New therapeutic approaches and novel alternatives for organophosphate toxicity

Abstract

Organophosphate compounds (OPCs) are commonly used as pesticides and were developed as nerve agents for chemical warfare. Exposure to OPCs results in toxicity due to their covalent binding and inhibition of acetylcholinesterase (AChE). Treatment for toxicity due to OPC exposure has been largely focused on the reactivation of AChE by oxime-based compounds via direct nucleophilic attack on the phosphorous center. However, due to the disadvantages to existing oxime-based reactivators for treatment of OPC poisoning, we considered non-oxime mechanisms of reactivation. A high throughput screen of compound libraries was performed to discover previously unidentified reactivation compounds, followed by studies on their analogs. In the process, we discovered multiple non-oxime classes of compounds, the most robust of which we have already reported [1]. Herein, we report other classes of compounds we identified in our screen that are efficient at reactivation. During biochemical characterization, we also found some compounds with other activities that may inspire novel therapeutic approaches to OPC toxicity. Specifically, we found compounds that [1] increase the rate of substrate hydrolysis by AChE and, [2] protect the enzyme from inhibition by OPC. Further, we discovered that a subset of reactivator compounds recover activity from both AChE and the related enzyme butyrylcholinesterase (BuChE). We now report these compounds, their activities and discuss how each relates to therapeutic approaches that would provide alternatives to traditional oxime-based reactivation.

Resurrection and Reactivation of Acetylcholinesterase and Butyrylcholinesterase

“…[160] pletely recovered the activity of the enzyme up to the level of the positive controla nd the other two reached 60-70%.T hese results are in good agreement with an umber of studies which found that Mannich bases are reactivators of OP-inhibited AChE. [91,113,114,116] While the kinetic assays confirmed recovery of the native enzyme,b ottom-up proteomics were conducted to try and isolate the enzyme in the native, inhibited (re-alkylated), and aged states. As olution of the lead compound 103 wasi ncubated with aged eeAChE for 11 days beforeb eing digested with trypsin.…”

“…Along with testing their non-oxime reactivators with AChE, Stojanovic and co-workers testedt he same library of compoundsw ith paraoxon-inhibitedB ChE. [91] Indeed,s omeo ft he Figure 30. Imidazolium-based oximes for the reactivation of OP-inhibited BChE.…”

“…Mannich base for the selective reactivationo f paraoxon-inhibitedB ChE. [91] Figure 33. Edrophonium-derived derivative 53 for the reactivation of hBChE.…”

“…Stojanovic and co-workersf ollowedu pt heir discovery of two classes of non-oxime reactivators in an additional expansion study.T he first portion of the study focused on analyzing an analogue of amodiaquine called isoquine ( Figure 28). [91] Isoquine wass hown to have reduced toxicitya si td oes not form the iminoquinone;h owever,t he structure still remains aM annich base. Isoquine, like amodiaquine, showedr eactivation potential versus paraoxon-inhibited AChE and actually performed at al evel equal to that of amodiaquine.…”

“…Twop romising families of compounds have been most notable,i ncludingt hose with ap endant base attached to an aryl anchora nd then Mannich bases. [91,113,114,116,160] While all of these compounds were discovered independently,the structuralfeatures continuetobesimilar.W hat is conserved between both types of compounds is the basic nitrogen attached to an aromaticc ore;f or the Mannich bases, ortho substitution relative to ap henol is the most effective. Continued efforts are needed to better understand the mechanism of action for these particular types of molecules to aid in future drug design,w hether those efforts are computational and crystallographic studies or structure-activity relationships.…”