Enhancement of organophosphinate hydrolase activity therough mechanistic evaluati

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

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Abstract

A critical barrier to using enzymes as catalytic human therapeutics against organophosphorous (OP) nerve[unreadable] agents is their poor activity and specificity. Using computational, mechanistic, spectroscopic, and protein[unreadable] mutagenic approaches, we propose the production and optimization of mutants of human enzymes[unreadable] (paraoxonase I, butyrylcholine esterase, and acetylcholinesterase) to catalyze the hydrolysis of chemical[unreadable] warfare nerve agents with greater activity and specificity than those identified to date. Since mutated[unreadable] BuChE, AChE and HuPONI are based on human proteins, the expectation is that these proteins would have[unreadable] few or no immunological and behavioral side effects, making these reasonable human therapeutic[unreadable] candidates. We will elucidate the underlying chemical mechanisms of action necessary for catalytic[unreadable] hydrolysis of chemical warfare nerve agents, design mutants of human proteins with enhanced activity[unreadable] toward nerve agents, and appropriately design recombinant proteins of human origin which can then be[unreadable] expressed in sufficient quantities for subsequent in vivo validation of efficacy. The overall expectation is to[unreadable] develop a sufficient body of scientific data to allow for a selection to be made of one or two protein products[unreadable] for the transition to advanced development as a new generation of prophylactic biological agents with the[unreadable] potential to be granted NDA status and that these biological agents will provide enhanced protection against[unreadable] nerve agent poisoning in a military or civilian setting. Computational, mechanistic, spectroscopic,[unreadable] photoaffinity labeling, mass spectrometric, proteomic, and biochemical tools will be used with wild-type and[unreadable] recombinant mutant forms of cholinesterase and esterase enzymes for the development of these novel[unreadable] therapeutics against organophosphorous (OP) nerve agents.[unreadable] The development of these novel forms of cholinesterase enzymes will provide a biological therapeutic[unreadable] against the use of organophosphorous nerve agents in military and civilian settings. These therapeutics,[unreadable] being of human origin, will have the desired chemical specificity and also few or no immunological and[unreadable] behavioral side effects