Stenzel J, Worek F, Eyer P
Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians-University, Goethestrasse 33, D-80336 Munich, Germany.
Biochem Pharmacol. 2007 Nov 1;74(9):1390-400. doi: 10.1016/j.bcp.2007.07.013. Epub 2007 Jul 17.
The potential of the most active pyridinium-4-aldoximes, such as obidoxime and trimedoxime, to reactivate phosphorylated acetylcholinesterase is not fully exploited because of inevitable formation of phosphoryloximes (POXs) with extremely high anticholinesterase activity. Hence, a topochemical equilibrium is expected at the active site, with the freshly reactivated enzyme being rapidly re-inhibited by POX produced during reactivation. In the present study, dimethylphosphoryl-, diethylphosphoryl-, and diisopropyl-obidoxime conjugates were generated and isolated in substance. Their inhibition rate of acetylcholinesterase from human red cell membranes was by a factor of 2250, 480 and 600 higher than that observed with paraoxon-methyl, paraoxon-ethyl, and diisopropyl phosphorofluoridate, respectively. All three POXs were hydrolyzed by human paraoxonase (PON1), with the alloenzyme PON1192Q being about 50-fold more active than PON1192R. The rate of hydrolysis, yielding obidoxime, was 1:6:0.03 for the three POXs, respectively. The rate of non-enzymic degradation, yielding obidoxime mononitrile, was similar with the three POXs and showed a high dependency on the reaction temperature (activation energy 83 kJ/mol), while enzymic hydrolysis required less energy (16 kJ/mol). To determine POX-hydrolase activity, we preferred a reaction temperature of 20 degrees C to reduce the noise of spontaneous degradation. A plot of POX-hydrolase versus salt-stimulated paraoxonase activity showed a highly discriminating power towards the PON1Q192R alloenzymes, which may be based on repulsive forces of the quaternary nitrogen atoms of the protonated arginine subtype and the bisquaternary POXs. It is concluded that the pharmacogenetic PON1Q192R polymorphism may be another contributor to the large variability of susceptible subjects seen in obidoxime-treated patients.
由于不可避免地会形成具有极高抗胆碱酯酶活性的磷酰肟（POXs），最具活性的吡啶鎓-4-醛肟（如双复磷和三甲肟）重新激活磷酸化乙酰胆碱酯酶的潜力尚未得到充分利用。因此，预计在活性位点会存在拓扑化学平衡，新重新激活的酶会迅速被重新激活过程中产生的POX重新抑制。在本研究中，生成并分离出了二甲基磷酰基、二乙基磷酰基和二异丙基双复磷共轭物。它们对人红细胞膜乙酰胆碱酯酶的抑制率分别比对氧磷甲基、对氧磷乙基和二异丙基氟磷酸酯高2250倍、480倍和600倍。所有三种POX都能被人对氧磷酶（PON1）水解，同工酶PON1192Q的活性比对PON1192R高约50倍。三种POX水解生成双复磷的速率分别为1:6:0.03。三种POX生成双复磷一腈的非酶促降解速率相似，且对反应温度高度依赖（活化能83 kJ/mol），而酶促水解所需能量较少（16 kJ/mol）。为了测定POX水解酶活性，我们选择20℃的反应温度以减少自发降解的干扰。POX水解酶与盐刺激的对氧磷酶活性的关系图显示，对PON1Q192R同工酶具有高度鉴别能力，这可能基于质子化精氨酸亚型的季铵氮原子与双季铵POX之间的排斥力。结论是，药物遗传学PON1Q192R多态性可能是双复磷治疗患者中易感个体差异大的另一个原因。
