In silico approaches to evaluate the molecular properties of organophosphate compounds to inhibit acetylcholinesterase activity in housefly

Organophosphate compounds (OPC) have become the primary choice as insecticides and are widely used across the world. Additionally, OPCs were also commonly used as a chemical warfare agent that triggers a great challenge to public safety. Exposure of OPCs to human causes immediate excitation of cholinergic neurotransmission through transient elevation of synaptic acetylcholine (ACh) levels and accumulations. Likewise, prolonged exposure of OPCs can affect the processes in immune response, carbohydrate metabolism, cardiovascular toxicity, and several others. Studies revealed that the toxicity of OPCs was provoked by inhibition of acetylcholinesterase (AChE). Therefore, combined in silico approaches – pharmacophore-based 3D-QSAR model; docking and Molecular Dynamics (MD) – were used to assess the precise and comprehensive effects of series of known OP-derived compounds together with its ?log LD₅₀ values. The selected five-featured pharmacophore model – AAHHR.61 – displayed the highest correlation (R² = .9166), cross-validated coefficient (Q² = .8221), F = 63.2, Pearson-R = .9615 with low RMSE = .2621 values obtained using five component PLS factors. Subsequently, the well-validated model was then used as a 3D query to search novel OPCs using a high-throughput virtual screening technique. Simultaneously, the docking studies predicted the binding pose of the most active OPC in the MdAChE binding pocket. Additionally, the stability of docking was verified using MD simulation. The results revealed that OP22 and predicted lead compounds bound tightly to S315 of MdAChE through potential hydrogen bond interaction over time. Overall, this study might provide valuable insight into binding mode of OPCs and hit compounds to inhibit AChE in housefly.     

Non-calcium dependent activity hydrolysing organophosphorus compounds in hen plasma

O-Hexyl O-25, dichlorophenyl phosphoramidate (HDCP) is a chiral compound that induces delayed neuropathy in hens. The chicken has very low activity of Ca-dependent organophosphorus-hydrolases (OP-hydrolases) such as paraoxonase. HDCP is degraded at a similar rate in rat and hen plasma (16 and 21 nmol/min/μl plasma, respectively) when measured by the loss of its anti-cholinesterase potency (Díaz-Alejo et al., 1990). The time course of the HDCP hydrolysis was not significantly affected by the following treatments: (a) 0.5–1 mM Ca²⁺ or 1–10 mM EDTA added at 30 min before starting the reaction at 37°C; (b) preincubation with a carboxylesterase inhibitor 100 μM diisopropyl phosphoros-fluoridate (DFP) for 60 min at 37°C; (c) preincubation with 100 μM HDCP for 60 min at 37°C; and (d) the presence of 50 μM DCP. However, the hydrolysis of HDCP was slightly modified by the other product of its hydrolysis. There is no contribution to the HDCP hydrolysis by covalent binding to carboxylesterase proteins. The course of the hydrolysis of HDCP was similar when measured by either the loss of anti-cholinesterase potency or the DCP liberated. HDCP is hydrolysed by an OP-hydrolase which is not Ca-dependent and is present in hen in contrast to the best known OP-hydrolases which are Ca-dependent and are undetectable in birds.     

Two types of sesamex-suppressible resistance in the housefly

In a resistant strain of housefly, Fc, derived from a Danish strain, DDT resistance and most of the diazinon resistance are under the control of the third chromosome. Resistance to both compounds is suppressed by sesamex. These two facts indicate that there is a single mechanism responsible for resistance to these (and many other) compounds. It appears that in this strain DDT is rapidly metabolized and that water soluble products are excreted. This metabolism is blocked by sesamex.A Dutch strain with high resistance to dithion did not show the low aliesterase activity characteristic of most organophosphate-resistant strains. Therefore a resistance mechanism different from that in most other strains was expected. The fact that sesamex can suppress the dithion resistance indicates that a detoxication mechanism that can be blocked by this synergist is responsible for the resistance. The gene for this resistance is on the fifth chromosome.

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Zusammenfassung In einem resistenten Stubenfliegenstamm, Fc, der aus einem dänischen Stamm hervorgegangen ist, wird die DDT-Resistenz und der grösste Teil der Diazinon-Resistenz von dem dritten Chromosom kontrolliert. Die Resistenz gegen beide Verbindungen wird durch Sesamex verringert. Diese beiden Tatsachen zeigen, dass für die Resistenz gegen diese (und viele andere) Stoffe ein einziger Mechanismus verantwortlich ist. Es ergibt sich, dass in diesem Stamm DDT schnell abgebaut wird und dass wasserlösliche Produkte ausgeschieden werden. Dieser Abbau wird durch Sesamex blockiert.Ein holländischer Stamm mit hoher Resistenz gegen Dithion zeigt die für die meisten organophosphatresistenten Stämme charakteristische niedrige Aliesterase-Aktivität nicht. Daher wurde ein anderer Resistenzmechanismus als in den meisten anderen Stämmen erwartet. Die Tatsache, dass Sesamex die Dithionresistenz senken kann, zeigt, dass ein Mechanismus für die Resistenz verantwortlich ist, der durch diesen Synergisten blockiert werden kann. Das Gen für diese Resistenz befindet sich auf dem fünften Chromosom.

     

Biochemistry and genetics of esterases in houseflies (Musca domestica) with special reference to the development of resistance to organophosphorus compounds

The paper reviews and reconsiders the experimental work on esterases in the housefly performed in the Laboratory for Research on Insecticides in Wageningen (formerly in Utrecht). It is attempted to integrate the different aspects of former studies, adding new data where necessary and possible. Two subjects that are intimately connected with esterases are briefly discussed, viz. the mode of action of organophosphorus insecticides and the mechanism of resistance to these compounds. A discussion on the possible functions of the esterases studied concludes the paper.

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Zusammenfassung Die Arbeit gibt eine Übersicht über die experimentellen Untersuchungen an Esterasen der Stubenfliege, die im Laboratorium für Insektizid-Forschung durchgeführt wurden. Es wurde versucht, die verschiedenen Aspekte früherer Arbeiten zu integrieren; nötigenfalls sind neue Daten beigefügt. Zwei Probleme, die sehr eng mit der Aktivität von Esterasen verknüpft sind, nämlich der Wirkungsmechanismus der Phosphorsäureester und die Resistenz gegen diese Substanzen werden ebenfalls besprochen. Eine Betrachtung über die Funktion der Esterasen beschließt die Arbeit.

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The material presented here was presented originally at the Symposium on the Function of Esterases in Animals, University of Recife, Recife, Pernambuco, Brazil, September 8–12. 1963.

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Deceased on October 3, 1964.     

Mutant of Bungarus fasciatus acetylcholinesterase with low affinity and low hydrolase activity toward organophosphorus esters

Enzymes hydrolysing highly toxic organophosphate esters (OPs) are promising alternatives to pharmacological countermeasures against OPs poisoning. Bungarus fasciatus acetylcholinesterase (BfAChE) was engineered to acquire organophosphate hydrolase (OPase) activity by reproducing the features of the human butyrylcholinesterase G117H mutant, the first mutant designed to hydrolyse OPs. The modification consisted of a triple mutation on the (122)GFYS(125) peptide segment, resulting in (122)HFQT(125). This substitution introduced a nucleophilic histidine above the oxyanion hole, and made space in that region. The mutant did not show inhibition by excess acetylthiocholine up to 80 mM. The k(cat)/K(m) ratio with acetylthiocholine was 4 orders of magnitude lower than that of wild-type AChE. Interestingly, due to low affinity, the G122H/Y124Q/S125T mutant was resistant to sub-millimolar concentrations of OPs. Moreover, it had hydrolysing activity with paraoxon, echothiophate, and diisopropyl phosphofluoridate (DFP). DFP was characterised as a slow-binding substrate. This mutant is the first mutant of AChE capable of hydrolysing organophosphates. However, the overall OPase efficiency was greatly decreased compared to G117H butyrylcholinesterase.     

Genetics and evolution of resistance to insecticides

The evolution of resistance to insecticides has become a serious problem world-wide. It is important to identify patterns of insecticide use whereby insecticides can be used in integrated pest management programmes to help control insect numbers, but in such a manner that the evolution of resistance to insecticides will be retarded. The principal mechanisms of insecticide action and of resistance to these are reviewed. Some generalizations that can be made about the evolution of resistance are examined. In general, to control resistance it appears better to use an intense dose of non-persistent pesticides over a circumscribed area. Some features of the problem where population genetics and evolutionary theory might contribute to controlling resistance are discussed.     

Studies on the biochemical basis of susceptibility and resistance of the housefly to fluoroacetate

1. Administration of fluoroacetate to sensitive houseflies in amounts close to the L.D.₅₀ range (0.25–0.3 μg/fly) brought about a prompt elevation of their citrate content. With about 10-fold higher doses of fluoroacetate a concurrent increase of both citrate and pyruvate levels took place in the fly tissues.

2. Incubation of sarcosomes of the sensitive housefly strain in the presence of oxidizable substrates and fluoroacetate resulted in accumulation of citrate, inhibition of respiration and uncoupling of oxidative phosphorylation. The magnitude of the effects varied considerably with the different substrates used, being particularly pronounced with pyruvate and malate and inappreciable with succinate and -glycerophosphate.

3. The respiratory inhibition induced by a brief exposure in the cold of housefly sarcosomes to fluoroacetate, persisted after the sarcosomes had been washed free from fluoroacetate. The toxic effect of fluoroacetate on the respiratory chain could be prevented by an excess of simultaneously added acetate.

4. The susceptibility of the respiratory function of the sarcosomes to fluoroacetate inhibition was abolished by sonication. The unresponsiveness of the sonicated sarcosomes to fluoroacetate was attended by a loss of their respiratory chain phosphorylation activity.

5. Sarcosomes derived from a partially resistant housefly strain, when incubated in the presence of fluoroacetate, failed to accumulate citrate, but displayed the characteristic respiratory-inhibition response. Sarcosomes from a highly resistant strain showed no impairment of their functional capacity by fluoroacetate. However, all the different housefly strains tested proved to be equally sensitive to the deleterious effect of fluorocitrate on sarcosomal respiration.

6. The possible biochemical mechanisms underlying the toxicity of fluoroacetate in the housefly are considered with particular reference to the altered response of the target systems exhibited by the fluoroacetate-resistant strains.     

Genetics of resistance to tetraethyllead.

A Drosophila melanogaster population was exposed for 25 generations to 60 micrograms tetraethyllead per gram of medium. Selection over this period resulted in an increase in fecundity, hatchability and larva-to-adult viability. Chromosome assay showed that response in these traits was generally under additive genetic control in conformity with existing results in the literature on the genetics of resistance to acute environmental stress in D. melanogaster.     

Genetics of susceptibility and resistance to disease in fishes

The genetics of disease resistance in fish is examined. Major genes may cause skeletal or other phenotypic deformities and have also been implicated in the development of neoplasia. Others may affect viability through pleiotropic effects, or induce stress and therefore predispose to invasion by pathogens. Resistance usually appears, however, to be of a multifactorial nature. Variations in resistance between related species and between populations of the same species are described. In order to obtain an idea of the within-population variation, heritability estimates have been obtained in some instances. Generally they are low, but may still indicate a sufficient level to be of use in a selection programme. The results of a limited number of selection programmes are described.     

DDT resistance in the housefly caused by microsomal degradation

In vitro microsomal detoxication of DDT was studied in five strains of houseflies. One of these, Fc, is resistant to DDT and diazinon, and this resistance can be overcome by sesamex. Microsomes of this strain degraded DDT at a rate of 0.5 g per fly per hour to at least four products of higher polarity. Resistance in this strain is due to a gene on the 3^rd chromosome, which we have called DDT-md (microsomal detoxication). Evidence is presented that some of the products from DDT are produced in more than one step, and that primary and secondary reactions occur in microsomes of strain Fc only and are CO sensitive. In the other strains tested there was only a much lower rate of detoxication. No evidence was found in Fc flies of an increased detoxication by microsomal induction with DDT.

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Zusammenfassung Entgiftung von DDT durch Mikrosomen in vitro wurde bei 5 Stubenfliegen-Stämmen studiert. Einer von ihnen, Fc, ist resistent gegen DDT und Diazinon; diese Resistenz kann durch Sesamex überwunden werden. Mikrosomen dieses Stammes bauten DDT mit einer Rate von 0,5 mg pro Fliege und Stunde zu wenigstens 4 Produkten höherer Polarität ab. Die Resistenz beruht in diesem Stamme auf einem Gen im dritten Chromosom, welches wir DDT-md (microsomal detoxication) genannt haben. Es wird wahrscheinlich gemacht, daß einige der Produkte aus DDT in mehr als einem Schritt erzeugt werden und daß primäre und sekundäre Reaktionen nur in Mikrosomen des Fc-Stammes auftreten und CO-empfindlich sind. In anderen geprüften Stämmen ergab sich nur eine geringere Entgiftungsrate. In Fc-Fliegen wer kein Nachweis für eine verstärkte Entgiftung durch mikrosomale Induktion mit DDT zu erbringen.

     

A liver arylamidase extremely sensitive to organophosphorus compounds

Inhibitory effect of 7 organophosphorus compounds on the liver isocarboxazid amidase was studied in rats. This enzyme is more sensitive to the relatively less toxic compounds, i.e., Sumithion^®, malathion and TOTP than to the more toxic ones susch as parathion, paraoxon and EPN. As little as 0.5 mg/kg of Sumithion inhibited this arylamidase activity by approx. 28% in male and 37% in female rats. TOTP completely inhibited the arylamidase in a dose of 20 mg/kg, moreover, a significant sex difference in the TOTP-induced inhibition of the enzyme was observed.     

Microbial degradation of organophosphorus compounds

Synthetic organophosphorus compounds are used as pesticides, plasticizers, air fuel ingredients and chemical warfare agents. Organophosphorus compounds are the most widely used insecticides, accounting for an estimated 34% of world-wide insecticide sales. Contamination of soil from pesticides as a result of their bulk handling at the farmyard or following application in the field or accidental release may lead occasionally to contamination of surface and ground water. Several reports suggest that a wide range of water and terrestrial ecosystems may be contaminated with organophosphorus compounds. These compounds possess high mammalian toxicity and it is therefore essential to remove them from the environments. In addition, about 200,000 metric tons of nerve (chemical warfare) agents have to be destroyed world-wide under Chemical Weapons Convention (1993). Bioremediation can offer an efficient and cheap option for decontamination of polluted ecosystems and destruction of nerve agents. The first micro-organism that could degrade organophosphorus compounds was isolated in 1973 and identified as Flavobacterium sp. Since then several bacterial and a few fungal species have been isolated which can degrade a wide range of organophosphorus compounds in liquid cultures and soil systems. The biochemistry of organophosphorus compound degradation by most of the bacteria seems to be identical, in which a structurally similar enzyme called organophosphate hydrolase or phosphotriesterase catalyzes the first step of the degradation. organophosphate hydrolase encoding gene opd (organophosphate degrading) gene has been isolated from geographically different regions and taxonomically different species. This gene has been sequenced, cloned in different organisms, and altered for better activity and stability. Recently, genes with similar function but different sequences have also been isolated and characterized. Engineered microorganisms have been tested for their ability to degrade different organophosphorus pollutants, including nerve agents. In this article, we review and propose pathways for degradation of some organophosphorus compounds by microorganisms. Isolation, characterization, utilization and manipulation of the major detoxifying enzymes and the molecular basis of degradation are discussed. The major achievements and technological advancements towards bioremediation of organophosphorus compounds, limitations of available technologies and future challenge are also discussed.     

Mutagenicity of organophosphorus compounds in bacteria and drosophila

140 Organophosphorus compounds (OP's) have been tested for mutagenic activity in bacteria, principally by using two specially constructed sets of tester strains of the bacteria Salmonella typhimurium and Escherichia coli. It was found that 20% gave positive mutagenic responses and that this group of chemicals produce base substitutions rather than frame-shift mutations. In most cases the DNA repair genes exrA⁺ and recA⁺ were for mutagenic activity.Seven compounds were further tested in Drosophila melanogaster for the ability to induce recessive lethal mutations. In some of these cases the doses administered to the flies had to be very low due to the highly toxic nature of the compounds. To overcome this problem, the accumulation of recessive lethal mutations was measured in populations which were continually exposed to the compounds over a period of some 18 months. During this time the populations developed increased resistance to the compound and so the dose administered could gradually be increased. Six of the compounds were mutagenic.Of the compounds tested in both systems, those showing mutagenic activity in bacteria were also mutaganic in Drosophila, those mutagenic in bacteria were not mutagenic in Drosophila.     

From metabolism to comparative biochemistry of toxic organophosphorus compounds

The main stages of scientific biography of Professor Viktor Iosifovich Rozengart are exposed: his works on muscle bioenergetics, discovery of the pathway of synthesis of creatinine, his development of new concepts of pathways of metabolism of organophosphorus xenobiotics, creation of biochemical grounds of selective toxicity as well as studies in the new field, of which he was one of founders,—comparative biochemistry of toxic organophosphorus compounds.     

Anticholinesterase activity (in the Mediterranean fruit fly) and mouse toxicity of some new organophosphorus compounds

Phenyl and thienyl substituted phosphinates and phosphonates, analogues of dichlorvos, trichlorfon and naled, were examined for their in vitro anticholinesterase activities to heads of the Mediterranean fruit fly, and for toxicities to mice. The data obtained are evaluated in comparison with toxicity results by topical application to adult flies.

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Résumé Les auteurs ont étudié la corrélation entre la toxicité de quelques nouveaux composés organo-phosphoriques envers les adultes de la mouche des fruits (Ceratitis capitata) d'une part, et leur pouvoir inhibiteur (in vitro) sur la cholinestérase de la tête et leur toxicité envers les souris d'autre part.Les composés étudiés sont les analogues phényl- et thiénylphosphoniques et-phosphiniques de dichlorvos (DDVP), trichlorfon (Dipterex) et naled (Dibrom).Tandis que les insecticides les plus puissants — dichlorvos, O-méthyl O-(2,2-dichlorovinyl) phénylphosphonate et naled — furent aussi les inhibiteurs les plus efficaces de la cholinestérase, les auteurs n'ont pas trouvé une pareille corrélation pour les autres composés. Ainsi, certains étaient assez bons comme insecticides par application topicale mais possédaient seulement un faible effect sur la cholinestérase. Contrairement, certains anti-cholinestératiques assez puissants ne montraient qu'un faible pouvoir insecticide envers les mouches adultes. Les nouveaux composés étaient cependant généralement plus actifs dans les essais in vivo que dans ceux in vitro.Une augmentation du poids moléculaire dans une série entraine aussi une perte d'activité. L'analogue thiényl-phosphonique du dichlorvos offre une exception intéressante.Les auteurs n'ont pas trouvé une corrélation entre le pouvoir anticholinestératique de ces composés et leur toxicité envers les souris.

     

Physiological activity of some organophosphorus compounds and their effects on the physico-chemical properties of model membranes

The effect of the newly synthesized phosphonic compound dibutyl 2-octylamino-2-propanephosphonate (DBOP) on the growth of the aquatic plant Spirodela oligorrhiza and stability of red blood cells (RBC) and planar lipid membranes (BLM) was studied to determine its physiological activity and, if possible, correlate this activity to compound-induced changes in the mechanical properties of the model membranes. The measure of the phytotoxicity was the DBOP concentration causing 50% plant growth retardation, while measures of stability of model membranes were 100% hemolysis of RBC and a critical concentration of DBOP causing BLM destruction in no more that 3 min. These data were compared with those for dibutyl 1-butylamino-1-cyclohexanephosphonate (DBBC) and diethyl 9-butylamino-9-fluorenephosphonate (DEBF) known for their physiological activities. Both DBBC and DEBF influenced Spirodela growth significantly less than DBOP Destabilization of the model membrane caused by DBBC and DBOP was similar whereas DEBF exerted a weak influence on RBC and BLM stability. The results indicate that the physiological activities of DBOP and DEBF are not limited to the lipid phase of biological membranes and may involve also disturbance of metabolic processes.     

A microassay-based procedure for measuring low levels of toxic organophosphorus compounds through acetylcholinesterase inhibition

Using a microtiter plate spectrophotometric system, an assay procedure was developed for the following toxic organophosphorus compounds: 1,2,2-trimethylpropyl ester of methylphosphonofluoridic acid (1, soman); ethyl N,N-dimethylphosphoramidocyanidate (3, tabun); O-ethyl S-[2-[bis(1-methylethyl)amino]ethyl]- methylphosphonothiolate (4, VX); the diethyl 4-nitrophenyl ester of phosphoric acid (5, paraoxon); and bis(1-methylethyl) phosphorofluoridate (6, DFP). The procedure, based on the Ellman assay method, uses inhibition of eel acetylcholinesterase (0.01 unit per well) to carry out the determination of inhibitor concentrations for both a standard curve and the unknown samples on a single 96-well microtiter plate. On a typical plate, samples of both unknowns and standards (a minimum of six concentrations were used per standard curve) were assayed five times per sample, with three control (uninhibited) enzyme activity points included for each sample. The time required for carrying out a single plate was approx 30 min. Sensitivity for the most potent acetylcholinesterase inhibitor tested was 0.4 nM under the conditions used for a typical assay. It should be noted, however, that no attempt was made to optimize the assay procedure for sensitivity.