Molecular Evolution of Human PON to Design Enhanced Catalytic Efficiency for Hydrolysis of Nerve Agents
Abstract
Project Summary: A technology was developed to identify proteins capable of intercepting both existing and emerging organophosphate-based chemical warfare nerve agents (CWNA). The 5 years of performance under this project have demonstrated the potential of directed evolution, combining random and designed mutations based on 3D structures, to generate mutants of a recombinant mammalian PON1 (rePON1) with catalytic proficiency well above that of the wild type (wt)1-3. We have evolved mutants that hydrolyze >105-fold faster the toxic isomers of the coumarin-based nerve agent surrogates, when compared to wt rePON1 and human PON1. The kcat/Km values for these variants when reacting with G-type CWNA are as high as 2-5x107 M-1min-1, a value that approaches the theoretical estimated minimal requirement for efficient prophylactic protection at a reasonable protein dose (~50 mg/70 kg). The most advanced mutants were evolved to completely reverse the stereo-preference of PON1 from the non-toxic to the toxic isomers of racemic nerve agents. PON1 variants evolved by selection with VX are capable of hydrolyzing the toxic isomer of VX >75,000-fold faster than the wt enzyme. Preliminary in vivo evaluation (in guinea pigs) at ICD, Aberdeen Proving Ground MD, demonstrated the ability of several PON1 variants to confer protection in a catalytic manner against multiple LD50 doses of all G-type nerve agents at protein dose of 1 mg/kg. Relevance: This technology permits rapid discovery of pretreatment and post challenge therapeutic drugs against existing and emerging CWNA threats at low protein doses. Existing libraries will shorten the time from emergence of a new CWNA threat to identification of potential counter-measures to a few days or weeks.
Document Details
- Document Type
- Technical Report
- Publication Date
- Dec 01, 2011
- Accession Number
- ADA561380
Entities
People
- Dan S Tawfik
- Joel L. Sussman
Organizations
- Weizmann Institute of Science