Tuning Butyrylcholinesterase Inactivation and Reactivation by Polymer‐Based Protein Engineering

Abstract

Organophosphate nerve agents rapidly inhibit cholinesterases thereby destroying the ability to sustain life. Strong nucleophiles, such as oximes, have been used as therapeutic reactivators of cholinesterase‐organophosphate complexes, but suffer from short half‐lives and limited efficacy across the broad spectrum of organophosphate nerve agents. Cholinesterases have been used as long‐lived therapeutic bioscavengers for unreacted organophosphates with limited success because they react with organophosphate nerve agents with one‐to‐one stoichiometries. The chemical power of nucleophilic reactivators is coupled to long‐lived bioscavengers by designing and synthesizing cholinesterase‐polymer‐oxime conjugates using atom transfer radical polymerization and azide‐alkyne “click” chemistry. Detailed kinetic studies show that butyrylcholinesterase‐polymer‐oxime activity is dependent on the electrostatic properties of the polymers and the amount of oxime within the conjugate. The covalent coupling of oxime‐containing polymers to the surface of butyrylcholinesterase slows the rate of inactivation of paraoxon, a model nerve agent. Furthermore, when the enzyme is covalently inhibited by paraoxon, the covalently attached oxime induced inter‐ and intramolecular reactivation. Intramolecular reactivation will open the door to the generation of a new class of nerve agent scavengers that couple the speed and selectivity of biology to the ruggedness and simplicity of synthetic chemicals.

Structure and therapeutic uses of butyrylcholinesterase: Application in detoxification, Alzheimer's disease, and fat metabolism

“…If rates of reactivation and inhibition are consistent, the enzyme and the reactivator could act as effective pseudo‐catalytic bioscavengers. In 2020, Zhang et al 81 connected BChE and oxime (1:1) by polymer to locally increase the concentration of reactivators. The BChE‐polymer‐oxime conjugates were insensitive to the inhibition of OPs and therefore extended the active time of BChE.…”

Non-quaternary oximes detoxify nerve agents and reactivate nerve agent-inhibited human butyrylcholinesterase

“…We have recently reported that oximes that have been coupled to polymer backbones via click chemistry can reactivate OP-inhibited cholinesterases 15 . We were therefore interested in whether a family of newly synthesized clickable alkyne-oximes (chemical structures are shown in Supplementary Scheme 6 ), derived from the HTS-identified leads, would also reactivate the inhibited enzyme.…”

“…In order to bridge this PK gap, we are interested in the direct conjugation of oximes to polymers. Such protein polymer–oxime conjugates were recently synthesized in our lab 15 . These polymer–oxime adducts tethered covalently to BChE by the atom transfer radical polymerization (ATRP) process were shown to reactivate OP-inhibited BChE by inter- and intramolecular interactions 15 .…”

Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P–S Bonded Organophosphorus as Monitored by Spectrofluorimetry

“…Phosphotriesterases (PTE) of various origins, in particular, can be used for detection of OPs [13], decontamination and remediation [8,[14][15][16][17][18] and for therapy of OP poisoning as catalytic bioscavengers [7,[19][20][21][22]. Because OPs are hemi-substrates of ChEs, research of novel human ChE mutants capable of hydrolyzing OPs is of great interest for improving catalytic bioscavenger-based medical countermeasures of OP poisoning [23][24][25] and implementation of pseudocatalytic bioscavenger systems composed of ChE-reactivator [26][27][28]. Enzymatic hydrolysis of P-S bonded OPs, such as V-agents, is currently monitored by the reaction of the OP thiol leaving group with DTNB [16,29].…”