Evaluation of natural and synthetic compounds according to their antioxidant activity using a multivariate approach

The antioxidant activity of natural and synthetic compounds was evaluated using five in vitro methods: ferric reducing/antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydradzyl (DPPH), oxygen radical absorption capacity (ORAL), oxidation of an aqueous dispersion of linoleic acid accelerated by azo-initiators (LAOX), and oxidation of a meat homogenate submitted to a thermal treatment (TBARS). All results were expressed as Trolox equivalents. The application of multivariate statistical techniques suggested that the phenolic compounds (caffeic acid, carnosic acid, genistein and resveratrol), beyond their high antioxidant activity measured by the DPPH, FRAP and TBARS methods, showed the highest ability to react with the radicals in the ORAC methodology, compared to the other compounds evaluated in this study (ascorbic acid, erythorbate, tocopherol, BHT, Trolox, tryptophan, citric acid, EDTA, glutathione, lecithin, methionine and tyrosine). This property was significantly correlated with the number of phenolic rings and catecholic structure present in the molecule. Based on a multivariate analysis, it is possible to select compounds from different clusters and explore their antioxidant activity interactions in food products.

The inhibitory activity of synthetic compounds and ions against transporters of multi-drug resistant bacteria

RESUMO: Sessenta e três derivados de hidantoína foram utilizados para avaliar possíveis efeitos de modulação na actividade das bombas de efluxo (BE) na Salmonella NCTC 13349 utilizando um método fluorimétrico semi-automático. Nenhum dos compostos apresentaram actividade anti-bacteriana até concentrações de 240 mg/L. Entre todos os compostos, SZ-7 demonstrou possuir propriedades de modulação de effluxo na presença de glucose. Para testar esta actividade, estirpes de Salmonella resistentes à ciprofloxacina, induzidas a elevados níveis de resistência com sobre-expressão de BE, foram expostas ao SZ-7. Este derivado afectou a susceptibilidade das estirpes à ciprofloxacina. Uma vez que os 63 compostos estudados apresentaram pouco efeito inibitório /cumulativo, apesar de serem conhecidos pelos seus efeitos moduladores de BE-dependentes de iões em eucariotas, foi questionado o papel dos iões na regulação de BE bacterianas, que poderão influenciar a eficácia de novos compostos. Para este estudo, utilizamos a Escherichia coli AG100 como modelo, devido ao extenso conhecimento no que respeita a estrutura e actividade das BE. Devido à importância de iões de cálcio (Ca2+) nos canais de transporte membranar e na actividade de ATPases, a sua actividade na modulação do efluxo foi investigada. De resultados anteriormente obtidos concluiu-se que a pH 5 o efluxo é independente de energia metabólica; contudo, a pH 8 é absolutamente dependente, sendo que o Ca2+ é indispensável para manter a actividade das ATPases bacterianas. A acumulação/effluxo de EtBr pela E. coli AG100 foi determinada na presença/ausência de Ca2+, clorpromazina (inibidor de ligação de Ca2+ a proteínas), e ácido etilenodiamino tetra-acético (quelante de Ca2+). Acumulação/effluxo aumentou a pH 8, contudo o Ca2+ reverte estes efeitos evidenciando a sua importância no funcionamento das BE bacterianas. Em resumo este trabalho colocou em evidência que muitos aspectos bioquímicos e bioenergéticos devem ser tomados em consideração no estudo da resistência bacteriana mediada por BE.------- ABSTRACT: Sixty-three hydantoin derivatives were evaluated for their modulating effects on efflux pump (EP) activity of Salmonella NCTC 13349 utilizing a semi-automatic fluorometric method. None of the compounds presented antibacterial activities at concentrations as high as 240 mg/L. Among all compounds, SZ-7 showed possible efflux modulating activity in the presence of glucose, indicative of a potential EP inhibitor. To verify its potential effects, ciprofloxacin-resistant Salmonella strains, induced to high level resistance with over-expressing EPs, were exposed to SZ-7. This derivative affected the susceptibility of the ciprofloxacin-resistant strains. Since the 63 compounds studied had very low inhibitory/accumulative effects, even though their known for being efficient in modulating ion-driven eukaryotic EPs, we questioned whether ions had a leading role in regulating bacterial EPs, influencing the effectiveness of new compounds. For this study we used Escherichia coli AG100 as a model, due to the extensive knowledge on its EPs structure and activity. Owing the importance of calcium ions (Ca2+) for membrane transport channels and activity of ATPases, the role of Ca2+ was investigated. From previous results we concluded that at pH 5 efflux is independent of metabolic energy; however, at pH 8 it is entirely dependent of metabolic energy and the Ca2+ ions are essential to maintain the activity of bacterial ATPases. Accumulation and efflux of ethidium bromide (EtBr) by E. coli AG100 was determined in the presence and absence of Ca2+, chlorpromazine (inhibitor of Ca2+-binding to proteins), and ethylenediaminetetraacetic acid (Ca2+ chelator). Accumulation of EtBr increased at pH 8; however Ca2+ reversed these effects providing information as to the importance of this ion in the regulation of bacterial EP systems. Overall this work puts in evidence that many biochemical and bioenergetic aspects related to the strains physiology need to be taken into consideration in bacterial drug resistance mediated by EPs.

Toxicity of synthetic piperonyl compounds to leaf-cutting ants and their symbiotic fungus

The development of Leucoagaricus gongylophorus, the fungus cultured by the leaf-cutting ant Atta sexdens was inhibited in vitro by synthetic compounds containing the piperonyl group. In addition, worker ants that were fed daily on an artificial diet to which these compounds were added had a higher mortality rate than the controls. The inhibition of the fungal growth increased with the size of the carbon side chain ranging from C1 through C8 and decreasing thereafter. 1-(3,4-Methylenedioxybenzyloxy)octane (compound 5) was the most active compound and inhibited the fungal development by 80% at a concentration of 15 μg m1-1. With worker ants the toxic effects started with compound 5 and increased with the number of carbons in the side chain. Thus, for the same concentration (100 μg m1-1) the mortality rates observed after 8 days of diet ingestion were 82%, 66% and 42%, for 1-(3,4-methylenedioxybenzyloxy)decane, 1-(3,4-methylenedioxybenzyloxy)dodecane and compound 5, respectively, whereas with commercial piperonyl butoxide the mortality was 68%. The latter compound, which is known as a synergist insecticide, was as inhibitory to the symbiotic fungus as the synthetic compound 5. The possibility of controlling these insects in the future using compounds that can target simultaneously both organisms is discussed. © 2001 Society of Chemical Industry.

New antitumoral agents I: In vitro anticancer activity and in vivo acute toxicity of synthetic 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one and derivatives

This paper describes a new method for the preparation of 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one 1 and its derivatives 2-5. This set of synthetic compounds exhibited high antitumoral activities regarding in vitro screening against several human tumor cell lines as lung carcinoma NCI-460, melanoma UACC-62, breast MCF-7, colon HT-29, renal 786-O, ovarian OVCAR-03 and ovarian expressing the resistance phenotype for adriamycin NCI-ADR/ RES, prostate PC-3, and leukemia K-562. Compounds were also tested against murine tumor cell line B16F10 melanoma and lymphocytic leukemia L1210 as well as to their effect toward normal macrophages. Specific activity against colon cancer cells HT-29 was observed for all tested compounds and suggests further studies with models of colon cancer. Compounds 1, 2, and 4 showed significant cytotoxic activity with IC(50) values <= 2.3 mu M for all human cancer cell lines. Intraperitoneal acute administration of compound 1 and 2 showed very low toxicity rate. (C) 2010 Elsevier Ltd. All rights reserved.

O2 activation at bioinspired complexes: dinuclear copper systems and mononuclear non-heme iron compounds. Mechanisms and catalytic applications in oxidative transformations

L'activació d'oxigen que té lloc en els éssers vius constitueix una font d'inspiració pel desenvolupament d'alternatives als oxidants tradicionals, considerats altament tòxics i nocius. En aquesta treball s'utilitzen compostos sintètics com a models del centre actiu de proteïnes dinuclears de coure i mononuclears de ferro de tipus no-hemo que participen en l'activació d'oxigen en els éssers vius. Els sistemes dinuclears de coure mostren un centre de tipus coure(III) bis(oxo) que és capaç de dur a terme l'ortho-hidroxilació de fenols de manera similar a la reacció que catalitza la proteïna tirosinasa. Per altra banda, els sistemes de ferro desenvolupats en aquest treball actuen com a models de les dioxigenases de Rieske i poden dur a terme l'hidroxilació estereoespecífica d'alcans i l'epoxidació i cis-dihidroxilació d'olefines utilitzant peròxid d'hidrogen com a agent oxidant. Tot plegat demostra que el desenvolupament de sistemes model constitueix una bona estratègia per l'estudi dels sistemes naturals.

A versatile synthetic dimerizer for the regulation of protein–protein interactions

The use of low molecular weight organic compounds to induce dimerization or oligomerization of engineered proteins has wide-ranging utility in biological research as well as in gene and cell therapies. Chemically induced dimerization can be used to activate intracellular signal transduction pathways or to control the activity of a bipartite transcription factor. Dimerizer systems based on the natural products cyclosporin, FK506, rapamycin, and coumermycin have been described. However, owing to the complexity of these compounds, adjusting their binding or pharmacological properties by chemical modification is difficult. We have investigated several families of readily prepared, totally synthetic, cell-permeable dimerizers composed of ligands for human FKBP12. These molecules have significantly reduced complexity and greater adaptability than natural product dimers. We report here the efficacies of several of these new synthetic compounds in regulating two types of protein dimerization events inside engineered cells—–induction of apoptosis through dimerization of engineered Fas proteins and regulation of transcription through dimerization of transcription factor fusion proteins. One dimerizer in particular, AP1510, proved to be exceptionally potent and versatile in all experimental contexts tested.

Topliss method in the optimization of salicylic acid derivatives as potential antimycobacterial agents

The Topliss method was used to guide a synthetic path in support of drug discovery efforts toward the identification of potent antimycobacterial agents. Salicylic acid and its derivatives, p-chloro, p-methoxy, and m-chlorosalicylic acid, exemplify a series of synthetic compounds whose minimum inhibitory concentrations for a strain of Mycobacterium were determined and compared to those of the reference drug, p-aminosalicylic acid. Several physicochemical descriptors (including Hammett`s sigma constant, ionization constant, dipole moment, Hansch constant, calculated partition coefficient, Sterimol-L and -B-4 and molecular volume) were considered to elucidate structure-activity relationships. Molecular electrostatic potential and molecular dipole moment maps were also calculated using the AM1 semi-empirical method. Among the new derivatives, m-chlorosalicylic acid showed the lowest minimum inhibitory concentration. The overall results suggest that both physicochemical properties and electronic features may influence the biological activity of this series of antimycobacterial agents and thus should be considered in designing new p-aminosalicylic acid analogs.

Postharvest shelf life extension of blueberries using a chitosan-based edible coating containing aloe vera juice

Poster

Estudos sobre a atividade dos extratos da suprarenal

Studies have been carried out on the method of Britton and Silvette modified by Reinecke and Kendall, for the evaluation of cortico-adrenal extracts, based on the deposition of glycogen in the liver of adrenaletomized rats. The test was performed in a total of 180 normal and adrenalectomized rats. The extracts tested were: a) an aqueous extract of cortico-adrenal cortex prepared by the Swingle and Pfiffner technique; b) the same extract added with ascorbic acid (Supracortin Labor); c) desoxycorticosterone acetate (Percortol Ciba and Syncortyl Roussell). Male rats were used, ranging from 40-200g, fed since the 18 th days old with a special diet, in which they were maintained until the day before the injection. Adrenalectomy was performed under urethane anesthesia. The fourth day after operation, food was removed and they were fasted for 24 hours. In the morning of the fifth day, injections of the material to be assayed were given at hourly and two hours intervals, during four to eight hours. One or two hours after the last injection, the animals were sacrified, the livers removed and dropped into a hot 30% solution of potassium hydroxide, and worked by Good, Kramer and Somogyi method. The glycogen was calculated as milligrams per lOOg of body and liver weight. The results obtained are shown in the tables I, II, and III. When several dosages of the same sample of extract were made (5 animals each dose), the amount of glycogen deposited in the liver per lOOg of body and liver weight, was found to be a positive function of the dose injected. The graph 2, shows these results. The synthetic compounds were ineffective. Our results are in agreement with those of Reinecke and Kendall and of Olson et al.

8-Methoxy-naphtho[2,3-b]thiophen-4,9-quinone, a non-competitive inhibitor of trypanothione reductase

The enzyme trypanothione reductase is a recognised drug target in trypanosomatids and has been used in the search of new compounds with potential activity against diseases such as leishmaniasis, Chagas disease and African trypanosomiasis. 8-Methoxy-naphtho [2,3-b] thiophen-4,9-quinone was selected in a screening of natural and synthetic compounds using an in vitro assay with the recombinant enzyme from Trypanosoma cruzi. Its mode of inhibition fits a non-competitive model with respect to the substrate (trypanothione) and to the co-factor (NADPH), with Ki-values of 5 and 3.6 µM, respectively. When tested against human glutathione reductase, this compound did not display any significant inhibition at 100 µM, indicating a good selectivity against the parasite enzyme.

In vitro and in vivo experimental models for drug screening and development for Chagas disease

Chagas disease, a neglected illness, affects nearly 12-14 million people in endemic areas of Latin America. Although the occurrence of acute cases sharply has declined due to Southern Cone Initiative efforts to control vector transmission, there still remain serious challenges, including the maintenance of sustainable public policies for Chagas disease control and the urgent need for better drugs to treat chagasic patients. Since the introduction of benznidazole and nifurtimox approximately 40 years ago, many natural and synthetic compounds have been assayed against Trypanosoma cruzi, yet only a few compounds have advanced to clinical trials. This reflects, at least in part, the lack of consensus regarding appropriate in vitro and in vivo screening protocols as well as the lack of biomarkers for treating parasitaemia. The development of more effective drugs requires (i) the identification and validation of parasite targets, (ii) compounds to be screened against the targets or the whole parasite and (iii) a panel of minimum standardised procedures to advance leading compounds to clinical trials. This third aim was the topic of the workshop entitled Experimental Models in Drug Screening and Development for Chagas Disease, held in Rio de Janeiro, Brazil, on the 25th and 26th of November 2008 by the Fiocruz Program for Research and Technological Development on Chagas Disease and Drugs for Neglected Diseases Initiative. During the meeting, the minimum steps, requirements and decision gates for the determination of the efficacy of novel drugs for T. cruzi control were evaluated by interdisciplinary experts and an in vitro and in vivo flowchart was designed to serve as a general and standardised protocol for screening potential drugs for the treatment of Chagas disease.

International Union of Pharmacology. LXI. Peroxisome proliferator-activated receptors.

The three peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear hormone receptor superfamily. They share a high degree of structural homology with all members of the superfamily, particularly in the DNA-binding domain and ligand- and cofactor-binding domain. Many cellular and systemic roles have been attributed to these receptors, reaching far beyond the stimulation of peroxisome proliferation in rodents after which they were initially named. PPARs exhibit broad, isotype-specific tissue expression patterns. PPARalpha is expressed at high levels in organs with significant catabolism of fatty acids. PPARbeta/delta has the broadest expression pattern, and the levels of expression in certain tissues depend on the extent of cell proliferation and differentiation. PPARgamma is expressed as two isoforms, of which PPARgamma2 is found at high levels in the adipose tissues, whereas PPARgamma1 has a broader expression pattern. Transcriptional regulation by PPARs requires heterodimerization with the retinoid X receptor (RXR). When activated by a ligand, the dimer modulates transcription via binding to a specific DNA sequence element called a peroxisome proliferator response element (PPRE) in the promoter region of target genes. A wide variety of natural or synthetic compounds was identified as PPAR ligands. Among the synthetic ligands, the lipid-lowering drugs, fibrates, and the insulin sensitizers, thiazolidinediones, are PPARalpha and PPARgamma agonists, respectively, which underscores the important role of PPARs as therapeutic targets. Transcriptional control by PPAR/RXR heterodimers also requires interaction with coregulator complexes. Thus, selective action of PPARs in vivo results from the interplay at a given time point between expression levels of each of the three PPAR and RXR isotypes, affinity for a specific promoter PPRE, and ligand and cofactor availabilities.

Estratégia de simplificação molecular no planejamento racional de fármacos: a descoberta de novo agente cardioativo

In this article are described examples of the successful use of molecular simplification strategy in the discovery of new drugs from bioactive natural products and synthetic compounds. The discovery of a new cardiotonic derivative (37, 2-thienylidene-3,4-methylenedioxybenzoylhydrazine; LASSBio-294), efficiently synthesized from Brazilian natural product and structurally designed by molecular simplification of active pyridazinone compounds reported in the literature, is described. A brief description of the pharmacological profile of this new cardiotonic lead-compound, belonging to the N-acylhydrazone (NAH) class, is also reported herein.

Sustainable drugs and global health care

Each day, Earth's finite resources are being depleted for energy, for material goods, for transportation, for housing, and for drugs. As we evolve scientifically and technologically, and as the population of the world rapidly approaches 7 billion and beyond, among the many issues with which we are faced is the continued availability of drugs for future global health care. Medicinal agents are primarily derived from two sources, synthetic and natural, or in some cases, as semi-synthetic compounds, a mixture of the two. For the developed world, efforts have been initiated to make drug production "greener", with milder reagents, shorter reaction times, and more efficient processing, thereby using less energy, and reactions which are more atom efficient, and generate fewer by-products. However, most of the world's population uses plants, in either crude or extract form, for their primary health care. There is relatively little discussion as yet, about the long term effects of the current, non-sustainable harvesting methods for medicinal plants from the wild, which are depleting these critical resources without concurrent initiatives to commercialize their cultivation. To meet future public health care needs, a paradigm shift is required in order to adopt new approaches using contemporary technology which will result in drugs being regarded as a sustainable commodity, irrespective of their source. In this presentation, several approaches to enhancing and sustaining the availability of drugs, both synthetic and natural, will be discussed, including the use of vegetables as chemical reagents, and the deployment of integrated strategies involving information systems, biotechnology, nanotechnology, and detection techniques for the development of medicinal plants with enhanced levels of bioactive agents.

The spindle assembly checkpoint as a drug target - Novel small-molecule inhibitors of Aurora kinases

Cell division (mitosis) is a fundamental process in the life cycle of a cell. Equal distribution of chromosomes between the daughter cells is essential for the viability and well-being of an organism: loss of fidelity of cell division is a contributing factor in human cancer and also gives rise to miscarriages and genetic birth defects. For maintaining the proper chromosome number, a cell must carefully monitor cell division in order to detect and correct mistakes before they are translated into chromosomal imbalance. For this purpose an evolutionarily conserved mechanism termed the spindle assembly checkpoint (SAC) has evolved. The SAC comprises a complex network of proteins that relay and amplify mitosis-regulating signals created by assemblages called kinetochores (KTs). Importantly, minor defects in SAC signaling can cause loss or gain of individual chromosomes (aneuploidy) which promotes tumorigenesis while complete failure of SAC results in cell death. The latter event has raised interest in discovery of low molecular weight (LMW) compounds targeting the SAC that could be developed into new anti-cancer therapeutics. In this study, we performed a cell-based, phenotypic high-throughput screen (HTS) to identify novel LMW compounds that inhibit SAC function and result in loss of cancer cell viability. Altogether, we screened 65 000 compounds and identified eight that forced the cells prematurely out of mitosis. The flavonoids fisetin and eupatorin, as well as the synthetic compounds termed SACi2 and SACi4, were characterized in more detail utilizing versatile cell-based and biochemical assays. To identify the molecular targets of these SAC-suppressing compounds, we investigated the conditions in which SAC activity became abrogated. Eupatorin, SACi2 and SACi4 preferentially abolished the tensionsensitive arm of the SAC, whereas fisetin lowered also the SAC activity evoked by lack of attachments between microtubules (MTs) and KTs. Consistent with the abrogation of SAC in response to low tension, our data indicate that all four compounds inhibited the activity of Aurora B kinase. This essential mitotic protein is required for correction of erratic MT-KT attachments, normal SAC signaling and execution of cytokinesis. Furthermore, eupatorin, SACi2 and SACi4 also inhibited Aurora A kinase that controls the centrosome maturation and separation and formation of the mitotic spindle apparatus. In line with the established profound mitotic roles of Aurora kinases, these small compounds perturbed SAC function, caused spindle abnormalities, such as multi- and monopolarity and fragmentation of centrosomes, and resulted in polyploidy due to defects in cytokinesis. Moreover, the compounds dramatically reduced viability of cancer cells. Taken together, using a cell-based HTS we were able to identify new LMW compounds targeting the SAC. We demonstrated for the first time a novel function for flavonoids as cellular inhibitors of Aurora kinases. Collectively, our data support the concept that loss of mitotic fidelity due to a non-functional SAC can reduce the viability of cancer cells, a phenomenon that may possess therapeutic value and fuel development of new anti-cancer drugs.