Investigation of evolved paraoxonase-1 variants for prevention of organophosphorous pesticide compound intoxication.
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.
Physiology and Immunology Branch, Research Division, United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland.
David G
DG
Mata
Peter E
PE
Rezk
Praveena
P
Sabnekar
Douglas M
DM
Cerasoli
Nageswararao
N
Chilukuri
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D043303:Aryldialkylphosphatase / Q000097:blood / Q000235:genetics* / Q000378:metabolism*; D055162:Biocatalysis; D004926:Escherichia coli / Q000235:genetics; D018014:Gene Transfer Techniques; D014644:Genetic Variation; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D007928:Lethal Dose 50; D008297:Male; D051379:Mice; D062025:Organophosphate Poisoning / Q000201:enzymology / Q000517:prevention & control*; D010575:Pesticides / Q000493:pharmacokinetics / Q000633:toxicity*; D015202:Protein Engineering
D010575:Pesticides; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human