engleski

Recombinant DNA approaches to therapeutic treatments of toxicity associated with acetylcholinesterase inhibitors

It has been nearly two decades since the first acetylcholinesterase (AChE) gene was cloned, and nearly 15 years elapsed since the three-dimensional structure of the gene product was elucidated. These major events describing gene and protein sequence and structure have greatly enhanced our understanding of the molecular basis of inhibitor interactions with the enzyme and the basis for organophosphate toxicity. Given this molecular-based knowledge of the target of toxicity itself, we may be able to turn the tables and use it appropriately in the prophylaxis or treatment of organophosphate poisoning. One approach that has met with some success and FDA approval in the U.S. is the administration of human butyrylcholinesterase as a blood-borne scavenger for the treatment of organophosphate toxicity. However, limitations of dose and amounts of material required exist, since butyrylcholinesterase with its mass of ~80kD is a stoichiometric rather than catalytic scavenger. Butyrylcholinesterase has also been mutated to hydrolyze organophosphates. However, the known mutations that lead to catalysis also result in a diminished rate of reaction with the offending organophosphate, therein compromising the initial scavenging rate. To circumvent these limitations, we have investigated an alternative of using oxime-assisted AChE catalysis of organophosphates. AChE in the presence of oximes will turnover organophosphates, but the rate is limited by the slow rates of oxime attack. These rates may be enhanced by through mutation of AChE to enlarge slightly the active center gorge and reduce steric impaction within the gorge structure. This yields up to a 120-fold improvement of rate of oxime reactivation (1). Through additional refining of the active center gorge structure, it may be possible to enhance reactivation rates even further. To this end we are developing a high through-put assay and mutagenesis screening methods to enhance oxime reactivation rates. The typical organophosphate has the requisite hydrophobicity to become sequestered in lipid stores, hence prolonged treatment with oximes could be enhanced by the catalytic scavenging potential of co-administered AChE. The AChE target may also serve as a basis for developing improved reactivating agents over the oximes, 2-PAM and Hl-6, that are currently available. Our experience with freeze-frame, "click-chemistry" has shown that inhibitors can be produced with high selectivity for AChE's from different species, when electric fish, mammalian and Drosophila AChE's are compared (2-4). This approach can be applied to optimizing the design of new nucleophiles for reactivation of organophosphate conjugated cholinesterases. 1. Kovarik Z, Radic Z, Berman HA, Simeon-Rudolf V, Reiner E, and Taylor P. Biochemistry 43:3222-3229(2004). 2. Lewis WG, Green LG, Grynszpan F, Radic Z, Carter PR, Taylor P, Finn MG and Sharpless KB Anqew Chem. 41:1053-1057 (2002). 3. Bourne Y, Kolb HC, Radic Z, Sharpless KB, Taylor P and Marchot P. Proc. Natl. Acad. Sci 101:1449-1454(2004). 4. Krasinski A, Radic Z, Manetsch R, Raushel J, Taylor P, Sharpless KB and Kolb HC J. Amer. Chem. Soc. In press - On line version available (2005).

acetylcholinesterase; oxime; organophosphorus compounds

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