Abstract
The biologically stable and highly toxic organophosphorus nerve agent (OP) VX poses a major health threat. Standard medical therapy, consisting of reactivators and competitive muscarinic receptor antagonists, is insufficient. Recently, two engineered mutants of the Brevundimonas diminuta phosphotriesterase (PTE) with enhanced catalytic efficiency (kcat/KM = 21 to 38 × 106 M−1 min−1) towards VX and a preferential hydrolysis of the more toxic P(−) enantiomer were described: PTE-C23(R152E)-PAS(100)-10-2-C3(I106A/C59V/C227V/E71K)-PAS(200) (PTE-2), a single-chain bispecific enzyme with a PAS linker and tag having enlarged substrate spectrum, and 10-2-C3(C59V/C227V)-PAS(200) (PTE-3), a stabilized homodimeric enzyme with a double PASylation tag (PAS-tag) to reduce plasma clearance. To assess in vivo efficacy, these engineered enzymes were tested in an anesthetized rat model post-VX exposure (~ 2LD50) in comparison with the recombinant wild-type PTE (PTE-1), dosed at 1.0 mg kg−1 i.v.: PTE-2 dosed at 1.3 mg kg−1 i.v. (PTE-2.1) and 2.6 mg kg−1 i.v. (PTE-2.2) and PTE-3 at 1.4 mg kg−1 i.v. Injection of the mutants PTE-2.2 and PTE-3, 5 min after s.c. VX exposure, ensured survival and prevented severe signs of a cholinergic crisis. Inhibition of erythrocyte acetylcholinesterase (AChE) could not be prevented. However, medulla oblongata and diaphragm AChE activity was partially preserved. All animals treated with the wild-type enzyme, PTE-1, showed severe cholinergic signs and died during the observation period of 180 min. PTE-2.1 resulted in the survival of all animals, yet accompanied by severe signs of OP poisoning. This study demonstrates for the first time efficient detoxification in vivo achieved with low doses of heterodimeric PTE-2 as well as PTE-3 and indicates the suitability of these engineered enzymes for the development of highly effective catalytic scavengers directed against VX.
Highlights
Despite ban by the Chemical Weapons Convention, highly toxic organophosphorus nerve agents (OP) were repeatedly used in military conflicts, terrorist attacks or assassinationThe high toxicity of OP results from covalent binding to and subsequent inhibition of the pivotal enzyme acetylcholinesterase (AChE)
Degradation half-life was calculated with Eq (1) and time to 96% VX degradation was estimated as 5 × t1/2 to wild-type PTE-1, against VX exposure in rats their pharmacokinetics (PK) after i.v. injection was studied in the same species (Fig. 1a)
This study demonstrates the efficacy of post-exposure therapy of VX poisoned animals with the new PTE mutants PTE-2 and PTE-3
Summary
The high toxicity of OP results from covalent binding to and subsequent inhibition of the pivotal enzyme acetylcholinesterase (AChE). This leads to a synaptic overflow of acetylcholine (ACh), followed by overstimulation and desensitization of muscarinic and nicotinic cholinergic receptors causing a broad spectrum of clinical signs including miosis, salivation and cardiac arrhythmia (Aldridge and Davison 1953; Aldridge and Reiner 1972; Holmstedt 1959). Multiple in vitro and in vivo studies have shown that this treatment has limited effectiveness against different OP and cannot prevent cholinergic signs (Thiermann et al 2013; Worek and Thiermann 2013) This triggered extensive research on enzyme-based scavengers to detoxify OP via hydrolysis to less toxic products in the blood compartment, preventing the distribution into target tissues and reducing toxicity (Masson and Nachon 2017; Worek et al 2016a)
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