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Acetylcholinesterase (AChE) is a serine hydrolase found in brain synapses, neuromuscular junctions (NMJs) and erythrocytes. Its role is to silence nerve impulses by selectively hydrolyzing acetylcholine, a neurotransmitter. Inhibition of AChE can lead to accumulation of acetylcholine at synapses and NMJs; if left untreated, the symptoms can lead to death. Organophosphorus (OP) chemical nerve agents are a type of suicide inhibitors for AChE, leading to phosphylation of the catalytic serine; such phosphylation blocks the critical nucleophilic serine residue in the active site. OPs have been used as pesticides and chemical warfare agents, and exposure to these compounds result in the death of thousands of people every year. Clinically, OP poisoning can be treated by a combination of anti-cholinergic drugs and oximes. However, a dealkylation process referred to as aging can follow inhibition. To date, the aged form of AChE has been recalcitrant to reactivation by any oxime. A straightforward post-aging treatment is to reverse aging by realkylation of the oxyanion on the phosphylated adduct. Quinone methides (QMs) and quinone methide precursor (QMP) have been reported as alkylators of proteins and phosphates. These previous reports imply the possibility to realkylate aged AChE using a QM or QMP. We therefore chose QMPs for realkylation of aged AChE.Our preliminary studies revealed the binding affinity and selectivity of QMPs to AChE active site. We also estimated whether or not the realkylated AChE could be stable enough to prevent rapid re-aging. A series of nine benzyl OPs were synthesized and characterized, particularly in terms of their aging kinetics. The structures of the corresponding inhibited AChE adducts resemble those of QMP-realkylated AChE. Their aging rates were measured and provided insight into the re-aging rates of such QMP-realkylated AChE adducts. Guided by molecular docking and molecular dynamics simulations, we designed and synthesized a library of QMPs. They were screened against aged forms of electric eel AChE. Several candidates derived from 3-hydroxypyridine successfully realkylated some aged AChE. The lead compound was further investigated in subsequent kinetic studies. Particularly, our lead compound resurrected 30.8% of isopropyl phosphate-aged electric eel AChE in a 30-day reaction, and 9.5% of methylphosphonate-aged AChE after 3 d of reaction. Its efficacy was confirmed by bottom-up proteomics of AChE. We also discovered that such 3-hydroxypyridines have a second function, i.e. reactivation of inhibited or realkylated AChE, but they are less effective than oximes.Offsite alkylation is an undesirable side reaction. By means of a lysozyme alkylation test, we confirmed that our lead compound and other four candidates would not show observable offsite alkylation. The lead compound also proved compatible with Ellman’s assay without causing any false positive signal.To date, despite over 50 years of study, no successful AChE realkylators have been published in any peer-reviewed journals. Our discovery is encouraging and will be a lead discovery on which future structure-activity relationship studies will be based. Follow-up studies will be conducted aiming to improve the performances of the current lead, and in due course, the safety and potency of the realkylators will also be tested in vivo. |
