Application of recombinant DNA methods for production of cholinesterases as organophosphate antidotes and detectors.
To develop new avenues for synthesizing novel antidotes for organophosphate poisoning and for detection of the organophosphates, we have turned to recombinant DNA methods to synthesize cholinesterases with unusual properties. For antidotal therapy we describe mutations of the native mouse and human enzymes that allow for enhanced rates of oxime reactivation. Such enzymes, when localized in the circulation, would enable the circulating cholinesterase to become a catalytic rather than simply a stoichiometric scavenger. Hence, "oxime-assisted catalysis" provides a means for scavenging the organophosphates in the circulation thereby minimizing their tissue penetration and toxicity. Accordingly, the oxime antidote or prophylactic agent has a dual action within the circulation and at the tissue level. Second, through a novel chemistry, termed freeze-frame, click chemistry, we have used organophosphate conjugates of acetylcholinesterase as templates for the synthesis of novel nucleophilic reactivating agents. Finally, acetylcholinesterase can be modified through cysteine substitution mutagenesis and attachment of fluorophores at the substitution positions. When linked at certain locations in the molecule, the attached fluorophore is sensitive to organophosphate conjugation with acetylcholinesterase, and thus the very target of insecticide or nerve agent action becomes a detection molecule for organophosphate exposure.
Taylor
Palmer
P
Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093-0657, USA. pwtaylor@ucsd.edu
Reiner
Elsa
E
Kovarik
Zrinka
Z
Radić
Zoran
Z
D000931:Antidotes / Q000138:chemical synthesis*; D002800:Cholinesterase Inhibitors / Q000138:chemical synthesis*; D002801:Cholinesterase Reactivators / Q000138:chemical synthesis*; D004274:DNA, Recombinant; D009943:Organophosphorus Compounds / Q000819:agonists* / Q000633:toxicity; D010091:Oximes
D000931:Antidotes; D002800:Cholinesterase Inhibitors; D002801:Cholinesterase Reactivators; D004274:DNA, Recombinant; D009943:Organophosphorus Compounds; D010091:Oximes