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

Abstract

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 k cat /K m of 2.1 Â 10 6 M 21 min 21 and 1.6 Â 10 6 M 21 min 21 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 Â LD 50 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 Â LD 50 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.

Targeting of organophosphorus compound bioscavengers to the surface of red blood cells

“…To confirm that the PON1 fusion proteins still retained enzymatic activity following the addition of the Ter119 scFv sequence we performed paraoxonase activity assays[27]. While all of the PON1 variants expressed by Trichoplusia ni larvae had lower catalytic efficiencies than previously reported[23] (10-25%), the addition of the Ter119 scFv did not appear to have any significant deleterious effect on activity(Figure 3. ).Ter119 scFv-PON1 in vitro bindingTo test whether the scFv-PON1 fusion proteins could bind RBCs, we incubated murine RBCs with increasing concentrations of AB743, AB771, and AB772 in vitro.…”

“…Fusion Proteins: Fusion proteins of a scFv for Ter119 [19] and the VIID11 [23,24] variant of PON1 were purchased from Chesapeake PERL Inc. (Savage, MD). The enzymes were expressed by Chesapeake PERL using their Trichoplusia ni larval expression system [25].…”

A new post-intoxication treatment of paraoxon and parathion poisonings using an evolved PON1 variant and recombinant GOT1

“…They contribute to the increase in food production and to the prevention of insect and pest infestations, but they are also toxic compounds. The US Environmental Protection Agency (EPA) estimates that 10,000e20,000 pesticide poisonings occur each year among the 2 million agricultural workers in the United States [1]. According to World Health Organization (WHO) data, it is estimated that~3 million pesticide poisonings occur each year worldwide.…”

Gene Therapy Leaves a Vicious Cycle

“…In fact, both protein and peptide-based drugs have emerged as a major class of therapeutics with nearly 380 marketed pharmaceuticals available in the world (9). However, these protein-based therapies are facing many challenges including low solubility and bioavailability, in vivo physicochemical instability, short circulating half-life, penetrability in vivo , biodistribution, and causing toxicity in large amounts (10–15). Another adverse effect of introducing therapeutic proteins into a patient's body is that it may result in severe immune responses, inflammation, and fever (16).…”