Anticholinesterase activity of some major intermediates in carbacylamidophosphate synthesis: preparation, spectral characterization and inhibitory potency determination

Carbacylamidophosphates with the general formula RC(O)NHP(O)R1R2 constitute organophosphorus compounds that are used as insecticides, pesticides and ureas inhibitors. In this work, we studied the inhibition potency of CCl3-C(O)NHP(O)Cl21, CHCl2C(O)NHP(O)Cl(2)2, CH2ClC(O)NHP(O)Cl23 and CF3C(O)NHP(O)Cl(2)4, which are the major intermediates for carbacylamidophosphates synthesis towards human erythrocyte acetylcholinesterase (hAChe) activity using Ellman's modified kinetic method. Unexpectedly, it was observed that they were not only hydrolytically unstable but also inhibited hAChE in a similar manner to that produced by organophosphorus insecticides. Enzymatic data, bimolecular inhibition rate constants (ki) and IC50 values for inhibition of hAChE demonstrated that they are irreversible inhibitors and the inhibition potency of compound 2 (IC50 = 88 microM) was the greatest in comparison with compounds 1, 3 and 4. Also the electropositivity of the phosphorus atom and the hydrophobicity of the compounds demonstrated that these two factors play an additional effect and different role in the inhibitory activity of these compounds. Hydrolytic stability of the compounds was determined by 31P NMR monitoring of the loss of the parent molecules with D2O as a function of time. This study considers antiacetylcholinesterase activity according to the structural and the electronic aspects of compounds 1-4, according to IR, 1H, 13C and 31P NMR spectral data.     

Acetylcholinesterase/Butyrylcholinesterase inhibition activity of some new carbacylamidophosphate deriviatives

Eight newly synthesized carbacylamidophosphates with the general formula RC(O)NHP(O)Cl₂ with R = pCl–C₆H₄ 1a, pBr–C₆H₄ 2a, C₆H₅ 3a, and pMe–C₆H₄ 4a and RC(O)NHP(O)(NC₄H₈O)₂ R = pCl–C₆H₄ 1b, pBr–C₆H₄ 2b, C₆H₅ 3b, pMe–C₆H₄ 4b, were selected to compare the inhibition kinetic parameters, IC₅₀, K_i, k_p and K_D, on human erythrocyte acetylcholinesterase (hAChE) and bovine serum butyrylcholinesterase (BuChE), Also, the in vivo inhibition potency of compound 2a, 2b and 3a, were studied. The data demonstrates that compound 2a and compound 2b are the potent sensitive as AChE and BuChE inhibitors respectively, and the inhibition of hAChE is about 10-fold greater than that of BuChE.     

Synthesis,characterization and inhibitory potency of two oxono and thiono analogues of phosphoramidate compounds on acetylcholinesterase

Two novel structurally related phosphoramidate compounds, 1 and 2, with likely β-diketone system were synthesized and characterized by ¹H, ¹³C, ³¹P NMR, IR spectroscopy and elemental analysis. Compound 2 exhibited a ³¹P NMR signal which was significantly shielded (8 ppm) relative to compound 1. Determination of human erythrocyte acetylcholinesterase (hAChE) inhibitory activity was carried out according to Ellman's modified kinetic method and the IC₅₀ values of compounds 1 and 2 were 1.567 and 2.986 mM, respectively. The k_i values of 1 and 2 were 1.39 to 2.65 min^{? 1} respectively. A comparison of the bimolecular rate constant (k_i) and IC₅₀ values for the irreversible inhibitors 1 and 2 revealed that the oxono analogue has greater affinity for hAChE than the thiono compound. Furthermore effects of two conventional oximes paralidoxime (A) and obidoxime (B) on reactivation of the inhibited hAChE were studied but low reactivity was shown by both the oximes.     

Inhibitory activity of xanthine oxidase and superoxide-scavenging activity in some taxa of the lichen family Graphidaceae

Results on the screening of species of the lichen family Graphidaceae for superoxide-scavenging activity (SSA) and xanthine-oxidase inhibitory (IXO) activity have been presented. The potential of the extracts for scavenging of superoxide and inhibition of xanthine-oxidase under various physiological conditions has been evaluated. The methanolic extracts of the species of family Graphidaceae showed inhibitory properties of xanthine oxidase (IC50 = 2.0 to 5.26 microg/ml) with an additional superoxide scavenging capacity (IC50 = 3.63 to 13.88 microg/ml). The potential of the methanolic extracts for scavenging of superoxide and inhibition of xanthine oxidase remained stable at 4 degrees C. Thus the extracts can be maintained for longer periods for their therapeutic uses.     

Differential effects of some antibiotics on paraoxonase enzyme activity on human hepatoma cells (HepG2) in vitro

Serum paraoxonase (aryldialkylphosphatase, EC 3.1.8.1., PON1) is an esterase protein synthesised by the liver and released into the serum, where it is associated with HDL lipoproteins. In this study, we have determined the in vitro effects of the following antibiotics: sodium ampicillin, ciprofloxacin, Rifamycin SV and clindamiycin phosphate, on human hepatoma (HepG2) cells (liver hPON1). All the antibiotics caused a dose-dependent and time-dependent decrease in the paraoxonase activity while Rifamycin SV was the most effective antibiotic due to its low 50% inhibition concentration (IC₅₀) value. Liver hPON1 activity was determined using paraoxon as a substrate. The IC₅₀ values of the drugs were calculated from graphs of hydratase activity (%) by plotting concentration of the drugs that showed an inhibition effect.     

Synthesis and in vitro antiproliferative and antibacterial activity of new thiazolidine-2,4-dione derivatives

In our present research, we synthesised new thiazolidine-2,4-diones (12–28). All the newly synthesised compounds were evaluated for antiproliferative and antibacterial activity. Antiproliferative evaluation was carried out using normal human skin fibroblasts and tumour cell lines: A549, HepG2, and MCF-7. The IC₅₀ values were determined for tested compounds revealing antiproliferative activity. Moreover, safety index (SI) was calculated. Among all tested derivatives, the compound 18 revealed the highest antiproliferative activity against human lung, breast, and liver cancer cells. More importantly, the derivative 18 showed meaningfully lower IC₅₀ values when compared to the reference substance, irinotecan, and relatively high SI values. Moreover, newly synthesised compounds were screened for the bacteria growth inhibition in vitro. According to our screening results, most active compound was the derivative 18 against Gram-positive bacteria. Therefore, it may be implied that the novel compound 18 appears to be a very promising agent for anticancer treatment.     

Synthesis and antibacterial activity of alaremycin derivatives for the porphobilinogen synthase

The preparation and the antibacterial activity of alaremycin derivatives such as their CF₃-derivatives and (R)- and (S)-4-oxo-5-acetylaminohexanoic acid for the porphobilinogen synthase (PBGS), were described. The IC₅₀ values of the antibacterial activity of the prepared materials for the inhibitor of PBGS, were determined using PBGS assay.     

Formation of 2,5-Bis-(d-threo-trihydroxypropyl)pyrazine and Its 6-Isomer by the Oxidative Browning Reaction of d-Xylose with Ammonium Acetate in a Methanolic Medium

A pyruvate kinase-lacking mutant of Brevibacterium flavum produced 22.6 g/liter of l-aspartic acid with glutamic acid as a by-product, when cultured for 48 hr in a medium containing 100 g/liter of glucose. The production clearly depended on the amount of biotin added. This strain, 70, was derived by several steps of mutation from wild strain 2247 producing glutamate, successively via a citrate synthase-defective glutamate auxotroph, strain 214, a prototrophic revertant, strain 15-8, producing 10 g/liter of l-aspartic acid, and an S-(2-aminoethyl)-l-cysteine-resistant mutant, strain 1-231, having low pyruvate kinase and homoserine dehydrogenase and producing lysine. Strain 70, a methionine-insensitive revertant from strain 1-231, had a normal level of homoserine dehydrogenase but no pyruvate kinase. Its citrate synthase activity was about half that of the wild strain at saturated concentrations of the substrates with Michaelis constants for oxalacetate and acetyl-CoA of 110 and 6 times as high as those of the wild-type enzyme, respectively. The mutational step for these alterations in citrate synthase was strain 15-8. Phosphoenolpyruvate carboxylase of strain 70 showed 1.5-fold higher activity in the crude extract at saturated concentrations of phosphoenolpyruvate, a lower Michaelis constant (1.5mM).for the substrate, phosphoenolpyruvate, less sensitivity to the feedback inhibition by aspartate, and higher sensitivities to the activators, acetyl-CoA and fructose-1,6-bisphosphate, than those of the wild strain. The concentrations of aspartate giving 50% inhibition were 6.2- and 4.5-fold higher in the absence and presence of acetyl-CoA, respectively.     

Inhibition of rat liver cathepsins B and L by the peptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal and its analogues

Cathepsins B and L belong to the papain superfamily of cysteine proteases and play important roles in various physiological and pathological processes. In the course of studies on their inhibitors, we examined the inhibitory effects of the peptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLal) and its analogues. As a result, rat liver cathepsins B and L were shown to be strongly inhibited by them. The concentration required for 50% inhibition (IC₅₀) by ZLLLal was 88 nM for cathepsin B and 163 nM for cathepsin L. Moreover, various analogues of ZLLLal, including 2-furancarbonyl-, nicotinyl-, isonicotinyl- and 4-morpholinylsuccinyl-LLLals, and some acetyl-Pro (AcP)-containing analogues, AcPLLLal and AcPPLLLal, were shown to inhibit both enzymes more strongly than ZLLLal. Among them, isonicotinyl-LLLal was most inhibitory against both cathepsins B (IC₅₀, 12 nM) and L (IC₅₀, 20 nM). Several of these inhibitors were indicated to be somewhat more soluble in aqueous media than ZLLLal.     

Cytotoxic activity induced by crude extracts of Ganoderma lucidum (W. Curt.: Fr.) P. Karst. on mouse myeloma cancer cell-line

Ganoderma lucidum powder using hot water and methanol extraction methods indicated a twofold more active cytotoxic activity with IC₅₀ of 44 ± 3.8 μg/ml in the latter method. The representative dose-response curves of the G. lucidum crude extracts on J558 cell-lines revealed that there were great similarities between the curves which reflected rapid killing activities. The percentage viability of the J558 cell exposed to these crude extracts was dose dependent only up to 150 μg/ml. After which, there was no significant reduction when the dose was increased to 200 or 400 μg/ml. The morphological alterations induced by the crude extract were examined under the phase contrast, fluorescent and electron microscopy. When J558 cells were treated with doses higher than 50 μg/ml of the crude extract, obvious morphological changes and apoptosis occurred after 72 h. At 400 μg/ml, most of the cells showed necrosis characterized as small fragments with uniformly stained red nuclei. The apoptotic and necrotic cells increased by 16.5 and 29.1%, respectively whereas the viable cells decreased by as much as 45.6. The mode of cell death via apoptosis was 3.6% higher than necrosis. However, these morphological changes were not observed in the case of 3T3 cells. Results obtained from scanning electron microscopy and transmission electron microscopy further confirmed the occurrence of various apoptotic and necrotic features.     

Bioinspired FeIIICdII and FeIIIHgII complexes: synthesis, characterization and promiscuous catalytic activity evaluation

In this work we report on the synthesis, crystal structure, and physicochemical characterization of the novel dinuclear [Fe^IIICd^II(L)(μ-OAc)₂]ClO₄·0.5H₂O (1) complex containing the unsymmetrical ligand H₂L = 2-bis[{(2-pyridyl-methyl)-aminomethyl}-6-{(2-hydroxy-benzyl)-(2-pyridyl-methyl)}-aminomethyl]-4-methylphenol. Also, with this ligand, the tetranuclear [Fe₂^IIIHg₂^II(L)₂(OH)₂](ClO₄)₂·2CH₃OH (2) and [Fe^IIIHg^II(L)(μ-CO₃)Fe^IIIHg^II(L)](ClO₄)₂·H₂O (3) complexes were synthesized and fully characterized. It is demonstrated that the precursor [Fe^III₂Hg^II₂(L)₂(OH)₂](ClO₄)₂·2CH₃OH (2) can be converted to (3) by the fixation of atmospheric CO₂ since the crystal structure of the tetranuclear organometallic complex [Fe^IIIHg^II(L)(μ-CO₃)Fe^IIIHg^II(L)](ClO₄)₂·H₂O (3) with an unprecedented {Fe^III(μ-O_phenoxo)₂(μ-CO₃)Fe^III} core was obtained through X-ray crystallography. In the reaction 2 → 3 a nucleophilic attack of a Fe^III-bound hydroxo group on the CO₂ molecule is proposed. In addition, it is also demonstrated that complex (3) can regenerate complex (2) in aqueous/MeOH/NaOH solution. Magnetochemical studies reveal that the Fe^III centers in 3 are antiferromagnetically coupled (J = − 7.2 cm^− 1) and that the Fe^III-OR-Fe^III angle has no noticeable influence in the exchange coupling. Phosphatase-like activity studies in the hydrolysis of the model substrate bis(2,4-dinitrophenyl) phosphate (2,4-bdnpp) by 1 and 2 show Michaelis-Menten behavior with 1 being ~ 2.5 times more active than 2. In combination with k_H/k_D isotope effects, the kinetic studies suggest a mechanism in which a terminal Fe^III-bound hydroxide is the hydrolysis-initiating nucleophilic catalyst for 1 and 2. Based on the crystal structures of 1 and 3, it is assumed that the relatively long Fe^III…Hg^II distance could be responsible for the lower catalytic effectiveness of 2.     

Peptide microarrays for detailed, high-throughput substrate identification, kinetic characterization, and inhibition studies on protein kinase A

A microarray-based mix-and-measure, nonradioactive multiplex method with real-time detection was used for substrate identification, assay development, assay optimisation, and kinetic characterization of protein kinase A (PKA). The peptide arrays included either up to 140 serine/threonine-containing peptides or a concentration series of a smaller number of peptides. In comparison with existing singleplex assays, data quality was high, variation in assay conditions and reagent consumption were reduced considerably, and assay development could be accelerated because phosphorylation kinetics were monitored simultaneously on 4, 12, or 96 arrays. PKA was shown to phosphorylate many peptides containing known PKA phosphorylation sites as well as some new substrates. The kinetic behavior of the enzyme and the mechanism of inhibition by AMP-PNP, staurosporin, and PKA inhibitor peptide on the peptide microarray correlated well with data from homogeneous assays. Using this multiplex setup, we showed that the kinetic parameters of PKA and the potency of PKA inhibitors can be affected by the sequence of the peptide substrate. The technology enables kinetic monitoring of kinase activity in a multiplex setting such as a cell or tissue lysate. Finally, this high-throughput method allows fast identification of peptide substrates for serine/threonine kinases that are still uncharacterized.     

Heterologous expression and purification of neurotoxic Hainantoxin-III in E. coli

In the present study, we used Escherichia coli to produce recombinant Hainantoxin-III (rHNTX-III), a 33-amino acid peptic toxin from the tarantula spider Haplopelma hainanum. The toxin has three pairs of disulfide bonds. A pET-HS-HNTX-III vector was constructed and transformed into the E. coli strain SHuffle^TM. rHNTX-III was expressed using auto-induction medium. After using a Ni–NTA column, the expressed fusion protein was digested using SUMO protease (ULP1) to remove the HIS–SUMO tag, and then RP–HPLC and ultrafiltration were used for further purification. Then the rHNTX-III was identified by MALDI–TOF/TOF mass spectrometry. The purified rHNTX-III was further analyzed using a whole-cell patch-clamp assay. It was shown that the rHNTX-III was able to block currents generated by human Na_v1.7 (hNa_v1.7) at an IC₅₀ of 225?nM and also have high selectivity for different voltage-gated sodium channels. Therefore, it has very similar activity to the natural one.     

Parallel genetic changes and nonparallel gene-environment interactions characterize the evolution of drug resistance in yeast

Beneficial mutations are required for adaptation to novel environments, yet the range of mutational pathways that are available to a population has been poorly characterized, particularly in eukaryotes. We assessed the genetic changes of the first mutations acquired during adaptation to a novel environment (exposure to the fungicide, nystatin) in 35 haploid lines of Saccharomyces cerevisiae. Through whole-genome resequencing we found that the genomic scope for adaptation was narrow; all adapted lines acquired a mutation in one of four late-acting genes in the ergosterol biosynthesis pathway, with very few other mutations found. Lines that acquired different ergosterol mutations in the same gene exhibited very similar tolerance to nystatin. All lines were found to have a cost relative to wild type in an unstressful environment; the level of this cost was also strongly correlated with the ergosterol gene bearing the mutation. Interestingly, we uncovered both positive and negative effects on tolerance to other harsh environments for mutations in the different ergosterol genes, indicating that these beneficial mutations have effects that differ in sign among environmental challenges. These results demonstrate that although the genomic target was narrow, different adaptive mutations can lead populations down different evolutionary pathways, with respect to their ability to tolerate (or succumb to) other environmental challenges.     

Toxicity and bioaccumulation of chromium in some freshwater fish

Chromium (Cr) is a common pollutant of freshwater bodies in India, and is frequently detected in high concentrations in edible fish. Bioassays were carried out in the laboratory to determine acute the toxicity and pattern of accumulation of Cr in three species of freshwater fish. The 96-h LC₅₀ value of Cr for Labeo bata, Puntius sarana, and Catla catla was found respectively as 7.33, 10.37, and 31.61 mg/L. Concentrations of Cr in water, sediments, and fish, during a period of 28 d of exposure to 0, 0.73, and 2.19 mg/L of Cr, varied with exposure period, concentration of Cr, presence of weed, and species of fish exposed. Polynomial regressions obtained by drawing polynomial curves revealed that the aquatic weed Eichhornia crassipes prevented gradual decrease of Cr concentrations in water, but reduced the accumulation of Cr in L. bata and Catla catla. However, the effect of the weed on Puntius sarana was not apparent. The pattern of Cr deposition on sediments was also inconsistent. To explain interactions between environment and fish in a very precise manner, polynomial and multiple regression curves were simultaneously used. When polynomial curves were replaced by multiple regression curves, it was revealed that the aquatic weed E. crassipes could reduce Cr accumulation in Puntius sarana also.     

N-terminal amphipathic helix as a trigger of hemolytic activity in antimicrobial peptides: A case study in latarcins

In silico structural analyses of sets of α-helical antimicrobial peptides (AMPs) are performed. Differences between hemolytic and non-hemolytic AMPs are revealed in organization of their N-terminal region. A parameter related to hydrophobicity of the N-terminal part is proposed as a measure of the peptide propensity to exhibit hemolytic and other unwanted cytotoxic activities. Based on the information acquired, a rational approach for selective removal of these properties in AMPs is suggested. A proof of concept is gained through engineering specific mutations that resulted in elimination of the hemolytic activity of AMPs (latarcins) while leaving the beneficial antimicrobial effect intact.     

Metalloprobes: synthesis, characterization, and potency of a novel gallium(III) complex in human epidermal carcinoma cells

Multidrug resistance (MDR) mediated by overexpression of the MDR1 gene product, P-glycoprotein (Pgp), represents one of the best characterized barriers to chemotherapeutic treatment in cancer and may be a pivotal factor in progression of Alzheimer's disease (AD). Thus, agents capable of probing Pgp-mediated transport could be beneficial in biomedical imaging. Herein, we synthesized and structurally characterized a gallium(III) complex (5) of the naphthol-Schiff base ligand. The crystal structure revealed octahedral geometry for the metallodrug. Cytotoxicity profiles of 5 were evaluated in KB-3-1 (Pgp-) and KB-8-5 (Pgp+) human epidermal carcinoma cell lines. Compared with an LC(50) (the half-maximal cytotoxic concentration) value of 1.93 microM in drug-sensitive (Pgp-) cells, the gallium(III) complex 5 demonstrated an LC(50) value>100 microM in drug-resistant (Pgp+) cells, thus indicating that 5 was recognized by the Pgp as its substrate, thereby extruded from the cells and sequestered away from their cytotoxic targets. Radiolabeled analogues of 5 could be beneficial in noninvasive imaging of Pgp-mediated transport in vivo.     

Design and synthesis of substituted imidazole and triazole N-phenylbenzo[d]oxazolamine inhibitors of retinoic acid metabolizing enzyme CYP26

The design of N-phenylbenzo[d]oxazolamines as CYP26A1 inhibitors involved ligand docking experiments using molecular modeling (FlexX) and analysis of ligand interactions at the binding domain. The synthesis of the benzooxazol-2-yl-[phenyl-imidazol-1-yl-methyl)phenyl]amines was achieved by cyclisation of the corresponding isothiocyanates with subsequent introduction of the haem-binding heterocycle. Triazole and tetrazole derivatives were also prepared for comparison with the lead imidazole derivative. The benzooxazol-2-yl-[phenyl-imidazol-1-yl-methyl)phenyl]amines with small substituents in the phenyl ring were moderately potent CYP26A1 inhibitors (IC₅₀ 8 and 12 μM) and comparable with liarozole (IC₅₀ 7 μM).     

Synthesis and biological activities of 4-N-(anilinyl-n-[oxazolyl])-7-chloroquinolines (n=3' or 4') against Plasmodium falciparum in in vitro models

The synthesis (Pd-mediated coupling strategy) and characterization (NMR, IR, elemental analysis, etc.) of a short series of quinoline-oxazole hybrid compounds has been carried out. These materials are found to be moderately active against Plasmodium falciparum in vitro, with activities in the sub-micromolar range, and to display acceptable cytotoxicity to mononuclear leukocytes. Chemical modification strategies, with the intention to increase the biological potency of this new class of anti-malarial agents, are discussed.     

Toxicity effect of the acaricide fipronil in semi-engorged females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae): preliminary determination of the minimum lethal concentration and LC(50)

Chemical acaricides, especially fipronil (active ingredient of Frontline®), are still the most effective method to control tick populations. In this study, the effectiveness of fipronil was assessed in semi-engorged females of the tick Rhipicephalus sanguineus. A protocol for an in vitro bioassay (AIT) was developed, and the LC₅₀ (lethal concentration 50%) and 95% confidence interval were determined. Ticks were immersed in Petri dishes with different concentrations of fipronil or distilled water for 2 min, dried, and placed in an incubator for 7 days. Dead R. sanguineus females treated with the 14 concentrations of fipronil were counted daily. Mortality results were compared with the Probit analysis, and the LC₅₀ and 95% confidence interval were calculated, g (95): LC₅₀ = 9.647 (4.711 to 13.470). This study was aimed at developing a more appropriate and updated protocol for an in vitro bioassay (AIT – adult immersion test), and providing information on the toxic potential of fipronil (elimination of ectoparasites with lower concentrations) and sensitivity of ticks, especially R. sanguineus, a pest of great interest, due to its occurrence in urban environments.