Carvacrol derivatives as mushroom tyrosinase inhibitors; synthesis, kinetics mechanism and molecular docking studies

Abstract

The present work describesthe development of highly potent mushroom tyrosinase inhibitor better than the standard kojic acid. Carvacrol derivatives 4a-f and 6a-d having substituted benzoic acid and cinnamic acidresidues were synthesized with the aim to possess potent tyrosinase inhibitory activity.The structures of the synthesized compounds were ascertained by their spectroscopic data (FTIR, 1HNMR, 13CNMR and Mass Spectroscopy).Mushroom tyrosinase inhibitory activity of synthesized compounds was determined and it was found that one of the derivative 6c possess higher activity (IC50 0.0167μM) than standard kojic acid (IC50 16.69μM). The derivatives 4c and 6b also showed good tyrosinase inhibitory activity with (IC50 16.69μM) and (IC50 16.69μM) respectively.Lineweaver-Burk and Dixon plots were used for the determination of kinetic mechanism of the compounds 4c and 6b and 6c. The kinetic analysis revealed that compounds 4c and 6b showed mixed-type inhibition while 6c is a non-competitive inhibitor having Ki values19 μM, 10 μM, and 0.05 μMrespectively. The enzyme inhibitory kinetics further showed thatcompounds 6b and 6c formed irreversible enzyme inhibitor complex while 4c bind reversibly with mushroom tyrosinase.The docking studies showed that compound 6c have maximum binding affinity against mushroom tyrosinase (PDBID: 2Y9X) with binding energy value (-7.90 kcal/mol) as compared to others.The 2-hydroxy group in compound 6c interacts with amino acid HIS85 which is present in active binding site. The wet lab results are in good agreement with the dry lab findings.Based upon our investigation we may propose that the compound 6c is promising candidate for the development of safe cosmetic agent.

Fig 1

a) Determination of inhibition type of compound (6b) by Lineweaver-Burk plots c,d) the insets represent the plot of the slope & intercept respectively, from Lineweaver-Burk plot versus inhibitor of (6b) with concentrations (0.0, 7.0 and 28 μM). b) Determination of inhibition constant of compound (6b) with concentrations (0.0, 1.75, 7.0, 14.0 and 28 μM) by Dixon plots.The concentration of L-DOPA was 0.125 to 2.00 mM in both cases.

Fig 2. a) Determination of inhibition type of compound (6c) by Lineweaver-Burk plots b) the insets represents the plot of the slope from Lineweaver-Burk plot versus inhibitor of (6c)c) Determination of inhibition constant of compound (6c) by Dixon plots.

The concentrations of compound (6c) (0.0, 0.01, 0.02 and 0.04μM) and the concentrations of L-DOPA were (0.125, 0.25, 0.5, 1.0 and 2.00 mM) used in both cases.

Fig 3. a) Determination of inhibition type of compound (4c)by Lineweaver-Burk plots b) Determination of inhibition constant of compound (4c) by Dixon plots.

The concentrations of compound (4c) (0.0, 1.68, 3.37, 6.75 and 13.5μM) and the concentrations of L-DOPA were (0.25, 0.5, 1.0 and 2.00 mM) used in both cases.

Fig 4. a,b,c) Relationship between the catalytic activity of tyrosinase and various concentrations of compounds (6b), (6c) and (4c) respectively.

Fig 5. Binding interactions of compound (6c) with target protein.

The (6c) is mentioned in green color with embedded oxygen functional groups in red. The target protein is highlighted in line ribbon format with purple color. The active binding site amino acids are highlighted in dark mahroon color around the ligand molecule. Two copper ions are also present in black color.

Fig 6. Binding interactions of compound (4c) with target protein.

The (4c) is mentioned in green color with embedded oxygen functional groups in red. The target protein is highlighted in line ribbon format with purple color. The active binding site amino acids are highlighted in dark mahroon color around the ligand molecule. Two copper ions are also present in black color.

Fig 7. Docking interactions between 6b and target protein.

The 6b is mentioned in green color with embedded oxygen functional groups in red. The target protein is highlighted in line ribbon format with purple color. The active binding site amino acids are highlighted in dark mahroon color around the ligand molecule. Two copper ions are also present in black color.