Investigation of Evolved Paraoxonase-1 Variants for Prevention of Organophosphorous Pesticide Compound Intoxication

• Published in: The Journal of pharmacology and experimental therapeutics 2014-06, Vol.349 (3), p.549-558
• Main Authors: Mata, David G., Rezk, Peter E., Sabnekar, Praveena, Cerasoli, Douglas M., Chilukuri, Nageswararao
• Format: Article
• Language: English
• Subjects: Adenoviridae - genetics; Animals; Aryldialkylphosphatase - blood; Aryldialkylphosphatase - genetics; Aryldialkylphosphatase - metabolism; Biocatalysis; Escherichia coli - genetics; Gene Transfer Techniques; Genetic Variation; Genetic Vectors; HEK293 Cells; Humans; Lethal Dose 50; Male; Mice; Organophosphate Poisoning - enzymology; Organophosphate Poisoning - prevention & control; Pesticides - pharmacokinetics; Pesticides - toxicity; Protein Engineering
• ISSN: 0022-3565; 1521-0103
• DOI: 10.1124/jpet.114.213645

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 × 106 M−1 min−1 and 1.6 × 106 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.