Molecular Evolution of Human PON to Design Enhanced Catalytic Efficiency for Hydrolysis of Nerve Agents

The long-term objective of this effort is to develop a generic gene shuffling- based technology to rapidly screen libraries of 1010 proteinspeptides encoded by DNA libraries, for identifying biomolecules that can intercept both existing and emerging organophosphate-based chemical warfare nerve agents CWNA. All 3rd year milestones have been met, and additional tasks were performed to advance the goals of the project1 Plasmids of mutants 8C8, 2H4, and 1G3 were delivered for further evaluation against authentic nerve agents at USAMRICD. In addition, on early March 2009, 9 mutants were expressed and purified in our laboratory and transferred to USAMRICD. 2 Two mutants which displayed enhanced hydrolysis properties were over expressed, crystallized, and their 3D structure revealed movements of the catalytic Ca2 and amino acid side chains coordinating to the catalytic calcium. 3In vitro and in vivo studies demonstrated improved thermostability and antidotal efficacy of wt rePON1 compared to human PON1. 4 Stereo-specific protocols were developed for the synthesis of enantiomers of coumarin surrogates of nerve agents 5 A Protocol was developed for a safe in situ procedure for fluoridation of coumarin analogs of nerve agents thus permitting direct selection of clones by use of G-agents and the interception technique. This technology is envisaged to provide rapid discovery of pretreatment and post challenge therapeutic drugs against existing emerging CWNA threats and will shorten the time from emergence of a threat to identification of potential counter-measures to a few days or weeks.