Novel Nucleophiles Enhance the Human Serum Paraoxonase 1 (PON1)-mediated Detoxication of Organophosphates

Abstract

Paraoxonase 1 (PON1) is a calcium-dependent hydrolase associated with serum high-density lipoprotein particles. PON1 hydrolyzes some organophosphates (OPs), including some nerve agents, through nucleophilic attack of hydroxide ion (from water) in the active site. Most OPs are hydrolyzed inefficiently. This project seeks to identify nucleophiles that can enhance PON1-mediated OP degradation. A series of novel nucleophiles, substituted phenoxyalkyl pyridinium oximes, has been synthesized which enhance the degradation of surrogates of sarin (nitrophenyl isopropyl methylphosphonate; NIMP) and VX (nitrophenyl ethyl methylphosphonate; NEMP). Two types of in vitro assays have been conducted, a direct assay using millimolar concentrations of substrate with direct spectrophotometric quantitation of a hydrolysis product (4-nitrophenol) and an indirect assay using submicromolar concentrations of substrate with quantitation by the level of inhibition of an exogenous source of acetylcholinesterase from non-hydrolyzed substrate. Neither NIMP nor NEMP is hydrolyzed effectively by PON1 if one of these novel oximes is absent. However, in the presence of eight novel oximes, PON1-mediated degradation of both surrogates occurs. Computational modeling has created a model of PON1 embedded in phospholipid and has indicated general agreement of the binding enthalpies with the relative efficacy as PON1 enhancers. PON1 enhancement of degradation of OPs could be a unique and unprecedented mechanism of antidotal action.

The effect of PON1 enhancers on reducing acetylcholinesterase inhibition following organophosphate anticholinesterase exposure in rats

“…were selected from results of previous in vitro analyses (Chambers et al, 2015). The nucleophiles tested were substituted phenoxyalkyl pyridinium oximes, synthesized as previously described (Chambers et al, 2015) and are illustrated in Figure 1.…”

“…The nucleophiles tested were substituted phenoxyalkyl pyridinium oximes, synthesized as previously described (Chambers et al, 2015) and are illustrated in Figure 1. The oximes contained a linker chain length of 6 and were prepared by 2-step reactions.…”

“…This PON1 mediated-OP hydrolysis is relatively slow and inefficient in part because hydroxide is only a moderately strong nucleophile. In previous work from our laboratory, a series of potent nucleophiles was synthesized, computationally modeled and tested in an in vitro system for their ability to enhance PON1's degradation of some organophosphates (Chambers et al, 2015). These PON1 enhancers are small molecules containing an oxime moiety as the nucleophilic group.…”

“…From our previous in vitro study (Chambers et al, 2015), a subset of the most efficacious enhancers was selected for these in vivo tests. The OPs selected as substrates for PON1 were surrogates for the nerve agents sarin and VX that our laboratories have synthesized and used in AChE reactivation studies (Meek et al, 2012;Chambers et al, 2013) and in the in vitro PON1 enhancer studies (Chambers et al, 2015).…”

“…From our previous in vitro study (Chambers et al, 2015), a subset of the most efficacious enhancers was selected for these in vivo tests. The OPs selected as substrates for PON1 were surrogates for the nerve agents sarin and VX that our laboratories have synthesized and used in AChE reactivation studies (Meek et al, 2012;Chambers et al, 2013) and in the in vitro PON1 enhancer studies (Chambers et al, 2015). The goal of the present study was to determine if nucleophiles that can enhance PON1-mediated detoxication of OPs in vitro can provide protection from circulating toxic OPs in an in vivo system, thereby identifying compounds with the potential for being developed into nerve agent therapeutics possessing a novel mechanism of action.…”