Time Course of Aldehyde Oxidase and Why It Is Nonlinear

Abstract

Many promising drug candidates metabolized by aldehyde oxidase (AOX) fail during clinical trial owing to underestimation of their clearance. AOX is species-specific, which makes traditional allometric studies a poor choice for estimating human clearance. Other studies have suggested using half-life calculated by measuring substrate depletion to measure clearance. In this study, we proposed using numerical fitting to enzymatic pathways other than Michaelis-Menten (MM) to avoid missing the initial high turnover rate of product formation. Here, product formation over a 240-minute time course of six AOX substrates-O⁶-benzylguanine, N-(2-dimethylamino)ethyl)acridine-4-carboxamide, zaleplon, phthalazine, BIBX1382 [N8-(3-Chloro-4-fluorophenyl)-N2-(1-methyl-4-piperidinyl)-pyrimido[5,4-d]pyrimidine-2,8-diamine dihydrochloride], and zoniporide-have been provided to illustrate enzyme deactivation over time to help better understand why MM kinetics sometimes leads to underestimation of rate constants. Based on the data provided in this article, the total velocity for substrates becomes slower than the initial velocity by 3.1-, 6.5-, 2.9-, 32.2-, 2.7-, and 0.2-fold, respectively, in human expressed purified enzyme, whereas the K _m remains constant. Also, our studies on the role of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, show that ROS did not significantly alter the change in enzyme activity over time. Providing a new electron acceptor, 5-nitroquinoline, did, however, alter the change in rate over time for mumerous compounds. The data also illustrate the difficulties in using substrate disappearance to estimate intrinsic clearance.

Fig. 1.

Summary of AOX-catalyzed reactions for the substrates used in this study.

Fig. 2.

Five times the K_m amount of the substrates: (A) O6BG, (B) DACA, (C) zaleplon, (D) phthalazine (E) BIBX1382, and (F) zoniporide were incubated with HLC (0.0051 μM AO) at 37°C, and samples were quenched at different time points over 240 minutes (n = 3, P < 0.001). Data fitting was performed using the MAM. Based on the results, unlike the MM assumption, enzyme activity is not linear over time.

Fig. 3.

Five times the K_m amount of the substrates: (A) O6BG, (B) DACA, (C) zaleplon, (D) phthalazine, (E) BIBX1382, and (F) zoniporide were incubated with purified expressed human AOX (HAO, 0.0192 μM) in the same way as HLC to compare the time-course plots and ensure that AOX is the enzyme responsible for metabolism. Fitting to product formation was performed using the MAM (n = 3, P < 0.001).

Fig. 4.

Two different kinetic models were developed to compare the numerical fitting of time-course data with MM. (A) MM model, (B) dead enzyme model, (C) MAM. Akaike values were provided by Mathematica to compare the goodness of fit, proving that MAM is the best model to fit the time-course data for most substrates except for zoniporide. Data- fitting plots for O6BG are provided next to each kinetic scheme for comparison. The Akaike values for O6BG are provided on the bottom right side of each plot.

Fig. 5.

Catalase and SOD (250 U/ml) were used to remove ROS that were suspected to affect the enzymatic activity. HLC (0.0051 M AO) was used to start the reactions with A) O6BG, B) DACA, C) zaleplon, D) phthalazine E) BIBX1382, and F) zoniporide, (n = 3, P < 0.001). No significant change in linearity of the enzyme was observed once these reagents were used, suggesting that ROS are not the reason behind the decrease in enzymatic efficacy.

Fig. 6.

Catalase and SOD (250 U/ml) were used to remove ROS suspected to affect the enzymatic activity for purified HAO incubated with A) O6BG, B) DACA, C) zaleplon, D) phthalazine E) BIBX1382, and F) zoniporide. No significant change in linearity of the enzyme was observed once these reagents were used, suggesting that ROS are not the reason for the decrease in enzymatic efficacy.

Fig. 7.

Substrate consumption simulation for (A) O6BG, (B) DACA, (C) zaleplon, and (D) phthalazine were done using the kinetic parameters obtained from fitting the product formation data in purified expressed human AOX (HAO) to the MAM using Mathematica. This could not be done for the substrates for which we do not have the product standards.

Fig. 8.

Five times the Km amount of the substrates including A) O6BG, B) DACA, C) zaleplon, D) phthalazine E) BIBX1382, and F) zoniporide were used to start the reaction (n = 3, P < 0.001).