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.     

Inhibition of alpha-glucosidase by oleanolic acid and its synthetic derivatives

Oleanolic acid (1) and five synthetic derivatives (2-6) were tested spectrophotometrically for inhibition of urease, beta-lactamase, acetyl cholinesterase and alpha-glucosidase. All products showed a positive response only against alpha-glucosidase but not against the other enzymes; IC(50) calculations showed that the dihydroxy-olide derivative (4) was the most potent among all tested samples.     

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.     

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.     

Effects of bacillamide and newly synthesized derivatives on the growth of cyanobacteria and microalgae cultures

The antialgal activity of newly synthesized bacillamides against several cyanobacteria and microalgae isolates was screened using a rapid 96-well microplate bioassay. Cultures were exposed to serial dilutions of each bacillamide derivative (0–160 μg mL⁻¹) in the microplate wells and daily optical measurements were used to estimate growth over a 216 h period. Inhibition values (%) were calculated from the estimated growth curves and inhibitory concentrations (IC₅₀-216 h) were obtained from the sigmoidal inhibition curves fitted by regression analysis. The effects of bacillamides on cell morphology and ultrastructure were also analysed by light and transmission electron microscopy. In general, the toxic cyanobacteria Microcystis aeruginosa, Aphanizomenon gracile, Anabaena circinalis and Anabaenopsis circularis were much more sensitive to bacillamides then the chlorophytes Ankistrodesmus falcatus and Scenedesmus obliquus. However, clear signs of morphological and ultrastructural changes induced by bacillamide were observed on both cyanobacteria and chlorophytes. Other cyanobacteria, namely the nostocalean Nodularia spumigena and the oscillatorialeans Leptolyngbya sp. and Planktothrix rubescens, exhibit higher tolerances to bacillamides, similar to that shown by different eukaryotic microalgae. Diatoms, on the other hand, proved to be quite as sensitive to most bacillamides as the most affected cyanobacteria. The properties of 5-iodo-Bacillamide (algicide or algistatic) were further investigated. This compound acted as an algistactic agent against eukaryotic algae and, depending on its concentration, acted as either an algicide or algistactic agent against most of the cyanobacteria tested. Although bacillamides cannot be considered as broad spectrum cyanobacterial algicides, different bacillamides might be of use in selectively controlling the growth of particular species of cyanobacteria.     

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.     

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.     

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.     

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.     

Intracellular lactate dehydrogenase concentration as an index of cytotoxicity in rat hepatocyte primary culture

In searching for a reliable index for cytotoxicity testing in rat hepatocyte primary culture, lactate dehydrogenase (LDH) concentrations in lysates of attached hepatocytes and LDH released into the culture medium were compared under conditions of exposure to various dosages of sodium chloride, sodium salicylate, R-warfarin, acetaminophen, phenylbutazone, and furosemide (frusemide). The amount of intracellular LDH was assessed by inducing the cells to release the enzyme with 0.1% Tritron X-100. The induced LDH leakage was completed in 1 hr and the LDH activity was stable in storage at 10° for 2 weeks. We found that intracellular LDH is a direct indicator of the number of viable hepatocytes in contrast to the LDH released, because released LDH does not account for the significant number of cells detached from monolayer but which are not leaky, during the 6-hr test period. Based on IC₅₀ values (50% inhibitory concentration), the relative cytotoxicities are R-warfarin > phenylbutazone > furosemide > acetaminophen > sodium salicylate > sodium chloride.Abbreviations DMSO dimethyl sulfoxide - HPC hepatocyte primary culture - IC₅₀ 50% inhibitory concentration - LDH lactate dehydrogenase     

醛酮还原酶AKR1Cs的表达纯化及其催化还原福美司坦的作用研究

乳腺癌是女性最常见的恶性肿瘤之一,芳香化酶抑制剂（AIs）是辅助治疗乳腺癌的重要手段. 福美司坦（4-OHA）作为固醇类AIs,不可逆地抑制芳香化酶的活性,可有效治疗乳腺癌. 多项研究表明,醛酮还原酶（AKR1Cs）参与许多固醇类及其衍生物的代谢而间接治疗多种激素依赖性疾病,从而成为治疗靶点. 我们推测AKR1Cs可能参与4-OHA的特异性代谢而影响其疗效. 本文通过体外原核表达纯化成功得到4种具有酶活性的AKR1Cs蛋白亚型,采用荧光法检测AKR1Cs对4-OHA的催化效率,并检测AKR1Cs酶抑制剂对其催化还原4-OHA的影响. 结果发现4种AKR1Cs蛋白亚型均能催化还原 4-OHA,其中AKR1C4能快速还原4-OHA,转化率几乎为100%,其次是AKR1C3和AKR1C1,转化效率为30%左右,AKR1C2对4-OHA的还原性最低,转化效率只有20%左右. 同时抑制剂对AKR1Cs表现出明显的剂量-效应关系,非线性回归分析得到抑制剂对AKR1C3和AKR1C4的亲和性较强,IC₅₀值分别为47.4 μmol/L和54.68 μmol/L,而对AKR1C1和AKR1C2的抑制力相对较弱,IC₅₀值分别为77.37 μmol/L和82.24 μmol/L. 以上结果表明,4-OHA能被肝脏中特异性表达的AKR1C4快速代谢,从代谢角度揭示了4-OHA口服用药效果不理想的原因. 因此,本研究阐明了4-OHA肠胃外或经皮给药的优势并奠定了理论基础,也为今后利用纳米药物载体和开展该药物衍生物的深入研究提供了新思路.     

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 CCl₃C(O)NHP(O)Cl₂1, CHCl₂C(O)NHP(O)Cl₂2, CH₂ClC(O)NHP(O)Cl₂3 and CF₃C(O)NHP(O)Cl₂4, 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 (k_i) and IC₅₀ values for inhibition of hAChE demonstrated that they are irreversible inhibitors and the inhibition potency of compound 2 (IC₅₀ = 88 μM) 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.     

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.     

In vitro and in vivo interaction of synthetic peroxide RBx11160 (OZ277) with piperaquine in Plasmodium models

RBx11160 (OZ277) is a promising antimalarial drug candidate that Ranbaxy Laboratories Limited and Medicines for Malaria Venture (MMV) are currently developing as a fixed combination with piperaquine. Here, we describe the in vitro (Plasmodium falciparum) and in vivo (Plasmodium berghei) activities of piperaquine in combination with RBx11160 and artemether. In vitro, both combinations demonstrated a slight tendency towards antagonism with mean sums of fractional inhibitory concentrations (mean Sigma FICs) of 1.5. In vivo, piperaquine and artemether were borderline antagonistic (mean Sigma FIC of 1.4). However, an additive in vivo interaction of piperaquine and RBx11160 (mean Sigma FIC of 1.1) was identified, suggesting that a RBx11160-piperaquine combination therapy in humans should allow each molecule to exert its full antimalarial effect.     

Plasmodium vivax: in vitro susceptibility of blood stages to synthetic trioxolane compounds and the diamidine DB75

Plasmodium vivax is an important human pathogen causing malaria in more temperate climates of the world. Similar to Plasmodium falciparum, the causative agent for malaria tropica, drug resistance is beginning to emerge for this parasite species and this hampers adequate treatment of infection. We have used a short-term ex vivo drug assay to monitor activity of OZ277 (RBx-11160), a fully synthetic anti-malarial peroxide, and the diamidine DB75 against P. vivax. For both compounds as well as the anti-malarial reference compounds artesunate, artemether, and chloroquine, the in vitro IC(50) values were determined in one-cycle hypoxanthine incorporation assays. Results from such assays were found to be very similar compared to IC(50) values obtained from one-cycle P. falciparum hypoxanthine assays. We demonstrate the anti-parasite activity of OZ277 and the reference compounds to be faster than that of DB75. These data warrant clinical testing of OZ277 against P. vivax malaria and support recent data on clinical activity against P. vivax for DB75.     

Comparison of cytotoxic activities betweenin vitro and field grown plants ofTyphonium flagelliforme (Lodd.) blume

Anin vitro cytotoxicity screening of theTyphonium flagelliforme extracts indicated high cytotoxicity effect on human lung carcinoma NCl-H23 cells and human mammary gland carcinoma T-47D cells, but the extracts were not active on human liver carcinoma HepG2 cells. NCl-H23 cells were more susceptible toT. flagelliforme extracts than T-47D cells. EDP₅₀ values of the hexane fractions of the mature plant and thein vitro plantlet ofT. flagelliforme on NCl-H23 cells were less than 2 μg/mL Extract from the mature plant was relatively more cytotoxic than the one fromin vitro plantlet except for the hexane fraction. The chloroform and butanol fraction of the mature plant had higher cytotoxicity effect than the fraction fromin vitro plantlet on NCl-H23 cells. All the 3 fractions (hexane, chloroform, and butanol) of the mature plant exhibited higher cytotoxicity effects on human mammary gland carcinoma T-47D cells than the 3 fractions ofin vitro plantlet. However, the human liver carcinoma cells were resistant toT. flagelliforme extracts except for higher concentration of hexane fractions of both the mature and thein vitro plants and the chloroform fraction of the mature plant. Micropropagated plantlets ofT. flagelliforme could hence be used as herbal materials for the treatment of human lung and breast cancers.     

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).     

Effects of polyamines on the functionality of photosynthetic membrane in vivo and in vitro

The three major polyamines are normally found in chloroplasts of higher plants and are implicated in plant growth and stress response. We have recently shown that putrescine can increase light energy utilization through stimulation of photophosphorylation [Ioannidis et al., (2006) BBA-Bioenergetics, 1757, 821-828]. We are now to compare the role of the three major polyamines in terms of chloroplast bioenergetics. There is a different mode of action between the diamine putrescine and the higher polyamines (spermidine and spermine). Putrescine is an efficient stimulator of ATP synthesis, better than spermidine and spermine in terms of maximal % stimulation. On the other hand, spermidine and spermine are efficient stimulators of non-photochemical quenching. Spermidine and spermine at high concentrations are efficient uncouplers of photophosphorylation. In addition, the higher the polycationic character of the amine being used, the higher was the effectiveness in PSII efficiency restoration, as well as stacking of low salt thylakoids. Spermine with 50 μM increase F_V as efficiently as 100 μM of spermidine or 1000 μM of putrescine or 1000 μM of Mg²⁺. It is also demonstrated that the increase in F_V derives mainly from the contribution of PSIIα centers. These results underline the importance of chloroplastic polyamines in the functionality of the photosynthetic membrane.