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1.
Chem Biol Interact ; 308: 323-331, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31173750

RESUMO

Organophosphorus compounds have been widely employed to the development of warfare nerve agents and pesticides, resulting in a huge number of people intoxicated annually, being a serious problem of public health. Efforts worldwide have been done in order to design new technologies that are capable of combating or even reversing the poisoning caused by these OP nerve agents. In this line, the bioremediation arises as a promising and efficient alternative for this purpose. As an example of degrading enzymes, there is the organophosphate-degrading (OpdA) enzyme from Agrobacterium radiobacter, which has been quite investigated experimentally due to its high performance in the degradation of neurotoxic nerve agents. This work aims to look into the structural and electronic details that govern the interaction modes of these compounds in the OpdA active site, with the posterior hydrolysis reaction prediction. Our findings have brought about data about the OpdA performance towards different nerve agents, and among them, we may realize that the degradation efficiency strongly depends on the nerve agent structure and its stereochemistry, being in this case the compound Tabun the one more effectively hydrolyzed. By means of the chemical bonds (AIM) and orbitals (FERMO) analysis, it is suggested that the initial reactivity of the OP nerve agents in the OpdA active site does not necessarily dictate the reactivity and interaction modes over the reaction coordinate.


Assuntos
Biodegradação Ambiental , Agentes Neurotóxicos/metabolismo , Agrobacterium tumefaciens/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biocatálise , Domínio Catalítico , Humanos , Simulação de Acoplamento Molecular , Agentes Neurotóxicos/química , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Teoria Quântica , Sarina/química , Sarina/metabolismo
2.
Chem Biol Interact ; 307: 16-20, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31004594

RESUMO

Bispyridinium oximes with one (K865, K866, K867) or two (K868, K869, K870) ortho-positioned chlorine moiety, analogous to previously known K027, K048 and K203 oximes, and potent reactivators of human acetylcholinesterase (AChE) inhibited by nerve agents, were tested in the reactivation of human butyrylcholinesterase (BChE) inhibited by sarin, cyclosarin, VX, and tabun. A previously highlighted AChE reactivator, dichlorinated bispyridinium oxime with propyl linker (K868), was tested in more detail for reactivation of four nerve agent-BChE conjugates. Its BChE reactivation potency was showed to be promising when compared to the standard oximes used in medical practice, asoxime (HI-6) and pralidoxime (2-PAM), especially in case of sarin and tabun. This finding could be used in the pseudo-catalytic scavenging of the most nerve agents due to its cumulative capacity to reactivate both AChE and BChE.


Assuntos
Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/metabolismo , Agentes Neurotóxicos/metabolismo , Oximas/metabolismo , Butirilcolinesterase/química , Inibidores da Colinesterase/química , Ativação Enzimática/efeitos dos fármacos , Halogenação , Humanos , Cinética , Agentes Neurotóxicos/química , Oximas/química , Oximas/farmacologia , Compostos de Piridínio/química , Sarina/química , Sarina/metabolismo
3.
J Chromatogr A ; 1583: 108-116, 2019 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-30470454

RESUMO

A sensitive method for determination of fluoridated phosphonates produced by fluoride-mediated regeneration of nerve agent adduct in human serum was developed using gas chromatography-mass spectrometry (GCMS) with large-volume injection. The GC injection was administered using stomach-type spiral injector (LVI, AiSTI SCIENCE) enabling introduction of only target compounds from 50 µL ethyl acetate extract after purging the solvent. For GCMS analysis of sarin (GB), 670 times higher sensitivity, based on limit of detection (LOD, S/N = 3, on extracted ion chromatogram (EIC) at m/z 99), was achieved using this injection (50 µL) compared to that achieved using 1 µL split injection (ratio 20:1). Ethyl (EtGB), isopropyl (GB), n-propyl (nPrGB), isobutyl (iBuGB), pinacolyl (GD), cyclohexyl (GF) methylphosphonofluoridates, and O-ethyl N, N-dimethylphosphoramidofluoridate (GAF) were detected with low LOD (15-75 pg/mL) and sharp peak shapes (high practical plate number (defined as 5.54 x (tR/Wh)2, where tR is the retention time and Wh is the bandwidth at half-height): 1100000-2400000) in GCMS using a polar separation column, electron ionization, and quadruple mass analyzer. During the analysis of fluoridated phosphonate-spiked ethyl acetate extract of solid phase extraction (SPE, Bond Elut NEXUS) from fluoride-mediated regeneration of blank human plasma, LOD (on EIC at m/z 99 except for GAF (m/z 126)) were 25-140 pg/mL with sharp peak shapes. The reaction recoveries in fluoride-mediated regeneration of plasma, which was inhibited by GB, GD, GA, GF, VX, and Russian VX (10 ng/mL), were 49-114% except for GD (10%). The concentration levels of 0.3-1 ng/mL of nerve agents in plasma could be determined.


Assuntos
Fluoretos/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Agentes Neurotóxicos/química , Organofosfonatos/sangue , Acetatos/química , Humanos , Compostos Organotiofosforados/química , Sarina/química , Extração em Fase Sólida , Soluções
4.
Comput Biol Chem ; 75: 74-81, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29747078

RESUMO

Organophosphorus nerve agents (NAs) irreversibly inhibit acetylcholinesterase (AChE), the enzyme responsible for breaking down the neurotransmitter acetylcholine (ACh). The over accumulation of ACh after NA exposure leads to cholinergic toxicity, seizure, and death. Current medical countermeasures effectively mitigate peripheral symptoms, however; the brain is often unprotected. Alternative acute treatment with the adenosine A1 receptor agonist N6-cyclopentyladensosine (CPA) has previously been demonstrated to prevent AChE inhibition as well as to suppress neuronal activity. The mechanism of AChE protection is unknown. To elucidate the feasibility of potential CPA-AChE interaction mechanisms, we applied a truncated molecular model approach and density functional theory. The candidate mechanisms studied are reversible enzyme inhibition, enzyme reactivation, and NA blocking prior to enzyme conjugation. Our thermodynamic data suggest that CPA can compete with the NAs sarin and soman for the active site of AChE, but may, in contrast to NAs, undergo back-reaction. We found a strong interaction between CPA and NA conjugated AChE, making enzyme reactivation unlikely but possibly allowing for CPA protection through the prevention of NA aging. The data also indicates that there is an affinity between CPA and unbound NAs. The results from this study support the hypothesis that CPA counters NA toxicity via multiple mechanisms and is a promising therapeutic strategy that warrants further development.


Assuntos
Acetilcolinesterase/metabolismo , Agonistas do Receptor A1 de Adenosina/metabolismo , Adenosina/análogos & derivados , Agentes Neurotóxicos/metabolismo , Compostos Organofosforados/metabolismo , Sarina/metabolismo , Soman/metabolismo , Adenosina/química , Adenosina/metabolismo , Adenosina/farmacologia , Agonistas do Receptor A1 de Adenosina/química , Agonistas do Receptor A1 de Adenosina/farmacologia , Animais , Estrutura Molecular , Agentes Neurotóxicos/química , Agentes Neurotóxicos/farmacologia , Compostos Organofosforados/química , Compostos Organofosforados/farmacologia , Teoria Quântica , Ratos , Sarina/química , Sarina/farmacologia , Soman/química , Soman/farmacologia , Termodinâmica
5.
J Mass Spectrom ; 53(2): 138-145, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29134720

RESUMO

Electron ionization mass spectrum of sarin(Se) was interpreted in compare of sarin MS spectrum. Inhibition of butyrylcholinesterase of human plasma by sarin and sarin(Se) was determined spectrophotometrically using modified Ellman method. It appeared that after incubation with sarin and sarin(Se), cholinesterase inhibition were 93% and 83%, respectively. Sarin, sarin(Se), and sarin(Se)-d7 were spiked into a vial containing human plasma, and albumin adduct metabolites were identified using liquid chromatography-tandem mass spectrometry. The experiments show that these agents are attached to tyrosine on albumin in human blood. Corresponding deuterated adducts were used to confirm the proposed mechanisms for the formation of the fragments in mass spectrometry experiments.


Assuntos
Proteínas Sanguíneas/química , Substâncias para a Guerra Química/química , Inibidores da Colinesterase/química , Compostos Organosselênicos/química , Sarina/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas Sanguíneas/metabolismo , Butirilcolinesterase/metabolismo , Substâncias para a Guerra Química/metabolismo , Inibidores da Colinesterase/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Ligação Proteica , Espectrometria de Massas em Tandem , Tirosina/química
6.
J Biomol Struct Dyn ; 36(13): 3444-3452, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29019446

RESUMO

The oximes 4-carbamoyl-1-[({2-[(E)-(hydroxyimino) methyl] pyridinium-1-yl} methoxy) methyl] pyridinium (known as HI-6) and 3-carbamoyl-1-[({2-[(E)-(hydroxyimino) methyl] pyridinium-1-yl} methoxy) methyl] pyridinium (known as HS-6) are isomers differing from each other only by the position of the carbamoyl group on the pyridine ring. However, this slight difference was verified to be responsible for big differences in the percentual of reactivation of acetylcholinesterase (AChE) inhibited by the nerve agents tabun, sarin, cyclosarin, and VX. In order to try to find out the reason for this, a computational study involving molecular docking, molecular dynamics, and binding energies calculations, was performed on the binding modes of HI-6 and HS-6 on human AChE (HssAChE) inhibited by those nerve agents.


Assuntos
Acetilcolinesterase/metabolismo , Substâncias para a Guerra Química/química , Inibidores da Colinesterase/química , Agentes Neurotóxicos/química , Oximas/metabolismo , Compostos de Pralidoxima/metabolismo , Compostos de Piridínio/metabolismo , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Organofosfatos/química , Compostos Organofosforados/química , Compostos Organotiofosforados/química , Sarina/química
7.
J Chromatogr A ; 1512: 71-77, 2017 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-28712549

RESUMO

A methodology for sensitive determination of sarin (GB), soman (GD) and cyclosarin (GF) chemical warfare agents in aqueous media was developed. The method incorporates direct derivatization with 2-[(dimethylamino)methyl]phenol (2-DMAMP), a commercially available, water-soluble reagent, followed by LC-ESI-MS/MS analysis in the positive ion mode. Five derivatization agents were characterized for their MS/MS fragmentation pattern, and their reaction time, temperature and derivatization-reagent amount were optimized. The developed derivatization reaction is simple, fast (1min) and proceeds at ambient temperature. Sample preparation consists of only the addition of the reagent directly into an injection vial prior to LC-ESI(+)-MS/MS analysis. All 2-DMAMP derivatives were stable for at least 48h and had unique tandem mass spectra characterized by common product ions at m/z 230 and 185. Compared with conventional GC-MS or LC-MS methods, simplicity, better sensitivity and informative MS/MS spectra were achieved by this method. Limits of detection (LODs), identification (LOIs), and quantification (LOQs) were determined in tap water and found to be 1pg/ml, 4pg/ml and 4pg/ml respectively. The proposed methodology is appropriate for routine evaluation of contaminated water supplies and has the advantage of a simultaneous analysis of both derivatized G-nerve agents and their intact hydrolysis products within a single LC-MS analysis.


Assuntos
Substâncias para a Guerra Química/química , Cromatografia Líquida/métodos , Agentes Neurotóxicos/química , Espectrometria de Massas em Tandem/métodos , Poluentes Químicos da Água/química , Cromatografia Gasosa-Espectrometria de Massas , Hidrólise , Limite de Detecção , Sarina/química , Soman/química , Espectrometria de Massas em Tandem/instrumentação
8.
J Biomol Struct Dyn ; 35(6): 1272-1282, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27125569

RESUMO

Butyryl cholinesterase (BChE) has been seen as a key enzyme in the search for new strategies in the treatment of poisoning by organophosphates (OPs), since human BChE (HssBChE), complexed with the appropriate oxime, can be a suitable scavenger and deactivator for OPs in the blood stream. However, the efficacy of HssBChE is limited by its strict stoichiometric scavenging, slow reactivation, and propensity for aging. The improvement of the reactivation rate by new and more efficient oximes could contribute to mitigate this problem and increase the HssBChE efficiency as scavenger. Several oximes have been synthesized and tested with this goal, some with promising results, but the mechanistic aspects of the reactivation reaction are not fully understood yet. In order to better investigate this mechanism, docking and mixed quantum and molecular mechanics combined with principal components analysis were performed here to evaluate the capacity of reactivation and determine the preferred route for the reactivation reaction of two new oximes on HssBChE inhibited by the neurotoxic agents cyclosarin and sarin. Plots of potential energies were calculated and all the transition states of the reactional mechanism were determined. Our results showed a good correlation with experimental data and pointed to the most efficient oxime with both OPs. The protocol used could be a suitable tool for a preliminary evaluation of the HssBChE reactivation rates by new oximes.


Assuntos
Butirilcolinesterase/química , Inibidores da Colinesterase/química , Reativadores da Colinesterase/química , Compostos Organofosforados/química , Oximas/química , Sarina/química , Inibidores da Colinesterase/farmacologia , Reativadores da Colinesterase/farmacologia , Humanos , Modelos Químicos , Modelos Moleculares , Compostos Organofosforados/farmacologia , Oximas/farmacologia , Ligação Proteica , Sarina/farmacologia
9.
Chem Res Toxicol ; 29(9): 1439-57, 2016 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-27509164

RESUMO

A mechanistic investigation has been carried out to explore all possible gas phase unimolecular isomerization as well as decomposition pathways of toxic organophosphorus compounds (OPCs), namely, sarin (GB) and soman (GD), which are better known as nerve agents. We have identified a total of 13 detoxication pathways for sarin, where the α-H, ß-H, and γ-H take part in the H-transfer process. However, for soman, due to the presence of ω-H, three additional detoxication pathways are obtained, where the ω-H is involved in the H-transfer process. Among all the pathways, the D3 decomposition pathway, where the phosphorus oxoacid derivative and alkene are generated via the formation of a six-membered ring in the transition state, is identified as the most feasible pathway from the perspective of both activation barrier and reaction enthalpy values. Moreover, we have studied the feasibility of the isomerization and decomposition pathways by performing the reaction kinetics in the temperature range of 300 K-1000 K using the one-dimensional Rice-Ramsperger-Kassel-Marcus (RRKM) master equation. From the RRKM calculation also, D3 pathway is confirmed as the most feasible pathway for both OPCs. The rate constant values associated with the D3 pathway within the temperature range of 600 K-700 K imply that the degradation of the OPCs is possible within this temperature range via the D3 pathway, which is in good agreement with the earlier reported experimental result. It is also observed that at higher temperature range (∼900 K), the increased rate constant values of other detoxication pathways indicate that along with D3, all other pathways become more or less equally feasible. Therefore, the entire work provides a widespread idea about the kinetic as well as thermodynamic feasibility of the explored detoxication pathways of the titled OPCs.


Assuntos
Sarina/metabolismo , Soman/metabolismo , Termodinâmica , Gases , Cinética , Estrutura Molecular , Transição de Fase , Sarina/química , Sarina/toxicidade , Soman/química , Soman/toxicidade
10.
Bioorg Med Chem ; 24(18): 4171-4176, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27450532

RESUMO

Previously (Karade et al., 2014), we have reported the synthesis and in vitro evaluation of bis-pyridinium derivatives of pyridine-3-yl-(2-hydroxyimino acetamide), as reactivators of sarin and VX inhibited hAChE. Few of the molecules showed superior in vivo protection efficacy (mice model) (Kumar et al., 2014; Swami et al., 2016) in comparison to 2-PAM against DFP and sarin poisoning. Encouraged by these results, herein we report the synthesis and in vitro evaluation of isonicotinamide derivatives of pyridine-3-yl-(2-hydroxyimino acetamide) (4a-4d) against sarin and VX inhibited erythrocyte ghost hAChE. Reactivation kinetics of these compounds was studied and the determined kinetic parameters were compared with that of commercial reactivators viz. 2-PAM and obidoxime. In comparison to 2-PAM and obidoxime, oxime 4a and 4b exhibited enhanced reactivation efficacy toward sarin inhibited hAChE while oxime 4c showed far greater reactivation efficacy toward VX inhibited hAChE. The acid dissociation constant and IC50 values of these oximes were determined and correlated with the observed reactivation potential.


Assuntos
Acetamidas/química , Aminopiridinas/química , Reativadores da Colinesterase/química , Niacinamida/análogos & derivados , Niacinamida/química , Oximas/química , Acetamidas/síntese química , Aminopiridinas/síntese química , Inibidores da Colinesterase/química , Reativadores da Colinesterase/síntese química , Membrana Eritrocítica/enzimologia , Humanos , Cinética , Niacinamida/síntese química , Cloreto de Obidoxima/química , Compostos Organotiofosforados/química , Oximas/síntese química , Compostos de Pralidoxima/química , Sarina/química
11.
Proc Natl Acad Sci U S A ; 113(20): 5514-9, 2016 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-27140636

RESUMO

Organophosphorus nerve agents interfere with cholinergic signaling by covalently binding to the active site of the enzyme acetylcholinesterase (AChE). This inhibition causes an accumulation of the neurotransmitter acetylcholine, potentially leading to overstimulation of the nervous system and death. Current treatments include the use of antidotes that promote the release of functional AChE by an unknown reactivation mechanism. We have used diffusion trap cryocrystallography and density functional theory (DFT) calculations to determine and analyze prereaction conformers of the nerve agent antidote HI-6 in complex with Mus musculus AChE covalently inhibited by the nerve agent sarin. These analyses reveal previously unknown conformations of the system and suggest that the cleavage of the covalent enzyme-sarin bond is preceded by a conformational change in the sarin adduct itself. Together with data from the reactivation kinetics, this alternate conformation suggests a key interaction between Glu202 and the O-isopropyl moiety of sarin. Moreover, solvent kinetic isotope effect experiments using deuterium oxide reveal that the reactivation mechanism features an isotope-sensitive step. These findings provide insights into the reactivation mechanism and provide a starting point for the development of improved antidotes. The work also illustrates how DFT calculations can guide the interpretation, analysis, and validation of crystallographic data for challenging reactive systems with complex conformational dynamics.


Assuntos
Acetilcolinesterase/química , Antídotos/química , Reativadores da Colinesterase/química , Agentes Neurotóxicos/química , Oximas/química , Compostos de Piridínio/química , Sarina/química , Cristalografia por Raios X , Cinética , Conformação Molecular
12.
J Chromatogr A ; 1450: 86-93, 2016 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-27179675

RESUMO

The terroristic availability of highly toxic nerve agents (NAs) highlights the necessity for a deep understanding of their toxicities and effective medical treatments. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for a characterization of the NAs poisoning and an evaluation on the efficacy of reactivators in in vitro was developed for the first time. After exposure to sarin or VX and pepsin digestion, the specific peptides of acetylcholinesterase (AChE) in a purified status, i.e. undecapeptide "GESAGAASVGM" in free, unaged, or aged status was identified and quantified. A key termination procedure is focused to make the reaction system "frozen" and precisely "capture" the poisoning, aging and spontaneous reactivation status of AChE, and the abundance of such specific peptides can thus be simultaneously measured. In our established method, as low as 0.72% and 0.84% inhibition level of AChE induced by 0.5nM sarin and VX can be detected from the measurement of peptide adducts, which benefits a confirmation of NAs exposure, especially at extremely low levels. Comparing with conventional colorimetric Ellman assays, our method provides not only enzyme activity and inhibition rate, but also the precise poisoning status of NAs exposed AChE. Based on the full information provided by this method, the efficacy of reactivators, such as HI-6, obidoxime and pralidoxime, in the typical treatment of NAs poisoned AChE in in vitro was further evaluated. Our results showed that this method is a promising tool for the characterization of NAs poisoning and the evaluation of reactivator efficacy.


Assuntos
Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Reativadores da Colinesterase/farmacologia , Agentes Neurotóxicos/farmacologia , Agentes Neurotóxicos/envenenamento , Fragmentos de Peptídeos/análise , Espectrometria de Massas em Tandem/métodos , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/envenenamento , Cromatografia Líquida , Colorimetria , Ativação Enzimática/efeitos dos fármacos , Humanos , Técnicas In Vitro , Agentes Neurotóxicos/química , Cloreto de Obidoxima/farmacologia , Oximas/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Compostos de Pralidoxima/farmacologia , Compostos de Piridínio/farmacologia , Sarina/química , Sarina/farmacologia
13.
Chemistry ; 22(32): 11138-42, 2016 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-27124609

RESUMO

Test strips that in combination with a portable fluorescence reader or digital camera can rapidly and selectively detect chemical warfare agents (CWAs) such as Tabun (GA), Sarin (GB), and Soman (GD) and their simulants in the gas phase have been developed. The strips contain spots of a hybrid indicator material consisting of a fluorescent BODIPY indicator covalently anchored into the channels of mesoporous SBA silica microparticles. The fluorescence quenching response allows the sensitive detection of CWAs in the µg m(-3) range in a few seconds.


Assuntos
Compostos de Boro/química , Substâncias para a Guerra Química/química , Agentes Neurotóxicos/química , Organofosfatos/química , Tempo de Protrombina/métodos , Sarina/química , Dióxido de Silício/química , Soman/química , Substâncias para a Guerra Química/análise , Agentes Neurotóxicos/análise , Organofosfatos/análise , Sarina/análise , Soman/análise
14.
Chem Biol Interact ; 259(Pt B): 133-141, 2016 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-27062893

RESUMO

Currently fielded treatments for nerve agent intoxication include atropine, an acetylcholine receptor antagonist, and pralidoxime (2PAM), a small molecule reactivator of acetylcholinesterase (AChE). 2PAM reactivates nerve agent-inhibited AChE via direct nucleophilic attack by the oxime moiety on the phosphorus center of the bound nerve agent. Due to a permanently charged pyridinium motif, 2PAM is not thought to cross the blood brain barrier and therefore cannot act directly in the neuronal junctions of the brain. In this study, ADOC, a non-permanently charged, non-oxime molecule initially identified using pesticide-inhibited AChE, was characterized in vitro against nerve agent-inhibited recombinant human AChE. The inhibitory and reactivation potentials of ADOC were determined with native AChE and AChE inhibited with tabun, sarin, soman, cyclosarin, VX, or VR and then compared to those of 2PAM. Several structural analogs of ADOC were used to probe the reactivation mechanism of the molecule. Finally, guinea pigs were used to examine the protective efficacy of the compound after exposure to sarin. The results of both in vitro and in vivo testing will be useful in the design of future small molecule reactivators.


Assuntos
Acetilcolinesterase/metabolismo , Reativadores da Colinesterase/metabolismo , Agentes Neurotóxicos/metabolismo , Oximas/química , Fenóis/metabolismo , Compostos de Pralidoxima/metabolismo , Acetilcolinesterase/química , Acetilcolinesterase/genética , Animais , Reativadores da Colinesterase/química , Reativadores da Colinesterase/uso terapêutico , Eritrócitos/enzimologia , Cobaias , Meia-Vida , Humanos , Cinética , Masculino , Agentes Neurotóxicos/química , Agentes Neurotóxicos/envenenamento , Intoxicação por Organofosfatos/tratamento farmacológico , Organofosfatos/química , Organofosfatos/metabolismo , Fenóis/química , Fenóis/uso terapêutico , Compostos de Pralidoxima/química , Compostos de Pralidoxima/uso terapêutico , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Sarina/química , Sarina/metabolismo , Soman/química , Soman/metabolismo , Relação Estrutura-Atividade
15.
Phys Chem Chem Phys ; 18(14): 9838-46, 2016 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-27000635

RESUMO

The main treatment for organophosphorus (OP) compound poisoning in clinics is to restore the activity of acetylcholinesterase (AChE) through oxime-induced reactivation of the phosphorylated OP-AChE adduct. It suffers from a competitive and irreversible aging reaction of the phosphorylated OP-AChE adduct, resulting in permanent inactivity of AChE. However, it was recently reported that N-methyl-2-methoxypyridinium species can act as methylating agents to methylate the methyl methane-phosphonate monoanion, in which the reaction mimics the reverse of the aging reaction of the phosphorylated OP-AChE adduct. If the aging reaction could be really reversed, the efficiency for the OP detoxification should be significantly improved, bringing up the possibility to develop an agent to reverse the aging process of the phosphorylated OP-AChE adduct. However, such a reaction with the N-methyl-2-methoxypyridinium species in the enzyme is still not reported so far. It is of great interest to know whether or not this reaction is observable in the enzyme, and more importantly, if it turns out to be not observable in the enzyme, why such a reaction proceeds quickly in aqueous solution but not in the enzyme. In the present study, we performed DFT calculations and quantum mechanical/molecular mechanical (QM/MM) calculations to reveal the fundamental mechanism for the methylation of both the methyl methane-phosphonate monoanion and the aged sarin-AChE adduct by N-methyl-2-methoxypyridinium species, respectively. The obtained results support the SN2 reaction mechanism, not the stepwise mechanism, for the methylation of the methyl methane-phosphonate monoanion by 9 reported N-methyl-2-methoxypyridinium compounds. The calculated free energy barriers are in good agreement with the experimental data. The methylation of the aged sarin-AChE adduct by one N-methyl-2-methoxypyridinium compound (labeled as compound 2) also employs the SN2 reaction mechanism with an extremely high free energy barrier of 30.4 ± 3.5 (or 26.6) kcal mol(-1), implying that this reaction in the enzyme hardly occurs. Our results clearly show that compound 2 forms a strong π-π stacking interaction with the aromatic ring of the W86 residue of AChE, making itself unable to approach sarin for the reverse of the aging process. On the basis of the structure and mechanism, several possible strategies have been suggested for designing methylating agents with higher activity against the aged sarin-AChE adduct.


Assuntos
Acetilcolinesterase/química , Inibidores da Colinesterase/toxicidade , Metilação/efeitos dos fármacos , Compostos de Piridínio/química , Compostos de Piridínio/farmacologia , Sarina/antagonistas & inibidores , Sarina/toxicidade , Animais , Antídotos/química , Antídotos/farmacologia , Inibidores da Colinesterase/química , Humanos , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Organofosfonatos/química , Sarina/química , Relação Estrutura-Atividade
16.
Arch Toxicol ; 90(3): 603-16, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25743373

RESUMO

The hydroxyl oxygen of the catalytic triad serine in the active center of serine hydrolase acetylcholinesterase (AChE) attacks organophosphorus compounds (OPs) at the phosphorus atom to displace the primary leaving group and to form a covalent bond. Inhibited AChE can be reactivated by cleavage of the Ser-phosphorus bond either spontaneously or through a reaction with nucleophilic agents, such as oximes. At the same time, the inhibited AChE adduct can lose part of the molecule by progressive dealkylation over time in a process called aging. Reactivation of the aged enzyme has not yet been demonstrated. Here, our goal was to study oxime reactivation and aging reactions of human AChE inhibited by mipafox or a sarin analog (Flu-MPs, fluorescent methylphosphonate). Progressive reactivation was observed after Flu-MPs inhibition using oxime 2-PAM. However, no reactivation was observed after mipafox inhibition with 2-PAM or the more potent oximes used. A peptide fingerprinted mass spectrometry (MS) method, which clearly distinguished the peptide with the active serine (active center peptide, ACP) of the human AChE adducted with OPs, was developed by MALDI-TOF and MALDI-TOF/TOF. The ACP was detected with a diethyl-phosphorylated adduct after paraoxon inhibition, and with an isopropylmethyl-phosphonylated and a methyl-phosphonylated adduct after Flu-MPs inhibition and subsequent aging. Nevertheless, nonaged nonreactivated complexes were seen after mipafox inhibition and incubation with oximes, where MS data showed an ACP with an NN diisopropyl phosphoryl adduct. The kinetic experiments showed no reactivation of activity. The computational molecular model analysis of the mipafox-inhibited hAChE plots of energy versus distance between the atoms separated by dealkylation showed a high energy demand, thus little aging probability. However, with Flu-MPs and DFP, where aging was observed in our MS data and in previously published crystal structures, the energy demand calculated in modeling was lower and, consequently, aging appeared as a more likely reaction. We document here direct evidence for a phosphorylated hAChE refractory to oxime reactivation, although we observed no aging.


Assuntos
Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacocinética , Isoflurofato/análogos & derivados , Sarina/análogos & derivados , Sequência de Aminoácidos , Domínio Catalítico , Reativadores da Colinesterase/química , Reativadores da Colinesterase/farmacologia , Humanos , Isoflurofato/química , Isoflurofato/farmacocinética , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Compostos Organofosforados/química , Compostos Organofosforados/farmacocinética , Oximas/química , Paraoxon/farmacocinética , Fosforilação , Conformação Proteica , Sarina/química , Serina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
J Biomol Struct Dyn ; 34(12): 2632-2642, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26612005

RESUMO

In the present work, we performed docking and molecular dynamics simulations studies on two groups of long-tailored oximes designed as peripheral site binders of acetylcholinesterase (AChE) and potential penetrators on the blood brain barrier. Our studies permitted to determine how the tails anchor in the peripheral site of sarin-inhibited human AChE, and which aminoacids are important to their stabilization. Also the energy values obtained in the docking studies corroborated quite well with the experimental results obtained before for these oximes.


Assuntos
Acetilcolinesterase/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Oximas/química , Sarina/química , Acetilcolinesterase/metabolismo , Sítios de Ligação , Humanos , Ligação de Hidrogênio , Ligantes , Conformação Molecular , Oximas/farmacologia , Ligação Proteica , Sarina/farmacologia
18.
Bioorg Med Chem ; 23(15): 4899-910, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26043948

RESUMO

Presently available medications for treatment of organiphosphorus poisoning are not sufficiently effective due to various pharmacological and toxicological reasons. In this regard, herein we report the synthesis of a series of N-thiazolylacetamide monoquaternary pyridinium oximes and its analogs (1a-1b to 6a-6b) with diversely substituted thiazole ring and evaluation of their in vitro reactivation efficacies against nerve agent (sarin, O-ethylsarin and VX) inhibited human erythrocyte acetylcholinesterase (hAChE). Reactivation kinetics was performed to determine dissociation constant (KD), reactivity rate constant (kr) and the second order rate constant (kr2) for all the compounds and compared their efficacies with commercial antidotes viz. 2-PAM and obidoxime. All the newly synthesized oximes were evaluated for their physicochemical parameters (pKa) and correlated with their respective reactivation efficacies to assess the capability of the oxime reactivator. Three of these novel compounds showed promising reactivation efficacies toward OP inhibited hAChE. Molecular docking studies were performed in order to correlate the reactivation efficacies with their interactions in the active site of the AChE.


Assuntos
Acetilcolinesterase/química , Substâncias para a Guerra Química/química , Reativadores da Colinesterase/síntese química , Oximas/química , Acetilcolinesterase/metabolismo , Sítios de Ligação , Domínio Catalítico , Substâncias para a Guerra Química/metabolismo , Reativadores da Colinesterase/química , Reativadores da Colinesterase/metabolismo , Humanos , Cinética , Simulação de Acoplamento Molecular , Compostos Organotiofosforados/química , Compostos Organotiofosforados/metabolismo , Oximas/síntese química , Oximas/metabolismo , Compostos de Piridínio/química , Sarina/análogos & derivados , Sarina/química , Sarina/metabolismo , Tiazóis/química
19.
J Occup Environ Hyg ; 12(2): 130-7, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25192468

RESUMO

The effects of an eight-year natural aging of ASC impregnated activated carbon on the adsorption capacity and breakthrough times of model organic vapors and of the nerve agent sarin were investigated. Aging delayed methanol breakthrough from dry air on pre-dried carbon, but shortened the breakthrough time of both methanol and hexane under relative humidity (RH) of 30-85% on pre-humidified carbon. Aging also shortened the breakthrough time of the less volatile model compound 2-methoxyethanol, especially under RH of 60-85%. Aging significantly reduced the protection capacity against sarin at RH of 85%. The effects of aging on physisorption are attributed to enhanced hydrogen-bonding capability and strength of the interaction between water and adsorption sites on the carbon surface.


Assuntos
Poluentes Atmosféricos/química , Carbono/química , Substâncias Perigosas/química , Modelos Químicos , Compostos Orgânicos/química , Adsorção , Poluentes Ocupacionais do Ar/química , Substâncias para a Guerra Química/química , Umidade , Dispositivos de Proteção Respiratória , Sarina/química , Fatores de Tempo
20.
Chirality ; 26(12): 817-24, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25298066

RESUMO

Chemical warfare nerve agents (CWNAs) are extremely toxic organophosphorus compounds that contain a chiral phosphorus center. Undirected synthesis of G-type CWNAs produces stereoisomers of tabun, sarin, soman, and cyclosarin (GA, GB, GD, and GF, respectively). Analytical-scale methods were developed using a supercritical fluid chromatography (SFC) system in tandem with a mass spectrometer for the separation, quantitation, and isolation of individual stereoisomers of GA, GB, GD, and GF. Screening various chiral stationary phases (CSPs) for the capacity to provide full baseline separation of the CWNAs revealed that a Regis WhelkO1 (SS) column was capable of separating the enantiomers of GA, GB, and GF, with elution of the P(+) enantiomer preceding elution of the corresponding P(-) enantiomer; two WhelkO1 (SS) columns had to be connected in series to achieve complete baseline resolution. The four diastereomers of GD were also resolved using two tandem WhelkO1 (SS) columns, with complete baseline separation of the two P(+) epimers. A single WhelkO1 (RR) column with inverse stereochemistry resulted in baseline separation of the GD P(-) epimers. The analytical methods described can be scaled to allow isolation of individual stereoisomers to assist in screening and development of countermeasures to organophosphorus nerve agents.


Assuntos
Substâncias para a Guerra Química/isolamento & purificação , Cromatografia com Fluido Supercrítico/métodos , Substâncias para a Guerra Química/análise , Substâncias para a Guerra Química/química , Espectrometria de Massas , Organofosfatos/análise , Organofosfatos/química , Organofosfatos/isolamento & purificação , Compostos Organofosforados/análise , Compostos Organofosforados/química , Compostos Organofosforados/isolamento & purificação , Sarina/análise , Sarina/química , Sarina/isolamento & purificação , Soman/análise , Soman/química , Soman/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , Estereoisomerismo
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