Binding Kinetics And Target Occupancy Of Flavonoids To Xanthine Oxidase

Objective:Xanthine oxidase,catalyzing the xanthine to uric acid,is an important target for the treatment of gout and hyperuricemia.Topiroxostat and febuxostat are tightly binding inhibitors targeting it and have very strong affinity.For tightly binding inhibitors,almost all of the inhibitors are bound to enzyme molecules during enzymatic reactions,and the free inhibitor concentration in the system is extremely small,which is inconsistent with the assumption of classical steady-state kinetics.Therefore,there is a risk of making mistakes,if the classical method is used to determine its affinity and inhibition modality.In addition,studies in recent years have found that the binding kinetic process of drug and target is correlated with the efficacy of the drug in vivo.Therefore,this study compared the results of two methods determining the inhibition modality and affinity of the tight-binding inhibitors;measured the binding kinetic parameters;predicted the drug-time curve of free drugs in liver tissue;established and verified the target occupancy fraction model.And this study can provide correct methods for the accurate determination of affinity and inhibition modality of tight-binding inhibitors and provide new ideas and methods for evaluating the activity of xanthine oxidase inhibitors.Methods:Established an in vitro enzymatic reaction system;Classical steady-state method and Morrison quadratic equation were applied to determine affinity of tight binding inhibitors;Graphical method of IC50 versus substrate concentration and Lineweaver-Burk plots were utilized to measure inhibition modality of tight binding inhibitors;Reaction progress curves were conducted and were analyzed to determine binding mechanisms and binding rate constants(kon);Experiment of activity recovered of inhibitor-enzyme complex was used to determine the dissociation rate constant(koff);The surface plasmon resonance technology(SPR)was applied to measure binding kinetic parameters of flavonoids components;PK parameters and Rodgers mechanism equation were used to predict free drug concentration-time curve in the liver,and combined it with the kinetic parameters to establish in vivo target occupancy fraction model;verifed model with clinical efficacy,dose and dosing interval of positive drug;Used estabished model to predict in vivo target occupancy fraction after administration of flavonoids,and verifed prediction result by measuring the activity of xanthine oxidase target in the liver at different times after administration.Results:(1)With the uric acid concentration in the range of 2.5?M-150?M,the absorbance and uric acid concentration showed a good linear relationship,R2=0.9997.The key factors affecting velocity rate of reaction,substrate concentration and enzyme concentration were investigated.As a result,the final concentration of enzyme in the reaction system was selected to be 4nM,and the Km value of the substrate xanthine was measured to be 17?M.(2)With the increase of substrate concentration,IC50 of topiroxostat increased in a linear manner which is conformed to the competitive inhibiton model,while IC50 of febuxostat varied in an irregular manner,which is consistent with the noncompetitive inhibiton model.The use of Lineweaver-Burk plot can be very misleading when determine the inhibition modalities of tightly binding inhibitors.(3)In contrast to the values obtained by the classical steady-state method,the Kiapp value estimated by the Morrison quadratic equation for topiroxostat was only slightly difference,just with a 0.6-fold lower value,nevertheless for febuxostat was significantly difference,with a 10-fold lower value.Additionally,the Ki*,app obtained from two methods showed sharp differences.The values calculated by Morrison quadratic equation were comparably smaller with a 14-fold decrease for topiroxostat and with an about 10-fold reduction for febuxostat,respectively.(4)The experiment results of reaction progress curve and activity recovery of the inhibitor-enzyme complex showed that the binding of topiroxostat to enzyme were conformed to mechanism B and the kon value and koff value of topiroxostat were(4.3±1.1)×106 M-1s-1 and(1.0±0.2)×10-5 s-1,respectively.For febuxostat,the binding mechanism is mechanism B,the kon value and koff value were(133.3±3.5)×106M-1s-1 and<0.6×10-5s-1,respectively.The surface plasmon resonance experiment displayed that the kon values of quercetin,galangin and baicalein were 2.669×105M-1s-1,4.250×105M-1s-1,6.027×105M-1s-1,respectively;koff values were 0.054s-1,0.076s-1,and 0.057s-1,respectively.(5)The target occupancy process of xanthine oxidase in vivo predicted with model was basically consistent with its actual clinical efficacy and dosing interval(once a day).The results of animal experiments showed that measured occupancy fraction of target in vivo after administration of quercetin and baicalein was basically consistent with predicted curve,and measured occupancy fraction after administration of galangin showed a slower downtrend compared with predicted result.The maximum target occupancy fraction and duration of target occupancy more than 60% were close to predicted value,but the peak time was quite different.Conclusion:This study used two methods for determining the inhibition modality and affinity of tight-binding inhibitor;established and verified target occupancy fraction model.And this study can provide a correct method for accurate determination of affinity and inhibition modality of tight-binding inhibitors and provide new idea and method for evaluating the activity of xanthine oxidase inhibitors.