Research Article: Catalytic Bioscavengers Against Toxic Esters, an Alternative Approach for Prophylaxis and Treatments of Poisonings

Date Published: April , 2009

Publisher: A.I. Gordeyev

Author(s): Patrick Masson, Daniel Rochu.

http://

Abstract

Bioscavengers are biopharmaceuticals that specifically react with toxicants. Thus, enzymes reacting with poisonous esters can be used as bioscavengers for neutralization of toxic molecules before they reach physiological targets. Parenteral administration of bioscavengers is, therefore, intended for prophylaxis or pre-treatments, emergency and post-exposure treatments of intoxications. These enzymes can also be used for application on skin, mucosa and wounds as active components of topical skin protectants and decontamination solutions.

Partial Text

Enzyme systems located in skin, blood organs are involved in natural defences against endogenous and exogenous poisons. Detoxification processes occur through different types of reactions, including oxidation, hydrolysis, and conjugation. The role of liver, lung and kidney enzymes, cytochromes P450 (Brown et al., 2008), oxidases, transferases (Miners et al., 2006), amido-carboxylesterases (Redinbo and Potter, 2005; Potter and Wadkins, 2006; Satoh and Hosokawa, 2006) in the metabolism of drugs and xenobiotics is well known. The importance of plasma esterases in the inactivation of numerous toxicants has been recognized, too. Lastly, there is growing evidence that catalytic antibodies play also an efficient role in scavenging deleterious molecules and radicals (Belogurov et al., 2009). These multiple enzymes constitute cellular and circulating barriers that protect physiological machineries and systems against specific toxicants. Here we will examine endogenous and exogenous enzymes that react with poisonous carboxylic-, organophosphoryl- and carbamyl-esters. These enzymes act either as stoichiometric bioscavengers or catalytic bioscavengers. Catalytic bioscavengers are biocatalysts capable of degrading poisonous compounds with a turnover. Enzymes that are potential catalytic bioscavengers will be reviewed.

Unlike the plasma of most mammalians, there is no carboxylesterase in human plasma (Li et al., 2005). However, two enzymes are capable of degrading esters in the blood stream. Plasma paraoxonase (PON1; EC 3.1.8.1) displays an arylesterase activity, and butyrylcholinesterase (BChE; EC 3.1.1.8) — that has broad esterase specificity — plays a role in processing, catabolism and or detoxification of numerous poisonous esters: for instance, human BChE hydrolyzes ester-containing therapeutic and/or addictive drugs such as succinylcholine and its long-chain derivatives (Grigoryan et al., 2008), aspirin, irinotecan, heroin (Lockridge, 1990; Li et al., 2005). Plasma BChE also hydrolyzes prodrugs such as isosorbide diaspirinate, bambuterol (Li et al., 2005), and ISDA, a new aspirin prodrug (Moriarty et al., 2008).

Organophosphates are widely used as pesticides. Some OPs are drugs, others are potent chemical warfare agents. OPs are irreversible inhibitors of cholinesterases (ChEs): acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (Fig. 2). AChE plays a major role in the cholinergic system terminating the action of acetylcholine. Thus, synaptic AChEs are the primary targets of OPs. Irreversible inhibition of AChE is the main cause of acute toxicity of OPs (Maxwell et al., 2006). No clear physiological function has been ascribed to BChE. In the nervous system and at neuromuscular junctions, BChE may surrogate AChE under certain conditions (Lockridge et al., 2009). However, BChE is of pharmacological and toxicological importance as shown in the previous section.

Administration of human plasma has been used for treating OP poisoning. The effects of fresh frozen plasma on cholinesterase levels and outcomes in patients with OP-poisoning were evaluated (Güven et al. 2004). Results suggest that plasma therapy may be an effective alternative or adjunctive treatment method. Plasma BChE and possibly other abundant OP scavenging proteins in plasma (albumin and PON1) may have contributed to this result.

As said, the detoxification of OPs involves the hydrolysis of the phosphoester bond by organophosphorus acid anhydride hydrolases (OPAH), also called phosphotriesterases (PTE), or oxidation to less toxic compounds by degrading their alkyl/aryl chain. Though works on catalytic antibodies (Vayron et al., 2000; Jovic et al. 2005; Reshetnyak et al., 2007) have made some progress, the re-design and engineering of enzymes capable of degrading OPs is the most promising research field. These enzymes could be used as catalytic bioscavengers for prophylaxis and treatment of OP poisoning, for topical protection (Fisher et al., 2005), and for the decontamination of skin, mucosa and open wounds (Lejeune and Russell, 1999; Gill and Ballesteros, 2000). Immobilized OPAH in bioreactors can be used for the decontamination of water (Simo et al., 2008), as well as genetically engineered bacteria producing OPAH can be introduced in water effluents of decontamination units for the purification of contaminated water before recycling or washing up in the environment (Chen and Mulchandani, 1998).

There are several general requirements for the use of enzyme-degrading OPs as medical countermeasures against OP poisoning. Enzymes must have a wide spectra of activity, and ideally, enantioselectivity for toxic stereoisomers. Their mass production under GMP conditions must be realizable at a reasonable cost. Long-term storage without activity loss must be possible under field conditions. Conformational stability can be optimized by chemical modification or the addition of stabilizers.

Cholinesterases – OPs may be regarded as hemi-substrates of ChEs (Fig. 2). When the enzyme reacts with carboxyl-esters, it is transiently acylated, the acyl group being rapidly displaced by a water molecule. On the contrary, with phosphyl-esters, the stereochemistry of the phosphyl-enzyme intermediate restricts the accessibility of water to the phosphorus atom. Thus, the spontaneous hydrolysis of the phosphylated enzyme is very slow or even impossible. Jarv postulated that the introduction of a second nucleophile pole in the active center could activate a water molecule. This water molecule could subsequently attack the phosphorus atom on the back face, leading to breakage of the P-serine bond (Jarv, 1989). The resolution of the three-dimensional structure of Topedo californica AChE (Sussman et al., 1991) opened the way to the rational re-design of ChEs. Thus, the possibility to convert ChEs into OP hydrolases (OPHs) was hypothesized. Human BChE was chosen because its active center is larger (500 Å3) than that of AChE (300 Å3) and it is less stereospecific. Molecular modeling based on the structure of Torpedo AChE was used for making mutants of human BChE. The second nucleophile pole was created in the oxyanion hole of the active center where a glycine residue was replaced by a histidine. This first mutant, G117H, was capable of hydrolyzing paraoxon, sarin, echothiophate and VX (Millard et al., 1995; Lockridge et al., 1997). However, it was irreversibly inhibited by soman, because “aging” of the conjugate was faster than dephosphonylation. The mechanism of aging, i.e. dealkylation of an akyl chain on the phosphorus atom, is almost completely elucidated (cf. Fig. 2). This reaction involves a carbocationic transient that is stabilized by active site residues E197 and W82 and water molecules (Shafferman et al., 1996; Viragh et al., 1997; Masson et al., 1999; Nachon et al., 2005; Li et al. 2007). Mutation of E197 into D, Q or G brought doun the rate of aging. As expected, the double mutant G117H/E197Q was capable of hydrolyzing soman (Millard et al., 1999). However, the catalytic activity of the double mutant was too slow to be of pharmacological interest.

Enzymes that degrade OPs have been isolated from humans, different animal species, fungi and bacteria. Identification and isolation of new natural enzymes, in particular in insects resistant to OP pesticides and among secondary targets of OPs in humans is an active field of research. Also, potential extremozymes have been discovered in halophilic, thermophilic and piezophilic bacteria and other extreme environments (Merone et al., 2005; Feerer et al., 2007). All these enzymes can be purified from their natural sources. However, production of natural enzymes free of infectious agents under GMP is expensive. Thus, suitable biopharmaceuticals are mostly recombinant enzymes. These enzymes can be produced in prokaryotic expression systems (E. coli), eukaryotic expression systems (yeast, insect, mammalian cell cultures), transgenic animals (worm, rabbit, goat), transgenic plants (tomato, potato, tobacco), and also acellular biosynthetic systems. Current research’s goal in protein engineering is to improve mass production of stable enzymes at low cost.

 

Source:

http://