Physiology and Immunology Branch, Research Division, United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland.
J Pharmacol Exp Ther. 2014 Jun;349(3):549-58. doi: 10.1124/jpet.114.213645. Epub 2014 Apr 4.
We investigated the ability of the engineered paraoxonase-1 variants G3C9, VII-D11, I-F11, and VII-D2 to afford protection against paraoxon intoxication. Paraoxon is the toxic metabolite of parathion, a common pesticide still in use in many developing countries. An in vitro investigation showed that VII-D11 is the most efficient variant at hydrolyzing paraoxon with a kcat/Km of 2.1 × 10(6) M(-1) min(-1) and 1.6 × 10(6) M(-1) min(-1) for the enzyme expressed via adenovirus infection of 293A cells and mice, respectively. Compared with the G3C9 parent scaffold, VII-D11 is 15- to 20-fold more efficacious at hydrolyzing paraoxon. Coinciding with these results, mice expressing VII-D11 in their blood survived and showed no symptoms against a cumulative 6.3 × LD50 dose of paraoxon, whereas mice expressing G3C9 experienced tremors and only 50% survival. We then determined whether VII-D11 can offer protection against paraoxon when present at substoichiometric concentrations. Mice containing varying concentrations of VII-D11 in their blood (0.2-4.1 mg/ml) were challenged with doses of paraoxon at fixed stoichiometric ratios that constitute up to a 10-fold molar excess of paraoxon to enzyme (1.4-27 × LD50 doses) and were assessed for tremors and mortality. Mice were afforded complete asymptomatic protection below a paraoxon-to-enzyme ratio of 8:1, whereas higher ratios produced tremors and/or mortality. VII-D11 in mouse blood coeluted with high-density lipoprotein, suggesting an association between the two entities. Collectively, these results demonstrate that VII-D11 is a promising candidate for development as a prophylactic catalytic bioscavenger against organophosphorous pesticide toxicity.
我们研究了工程化的对氧磷酶 1 变体 G3C9、VII-D11、I-F11 和 VII-D2 对抗对氧磷中毒的保护能力。对氧磷是敌百虫的有毒代谢物，敌百虫仍然是许多发展中国家使用的一种常见农药。体外研究表明，VII-D11 是水解对氧磷最有效的变体，其 kcat/Km 值分别为 2.1×10(6) M(-1) min(-1)和 1.6×10(6) M(-1) min(-1)，分别通过腺病毒感染 293A 细胞和小鼠表达。与 G3C9 亲本支架相比，VII-D11 水解对氧磷的效力提高了 15-20 倍。与这些结果一致的是，在血液中表达 VII-D11 的小鼠在接受累积 6.3×LD50 剂量的对氧磷后存活且没有出现症状，而表达 G3C9 的小鼠则出现震颤，仅有 50%存活。然后，我们确定了 VII-D11 在亚化学计量浓度下是否可以提供对抗对氧磷的保护。在血液中含有不同浓度 VII-D11 的小鼠（0.2-4.1mg/ml）接受固定化学计量比的对氧磷剂量挑战，这些剂量构成了对酶的 10 倍摩尔过量（1.4-27×LD50 剂量），并评估了震颤和死亡率。当对氧磷与酶的比例低于 8:1 时，小鼠受到完全无症状的保护，而更高的比例则产生震颤和/或死亡率。VII-D11 在小鼠血液中与高密度脂蛋白共洗脱，表明两者之间存在关联。总的来说，这些结果表明 VII-D11 是开发对抗有机磷农药毒性的预防性催化生物清除剂的有前途的候选物。
