Catalytic Soman Scavenging by the Y337A/F338A Acetylcholinesterase Mutant Assisted with Novel Site-Directed Aldoximes

Abstract

Exposure to the nerve agent soman is difficult to treat due to the rapid dealkylation of soman-acetylcholinesterase (AChE) conjugate known as aging. Oxime antidotes commonly used to reactivate organophosphate inhibited AChE are ineffective against soman, while the efficacy of the recommended nerve agent bioscavenger butyrylcholinesterase is limited by strictly stoichiometric scavenging. To overcome this limitation, we tested ex vivo, in human blood, and in vivo, in soman exposed mice, the capacity of aging-resistant human AChE mutant Y337A/F338A in combination with oxime HI-6 to act as a catalytic bioscavenger of soman. HI-6 was previously shown in vitro to efficiently reactivate this mutant upon soman, as well as VX, cyclosarin, sarin and paraoxon inhibition. We here demonstrate that ex vivo, in whole human blood, 1 μM soman was detoxified within 30 minutes when supplemented with 0.5 μM Y337A/F338A AChE and 100 μM HI-6. This combination was further tested in vivo. Catalytic scavenging of soman in mice improved the therapeutic outcome and resulted in the delayed onset of toxicity symptoms. Furthermore, in a preliminary in vitro screen we identified an even more efficacious oxime than HI-6, in a series of forty-two pyridinium aldoximes, and five imidazole 2-aldoxime N-propyl pyridinium derivatives. One of the later imidazole aldoximes, RS-170B, was a 2–3 –fold more effective reactivator of Y337A/F338A AChE than HI-6 due to the smaller imidazole ring, as indicated by computational molecular models, that affords a more productive angle of nucleophilic attack.

HI-6 assisted catalytic scavenging of VX by acetylcholinesterase choline binding site mutants

“…and 280 mg/kg for i.v . application, classifying HI-6 as a reactivator of lower toxicity and confirming our previous results [18,29]. No toxicity symptoms were observed upon i.v.…”

“…Very slow aging makes it effective as a soman bioscavenger [18] and, as described here, VX toxicity and concentrations are also efficiently curtailed.…”

“…So far, the administration of butyrylcholinesterase (BChE, EC 3.1.1.8) purified from human plasma has been indicated as the most promising prophylaxis [7-10]. However, high dosing requirements for the large protein of BChE in the field [5,7] for stoichiometric scavenging, coupled with its laborious production [11-13], may be minimized by catalytic mutant human AChE (hAChE) based bioscavengers assisted by oximes [14-18]. …”

“…However, alterations of the active site configuration could improve not only the reactivation of phosphylated AChE assisted by an oxime, but also the rate of phosphylation to form OP-conjugates and their subsequent rate of ageing [18,21]. Our kinetic studies have demonstrated that mutations around the choline binding site in human AChE (hAChE), such as Y337A and Y337A/F338A possess enhanced capacity for the reactivation of their phosphylated conjugates [15,18,22-25]. In addition, the dealkylation (aging) of OP-conjugated Y337A/F338A hAChE is dramatically slowed, allowing efficient oxime assisted catalytic turnover for those OPs that rapidly dealkylate.…”

Translation of in vitro to in vivo pyridinium oxime potential in tabun poisoning / Translacija učinkovitosti piridinijevih oksima kod trovanja tabunom iz in vitro sustava u in vivo primjenu

“…Current treatment relies on the use of anticholinergic drug atropine to reduce the effects of accumulating acetylcholine usually combined with oximes that reactivate phosphylated AChE (3)(4)(5). However, the potential of the oximes used in medical practice today, such as 2-PAM, HI-6, and obidoxime, is limited primarily due to the very specific properties of each of the enzyme-OP conjugates (6)(7)(8)(9).…”