Engineering of Paraoxonases for Pre- and Post-treatment of Intoxication by a Broad Spectrum of Nerve Agents and Pesticides
Annual rept. 7 May 2012-6 May 2013
WEIZMANN INST OF SCIENCE REHOVOT (ISRAEL)
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Project Summary 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. In the first year of the renewal contract we focused on a Design and generation of libraries VXG6 and VXG7 by screening with VX, and b HDL association and stabilization of PONs. Libraries VXG6 and VXG7 provided variants that hydrolyzed VX at kcatKm approaching 3x105 M-1min-1. A thio effect was defined and raised as a potential barrier that slowed the improvement of PON1 variants towards Vagents. The most active variants found 167-mix1-F5 and 268-2-E8, were sent to OSU for large-scale production intended for future in-vivo experiments at ICD. Targeting key positions in helixes H1 and H2 revealed a H-bonding network responsible for structural changes that stimulate PON1 activity that occur upon binding to HDL. Positions 185 and 198 were found to be key residues associated with increased stability and activity stimulation. Relevance this technology is envisaged to provide rapid discovery of pre- and post-treatment therapeutic drugs against existing and emerging CWNA threats and may shorten the time from emergence of a threat to identification of potential counter-measures to a few days or weeks.
- Chemical, Biological and Radiological Warfare