Molecular evolution of human PON to design enhanced catalytic efficiency for hydr

U.S. Army Medical Research Inst Chem Def, Aberdeen Proving Ground MD

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Abstract

The long-term objective of this effort is to develop a generic gene shuffling-based[unreadable] technology to rapidly screen libraries of 10[10] proteins/peptides encoded by DNA libraries, for identifying[unreadable] biomolecules that can intercept both existing and emerging organophosphate-based chemical warfare[unreadable] nerve agents (CWNA). Enzymes identified in these screens should be capable of catalytically neutralizing[unreadable] the target agent under physiological conditions, thereby providing a basis for development of a new[unreadable] generation of therapeutic agents against CWNA. The major milestone is to integrate established[unreadable] components of enhanced molecular evolution techniques so as to provide a means of miniaturizing[unreadable] existing low-throughput assays, thereby dramatically increasing both sensitivity and throughput. Micro[unreadable] beads will be coated with multiple copies of recombinant human AChE (the CWNA physiological target).[unreadable] Genes of interest will be attached to the same beads. Gene libraries will be obtained by random[unreadable] mutagenesis of several genes that encode enzymes capable of hydrolyzing OPs (e.g., organophosphate[unreadable] hydrolases of both bacterial and mammalian origin, and a repertoire of AChEs, e.g. other vertebrate[unreadable] AChEs, insect AChEs). Beads coated with AChE and the corresponding genes will be compartmentalized[unreadable] in approximately 5fL emulsion droplets, and single genes transcribed and translated in individual droplets. The[unreadable] expressed biomolecules will be allowed to intercept the CWNA, preventing interaction with its target (viz.[unreadable] AChE), and genes that code for an effective interceptor will be isolated. Uniquely, this screen is for[unreadable] directly detoxifying the CWNA, not simply for binding it, thus allowing identification of biomolecules that[unreadable] prevent its action by degradation at the desired rate. Relevance: this technology is envisaged to provide[unreadable] rapid discovery of pretreatment and post challenge therapeutic drugs against existing and emerging[unreadable] CWNA threats and will shorten the time from emergence of a threat to identification of potential countermeasures[unreadable] to a few days or weeks. Once developed this technology can be extended to identification of[unreadable] interceptors for vesicants, pulmonary agents, metabolic/cellular poisons and biological warfare agents. .