engleski

Reactivation of tabun-phosphylated cholinesterases probed by mutagenesis and new oximes

The copper-catalyzed azide-alkyne cycloaddition reaction enables an efficient and reliable synthesis of libraries of new oximes that were screened for the reactivation activity of tabun-inhibited human recombinant AChE (wild type, AChE mutants Y337A and Y337A/F338A) and human BChE. Out of 100 oximes, at a concentration of 1.0 mM, 53 reactivated wild type AChE, but only 14 oximes restored full activity. Within this series, it appears that an approximate distance equivalent to 8 methylenes between two quaternary nitrogens achieved an optimal level of AChE reactivation. The mutant, Y337A, at the choline binding site was reactivated over 80% with only 13 of the oximes. The most efficient reactivators of Y337A appeared to be 2PAM analogs, with maximal reactivation rate constants kmax up to 10-times faster than those determined for the most efficient reactivator of AChE w.t. Although introducing an additional mutation into the Y337A choline binding site in double mutant Y337A/F338A reduced the enhancement observed in the Y337A mutant, the most efficient Y337A/F338A reactivators also contained the 8 methylene equivalence between two quaternary nitrogens as found for the wild type. It seems that the modification of the active site in the double mutant on average compromised molecular recognition reflected in the Kox constant and slightly improved the maximal reactivation rate constant, kmax. Since all oximes were designed as reactivators of phosphorylated AChE, a limited reactivation capacity for BChE was expected. However, 37 oximes reactivated tabun-inhibited BChE more efficiently than 2PAM, and five reached maximal reactivation of 70 %. In addition, toxicity and antidotal studies with lead reactivators in mice showed significantly improved protective indexes in therapy upon tabun exposure compared to the standard antidote, 2PAM. Therefore, our findings offer a platform for further development of more potent congenic antidotes in tabun and related phosphoramidate exposure.

azide-alkyne cycloadition; reactivation; oximes; kmax; Kox; 2PAM

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