Identification and Study of Biomarkers from Novichok-Inhibited Butyrylcholinesterase in Human Plasma

Abstract

To identify biomarkers of ethyl (1-(diethylamino)ethylidene)phosphoramidofluoridate (A234)- or methyl (1-(diethylamino)ethylidene)phosphoramidofluoridate (A232)-inhibited butyrylcholinesterase (BChE), we investigated nonapeptide adducts containing the active site serine, which plays a key role in enzyme activity, using LC-MS/HRMS. Biomarkers were acquired as expected, and they exhibited a significant amount of fragment ions from the inhibiting agent itself, in contrast to the MS2 spectra of conventional nerve agents. These biomarkers had a higher abundance of [M+2H]2+ ions than [M+H]+ ions, making doubly charged ions more suitable for trace analysis.

Identification of Potent and Selective Acetylcholinesterase/Butyrylcholinesterase Inhibitors by Virtual Screening

“…For example, three phosphoryl-containing features were found to have a significant association with AChE/BChE inhibition in this study, e.g., “bond: PO_phosphorus_oxo” for AChE inhibition ( P value = 3.73 × 10 –15 ), “bond: PO_phosphate_dithio” for selective AChE inhibition ( P value = 9.14 × 10 –5 ), and “bond: PO_phosphonate_aliphatic_ester” for BChE inhibition ( P value = 2.14 × 10 –2 ) (Table and Table S4). Consistent with these findings, organophosphate nerve agents containing a phosphoryl (PO bond) group have been reported to disrupt cholinergic neurotransmission by irreversibly inhibiting AChE and BChE activity. , The feature “ring:hetero_[6_6_6]_N_S_phenothiazine” was found significant for BChE selectivity ( P value = 5.00 × 10 –4 ) in this study (Tables and S4). Consistent with this finding, there have been cases in the literature that the phenothiazine core was the basis for selective inhibition of BChE. − For example, ethopropazine, which contains the “ring:hetero_[6_6_6]_N_S_phenothiazine” feature, is a highly selective inhibitor of BChE over AChE (∼1000-fold selectivity) …”

“…Consistent with these findings, organophosphate nerve agents containing a phosphoryl (P�O bond) group have been reported to disrupt cholinergic neurotransmission by irreversibly inhibiting AChE and BChE activity. 13,14 The feature "ring:hetero_[6_6_6] _N_S_phenothiazine" was found significant for BChE selectivity (P value = 5.00 × 10 −4 ) in this study (Tables 1 and S4). Consistent with this finding, there have been cases in the literature that the phenothiazine core was the basis for selective inhibition of BChE.…”

“…A well-known class of compounds that can act as AChE/BChE inhibitors is organophosphates, which are commonly used in pesticides. 13,14 Screening a large diverse compound library may reveal new chemical scaffolds that are more suitable for therapeutic development.…”

Recent research on Novichok

“…In the meantime, theoretical and mass spectrometry studies have appeared that ascertain the structures shown in Fig. 1 (Bhakkoa et al 2019 ; Lee et al 2021 ; Jeong et al 2021 ).…”

“…Rapid advances in mass spectrometry to acquire high-quality data with greater resolution will enable analysis of protein and DNA adducts of xenobiotics including chemical warfare agents. In this context, Jeong et al ( 2021 ) investigated nonapeptide adducts of Novichok A 232 an A 234 to the active serine site of butyrylcholine esterase, which plays a key role in enzyme activity, using LC–MS/HRMS. Biomarkers exhibited a significant amount of fragment ions from the inhibiting agent.…”