Accurate quantitative determination of affinity and binding kinetics for tight binding inhibition of xanthine oxidase

The accurate quantitative determination of affinity and binding kinetics (BK) for tight binding inhibition is extraordinary important from both the continuous optimization of compounds, particularly in developing structure-activity relationships (SAR), and the prediction of in vivo target occupancy (TO). Due to the unique properties for tight binding inhibition that the inhibitors are characterized by the ultrahigh-affinity, relatively fast association to the target enzyme combined with extremely slow dissociation of the inhibitor-enzyme binary complex, the classical steady state equilibrium methods are no longer valid. Here, we made several recommendations of how to design the optimal experiments and apply special mathematical calculation approaches to quantitatively evaluate the accurate affinity and BK as the examples of two tight binding inhibitors against the xanthine oxidase (XO), as well as compared the differences in the results calculated from the different data analytical methods and analyzed the influence of these differences on the XO engagement in human. Analysis of the results displayed that the accurate apparent dissociation constant (K_i^*,app) was 0.2 ± 0.06 nM for topiroxotstat and was 0.45 ± 0.2 nM for febuxostat; that on-rate (k_on) was (4.3 ± 1.1) × 10⁶ M^-1s^-1 for topiroxotstat and was(133.3 ± 3.5) × 10⁶ M^-1s^-1 for febuxostat, and off-rate (k_off) was (1.0±0.2) × 10^-5 s^-1 for topiroxotstat and was ≤ 0.16 × 10^-5 s^-1for febuxostat. Moreover, there were significant differences in the K_i^*,app and k_off values estimated using the appropriate specialized methods for tight binding inhibition versus classical steady state equilibrium methods, with the substantial differences of 14-fold and 32-fold reduction for topiroxostat, respectively, and of 9.6-fold and ≥ 213-fold reduction for febuxostat, while the k_on values remain the moderate differences for the two inhibitors. The obvious greater AUC of XO engagement time courses and longer durations of above 70% engagement by the appropriate specialized methods for tight binding inhibition were observed that the results display the differences of 70.1% and 88%, respectively for topiroxostat and of 38.1% and 35.0%, respectively for febuxostat in human liver cell than by classical steady state equilibrium methods. Again, our studies provide several valuable recommendations of the optimal experiment protocols and appropriate analytical approaches for accurately quantitatively assessing the affinity and BK parameters as well as demonstrate the ability of our recommended methods to generate reliable data for tight binding inhibitors against XO.