Plant-derived human butyrylcholinesterase, but not an organophosphorous-compound hydrolyzing variant thereof, protects rodents against nerve agents

Abstract

The concept of using cholinesterase bioscavengers for prophylaxis against organophosphorous nerve agents and pesticides has progressed from the bench to clinical trial. However, the supply of the native human proteins is either limited (e.g., plasma-derived butyrylcholinesterase and erythrocytic acetylcholinesterase) or nonexisting (synaptic acetylcholinesterase). Here we identify a unique form of recombinant human butyrylcholinesterase that mimics the native enzyme assembly into tetramers; this form provides extended effective pharmacokinetics that is significantly enhanced by polyethylene glycol conjugation. We further demonstrate that this enzyme (but not a G117H/E197Q organophosphorus acid anhydride hydrolase catalytic variant) can prevent morbidity and mortality associated with organophosphorous nerve agent and pesticide exposure of animal subjects of two model species.countermeasures | nonconventional warfare agents | organophosphorous pesticides | protein engineering | transgenic plants B utyrylcholinesterase (BChE) is the major cholinesterase (ChE) in the serum of humans (1, 2). Although the closely related enzyme acetylcholinesterase (AChE) is well described as the primary synaptic regulator of cholinergic transmission, a definitive physiological role for BChE has not yet been demonstrated (3). BChE is catalytically promiscuous and hydrolyzes not only acetylcholine (ACh), but also longer-chain choline esters (e.g., butyrylcholine, its preferred substrate, and succinylcholine) and a variety of non-choline esters, such as acetylsalicylic acid (aspirin) and cocaine (4, 5). Moreover, BChE binds most environmentally occurring ChE inhibitors as well as man-made organophosphorous (OP) pesticides and nerve agents (NAs) (6, 7-10).The systemic biodistribution and affinity for ChE inhibitors allow endogenous BChE to provide broad-spectrum protection against various toxicants by their sequestration before they reach cholinergic synapses. However, under realistic high-dose exposure scenarios, BChE serum levels are too low to afford adequate protection, resulting in persistent cholinergic excitation due to irreversible inhibition of AChE and subsequent accumulation of ACh. Sublethal manifestations of this state include unregulated exocrine secretion and gastrointestinal hypermotility. Death usually results from unregulated stimulation at neuromuscular junction leading to hemodynamic instability and tetanic contraction of the respiratory muscles (11,12).Current OP poisoning therapy consists of atropine for muscarinic ACh receptor blockade and diazepam for symptomatic management of convulsions (12). Additionally, oxime therapy with 2-pralidoxime (2-PAM) can effectively reactivate some but not all OP-AChE adducts (13)(14)(15). This standard therapeutic approach can reduce mortality, but insufficiently prevents the incapacitation associated with OP toxicity (12, 16).Prophylaxis by administration of exogenous ChEs has proven successful in reducing OP-associated morbidity and mortality, but requires the availability of rel...

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

“…A combination of both pathways also was observed for cl.15. The selected BChE mutants were of high interest because they revealed a previously unreported target (the BChE acylbinding loop) for the creation of catalytic bioscavengers based on human BChE, which are used extensively for protection against nerve agent poisoning and postexposure treatment (12,13).…”

Chemical polysialylation of human recombinant butyrylcholinesterase delivers a long-acting bioscavenger for nerve agents in vivo

“…These profiles of recombinant human BChEs have been found to show differences compared with native plasma enzyme (16,25,32). PEGylation has been proposed (16) for long-acting DNA-encoded ChE-based bioscavengers.…”

“…Unfortunately, use of the human plasma BChE is limited by current manufacturer protocol, which requires large amounts of outdated human plasma for generation of a single dose of bioscavenger, thus making the resulting drug cost too high for wide application. Recent publication by the Mor and colleagues (16) vividly describes that "with current technology, creating a small stockpile of plasma-derived BChE (1 kg ≈ 5,000 doses) would require dedicating the entire annual US supply of outdated plasma to a purification effort at a considerable cost." The other hidden problem of using outdated plasma is possible contamination with viruses.…”

“…Recombinant technology provides controlled contamination-free environment, fixed price, and yield, thus making possible the generation of a drug stockpile on demand. Numerous attempts to achieve transgenic expression of BChE have been developed in the past 10 y, including transgenic plants (16), mice, and goats (17). However, none of these has yet been economically effective or received approval for administration to humans.…”

“…To overcome difficulties in expression of the tetrameric form of rBChE, a natural tetramerization peptide (28,29) can be introduced into medium (30) or to the expression system in vivo (31). Alternatively or simultaneously, PEGylation can be used to improve pharmacokinetic stability of native and recombinant BChE (16,25,32). In additional to PEG, polysialic acids can be used to increase the molecular size of rBChE.…”