Mechanism Study On The Interaction Between Tyrosinase And Inhibitor By Molecular Simulation And Spectroscopy

Tyrosinase is a copper-containing oxidoreductase which hydroxylates L-tyrosine and 3,4-dihydroxyphenylalanine into levodopa and dopa quinone to produce melanin in vivo.Excessive accumulation of melanin is associated with various diseases,such as pigmentation,melanoma,and neurodegeneration.Inhibition of tyrosinase activity with inhibitors is expected to improve the symptoms of this disease.In the past,there have been many studies on tyrosinase inhibitors,such as hydroquinone,kojic acid and tranexamic acid,but these compounds have some problems of cytotoxicity or low efficiency.At the same time,with the rapid increase of the number of small molecules in various global databases,the method of discovering new inhibitors by traditional experimental means is obviousl y insufficient.Therefore,it is the current and future research trend to explore and discover new potential potent inhibitors based on computer simulation,which provide guidance to traditional experimental methods.In my paper,tyrosinase and its flavonoids,peptides and peptide derivatives inhibitors were studied through simulation and molecular spectroscopy method.It explained the interaction mechanism of protease and inhibitor,built reliable quantitative structure-activity relationship model and designed efficient screening strategies.It will provide certain theoretical guidance for the development of new tyrosinase inhibitor.The specific research results are as follows:1.Interaction of flavonoid molecules with tyrosinase.First,the combin ation model of four flavonoids molecule and tyrosinase was compared and analyzed by molecular docking,which proves the specific 3-position hydroxyl group of quercetin C ring forms a special metal-acceptor hydrogen bond with the copper ion in the active tyrosinase center.This group is the key reason why the binding energy of quercetin is significantly better than that of the other three molecules.The 3’4’-catechol group on the B ring of isorhamnetin,myricetin and quercetin also enhanced the binding abili ty of ligand and receptor.Secondly,molecular dynamics simulation method was used to show that all the four molecules could stably bind to the Cu²⁺-containing catalytic active region of tyrosinase.Due to the differences in their chemical structures,the binding sites and insertion modes of the four molecules with tyrosinase were significantly different.Finally,by calculating the binding free energy of the four complex systems,the semi-inhibitory concentration and binding energy values of flavonoids were compared and analyzed,which proved that the experimental results were consistent with the molecular simulation results.This study first proposed that the 3-hydroxyl group on the C ring and the 3’4’-catechol group on the B ring of flavonoids can effectively strengthen the binding ability of inhibitors to tyrosinase.It will provide explicit guidance for design and optimization of the subsequent flavonoids lead compounds.2.Comprehensive calculation and analysis of dipeptide containing cysteine.Firstly,the largest common substructure of the dipeptide compound was obtained by the calculation of molecular fingerprint similarity.The differences among the 39dipeptides containing semiluminine were explained from the perspective of molecular topology.And the diversity of amino acid R group was the key factor,because it affected the activity of the dipeptides.Second,cysteine in C and N of the two kinds of quantitative structure-activity relationship model were built respectively,and its half inhibitory concentration predicted values showed a good linear rel ationship with the experiment.Meanwhile,according to the multiple linear regression equation,the inhibitory effect of cysteine-containing dipeptide on tyrosinase was closely related to the three-dimensional structure,polarity and three-dimensional arrangement of the molecule.Finally,MCS algorithm combined with quantitative structure-activity relationship prediction was used to screen in the COCONUT’s natural products database and obtain three candidate inhibitors of natural products.This study is the first time to construct a quantitative structure-activity relationship model based on cysteine ligation sites,which can be used for rapid and automatic screening of tyrosinase inhibitors containing cysteine dipeptide derivatives.3.Spectroscopy and molecular dynamics methods were used to study the mechanism of interaction of natural dipeptide derivatives containing semiluminine and tyrosinase.Firstly,the effects of three natural product inhibitors on tyrosine monophenolase and diphenolase were characterized by enzyme kinetic curves.The experiments showed that all three inhibitors had significant inhibitory effects.Secondly,the Michaelis-Menten equation and Lineweaver-Burk double reciprocal plot were used to infer that the three inhibitors were mixed inhibition types containing competitive and non-competitive mode,and the inhibition constants of the inhibitors on free enzymes and on enzyme-substrate complexes were calculated.Finally,it was concluded that the quenching type of the three inhibitors was static quenching through fluorescence quenching analysis.The thermodynamic data analysis showed that the interaction forces were mainly electrostatic and hydrophobic,and the experimental values were basically consistent with the results of molecular dy namics binding energy calculation.By means of spectroscopy and molecular dynamics calculation,this study elucidates that the inhibition type of cysteine dipeptide derivative inhibitor is a mixed inhibition types containing competitive and non-competitive mode,and the quenching type is a static quenching,and the interaction force is dominated by electrostatic and hydrophobic interaction.4.Multistep parallel screening of inhibitors of tyrosinase compounds.Firstly,ZINC20 database was screened based on the maximum common substructure of molecules.Secondly,the pharmacokinetics,quantitative structure-activity relationship prediction,molecular docking and binding energy calculation methods were used for parallel screening and filtering.Finally,the screening results were verified by molecular dynamics simulation.The results showed that four candidate tyrosinase inhibitors were successfully obtained,and their semi-inhibitory concentrations were all less than 10 n M.In particular,the molecule ZINC000104269856 showed the best performance in the screening results,and this compound ranked first in the predicted value of the half-inhibitory concentration（7 n M）as predicted by the quantitative structure-activity relationship model.The multi-step parallel screening strategy in this study can be widely used in protease inhibitor screening.At the same time,four compounds with a concentration of less than 10 n M were successfully found as highly effective tyrosinase inhibitors.