Oxime-dipeptides as anticholinesterase,reactivator of phosphonylated-serine of AChE catalytic triad: probing the mechanistic insight by MM-GBSA,dynamics simulations and DFT analysis

Neuropathological cascades leading to reduced cholinergic transmission in Alzheimer’s disease led to development of AChE-inhibitors. Although lethal dose of some inhibitors cause interruption with AChE mediated mechanism but reversible AChE inhibitors can assist in protection from inhibition of AChE and hence in an aim to probe potential molecules as anticholinesterase and as reactivators, computationally structure-based approach has been exploited in this work for designing new 2-amino-3-pyridoixime-dipeptides conjugates. We have combined MD simulations with flexible ligand docking approach to determine binding specificity of 2-amino-3-pyridoixime dipeptides towards AChE (PDB 2WHP). PAS residues are found to be responsible for oxime-dipeptides binding along with π–π interactions with Trp86 and Tyr286, hydrogen bonding with side chains of Asp74 and Tyr341 (Gscore –10.801 and MM-GBSA free energy –34.89?kcal/mol). The docking results depicted complementary multivalent interactions along with good binding affinity as predicted from MM-GBSA analysis. The 2-amino-3-pyridoxime-(Arg-Asn) AChE systems subjected to MD simulations under explicit solvent systems with NPT and NVT ensemble. MD simulations uncovered dynamic behavior of 2-amino-3-pyridoxime-(Arg-Asn) and exposed its mobile nature and competence to form strong long range-order contacts towards active site residues to approach inhibited serine residue and facilitated via large contribution from hydrogen bonding and water bridges along with slow and large movements of adjacent important residues. In an effort to evaluate the complete potential surface profile, 2-amino-3-pyridoxime induced reactivation pathway of sarin–serine adduct has been investigated by the DFT approach at the vacuum MO6/6–311G (d, p) level along with the Poisson-Boltzmann solvation model and found to be of relatively low energy barrier. The pKa evaluation has revealed the major deprotonated 2-amino-3-pyridoixime species having pKa of 6.47 and hence making 2-amino-3-pyridoxime-(Arg-Asn) potential anticholinesterase and reactivator for AChE under the physiological pH.     

Novel isoquinoline derivatives as antimicrobial agents

The wide variety of potent biological activities of natural and synthetic isoquinoline alkaloids encouraged us to develop novel antimicrobial isoquinoline compounds. We synthesized a variety of differently functionalized 1-pentyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolines (THIQs), including dihydroisoquinolinium salts (2 and 5), methyl pentanoate-THIQ (6), 1-pentanol-THIQ (7), ester derivatives (8–15) and carbamate derivatives (16–23). We employed classic intramolecular Bischler–Napieralski cyclodehydration to generate the isoquinoline core. All the structures were characterized by nuclear magnetic resonance and mass spectrometry. The bactericide and fungicide activities were evaluated for all the synthesized compounds and structure-activity relationships were established. Many compounds exhibited high and broad-range bactericidal activity. Fluorophenylpropanoate ester 13 and the halogenated phenyl- (17, 18) and phenethyl carbamates (21, 22) exerted the most remarkable bactericidal activity. However, few compounds displayed antifungal activity against most of the fungi tested. Among them, chlorinated derivatives like chlorobenzoate and chlorophenylpropanoate esters (10 and 14, respectively) and chlorophenethyl carbamate 22, exhibited the greatest antifungal activity.     

Inhibition kinetics on carboxylesterase and acetylcholinesterase of Liposcelis bostrychophila and Liposcelis entomophila (Psocop., Liposcelididae) of two insecticides

Abstract: The inhibition kinetics on carboxylesterase (CarE) and acetylcholinesterase (AChE) of Liposcelis bostrychophila and Liposcelis entomophila of paraoxon and carbosulfan were compared. The results showed that L. entomophila exhibited significantly greater specific activity of CarE than L. bostrychophila [0.045 vs. 0.012 μmol of α‐naphthyl acetate (α‐NA) hydrolysed/mg protein/min]. Moreover, L. entomophila showed higher affinity (i.e. lower K_m value) to the substrate α‐NA than L. bostrychophila (0.29 vs. 0.67 mm ). For AChE‐specific activity and affinity, no significant differences between the two species were observed. Based on the I₅₀ values, AChE of L. bostrychophila was more sensitive to paraoxon and carbosulfan than that of L. entomophila. According to inhibition kinetics, the results revealed that AChE of L. bostrychophila was 3.8‐fold more sensitive to inhibition by paraoxon than that of L. entomophila, but L. entomophila was 1.5‐fold more susceptible to carbosulfan.     

Insecticide resistance in the malarial mosquito Anopheles arabiensis and association with the kdr mutation

A colony of Anopheles arabiensis Patton (Diptera: Culicidae) from the Sennar region of Sudan was selected for resistance to dichlorodiphenyltrichloroethane (DDT). Adults from the F-16 generation of the resistant strain were exposed to all four classes of insecticides approved for use in malaria vector control and showed high levels of resistance to them all (24-h mortalities: malathion, 16.7%; bendiocarb, 33.3%; DDT, 12.1%; dieldrin, 0%; deltamethrin, 24.0%; permethrin, 0%). Comparisons between the unselected base colony and the DDT-resistant strain showed elevated glutathione-S-transferase (P<0.05) in both sexes and elevated esterases (P<0.05) in males only. The Leu-Phe mutation in the sodium channel gene was detected by polymerase chain reaction and sequencing, but showed no correlation with the resistant phenotype. These results do not provide any explanation as to why this colony exhibits such widespread resistance and further studies are needed to determine the precise mechanisms involved. The implications for malaria vector control in central Sudan are serious and resistance management (e.g. through the rotational use of different classes of insecticides) is recommended.     

A single post-exposure oxime RS194B treatment rapidly reactivates acetylcholinesterase and reverses acute symptoms in macaques exposed to diethylphosphorothioate parathion and chlorpyrifos insecticides

Millions of individuals globally suffer from inadvertent, occupational or self-harm exposures from organophosphate (OP) insecticides, significantly impacting human health. Similar to nerve agents, insecticides are neurotoxins that target and inhibit acetylcholinesterase (AChE) in central and peripheral synapses in the cholinergic nervous system. Post-exposure therapeutic countermeasures generally include administration of atropine with an oxime to reactivate the OP-inhibited AChE. However, animal model studies and recent clinical trials using insecticide-poisoned individuals have shown minimal clinical benefits of the currently approved oximes and their efficacy as antidotes has been debated. Currently used oximes either reactivate poorly, do not readily cross the blood–brain barrier (BBB), or are rapidly cleared from the circulation and must be repeatedly administered. Zwitterionic oximes of unbranched and simplified structure, for example RS194B, have been developed that efficiently cross the BBB resulting in reactivation of OP-inhibited AChE and dramatic reversal of severe clinical symptoms in mice and macaques exposed to OP insecticides or nerve agents. Thus, a single IM injection of RS194B has been shown to rapidly restore blood AChE and butyrylcholinesterase (BChE) activity, reverse cholinergic symptoms, and prevent death in macaques following lethal inhaled sarin and paraoxon exposure. The present macaque studies extend these findings and assess the ability of post-exposure RS194B treatment to counteract oral poisoning by highly toxic diethylphosphorothioate insecticides such as parathion and chlorpyrifos. These OPs require conversion by P450 in the liver of the inactive thions to the active toxic oxon forms, and once again demonstrated RS194B efficacy to reactivate and alleviate clinical symptoms within 60 mins of a single IM administration. Furthermore, when delivered orally, the Tmax of RS194B at 1–2 h was in the same range as those administered IM but were maintained in the circulation for longer periods greatly facilitating the use of RS194B as a non-invasive treatment, especially in isolated rural settings.

     

Syntheses of 19-[O-(carboxymethyl)oxime] haptens of epipregnanolone and pregnanolone

O-(Carboxymethyl)oximes 1 and 2 derived from two epimeric 5beta-pregnanolones (3beta-hydroxy-5beta-pregnan-20-one and 3alpha-hydroxy-5beta-pregnan-20-one) in position 19 were prepared. Two synthetic routes were employed, both using protection of the 20-keto group after reduction into the (20R)-alcohol in the form of acetate. In the first route, (20R)-19-hydroxy-5beta-pregnan-3beta,20-diyl diacetate (3) was transformed into the corresponding 19-[O-(carboxymethyl)oxime] methyl ester 6, then deacetylated by acid and partially silylated with tert-butyldimethylsilyl chloride. The desired 3-O-silylated derivative 8 was separated, oxidized to the 20-ketone and protecting groups were sequentially removed to give the first title hapten 1. The second route started from (20R)-19-hydroxy-3-oxopregn-4-en-20-yl acetate (11), which was hydrogenated in the presence of base to the 5beta-pregnan-3-one derivative 12, protected in position 19 with tert-butyldimethylsilyl group and reduced with borohydride. The prevailing 3alpha-alcohol 15 was separated, protected in position 3 with a methoxymethyl group, deprotected in position 19 and transformed into the 19-[O-(carboxymethyl)oxime] 19. After deacetylation, esterification with diazomethane and oxidation in position 20, the pregnanolone skeleton was regenerated. Final deprotection steps gave the second title hapten 2. Both haptens, i.e., (19E)-3beta- and -3alpha-hydroxy-20-oxo-5beta-pregnan-19-al 19-[O-(carboxymethyl)oxime], were designed for the development of immunoassays of the corresponding parent neuroactive steroids.     

Characterization of plasma cholinesterase activity in the Eurasian Griffon Vulture Gyps fulvus and its in vitro inhibition by carbamate pesticides

The legal and illegal use of organophosphorus and carbamate pesticides represents one of many threats to birds. The activity of the cholinesterase enzyme in plasma is used as a non‐destructive biomarker to diagnose the exposure of birds to these pesticides. Scavengers are one of the most important bird groups threatened by the use of baits poisoned with anticholinesterase pesticides. Knowledge of the characteristics of this enzyme in each bird species is crucial, as several studies indicate that more than one cholinesterase form may be present in the plasma of birds. In this study, cholinesterase activity was characterized in the plasma of the Eurasian Griffon Vulture Gyps fulvus by using several substrates and inhibitors of the enzyme, and its normal activity value was also determined. The in vitro sensitivity of Gyps fulvus plasma cholinesterase to carbamate insecticides (aldicarb, carbaryl and methomyl) was also investigated. The results indicated that propionylthiocholine iodide was the preferred substrate to determine plasma cholinesterase activity, followed by acetylcholine iodide and S‐butyrylcholine iodide, and acetylcholinesterase was the predominant enzymatic activity in Gyps fulvus plasma. Aldicarb was the most potent in vitro inhibitor of plasma cholinesterase activity in this species. However, cholinesterase enzymatic activity was significantly inhibited by all tested carbamates, providing further evidence that this biomarker is a suitable tool to monitor the exposure to these poisons in the field, highlighting its utility in conservation programmes.     

α-Glucosidase inhibitory activity and cytotoxic effects of some cyclic urea and carbamate derivatives

The inhibitory activities of selected cyclic urea and carbamate derivatives (1–13) toward α-glucosidase (α-Gls) in in vitro assay were examined in this study. All examined compounds showed higher inhibitory activity (IC₅₀) against α-Gls compared to standard antidiabetic drug acarbose. The most potent was benzyl (3,4,5-trimethoxyphenyl)carbamate (12) with IC₅₀?=?49.85?±?0.10?µM. In vitro cytotoxicity of the investigated compounds was tested on three human cancer cell lines HeLa, A549 and MDA-MB-453 using MTT assay. The best antitumour activity was achieved with compound 2 (trans-5-phenethyl-1-phenylhexahydro-1H-imidazo[4,5-c]pyridin-2(3H)-one) against MDA-MB-453 human breast cancer cell line (IC₅₀?=?83.41?±?1.60?µM). Cyclic ureas and carbamates showed promising anti-α-glucosidase activity and should be further tested as potential antidiabetic drugs. The PLS model of preliminary QSAR study indicated that, in planing the future synthesis of more potent compounds, the newly designed should have the substituents capable of polar interactions with receptor sites in various positions, while avoiding the increase of their lipophilicity.     

Charged pyridinium oximes with thiocarboxamide moiety are equally or less effective reactivators of organophosphate-inhibited cholinesterases compared to analogous carboxamides

The organophosphorus antidotes, so-called oximes, are able to restore the enzymatic function of acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) via cleavage of organophosphate from the active site of the phosphylated enzyme. In this work, the charged pyridinium oximes containing thiocarboxamide moiety were designed, prepared and tested. Their stability and pK_a properties were found to be analogous to parent carboxamides (K027, K048 and K203). The inhibitory ability of thiocarboxamides was found in low µM levels for AChE and high µM levels for BChE. Their reactivation properties were screened on human recombinant AChE and BChE inhibited by nerve agent surrogates and paraoxon. One thiocarboxamide was able to effectively restore function of NEMP- and NEDPA-AChE, whereas two thiocarboxamides were able to reactivate BChE inhibited by all tested organophosphates. These results were confirmed by reactivation kinetics, where thiocarboxamides were proved to be effective, but less potent reactivators if compared to carboxamides.     

Action of Systemic Nematicides in Control of Xiphinema iindex on Grape

In greenhouse tests using potted grape plants three nematicides, aldicarb 10 G at 4.5 ai/ha, phenamiphos 15 G at 22 kg ai/ha, and oxamyl liquid at 4.5 kg ai/ha, were tested against Xiphinema index on ''Thompson Seedless'' grape. Different timings for chemical treatments and X. index inoculations were used to determine some of the aspects of the mode of action. When nematodes and nematicides were applied simultaneously, nematodes were reduced from the initial 500 to the averages 5, 1, and 4, respectively, for aldicarb, phenamiphos, and oxamyl. Similar counts (respectively, 3, 1, and 2) were obtained when the nematicides were added first and the nematodes 14 d later. Nematode counts were 83, 112, and 1,346 when nematicides were applied first, and 14 days later plants were washed free of soil, repotted in untreated soil, and then inoculated. In untreated controls the population increased to an average of 2,703. Plant growth was inversely related to the level of nematode population resulting from the treatment.