addition of benzyl alcohol. The carbamates were determined to be very good inhibitors against for AChE and hCA I, and II isoenzymes. AChE inhibition was determined in the range 0.209–0.291?nM. On the other hand, tacrine, which is used in the treatment of Alzheimer’s disease possessed lower inhibition effect (K_i: 0.398?nM). Also, hCA I and II isoenzymes were effectively inhibited by the carbamates, with inhibition constants (K_i) in the range of 4.49–5.61?nM for hCA I, and 4.94–7.66?nM for hCA II, respectively. Acetazolamide, which was clinically used carbonic anhydrase (CA) inhibitor demonstrated K_i values of 281.33?nM for hCA I and 9.07?nM for hCA II. The results clearly showed that AChE and both CA isoenzymes were effectively inhibited by carbamates at the low nanomolar levels.     

Inhibition Profiles of Some Symmetric Sulfamides Derived from Phenethylamines on Human Carbonic Anhydrase I,and II Isoenzymes

In this work, the inhibitory effect of some symmetric sulfamides derived from phenethylamines were determined against human carbonic anhydrase (hCA) I, and II isoenzymes, and compared with standard compound acetazolamide. IC₅₀ values were obtained from the Enzyme activity (%)-[Symmetric sulfamides] graphs. Also, K_i values were calculated from the Lineweaver-Burk graphs. Some symmetric sulfamides compounds ( 11 – 18 ) demonstrated excellent inhibition effects against hCA I, and II isoenzymes. These compounds demonstrated effective inhibitory profiles with IC₅₀ values in ranging from 21.66–28.88 nM against hCA I, 14.44–30.13 nM against hCA II. Among these compounds, the best K_i value for hCA I (K_i: 8.34±1.60 nM) and hCA II (K_i: 16.40±1.00 nM) is compound number 11 . Besides, the IC₅₀ value of acetazolamide used as a standard was determined as hCA I, hCA II 57.75 nM, 49.50 nM, respectively. Moreover, in silico ADME-Tox study showed that all synthesized compounds ( 11 – 18 ) had good oral bioavailability in light of Jorgensen's rule of three, and of Lipinski's rule of five.     

Substrate share in the suicide inactivation of mushroom tyrosinase

To address the real cause of the suicide inactivation of mushroom tyrosinase (MT), under in vitro conditions, cresolase and catecholase reactions of this enzyme were investigated in the presence of three different pairs of substrates, which had been selected for their structural specifications. It was showed that the cresolase activity is more vulnerable to the inactivation. Acetylation of the free tyrosyl residues of MT did not cure susceptibility of the cresolase activity, but clearly decreased the inactivation rate of MT in the presence of 4-[(4-methylbenzo)azo]-1,2-benzenediol (MeBACat) as a catecholase substrate. Considering the results of the previous works and this research, some different possible reasons for the suicide inactivation of MT have been discussed. Accordingly, it was proposed that the interruption in the conformational changes in the tertiary and quaternary structures of MT, triggered by the substrate then mediated by the solvent molecules, might be the real reason for the suicide inactivation of the enzyme. However, minor causes like the toxic effect of the ortho-quinones on the protein body of the enzyme or the oxidation of some free tyrosyl residues on the surface of the enzyme by itself, which could boost the inactivation rate, should not be ignored.     

Inhibition of the catecholase and cresolase activity of mushroom tyrosinase by azide

The inhibition of mushroom tyrosinase by azide is examined as a function of the concentrations of l-tyrosine, l-3,4-dihydroxyphenylalanine (l-Dopa), and oxygen at pH 5.6 and 7.0. Mixed inhibition is observed with respect to l-tyrosine, l-Dopa, and oxygen. The data are interpreted in terms of azide combining with both the oxidized and reduced forms of the enzyme. A scheme is presented for the catecholase and cresolase reactions which explains the results of azide inhibition and also the effect of other inhibitors which complex with the copper of tyrosinase. Double-reciprocal plots of oxygen variation with l-tyrosine as the fixed substrate are nonlinear above about 500 μm oxygen. When l-Dopa is the fixed substrate, no curvature is observed. These results could be explained in terms of negative cooperativity or the presence of two kinetically distinct enzyme forms having different K_m values for oxygen. Although the kinetic data do not permit a choice between the two possibilities, the occurrence in all tyrosinase preparations of two forms, resting, bicupric enzyme and “intrinsic oxytyrosinase,” lends support to the latter suggestion.     

1H‐indazole molecules reduced the activity of human erythrocytes carbonic anhydrase I and II isoenzymes

Carbonic anhydrase (CA) is an important metabolic enzyme family closely related to many physiological and pathological processes. Currently, carbonic anhydrase inhibitors are the target molecules in the treatment and diagnosis of many diseases. In present study, we investigated the inhibitory effects of some indazole molecules on the CA‐I and CA‐II isoenzymes isolated from human erythrocytes. We showed that human CA‐I and CA‐II activities were reduced by of some indazoles at low concentrations. IC₅₀ values, K_i constants, and inhibition types for each indazole molecule were determined. The indazoles showed K_i constants in a range of 0.383 ± 0.021 to 2.317 ± 0.644 mM, 0.409 ± 0.083 to 3.030 ± 0.711 mM against CA‐I and CA‐II, respectively. Each indazole molecule exhibited a noncompetitive inhibition effect. Bromine‐ and chlorine‐bonded indazoles were found to be more potent inhibitory effects on carbonic anhydrase isoenzymes. In conclusion, we conclude that these results may be useful in the synthesis of carbonic anhydrase inhibitors.     

(3,4-Dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone and its derivatives as carbonic anhydrase isoenzymes inhibitors

In this study, we have synthesised (3,4-dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone and a series of its derivatives (5, 13–16) and tested the ability of these compounds to inhibit two metalloenzyme human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, hCA I and hCA II. The synthesised compounds showed inhibitory effect on hCA I and hCA II isozymes. The results showed that synthesised compounds (5, 13–16) demonstrated the best inhibition activity against hCA I (IC₅₀: 3.22–54.28 μM) and hCA II (IC₅₀: 18.52–142.01 μM). The compound 14 showed the highest inhibiton effect against hCA I (IC₅₀: 3.22 μM; K_i: 1.19?±?1.4 μM). On the other hand, the compound 13 showed the highest inhibiton effect against hCA II (IC₅₀: 18.52 μM; K_i: 3.25?±?1.13 μM).     

Novel amides of 1,1‐bis‐(carboxymethylthio)‐1‐arylethanes: Synthesis,characterization, acetylcholinesterase,butyrylcholinesterase, and carbonic anhydrase inhibitory properties

The thiolation reaction was carried out in a benzene solution at 80°C and p‐substituted ketones and mercaptoacetic acid in a molar ratio (1:4) of in the presence of a catalytic amount of toluene sulfonic acids. The enzyme inhibition activities of the novel amides of 1,1‐bis‐(carboxymethylthio)‐1‐arylethanes derivatives were investigated. These novel amides of 1,1‐bis‐(carboxymethylthio)‐1‐arylethanes derivatives showed good inhibitory action against acetylcholinesterase (AChE) butyrylcholinesterase (BChE), and human carbonic anhydrase I and II isoforms (hCA I and II). AChE inhibitors, interacting with the enzyme as their primary target, are applied as relevant drugs and toxins. Many clinically established drugs are carbonic anhydrase inhibitors, and it is highly anticipated that many more will eventually find their way into the market. The novel synthesized compounds inhibited AChE and BChE with K_i values in the range of 0.64–1.47 nM and 9.11–48.12 nM, respectively. On the other hand, hCA I and II were effectively inhibited by these compounds, with K_i values between 63.27–132.34 and of 29.63–127.31 nM, respectively.     

Differential effect of phenylhydrazine on the catecholase and cresolase activities of Vitis catechol oxidase

The simultaneous addition of phenylhydrazine and p-cresol to grape catechol oxidase resulted in enhanced oxidation of p-cresol. Carbonyl reagents such as hydrazine, borohydride and semicarbazide also enhanced cresolase activity but had no effect on catecholase activity. Pretreatment of the enzyme with periodate abolished cresolase activity. The effects of periodate and ascorbate or semicarbazide on cresolase activity were mutually reversible. The simultaneous addition of phenylhydrazine and 4-methylcatechol to the enzyme did not result in inhibition of the initial rate of oxidation of the phenolic substrate. It is concluded that phenylhydrazine does not react with a carbonyl group on the enzyme. The possible involvement of conformational changes in the enzyme, determining phenylhydrazine inhibition is discussed.     

Spirobisnaphthalenes effectively inhibit carbonic anhydrase

This study explores the correlation between human carbonic anhydrase (CA, EC 4.2.1.1) isoforms I and II (hCA I, II) and the inhibitory features of some spirobisnaphthalene derivatives. A group of spirobisnaphthalenes was synthesized and their hCA I and II inhibitory effects was investigated. The K_i values were similar for both CA isoenzymes, the compounds showing good inhibitory activity. K_i values ranged between 1.60 and 460.42?µM for hCA I and between 0.39 and 419.42?µM for hCA II, respectively. The spirobisnaphthalenes derivatives might be useful for designing CA inhibitors belonging to novel chemotypes compared to the highly investigated sulfonamides, sulfamates or coumarins.     

Inhibition of mushroom tyrosinase by a newly synthesized ligand: inhibition kinetics and computational simulations

Alterations in the synthesis of melanin contribute to a number of diseases; therefore, the design of new tyrosinase inhibitors is very important. Mushroom tyrosinase (MT) is a metalloenzyme, which plays an important role in melanin biosynthesis. In this study, the inhibitory effect of a novel designed compound, i.e. 2-((1Z)-(2-(2,4-dinitrophenyl)hydrazin-1-ylidene)methyl)phenol, as a specific ligand which can bind to the copper ion of MT, has been assessed. The ligand was found to competitively inhibit both the cresolase and catecholase activities of MT, with small inhibition constants of 2.8 and 2.6?μM, respectively. Intrinsic fluorescence studies were performed to gain more information on the binding constants. Docking results indicated that the ligand binds to copper ions in the active site of MT via the OH group of the ligand. The ligand makes four hydrogen bonds with aspartic acid and one hydrogen bond with the histidine residue in the active site. Molecular dynamics results show that ligand binds to the MT via both electrostatic and hydrophobic interactions with its different parts.