A collaborative endeavor to design cholinesterase-based catalytic scavengers against toxic organophosphorus esters.

Wild-type human butyrylcholinesterase (BuChE) has proven to be an efficient bioscavenger for protection against nerve agent toxicity. Human acetylcholinesterase (AChE) has a similar potential. A limitation to their usefulness is that both cholinesterases (ChEs) react stoichiometrically with organophosphosphorus (OP) esters. Because OPs can be regarded as pseudo-substrates for which the dephosphylation rate constant is almost zero, several strategies have been attempted to promote the dephosphylation reaction. Oxime-mediated reactivation of phosphylated ChEs generates a turnover, but it is too slow to make pseudo-catalytic scavengers of pharmacological interest. Alternatively, it was hypothesized that ChEs could be converted into OP hydrolases by using rational site-directed mutagenesis based upon the crystal structure of ChEs. The idea was to introduce a nucleophile into the oxyanion hole, at an appropriate position to promote hydrolysis of the phospho-serine bond via a base catalysis mechanism. Such mutants, if they showed the desired catalytic and pharmacokinetic properties, could be used as catalytic scavengers. The first mutant of human BuChE that was capable of hydrolyzing OPs was G117H. It had a slow rate. Crystallographic study of the G117H mutant showed that hydrolysis likely occurs by activation of a water molecule rather than direct nucleophilic attack by H117. Numerous BuChE mutants were made later, but none of them was better than the G117H mutant at hydrolyzing OPs, with the exception of soman. Soman aged too rapidly to be hydrolyzed by G117H. Hydrolysis was however accomplished with the double mutant G117H/E197Q, which did not age after phosphonylation with soman. Multiple mutations in the active center of human and Bungarus AChE led to enzymes displaying low catalytic activity towards OPs and unwanted kinetic complexities. A new generation of human AChE mutants has been designed with the assistance of molecular modelling and computational methods. According to the putative water-activation mechanism of G117H BChE, a new histidine/aspartate dyad was introduced into the active center of human AChE at the optimum location for hydrolysis of the OP adduct. Additional mutations were made for optimizing activity of the new dyad. It is anticipated that these new mutants will have OP hydrolase activity.