Assessing the stoichiometric efficacy of mammalian expressed paraoxonase-1 variant I-F11 to afford protection against G-type nerve agents.

We evaluated the ability of evolved paraoxonase-1 (PON1) to afford broad spectrum protection against G-type nerve agents when produced in mammalian cells via an adenovirus expression system. The PON1 variants G3C9, VII-D11, I-F11, VII-D2 and II-G1 were screened in vitro for their ability to hydrolyze G-agents, as well as for their preference towards hydrolysis of the more toxic P(-) isomer. I-F11, with catalytic efficiencies of (1.1 ± 0.1) × 106 M-1 min-1, (2.5 ± 0.1) × 106 M-1 min-1, (2.3 ± 0.5) × 107 M-1 min-1and (9.2 ± 0.1) × 106 M-1 min-1 against tabun (GA), sarin (GB), soman (GD) and cyclosarin (GF), respectively, was found to be a leading candidate for further evaluation. To demonstrate the broad spectrum efficacy of I-F11 against G-agents, a sequential 5 × LD50 dose of GD, GF, GB and GA was administered to ten mice expressing I-F11 on days 3, 4, 5 and 6 following virus injection, respectively. At the conclusion of the experiment, 80% of the animals survived exposure to all four G-agents. Using the concept of stoichiometric efficacy, we determined that I-F11 affords protection from lethality against an administered dose of 10, 15, 90 and 80 molar equivalents of GA, GB, GD and GF, respectively, relative to the molar equivalents of I-F11 in circulation. It also appears that I-F11 can associate with high density lipoprotein in circulation, suggesting that I-F11 retained this function of native PON1. This combination of attractive attributes demonstrates that I-F11 is an attractive candidate for development as a broad-therapeutic against G-type nerve agent exposure.

An in vitro and in vivo evaluation of the efficacy of recombinant human liver prolidase as a catalytic bioscavenger of chemical warfare nerve agents.

In this study, we determined the ability of recombinant human liver prolidase to hydrolyze nerve agents in vitro and its ability to afford protection in vivo in mice. Using adenovirus containing the human liver prolidase gene, the enzyme was over expressed by 200- to 300-fold in mouse liver and purified to homogeneity by affinity and gel filtration chromatography. The purified enzyme hydrolyzed sarin, cyclosarin and soman with varying rates of hydrolysis. The most efficient hydrolysis was with sarin, followed by soman and by cyclosarin {apparent kcat/Km [(1.9 ± 0.3), (1.7 ± 0.2), and (0.45 ± 0.04)] × 10(5 )M(-1 )min(-1), respectively}; VX and tabun were not hydrolyzed by the recombinant enzyme. The enzyme hydrolyzed P (+) isomers faster than the P (-) isomers. The ability of recombinant human liver prolidase to afford 24 hour survival against a cumulative dose of 2 × LD50 of each nerve agent was investigated in mice. Compared to mice injected with a control virus, mice injected with the prolidase expressing virus contained (29 ± 7)-fold higher levels of the enzyme in their blood on day 5. Challenging these mice with two consecutive 1 × LD50 doses of sarin, cyclosarin, and soman resulted in the death of all animals within 5 to 8 min from nerve agent toxicity. In contrast, mice injected with the adenovirus expressing mouse butyrylcholinesterase, an enzyme which is known to afford protection in vivo, survived multiple 1 × LD50 challenges of these nerve agents and displayed no signs of toxicity. These results suggest that, while prolidase can hydrolyze certain G-type nerve agents in vitro, the enzyme does not offer 24 hour protection against a cumulative dose of 2 × LD50 of G-agents in mice in vivo.

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.