PROJECT SUMMARY/ABSTRACT Exposure to toxic organophosphorus (OP) compounds in the form of insecticides or chemical warfare nerve agents (CWNAs) remains a persistent concern for both civilian and military populations. One countermeasure to OP exposure that has been shown to be effective in animal models is the use of intravenously administered butyrylcholinesterase (BChE), which scavenges free OP molecules in the bloodstream. A significant limitation of BChE, however, is that it simply binds OP compounds stoichiometrically, resulting in the inactivation of both molecules. The resulting requirement for large doses of BChE is exacerbated by difficulties associated with its manufacture, thereby complicating its use as an OP defense measure. An important advance would therefore be the modification of the human BChE enzyme to enable catalytic degradation of OP compounds, thus providing a therapeutic enzyme with minimal potential immunogenicity. Several lines of evidence support the feasibility of this approach: microbial enzymes that exhibit high OP hydrolysis activity are known, and studies by our lab and others have shown that BChE can be made modestly catalytic for OP hydrolysis through the introduction of mutations or by adding certain oxime compounds. With this in mind, our ultimate goal is to develop catalytic human BChE enzymes that can functionally mimic microbial or oxime-coupled OP degradation reactions, but which also exhibit long serum half-lives, negligible side effects, and minimal immunogenicity. A critical and novel aspect of our work is the rational introduction of a zinc-binding site within the substrate gorge of the BChE enzyme, which is designed to mimic the catalytic center of the highly efficient bacterial organophosphate hydrolase (OPH) and other engineered hydrolytic zinc enzymes. Three basic design strategies are described for initial evaluation, while additional and subsequent optimized mutants will be developed through multiscale computer-guided design. BChE mutants will be expressed in HEK293E cells, which we have successfully used to produce properly glycosylated BChE tetramers in quantities sufficient for characterization. Evaluation of the metal binding site will be assessed through spectroscopic and analytical techniques, while hydrolytic reactivity towards a range of OP model compounds will be measured through colorimetric assays. Due to their enhanced safety profiles, these engineered enzymes would provide enhancements to conventional clinical interventions for acute OP exposures, especially when combined with post-exposure oxime treatments. In future work, these studies will be used to optimize BChE metalloenzymes that are effective against highly toxic and difficult-to-assay targets, for which evolutionary approaches are intractable, and ultimately develop effective formulations for storage and delivery for post-exposure indications. The goal of the introductory work proposed here is the initial development and valid...