Department of Chemistry, Washington State University, Pullman, Washington.
Department of Chemistry, Washington State University, Pullman, Washington
Drug Metab Dispos. 2019 May;47(5):473-483. doi: 10.1124/dmd.118.085787. Epub 2019 Feb 20.
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-O-benzylguanine, N-(2-dimethylamino)ethyl)acridine-4-carboxamide, zaleplon, phthalazine, BIBX1382 [8-(3-Chloro-4-fluorophenyl)-2-(1-methyl-4-piperidinyl)-pyrimido[5,4-]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 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.
许多被醛氧化酶（AOX）代谢的有前途的药物候选物在临床试验中失败，原因是低估了它们的清除率。AOX 具有物种特异性，这使得传统的比例研究成为估算人体清除率的不佳选择。其他研究表明，可以使用通过测量底物耗用来计算半衰期来测量清除率。在这项研究中，我们提出使用除米氏-门坦（MM）之外的酶途径的数值拟合来避免错过产物形成的初始高周转率。在这里，我们使用了六个 AOX 底物-O-苄基鸟嘌呤、N-(2-二甲基氨基)乙基)吖啶-4-羧酰胺、扎来普隆、邻苯二甲嗪、BIBX1382[8-(3-氯-4-氟苯基)-2-(1-甲基-4-哌啶基)嘧啶并[5,4-]嘧啶-2,8-二胺二盐酸盐]和 zoniporide 的 240 分钟时间过程中的产物形成来举例说明随时间的酶失活，以帮助更好地理解为什么 MM 动力学有时会导致低估速率常数。基于本文提供的数据，在人表达纯化酶中，底物的总速度分别比初始速度慢 3.1 倍、6.5 倍、2.9 倍、32.2 倍、2.7 倍和 0.2 倍，而保持不变。此外，我们对活性氧（ROS）（如超氧阴离子和过氧化氢）的作用的研究表明，ROS 并没有显著改变酶活性随时间的变化。然而，提供新的电子受体 5-硝基喹啉确实改变了许多化合物随时间变化的速率。该数据还说明了使用底物消失来估计内在清除率的困难。
