New Structural Scaffolds for Centrally Acting Oxime Reactivators of Phosphylated Cholinesterases

Rakesh K Sit,Zoran Radić,Valeria Gerardi,Limin Zhang,Edzna Garcia,Maja Katalinić,Gabriel Amitai,Zrinka Kovarik,Valery V Fokin,K Barry Sharpless,Palmer Taylor

doi:10.1074/jbc.m111.230656

Abstract

We describe here the synthesis and activity of a new series of oxime reactivators of cholinesterases (ChEs) that contain tertiary amine or imidazole protonatable functional groups. Equilibration between the neutral and protonated species at physiological pH enables the reactivators to cross the blood-brain barrier and distribute in the CNS aqueous space as dictated by interstitial and cellular pH values. Our structure-activity analysis of 134 novel compounds considers primarily imidazole aldoximes and N-substituted 2-hydroxyiminoacetamides. Reactivation capacities of novel oximes are rank ordered by their relative reactivation rate constants at 0.67 mm compared with 2-pyridinealdoxime methiodide for reactivation of four organophosphate (sarin, cyclosarin, VX, and paraoxon) conjugates of human acetylcholinesterase (hAChE). Rank order of the rates differs for reactivation of human butyrylcholinesterase (hBChE) conjugates. The 10 best reactivating oximes, predominantly hydroxyimino acetamide derivatives (for hAChE) and imidazole-containing aldoximes (for hBChE) also exhibited reasonable activity in the reactivation of tabun conjugates. Reactivation kinetics of the lead hydroxyimino acetamide reactivator of hAChE, when analyzed in terms of apparent affinity (1/K(ox)) and maximum reactivation rate (k(2)), is superior to the reference uncharged reactivators monoisonitrosoacetone and 2,3-butanedione monoxime and shows potential for further refinement. The disparate pH dependences for reactivation of ChE and the general base-catalyzed oximolysis of acetylthiocholine reveal that distinct reactivator ionization states are involved in the reactivation of ChE conjugates and in conferring nucleophilic reactivity of the oxime group.

Highlights

Current therapy directed to reactivating inhibited AChE is limited to the peripheral circulation because commonly used quaternary pyridinium aldoxime reactivators do not cross the bloodbrain barrier (BBB) at therapeutically relevant levels [5]
The uncharged species of reactivators are expected to diffuse across the BBB into the CNS, and upon establishing the pH-dependent equilibrium between the neutral and ionized forms in extracellular CNS space, the protonated form would efficiently interact with the electron-rich active center environbutyrylcholinesterase; MINA, monoisonitrosoacetone; OP, organophosphate; 2PAM, 2-pyridinealdoxime methiodide
Reactivation trends and enhancement over 2PAM reactivation were similar for different OP conjugates (Fig. 1) with cyclosarin and VX conjugates being most amenable to reactivation, and paraoxon and tabun conjugates being noticeably more resistant to reactivation

Summary

Introduction

Current therapy directed to reactivating inhibited AChE is limited to the peripheral circulation because commonly used quaternary pyridinium aldoxime reactivators do not cross the BBB at therapeutically relevant levels [5]. A handful of studies involving centrally acting reactivators have been reported in the literature, including nonspecific acetyloxime derivatives 2,3-butanedione monoxime (DAM) and monoisonitrosoacetone (MINA) (6 – 8), glycosylated pyridinium oximes [9, 10], and dihydropyridine prodrug, pro-2PAM (2-pyridinealdoxime methiodide) derivatives [11, 12]. The uncharged species of reactivators are expected to diffuse across the BBB into the CNS, and upon establishing the pH-dependent equilibrium between the neutral and ionized forms in extracellular CNS space, the protonated form would efficiently interact with the electron-rich active center environbutyrylcholinesterase; MINA, monoisonitrosoacetone; OP, organophosphate; 2PAM, 2-pyridinealdoxime methiodide. The majority of oximes were screened for reactivation of paraoxon-, sarin-, VX-, and cyclosarin-inhibited human butyrylcholinesterase (hBChE) to assess the breadth of conjugates that show significant reactivation

Methods

Results

Conclusion