Chemical warfare nerve agents of the G and V-type groups are extremely toxic organophosphates (OPs) and currently available treatments of their intoxications are limited since they are symptomatic. The promiscuous nerve-agent hydrolyzing activities of enzymes such as human serum paraoxonase 1 (hPON1) or bacterial phosphotriesterase (PTE) make them prime candidates both for prophylactic and post exposure treatments of nerve-agent intoxications. However, efficient in-vivo detoxification using low doses of enzymes (≤ 50mg/70kg), following exposure to toxic doses of nerve agents, requires that their catalytic efficiencies towards the toxic nerve agent isomers will be greater than 1x107 M-1min-1. Using directed evolution methods and E.coli cells for expression and variant selection, we previously obtained recombinant PON1 (rePON1) variants that efficiently hydrolyze the toxic isomers of all the major G-type nerve agents and demonstrated the in-vivo prophylactic activity of an evolved variant in mice and guinea pigs. Our current efforts focus on generating PTE variants that will efficiently hydrolyze V-type nerve agents. Using targeted mutagenesis and DNA shuffling guided by structural and phylogenetic analyses, combined with computational design, we increased the efficiency of PTE towards the toxic isomer of VX to a level that is just 5-fold bellow our catalytic goal. We were then able to show efficient antidotal treatment of VX intoxicated animals using an evolved variant. Thus, both previous and current efforts are expected to provide advanced candidates for drug development and also effective means for bio-remediation.
