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Enzymes Reacting with Organophosphorus Compounds

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A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Chemical Biology".

Deadline for manuscript submissions: closed (31 December 2020) | Viewed by 26031

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Prof. Dr. Zrinka Kovarik

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Guest Editor

Institute for Medical Research and Occupational Health, Zagreb, Croatia
Interests: cholinergic mechanisms in health and neurological disorders; inhibition and reactivation of acetylcholinesterase; enzyme kinetics; antidotes for nerve agents poisoning

Special Issue Information

Dear Colleagues,

It is a great pleasure and honor to invite you to contribute to the Special Issue of Molecules entitled “Enzymes Reacting with Organophosphorus Compounds”. This Special Issue welcomes manuscripts describing original work, as well as review articles on structures, modes of action, and biological roles of enzymes reacting with organophosphorus compounds: acetylcholinesterase, butyrylcholinesterase, paraoxonase, neuropathy target esterases, and other OP hydrolyses. The Guest Editor will be pleased to accept and review manuscripts that address but are not restricted to the topics listed below:

Advances in research on kinetics and the mechanism of the enzymes action;
Cholinesterase inactivation by natural and synthetic ligands;
The reactivation of cholinesterases inhibited by nerve agents and insecticides;
Structural studies on native enzymes, their complexes with ligands, and their conjugates with organophosphorus compounds;
Advances in the biology of the enzymes and their cholinergic cross talk and involvement in neurological diseases;
The regulation and alternative roles of acetylcholinesterases.

Prof. Dr. Zrinka Kovarik
Guest Editor

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Keywords

Acetylcholinesterase
Paraoxonase
OP hydrolase
Nerve agents
Cholinergic mechanisms

Published Papers (10 papers)

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Research

14 pages, 10174 KiB

Open AccessFeature PaperArticle

A Thermophilic Bacterial Esterase for Scavenging Nerve Agents: A Kinetic, Biophysical and Structural Study

by Janek Bzdrenga, Elodie Trenet, Fabien Chantegreil, Kevin Bernal, Florian Nachon and Xavier Brazzolotto

Molecules 2021, 26(3), 657; https://doi.org/10.3390/molecules26030657 - 27 Jan 2021

Cited by 2 | Viewed by 1795

Abstract

Organophosphorous nerve agents (OPNA) pose an actual and major threat for both military and civilians alike, as an upsurge in their use has been observed in the recent years. Currently available treatments mitigate the effect of the nerve agents, and could be vastly improved by means of scavengers of the nerve agents. Consequently, efforts have been made over the years into investigating enzymes, also known as bioscavengers, which have the potential either to trap or hydrolyze these toxic compounds. We investigated the previously described esterase 2 from Thermogutta terrifontis (TtEst2) as a potential bioscavenger of nerve agents. As such, we assessed its potential against G-agents (tabun, sarin, and cyclosarin), VX, as well as the pesticide paraoxon. We report that TtEst2 is a good bioscavenger of paraoxon and G-agents, but is rather slow at scavenging VX. X-ray crystallography studies showed that TtEst2 forms an irreversible complex with the aforementioned agents, and allowed the identification of amino-acids, whose mutagenesis could lead to better scavenging properties for VX. In conjunction with its cheap production and purification processes, as well as a robust structural backbone, further engineering of TtEst2 could lead to a stopgap bioscavenger useful for in corpo scavenging or skin decontamination. Full article

(This article belongs to the Special Issue Enzymes Reacting with Organophosphorus Compounds)

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16 pages, 2320 KiB

Open AccessArticle

The Cholinesterase Inhibitory Properties of Stephaniae Tetrandrae Radix

by Xiang-Peng Kong, Hai-Qin Ren, Etta Y. L. Liu, Ka-Wing Leung, Shu-Chen Guo, Ran Duan, Tina T. X. Dong and Karl W. K. Tsim

Molecules 2020, 25(24), 5914; https://doi.org/10.3390/molecules25245914 - 14 Dec 2020

Cited by 4 | Viewed by 2040

Abstract

Stephaniae tetrandrae radix (STR) is a commonly used traditional Chinese medicine in alleviating edema by inducing diuresis. In the clinic, STR extracts or its components are widely used in the treatment of edema, dysuria, and rheumatism for the regulation of water metabolism. Furthermore, STR has been used in treating emotional problems for years by combining with other Chinese herbs. However, the material basis and mechanism of STR on the nervous system have not been revealed. Here, the main components of STR extracts with different extracting solvents were identified, including three major alkaloids, i.e., cyclanoline, fangchinoline, and tetrandrine. The cholinesterase inhibitory activity of STR extracts and its alkaloids was determined using the Ellman assay. Both cyclanoline and fangchinoline showed acetylcholinesterase (AChE) inhibitory activity, demonstrating noncompetitive enzyme inhibition. In contrast, tetrandrine did not show enzymatic inhibition. The synergism of STR alkaloids with huperzine A or donepezil was calculated by the median-effect principle. The drug combination of fangchinoline–huperzine A or donepezil synergistically inhibited AChE, having a combination index (CI) < 1 at F_a = 0.5. Furthermore, the molecular docking results showed that fangchinoline bound with AChE residues in the peripheral anionic site, and cyclanoline bound with AChE residues in the peripheral anionic site, anionic site, and catalytic site. In parallel, cyclanoline bound with butyrylcholinesterase (BChE) residues in the anionic site, catalytic site, and aromatic site. The results support that fangchinoline and cyclanoline, alkaloids derived from STR, could account for the anti-AChE function of STR. Thus, STR extract or its alkaloids may potentially be developed as a therapeutic strategy for Alzheimer’s patients. Full article

(This article belongs to the Special Issue Enzymes Reacting with Organophosphorus Compounds)

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24 pages, 6237 KiB

Open AccessFeature PaperArticle

Benzobicyclo[3.2.1]octene Derivatives as a New Class of Cholinesterase Inhibitors

by Tena Čadež, Ana Grgičević, Ramiza Ahmetović, Danijela Barić, Nikolina Maček Hrvat, Zrinka Kovarik and Irena Škorić

Molecules 2020, 25(21), 4872; https://doi.org/10.3390/molecules25214872 - 22 Oct 2020

Cited by 4 | Viewed by 2558

Abstract

A library of amine, oxime, ether, epoxy and acyl derivatives of the benzobicyclo[3.2.1]octene were synthesized and evaluated as inhibitors of both human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The majority of the tested compounds exhibited higher selectivity for BChE. Structural adjustment for AChE seems to have been achieved by acylation, and the furan ring opening of furo-benzobicyclo[3.2.1]octadiene results for compound 51 with the highest AChE affinity (IC₅₀ = 8.3 µM). Interestingly, its analogue, an oxime ether with a benzobicyclo[3.2.1]-skeleton, compound 32 was one of the most potent BChE inhibitors in this study (IC₅₀ = 31 µM), but not as potent as endo-43, an ether derivative of the benzobicyclo[3.2.1]octene with an additional phenyl substituent (IC₅₀ = 17 µM). Therefore, we identified several cholinesterase inhibitors with a potential for further development as potential drugs for the treatment of neurodegenerative diseases. Full article

(This article belongs to the Special Issue Enzymes Reacting with Organophosphorus Compounds)

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14 pages, 3111 KiB

Open AccessArticle

Solvent Deuterium Oxide Isotope Effects on the Reactions of Organophosphorylated Acetylcholinesterase

by Terrone L. Rosenberry

Molecules 2020, 25(19), 4412; https://doi.org/10.3390/molecules25194412 - 25 Sep 2020

Cited by 5 | Viewed by 1838

Abstract

Organophosphates (OPs) are esters of substituted phosphates, phosphonates or phosphoramidates that react with acetylcholinesterase (AChE) by initially transferring the organophosphityl group to a serine residue in the enzyme active site, concomitant with loss of an alcohol or halide leaving group. With substituted phosphates, this transfer is followed by relatively slow hydrolysis of the organophosphoryl AChE, or dephosphorylation, that is often accompanied by an aging reaction that renders the enzyme irreversibly inactivated. Aging is a dealkylation that converts the phosphate triester to a diester. OPs are very effective AChE inhibitors and have been developed as insecticides and chemical warfare agents. We examined three reactions of two organophosphoryl AChEs, dimethyl- and diethylphosphorylated AChE, by comparing rate constants and solvent deuterium oxide isotope effects for hydrolysis, aging and oxime reactivation with pralidoxime (2-PAM). Our study was motivated (1) by a published x-ray crystal structure of diethylphosphorylated AChE, which showed severe distortion of the active site that was restored by the binding of pralidoxime, and (2) by published isotope effects for decarbamoylation that decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.1 for N,N-diethylcarbamoyl AChE. We previously reconciled these results by proposing a shift in the rate-limiting step from proton transfer for the small carbamoyl group to a likely conformational change in the distorted active site of the large carbamoyl enzyme. This proposal was tested but was not supported in this report. The smaller dimethylphosphoryl AChE and the larger diethylphosphoryl AChE gave similar isotope effects for both oxime reactivation and hydrolysis, and the isotope effect values of about two indicated that proton transfer was rate limiting for both reactions. Full article

(This article belongs to the Special Issue Enzymes Reacting with Organophosphorus Compounds)

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21 pages, 1518 KiB

Open AccessArticle

New Hybrids of 4-Amino-2,3-polymethylene-quinoline and p-Tolylsulfonamide as Dual Inhibitors of Acetyl- and Butyrylcholinesterase and Potential Multifunctional Agents for Alzheimer’s Disease Treatment

by Galina F. Makhaeva, Nadezhda V. Kovaleva, Natalia P. Boltneva, Sofya V. Lushchekina, Tatiana Yu. Astakhova, Elena V. Rudakova, Alexey N. Proshin, Igor V. Serkov, Eugene V. Radchenko, Vladimir A. Palyulin, Sergey O. Bachurin and Rudy J. Richardson

Molecules 2020, 25(17), 3915; https://doi.org/10.3390/molecules25173915 - 27 Aug 2020

Cited by 27 | Viewed by 3273

Abstract

New hybrid compounds of 4-amino-2,3-polymethylene-quinoline containing different sizes of the aliphatic ring and linked to p-tolylsulfonamide with alkylene spacers of increasing length were synthesized as potential drugs for treatment of Alzheimer’s disease (AD). All compounds were potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with selectivity toward BChE. The lead compound 4-methyl-N-(5-(1,2,3,4-tetrahydro-acridin-9-ylamino)-pentyl)-benzenesulfonamide (7h) exhibited an IC₅₀ (AChE) = 0.131 ± 0.01 µM (five times more potent than tacrine), IC₅₀(BChE) = 0.0680 ± 0.0014 µM, and 17.5 ± 1.5% propidium displacement at 20 µM. The compounds possessed low activity against carboxylesterase, indicating a likely absence of unwanted drug-drug interactions in clinical use. Kinetics studies were consistent with mixed-type reversible inhibition of both cholinesterases. Molecular docking demonstrated dual binding sites of the conjugates in AChE and clarified the differences in the structure-activity relationships for AChE and BChE inhibition. The conjugates could bind to the AChE peripheral anionic site and displace propidium, indicating their potential to block AChE-induced β-amyloid aggregation, thereby exerting a disease-modifying effect. All compounds demonstrated low antioxidant activity. Computational ADMET profiles predicted that all compounds would have good intestinal absorption, medium blood-brain barrier permeability, and medium cardiac toxicity risk. Overall, the results indicate that the novel conjugates show promise for further development and optimization as multitarget anti-AD agents. Full article

(This article belongs to the Special Issue Enzymes Reacting with Organophosphorus Compounds)

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8 pages, 690 KiB

Open AccessArticle

In Vitro Interaction of Organophosphono- and Organophosphorothioates with Human Acetylcholinesterase

by Franz Worek, Horst Thiermann, Marianne Koller and Timo Wille

Molecules 2020, 25(13), 3029; https://doi.org/10.3390/molecules25133029 - 02 Jul 2020

Cited by 8 | Viewed by 1842

Abstract

The implementation of the Chemical Weapons Convention (CWC) in 1997 was a milestone in the prohibition of chemical warfare agents (CWA). Yet, the repeated use of CWA underlines the ongoing threat to the population. Organophosphorus (OP) nerve agents still represent the most toxic CWA subgroup. Defensive research on nerve agents is mainly focused on the “classical five”, namely tabun, sarin, soman, cyclosarin and VX, although Schedule 1 of the CWC covers an unforeseeable number of homologues. Likewise, an uncounted number of OP pesticides have been produced in previous decades. Our aim was to determine the in vitro inhibition kinetics of selected organophosphono- and organophosphorothioates with human AChE, as well as hydrolysis of the agents in human plasma and reactivation of inhibited AChE, in order to derive potential structure–activity relationships. The investigation of the interactions of selected OP compounds belonging to schedule 1 (V-agents) and schedule 2 (amiton) of the CWC with human AChE revealed distinct structural effects of the P-alkyl, P-O-alkyl and N,N-dialkyl residues on the inhibitory potency of the agents. Irrespective of structural modifications, all tested V-agents presented as highly potent AChE inhibitors. The high stability of the tested agents in human plasma will most likely result in long-lasting poisoning in vivo, having relevant consequences for the treatment regimen. In conclusion, the results of this study emphasize the need to investigate the biological effects of nerve agent analogues in order to assess the efficacy of available medical countermeasures. Full article

(This article belongs to the Special Issue Enzymes Reacting with Organophosphorus Compounds)

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14 pages, 2429 KiB

Open AccessArticle

Signature Ions in MS/MS Spectra for Dansyl-Aminohexyl-QQIV Adducts on Lysine

by Lawrence M. Schopfer and Oksana Lockridge

Molecules 2020, 25(11), 2659; https://doi.org/10.3390/molecules25112659 - 08 Jun 2020

Cited by 2 | Viewed by 3079

Abstract

Bacterial transglutaminase was used to label human plasma proteins with fluorescent tags. Protein lysines were modified with dansyl-epsilon-aminohexyl-Gln-Gln-Ile-Val-OH (dansylQQIV), while protein glutamines were modified with dansyl cadaverine. Labeled proteins included human butyrylcholinesterase, apolipoprotein A-1, haptoglobin, haptoglobin-related protein, immunoglobulin heavy chain, and hemopexin. Tryptic peptides were analyzed by LC-MS/MS on an Orbitrap Fusion Lumos mass spectrometer. Modified residues were identified in Protein Prospector and Proteome Discoverer searches of mass spectrometry data. The MS/MS fragmentation spectra from dansylQQIV-modified peptides gave intense peaks at 475.2015, 364.1691, 347.1426, 234.0585, and 170.0965 m/z. These signature ions are useful markers for identifying modified peptides. Human butyrylcholinesterase retained full activity following modification by dansylQQIV or dansyl cadaverine. Full article

(This article belongs to the Special Issue Enzymes Reacting with Organophosphorus Compounds)

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15 pages, 3627 KiB

Open AccessArticle

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

by Irina V. Zueva, Sofya V. Lushchekina, David Daudé, Eric Chabrière and Patrick Masson

Molecules 2020, 25(6), 1371; https://doi.org/10.3390/molecules25061371 - 17 Mar 2020

Cited by 8 | Viewed by 2904

Abstract

Enzyme-catalyzed hydrolysis of echothiophate, a P–S bonded organophosphorus (OP) model, was spectrofluorimetrically monitored, using Calbiochem Probe IV as the thiol reagent. OP hydrolases were: the G117H mutant of human butyrylcholinesterase capable of hydrolyzing OPs, and a multiple mutant of Brevundimonas diminuta phosphotriesterase, GG1, designed to hydrolyze a large spectrum of OPs at high rate, including V agents. Molecular modeling of interaction between Probe IV and OP hydrolases (G117H butyrylcholinesterase, GG1, wild types of Brevundimonas diminuta and Sulfolobus solfataricus phosphotriesterases, and human paraoxonase-1) was performed. The high sensitivity of the method allowed steady-state kinetic analysis of echothiophate hydrolysis by highly purified G117H butyrylcholinesterase concentration as low as 0.85 nM. Hydrolysis was michaelian with K_m = 0.20 ± 0.03 mM and k_cat = 5.4 ± 1.6 min⁻¹. The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency (K_m = 2.6 ± 0.2 mM; k_cat = 53400 min⁻¹). With a k_cat/K_m = (2.6 ± 1.6) × 10⁷ M⁻¹min⁻¹, GG1 fulfills the required condition of potential catalytic bioscavengers. quantum mechanics/molecular mechanics (QM/MM) and molecular docking indicate that Probe IV does not interact significantly with the selected phosphotriesterases. Moreover, results on G117H mutant show that Probe IV does not inhibit butyrylcholinesterase. Therefore, Probe IV can be recommended for monitoring hydrolysis of P–S bonded OPs by thiol-free OP hydrolases. Full article

(This article belongs to the Special Issue Enzymes Reacting with Organophosphorus Compounds)

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14 pages, 2946 KiB

Open AccessArticle

Chlorpyrifos Oxon-Induced Isopeptide Bond Formation in Human Butyrylcholinesterase

by Kevser Biberoglu, Ozden Tacal, Lawrence M. Schopfer and Oksana Lockridge

Molecules 2020, 25(3), 533; https://doi.org/10.3390/molecules25030533 - 25 Jan 2020

Cited by 5 | Viewed by 3022

Abstract

A newly recognized action of organophosphates (OP) is the ability to crosslink proteins through an isopeptide bond. The first step in the mechanism is covalent addition of the OP to the side chain of lysine. This activates OP-lysine for reaction with a nearby glutamic or aspartic acid to make a gamma glutamyl epsilon lysine bond. Crosslinked proteins are high molecular weight aggregates. Our goal was to identify the residues in the human butyrylcholinesterase (HuBChE) tetramer that were crosslinked following treatment with 1.5 mM chlorpyrifos oxon. High molecular weight bands were visualized on an SDS gel. Proteins in the gel bands were digested with trypsin, separated by liquid chromatography and analyzed in an Orbitrap mass spectrometer. MSMS files were searched for crosslinked peptides using the Batch-Tag program in Protein Prospector. MSMS spectra were manually evaluated for the presence of ions that supported the crosslinks. The crosslink between Lys544 in VLEMTGNIDEAEWEWK₅₄₄AGFHR and Glu542 in VLEMTGNIDEAEWE₅₄₂WK satisfied our criteria including that of spatial proximity. Distances between Lys544 and Glu542 were 7.4 and 9.5 Å, calculated from the cryo-EM (electron microscopy) structure of the HuBChE tetramer. Paraoxon ethyl, diazoxon, and dichlorvos had less pronounced effects as visualized on SDS gels. Our proof-of-principle study provides evidence that OP have the ability to crosslink proteins. If OP-induced protein crosslinking occurs in the brain, OP exposure could be responsible for some cases of neurodegenerative disease. Full article

(This article belongs to the Special Issue Enzymes Reacting with Organophosphorus Compounds)

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15 pages, 2008 KiB

Open AccessFeature PaperArticle

Interactions of Paraoxonase-1 with Pharmacologically Relevant Carbamates

by Anita Bosak, Aljoša Bavec, Tilen Konte, Goran Šinko, Zrinka Kovarik and Marko Goličnik

Molecules 2020, 25(1), 211; https://doi.org/10.3390/molecules25010211 - 04 Jan 2020

Cited by 9 | Viewed by 3031

Abstract

Mammalian paraoxonase-1 hydrolyses a very broad spectrum of esters such as certain drugs and xenobiotics. The aim of this study was to determine whether carbamates influence the activity of recombinant PON1 (rePON1). Carbamates were selected having a variety of applications: bambuterol and physostigmine are drugs, carbofuran is used as a pesticide, while Ro 02-0683 is diagnostic reagent. All the selected carbamates reduced the arylesterase activity of rePON1 towards the substrate S-phenyl thioacetate (PTA). Inhibition dissociation constants (K_i), evaluated by both discontinuous and continuous inhibition measurements (progress curves), were similar and in the mM range. The rePON1 displayed almost the same values of K_i constants for Ro 02-0683 and physostigmine while, for carbofuran and bambuterol, the values were approximately ten times lower and two times higher, respectively. The affinity of rePON1 towards the tested carbamates was about 3–40 times lower than that of PTA. Molecular modelling of rePON1-carbamate complexes suggested non-covalent interactions with residues of the rePON1 active site that could lead to competitive inhibition of its arylesterase activity. In conclusion, carbamates can reduce the level of PON1 activity, which should be kept in mind, especially in medical conditions characterized by reduced PON1 levels. Full article

(This article belongs to the Special Issue Enzymes Reacting with Organophosphorus Compounds)

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