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