Lin Hsia-Lien, Zhang Haoming, Walker Vyvyca J, D'Agostino Jaime, Hollenberg Paul F
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan.
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan
Drug Metab Dispos. 2017 Sep;45(9):990-999. doi: 10.1124/dmd.117.075846. Epub 2017 Jul 11.
The mechanism-based inactivation of human CYP2J2 by three terminal acetylenic compounds: -(methylsulfonyl)-6-(2-propargyloxyphenyl)hexanamide (MS), 17-octadecynoic acid (OD), and danazol (DZ) was investigated. The loss of hydroxyebastine (OHEB) carboxylation activity in a reconstituted system was time- and concentration-dependent and required NADPH for MS and OD, but not DZ. The kinetic constants for the mechanism-based inactivation of OHEB carboxylation activity were: of 6.1 μM and of 0.22 min for MS and of 2.5 μM and of 0.05 min for OD. The partition ratios for MS and OD were ∼10 and ∼20, respectively. Inactivation of CYP2J2 by MS or OD resulted in a loss of the native heme spectrum and a similar decrease in the reduced CO difference spectrum. A heme adduct was observed in the MS-inactivated CYP2J2. The possible reactive metabolite which covalently modified the prosthetic heme was characterized by analysis of the glutathione conjugates formed by MS or OD following oxygenation of the ethynyl moiety. Liquid chromatography-mass spectrometry showed that inactivation by MS or OD did not lead to modification of apoprotein. Interaction of CYP2J2 with DZ produced a type II binding spectrum with a of 2.8 μM and the IC for loss of OHEB carboxylation activity was 0.18 μM. In conclusion, heme modification by MS and OD was responsible for the mechanism-based inactivation of CYP2J2. The results suggest that the ethynyl moiety of MS and OD faces the heme iron, whereas the isoxazole ring of DZ is preferentially oriented toward the heme iron of CYP2J2.
研究了三种末端炔类化合物对人细胞色素P450 2J2（CYP2J2）的基于机制的失活作用：-(甲基磺酰基)-6-(2-炔丙氧基苯基)己酰胺（MS）、17-十八碳炔酸（OD）和达那唑（DZ）。在重组系统中，羟基依巴斯汀（OHEB）羧化活性的丧失具有时间和浓度依赖性，MS和OD需要NADPH，但DZ不需要。基于机制的OHEB羧化活性失活的动力学常数为：MS的(K_{I})为6.1 μM，(k_{inact})为0.22 min(^{-1})；OD的(K_{I})为2.5 μM，(k_{inact})为0.05 min(^{-1})。MS和OD的分配比分别约为10和20。MS或OD使CYP2J2失活导致天然血红素光谱的丧失以及还原型CO差光谱的类似下降。在MS失活的CYP2J2中观察到血红素加合物。通过分析MS或OD在乙炔基部分氧化后形成的谷胱甘肽缀合物，对共价修饰辅基血红素的可能反应性代谢物进行了表征。液相色谱-质谱分析表明，MS或OD失活不会导致脱辅基蛋白的修饰。CYP2J2与DZ的相互作用产生了II型结合光谱，(K_{d})为2.8 μM，OHEB羧化活性丧失的IC(_{50})为0.18 μM。总之，MS和OD对血红素的修饰是CYP2J2基于机制失活的原因。结果表明，MS和OD的乙炔基部分面向血红素铁，而DZ的异恶唑环优先朝向CYP2J2的血红素铁。
