Tyrosinase Inhibitory Activity , 3 D QSAR , andMolecular Docking Study of 2 , 5-Disubstituted-1 , 3 , 4-Oxadiazoles

In continuationwith our research program, in search of potent enzyme tyrosinase inhibitor, a series of synthesized 2,5-disubstituted 1,3,4-oxadiazoles have been evaluated for enzyme tyrosinase inhibitory activity. Subsequently, 3D QSAR and docking studies were performed to �nd optimum structural requirements for potent enzyme tyrosinase inhibitor from this series. e synthesized 20 compounds of 2,5-disubstituted-1,3,4-oxadiazole series were screened for mushroom tyrosinase inhibitory activity at various concentrations by enzyme inhibition assay. e percentage enzyme inhibition was calculated by recording absorbance at 492 nm with microplate reader. 3D QSAR and docking studies were performed using VLife MDS 3.5 soware. In the series 2,5disubstituted-1,3,4-oxadiazoles enzyme tyrosinase inhibitory activity was found to be dose dependent with maximum activity for compounds 4c, 4h, 4m, and 4r. 3D QSAR and docking studies revealed that more electropositive and less bulky substituents if placed on 1,3,4-oxadiazole nucleus may result in better tyrosinase inhibitory activity in the series.


Introduction
Tyrosinase (E.C. 1.14.18.1), known as polyphenol oxidase (PPO), is a multifunctional, glycosylated, and coppercontaining enzyme from the oxidase superfamily widely distributed in microorganisms, plants, and animals [1].It is a nonessential amino acid made by the body that is a building block for several important neurotransmitters like epinephrine, norepinephrine, serotonin, and dopamine.It plays a key role in melanin biosynthesis catalyzing two divergent reactions: the hydroxylation of monophenols (cresolase or monophenolase activity) and the oxidation of o-diphenols (catecholase or diphenolase activity) into reactive o-quinones [2][3][4].It is involved in human neuromelanin formation in the substantia nigra of the brain and dopamine neurotoxicity and contributes to Parkinson's disease-related neurodegeneration.In insects, tyrosinase is uniquely associated with different physiologically important biochemical processes, including sclerotization of the insect cuticle, defensive encapsulation and melanization of foreign organisms, and wound healing [5][6][7][8][9][10].
Tyrosinase inhibitors have become increasingly important in the �eld of medicine, agriculture, and cosmetics [11].Presently, tyrosinase inhibitors have been a great concern due to its role in both mammalian melanogenesis and fruit or fungi enzymatic browning.Although melanin has photoprotective function in human skin, the accumulation of an abnormal amount of melanin in different speci�c parts of the skin resulting in more pigmented patches (melasma, freckles, ephelides, senile lentigines, etc.) might become an esthetic problem [12,13].In addition, enzymatic browning in fruit and fungi is undesirable, for example, fresh fruits, beverages, vegetables, and mushrooms which decrease the commercial value of the products [14].ese phenomena prompted us to design novel tyrosinase inhibitors with higher bioactivities that could be useful in skin whitening and antibrowning of foods.
In continuation with our earlier work, here we report enzyme tyrosinase inhibitory activity, three-dimensional quantitative structure activity relationship (3D QSAR) analysis, and molecular docking study with enzyme tyrosinase of series of 2,5-disubstituted-1,3,4-oxadiazoles synthesized and reported by us [15].

Tyrosinase Inhibitory Activity.
As an extension of our work, synthesized 20 analogues of 2,5-disubstituted-1,3,4oxadiazole series (Table 1) were used to study their enzyme tyrosinase inhibitory activity.Tyrosinase inhibition activity was determined by the method described earlier [16].e title compounds were dissolved in DMSO.e 96 well plate was prepared by applying 140 L of phosphate buffer (pH 6.8), 20 L of mushroom tyrosinase (48 units/mL), 20 L of sample, and 20 L of DL-DOPA (0.85 mM) to make the concentrations of 10, 20, 40, 80, and 100 g/mL.Aer incubation for 10 minutes, the enzyme activity was determined by measuring the absorbance at 492 nm using the microplate reader (Biotek Company, US).Kojic acid (1 mg/mL) was used as positive control.e percentage of tyrosinase inhibition was calculated as follows: where,  and  are absorbance at 492 nm with and without test sample, respectively.e percentage tyrosinase inhibition at various concentrations of title compounds was calculated (Table 2), and logarithmic value of percentage inhibition at 100 g (Log % 100g ) was used as a dependant variable for the development of valid 3D-QSAR models.

Computational Details
2.2.1.Geometry Optimization.e 3D QSAR studies of 2,5-disubstituted-1,3,4-oxadiazole derivatives were carried out using VLife molecular design suite soware version 3.5 (MDS 3.5) running on Pentium IV processor.reedimensional structures of title compounds were constructed and optimized their geometries to make the conformations with least potential energy using merck molecular force �eld (MMFF) and MMFF charge for the atom [17] followed by considering distance-dependent dielectric constant of 1.0 and convergence criteria (rms gradient) of 0.01 kcal/mol.

Alignment of Molecules.
All molecules in the data set are aligned by template-based method where a template is built by considering common substructures in the series (Figure 1).Highly bioactive energetically stable conformation in the series is chosen as a reference molecule (Figure 2) on which other molecules are aligned (Figure 3).

Activity Prediction. e predictability of the QSAR model would be good if the values of biological activity
predicted by the QSAR model do not appreciably differ from the observed results of biological activity for the given data set.Quality of selected models was further ascertained by  2 ,  2 , and  test.e models were cross-validated by "leave one out" scheme and cross-validation corelation coefficient 2.2.4.3D QSAR.Several 3D QSAR techniques such as comparative molecular �eld analysis (COMFA), comparative molecular similarity analysis (COMSIA), and k-nearest neighbor (kNN) [18] are being used in modern QSAR research.In the present study, molecular �eld analysis coupled with partial least squares (PLS) was applied to obtain a 3D QSAR model [19].e calculated steric and electrostatic �eld descriptors were used as independent variables and log % 100g values were used as dependent variables to derive the 3D QSAR models.2.2.5.Docking.e crystal structure of mushroom tyrosinase (1wx2) was obtained from protein data bank and water molecules in the crystal structure were deleted.e optimized receptor was then saved as mol �le and used for docking simulation.
2.2.6.Ligand Preparation.e 2D structures of the compounds were built and then converted into the 3D.e 3D structures were then energetically minimized up to the rms gradient of 0.01 using MMFF.

2.2.�. �denti�cation o� �a�itie�.
By using cavity determination option of soware, cavities of enzyme were determined.e cavities in the receptor were mapped to assign an appropriate active site.e basic feature used to map the cavities were the surface mapping of the receptor and identifying the geometric voids as well as scaling the void for its hydrophobic characteristics.Hence, all the cavities that are present in receptor are identi�ed and ranked based on their size and hydrophobic surface area.Considering the dimensions and hydrophobic surface area, cavity-1 with    shows maximum where compounds 4b, 4f, 4g, and 4k shows minimum inhibition of enzyme tyrosinase from the series.

3D QSAR.
e model 1 describes the optimum structural feature for the tyrosinase inhibition activity as shown in Table 3. e training set of 16 molecules and test set of 4 molecules were used as described earlier.S_793, S_757, E_550 are the steric and electrostatic �eld energy of interactions between probe (CH 3 ) and compounds at their , and GLY198 amino acid residues as shown in Figure 6.e tyrosinase inhibitory assay and molecular docking study reveals that 2,5-disubstituted-1,3,4-oxadiazoles inhibits enzyme tyrosinase may be because of its interaction with SER142, SER146, GLY145, and GLY198 amino acid residues.

Conclusion
It can be concluded from the whole study that electropositivity is crucial for the inhibition of enzyme tyrosinase by 2,5-disubstituted-1,3,4-oxadiazoles.It would be worthwhile to synthesize a novel 1,3,4-oxadiazole analogues with less bulky and more electropositivity group as a better enzyme tyrosinase inhibitors.

3
and surface area 340.284790Å 2 was found to be the best void as an active site.2.2.8.Scoring Function.Distinction of good or bad dockedconformation is based on scoring or �tness function.MDS uses �tness functions on only electrostatic and both steric and electrostatic interactions between receptor ligand as well as dock score scoring function.e dock score compute binding affinity of a given protein-ligand complex with known 3D structure.

F 6 :
Interaction of compound 4r with mushroom tyrosinase.
T 2: Tyrosinase inhibition activity of title compounds.
T 4: Selected descriptors, actual and predicted activity of tyrosinase inhibitors.Docking studies of the title compounds with enzyme tyrosinase yielded docking score ranging from −5.8169 to −4.7232 (Table6) indicating stable enzyme-substrate interactions.e compound 4r with best docking score (−5.8169) has shown highest inhibition of enzyme tyrosinase in the dataset.e compounds interact with enzyme tyrosinase by binding with SER142, SER146, GLY145 T 6: Grid docking score of title compounds.