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-, 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.

Site-Directed Mutagenesis at the Molybdenum Pterin Cofactor Site of the Human Aldehyde Oxidase: Interrogating the Kinetic Differences Between Human and Cynomolgus Monkey

“…The goodness of fit was evaluated by AIC and RSquared commands. The models used were the Modified Activity Model (MAM) and the linear (Michaelis-Menten) model as described previously (Abbasi et al, 2019).…”

“…Traditional kinetic methods rely on several assumptions such as the steady state hypothesis that limits and occasionally misdirects our understanding of the kinetic data. Based on our previous studies on the human aldehyde oxidase (hAO), we have found that this enzyme shows a nonlinear behavior and should be assessed numerically to take the whole kinetic process into account instead of truncating the data to meet the Michaelis-Menten assumptions (Abbasi et al, 2019). Here, we use the same numerical approach to investigate the basis of interspecies kinetic differences between human and cynomolgus monkey.…”

“…We chose dantrolene as the reducing substrate in this study based on the previous reports of this compound being metabolized by AO even under normal oxygen concentration as well as its lack of competition with oxidative substrates and having a high turn-over rate among other structurally similar reductive substrates of AO (Amano et al, 2018;Ogiso et al, 2018). Furthermore, dantrolene has a similar K m to oxygen with the K m of oxygen for hAO being estimated to be close to 2 M (Abbasi et al, 2019) and the Km of dantrolene being 5 M (data not shown).…”

“…AO has proven to be a challenging drug metabolizing enzyme for clearance estimation in humans. Not only is AO both substrate and species-specific, it also exhibits nonlinear atypical kinetics (Dalvie et al, 2010;Barr et al, 2013a;Choughule et al, 2015;Abbasi et al, 2019).…”

Predicting liver cytosol stability of small molecules

“…Several well-known structure modification strategies exist to guide the medicinal chemist through CYP-mediated instabilities [5,6]. However, strategies to attenuate non-CYP-mediated liabilities are sparsely reported in the literature [7][8][9][10]. A few in-house projects at the National Center for Advancing Translational Sciences (NCATS) were recently discovered to have cytosol-mediated metabolic liabilities.…”