Potential sources of biodynamically active natural products in Brazil

In contrast to China where vegatation is predominantly herbaceous, vegetation in Brazil is commonly arboreous. This fact may explain why Chinese drugs are usually acetate derived, while actual and potential natural therapeutic agents from Brazil are mostly shikimate derived. Only relatively few compounds isolated from Brazilian plants have been submitted to adequate pharmacological testing

Evaluation of some organic compounds on bloodstream forms of Trypanosoma cruzi

Accidental transmission of Chagas' disease to man by blood transfusion is a serious problem in Latin-America. This paper describes the testing of several synthetic, semi-synthetic, and natural compounds for their activity against blood trypomastigotes in vitro at 4-C. The compounds embody several types of chemical structures: benzoquinone, naphthoquinone, anthracenequinone, phenanthrenequinone, imidazole, piperazine, quinoline, xanthene, and simple benzenic and naphthalenic derivates. Some of them are for the first time tested against Trypanosoma cruzi. The toxic effect these compounds on this parasite was done by two quite distinct sets of experiments. In one set, the compounds were added to infected blood as ethanolic solution. In this situation the most active one was a furan-1, 2-naphthoquinone, in the same range as gentian violet, a new fact to be considered in the assessment of structure-activity relationships in this class of compounds. In other set, we tentatively evaluated the biological activity of water insoluble compounds by adding them in a pure form without solvent into infected blood. In this way some appear to be very active and it was postulated that the effectiveness of such compounds must result from interactions between them and specific blood components.

Diterpenoids from Azorella compacta (Umbelliferae) active on Trypanosoma cruzi

The anti-Trypanosoma cruzi activity of natural products isolated from Azorella compacta was evaluated, with particular emphasis on their effect against intracellular amastigotes. Five diterpenoids from A. compacta derived from mulinane and azorellane were isolated and identified. Only two products, named azorellanol (Y-2) and mulin-11,3-dien-20-oic acid (Y-5), showed trypanocidal activity against all stages of T. cruzi including intracellular amastigotes. At 10 µM, these compounds displayed a strong lytic activity. It ranged from 88.4 ± 0.6 to 99.0 ± 1 % for all strains and stages evaluate, with an IC50 /18 h values of 20-84 µM and 41-87 µM, respectively. The development of intracellular amastigotes was also inhibited by nearly 60% at 25 µM. The trypanocidal molecules Y-2 and Y-5 did show different degrees of cytotoxicity depending on the cell line tested, with an IC50 /24 h ranging from 33.2 to 161.2 µM. We evaluated the effect of diterpenoids against intracellular T. cruzi forms by immunofluorescent identification of a specific membrane molecular marker (Ssp-4 antigen) of the T. cruzi amastigote forms. The accuracy and reproducibility of the measurements were found to be outstanding when examined by confocal microscopy.

Brazilian flora extracts as source of novel antileishmanial and antifungal compounds

Natural products have long been providing important drug leads for infectious diseases. Leishmaniasis is a protozoan parasitic disease found mainly in developing countries, and it has toxic therapies with few alternatives. Fungal infections have been the main cause of death in immunocompromised patients and new drugs are urgently needed. In this work, a total of 16 plant species belonging to 11 families, selected on an ethnopharmacological basis, were analyzed in vitro against Leishmania (L.) chagasi, Leishmania (L.) amazonensis, Candida krusei, and C. parapsilosis. Of these plant species, seven showed antifungal activity against C. krusei, five showed antileishmanial activity against L. chagasi and four against L. amazonensis, among them species of genus Plectranthus. Our findings confirm the traditional therapeutic use of these plants in the treatment of infectious and inflammatory disorders and also offer insights into the isolation of active and novel drug prototypes, especially those used against neglected diseases as Leishmaniasis.

Structured intermittent interruption of chronic HIV infection treatment with highly active antiretroviral therapy: Effects on leptin and TNF-alpha.

The changes in nutritional parameters and adipocytokines after structured intermittent interruption of highly active antiretroviral treatment of patients with chronic HIV infection are analyzed. Twenty-seven patients with chronic HIV infection (median CD4+ T cell count/microl: nadir, 394; at the beginning of structured interruptions, 1041; HIV viral load: nadir, 41,521 copies/ml; at the beginning of structured interruptions <50 copies/ml; median time of previous treatment: 60 months) were evaluated during three cycles of intermittent interruptions of therapy (8 weeks on/4 weeks off). CD4+ T cell count, HIV viral load, anthropometric measures, and serum concentrations of triglycerides, cholesterol, leptin, and tumor necrosis factor and its soluble receptors I and II were determined. After the three cycles of intermittent interruptions of therapy, no significant differences in CD4+ T cell count/microl, viral load, or serum concentrations of cholesterol or triglycerides with reference to baseline values were found. A near-significant higher fatty mass (skinfold thicknesses, at the end, 121 mm, at the beginning, 100 mm, p = 0.100), combined with a significant increase of concentration of leptin (1.5 vs. 4.7 ng/ml, p = 0,044), as well as a decrease in serum concentrations of soluble receptors of tumor necrosis factor (TNFRI, 104 vs. 73 pg/ml, p = 0.022; TNFRII 253 vs. 195 pg/ml, p = 0.098) were detected. Structured intermittent interruption of highly active antiretroviral treatment of patients with chronic HIV infection induces a valuable positive modification in markers of lipid turnover and adipose tissue mass.

Effects of highly active antiretroviral therapy with nelfinavir in vertically HIV-1 infected children: 3 years of follow-up. Long-term response to nelfinavir in children.

BACKGROUND Antiretroviral treatment (ART) in children has special features and consequently, results obtained from clinical trials with antiretroviral drugs in adults may not be representative of children. Nelfinavir (NFV) is an HIV-1 Protease Inhibitor (PI) which has become as one of the first choices of PI for ART in children. We studied during a 3-year follow-up period the effects of highly active antiretroviral therapy with nelfinavir in vertically HIV-1 infected children. METHODS Forty-two vertically HIV-infected children on HAART with NFV were involved in a multicentre prospective study. The children were monitored at least every 3 months with physical examinations, and blood sample collection to measure viral load (VL) and CD4+ cell count. We performed a logistic regression analysis to determinate the odds ratio of baseline characteristics on therapeutic failure. RESULTS Very important increase in CD4+ was observed and VL decreased quickly and it remained low during the follow-up study. Children with CD4+ <25% at baseline achieved CD4+ >25% at 9 months of follow-up. HIV-infected children who achieved undetectable viral load (uVL) were less than 40% in each visit during follow-up. Nevertheless, HIV-infected children with VL >5000 copies/ml were less than 50% during the follow-up study. Only baseline VL was an important factor to predict VL control during follow-up. Virological failure at defined end-point was confirmed in 30/42 patients. Along the whole of follow-up, 16/42 children stopped HAART with NFV. Baseline characteristics were not associated with therapeutic change. CONCLUSION NFV is a safe drug with a good profile and able to achieve an adequate response in children.

Clinical and Virological Efficacy of Etravirine Plus Two Active Nucleos(t)ide Analogs in an Heterogeneous HIV-Infected Population.

Etravirine (ETV) is recommended in combination with a boosted protease inhibitor plus an optimized background regimen for salvage therapy, but there is limited experience with its use in combination with two nucleos(t)ide reverse-transcriptase inhibitors (NRTIs). This multicenter study aimed to assess the efficacy of this combination in two scenarios: group A) subjects without virologic failure on or no experience with non-nucleoside reverse-transcriptase inhibitors (NNRTIs) switched due to adverse events and group B) subjects switched after a virologic failure on an efavirenz- or nevirapine-based regimen. The primary endpoint was efficacy at 52 weeks analysed by intention-to-treat. Virologic failure was defined as the inability to suppress plasma HIV-RNA to <50 copies/mL after 24 weeks on treatment, or a confirmed viral load >200 copies/mL in patients who had previously achieved a viral suppression or had an undetectable viral load at inclusion. Two hundred eighty seven patients were included. Treatment efficacy rates in group A and B were 88.0% (CI95, 83.9-92.1%) and 77.4% (CI95, 65.0-89.7%), respectively; the rates reached 97.2% (CI95, 95.1-99.3%) and 90.5% (CI95, 81.7-99.3), by on-treatment analysis. The once-a-day ETV treatment was as effective as the twice daily dosing regimen. Grade 1-2 adverse events were observed motivating a treatment switch in 4.2% of the subjects. In conclusion, ETV (once- or twice daily) plus two analogs is a suitable, well-tolerated combination both as a switching strategy and after failure with first generation NNRTIs, ensuring full drug activity. TRIAL REGISTRATION ClinicalTrials.gov NCT01437241.

Antifungal activity of extracts from Atacama Desert fungi againstParacoccidioides brasiliensis and identification ofAspergillus felis as a promising source of natural bioactive compounds

Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values≤ 125.0 µg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 µg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 µg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.

A quantitative analysis of antagonism and inverse agonism at wild-type and constitutively active hamster alpha1B-adrenoceptors.

In order to characterize inverse agonism at alpha1B-adrenoceptors, we have compared the concentration-response relationships of several quinazoline and non-quinazoline alpha1-adrenoceptor antagonists at cloned hamster wild-type (WT) alpha1B-adrenoceptors and a constitutively active mutant (CAM) thereof upon stable expression in Rat-1 fibroblasts. Receptor activation or inhibition thereof was assessed as [3H]inositol phosphate (IP) accumulation. Quinazoline (alfuzosin, doxazosin, prazosin, terazosin) and non-quinazoline alpha1-adrenoceptor antagonists (BE 2254, SB 216,469, tamsulosin) concentration-dependently inhibited phenylephrine-stimulated IP formation at both WT and CAM with Ki values similar to those previously found in radioligand binding studies. At CAM in the absence of phenylephrine, the quinazolines produced concentration-dependent inhibition of basal IP formation; the maximum inhibition was approximately 55%, and the corresponding EC50 values were slightly smaller than the Ki values. In contrast, BE 2254 produced much less inhibition of basal IP formation, SB 216,469 was close to being a neutral antagonist, and tamsulosin even weakly stimulated IP formation. The inhibitory effects of the quinazolines and BE 2254 as well as the stimulatory effect of tamsulosin were equally blocked by SB 216,469 at CAM. At WT in the absence of phenylephrine, tamsulosin did not cause significant stimulation and none of the other compounds caused significant inhibition of basal IP formation. We conclude that alpha1-adrenoceptor antagonsits with a quinazoline structure exhibit greater efficacy as inverse agonists than those without.

Pi*-pi* bonding interactions generated by halogen oxidation of zirconium(IV) redox-active ligand complexes.

The new complex, [Zr(pda)2]n (1, pda2- = N,N'-bis(neo-pentyl)-ortho-phenylenediamide, n = 1 or 2), prepared by the reaction of 2 equiv of pdaLi2 with ZrCl4, reacts rapidly with halogen oxidants to afford the new product ZrX2(disq)2 (3, X = Cl, Br, I; disq- = N,N'-bis(neo-pentyl)-ortho-diiminosemiquinonate) in which each redox-active ligand has been oxidized by one electron. The oxidation products 3a-c have been structurally characterized and display an unusual parallel stacked arrangement of the disq- ligands in the solid state, with a separation of approximately 3 A. Density functional calculations show a bonding-type interaction between the SOMOs of the disq- ligands to form a unique HOMO while the antibonding linear combination forms a unique LUMO. This orbital configuration leads to a closed-shell-singlet ground-state electron configuration (S = 0). Temperature-dependent magnetism measurements indicate a low-lying triplet excited state at approximately 750 cm-1. In solution, 3a-c show strong disq--based absorption bands that are invariant across the halide series. Taken together these spectroscopic measurements provide experimental values for the one- and two-electron energies that characterize the pi-stacked bonding interaction between the two disq- ligands.

Novel Macrocyclic Amidinoureas: Potent Non-Azole Antifungals Active against Wild-Type and Resistant Candida Species.

Novel macrocyclic amidinourea derivatives 11, 18, and 25 were synthesized and evaluated as antifungal agents against wild-type and fluconazole resistant Candida species. Macrocyclic compounds 11 and 18 were synthesized through a convergent approach using as a key step a ring-closing metathesis macrocyclization reaction, whereas compounds 25 were obtained by our previously reported synthetic pathway. All the macrocyclic amidinoureas showed antifungal activity toward different Candida species higher or comparable to fluconazole and resulted highly active against fluconazole resistant Candida strains showing in many cases minimum inhibitory concentration values lower than voriconazole.

EADock: docking of small molecules into protein active sites with a multiobjective evolutionary optimization.

In recent years, protein-ligand docking has become a powerful tool for drug development. Although several approaches suitable for high throughput screening are available, there is a need for methods able to identify binding modes with high accuracy. This accuracy is essential to reliably compute the binding free energy of the ligand. Such methods are needed when the binding mode of lead compounds is not determined experimentally but is needed for structure-based lead optimization. We present here a new docking software, called EADock, that aims at this goal. It uses an hybrid evolutionary algorithm with two fitness functions, in combination with a sophisticated management of the diversity. EADock is interfaced with the CHARMM package for energy calculations and coordinate handling. A validation was carried out on 37 crystallized protein-ligand complexes featuring 11 different proteins. The search space was defined as a sphere of 15 A around the center of mass of the ligand position in the crystal structure, and on the contrary to other benchmarks, our algorithm was fed with optimized ligand positions up to 10 A root mean square deviation (RMSD) from the crystal structure, excluding the latter. This validation illustrates the efficiency of our sampling strategy, as correct binding modes, defined by a RMSD to the crystal structure lower than 2 A, were identified and ranked first for 68% of the complexes. The success rate increases to 78% when considering the five best ranked clusters, and 92% when all clusters present in the last generation are taken into account. Most failures could be explained by the presence of crystal contacts in the experimental structure. Finally, the ability of EADock to accurately predict binding modes on a real application was illustrated by the successful docking of the RGD cyclic pentapeptide on the alphaVbeta3 integrin, starting far away from the binding pocket.

Comprehensive approach for the detection of antifungal compounds using a susceptible strain of Candida albicans and confirmation of in vivo activity with the Galleria mellonella model.

An efficient screening strategy for the identification of potentially interesting low-abundance antifungal natural products in crude extracts that combines both a sensitive bioautography assay and high performance liquid chromatography (HPLC) microfractionation was developed. This method relies on high performance thin layer chromatography (HPTLC) bioautography with a hypersusceptible engineered strain of Candida albicans (DSY2621) for bioactivity detection, followed by the evaluation of wild type strains in standard microdilution antifungal assays. Active extracts were microfractionated by HPLC in 96-well plates, and the fractions were subsequently submitted to the bioassay. This procedure enabled precise localisation of the antifungal compounds directly in the HPLC chromatograms of the crude extracts. HPLC-PDA-mass spectrometry (MS) data obtained in parallel to the HPLC antifungal profiles provided a first chemical screening about the bioactive constituents. Transposition of the HPLC analytical conditions to medium-pressure liquid chromatography (MPLC) allowed the efficient isolation of the active constituents in mg amounts for structure confirmation and more extensive characterisation of their biological activities. The antifungal properties of the isolated natural products were evaluated by their minimum inhibitory concentration (MIC) in a dilution assay against both wild type and engineered strains of C. albicans. The biological activity of the most promising agents was further evaluated in vitro by electron microscopy and in vivo in a Galleria mellonella model of C. albicans infection. The overall procedure represents a rational and comprehensive means of evaluating antifungal activity from various perspectives for the selection of initial hits that can be explored in more in-depth mode-of-action studies. This strategy is illustrated by the identification and bioactivity evaluation of a series of antifungal compounds from the methanolic extract of a Rubiaceae plant, Morinda tomentosa, which was used as a model in these studies.

Potential of antimicrobial volatile organic compounds to control Sclerotinia sclerotiorum in bean seeds

The objective of this work was to evaluate the potential of an artificial mixture of volatile organic compounds (VOCs), produced by Saccharomyces cerevisiae, to control Sclerotinia sclerotiorum in vitro and in bean seeds. The phytopathogenic fungus was exposed, in polystyrene plates, to an artificial atmosphere containing a mixture of six VOCs formed by alcohols (ethanol, 3-methyl-1-butanol, 2-methyl-1-butanol and phenylethyl alcohol) and esters (ethyl acetate and ethyl octanoate), in the proportions found in the atmosphere naturally produced by yeast. Bean seeds artificially contamined with the pathogen were fumigated with the mixture of VOCs in sealed glass flasks for four and seven days. In the in vitro assays, the compounds 2-methyl-1-butanol and 3-methyl-1-butanol were the most active against S. sclerotiorum, completely inhibiting its mycelial growth at 0.8 µL mL-1, followed by the ethyl acetate, at 1.2 µL mL-1. Bean seeds fumigated with the VOCs at 3.5 µL mL-1 showed a 75% reduction in S. sclerotiorum incidence after four days of fumigation. The VOCs produced by S. cerevisiae have potential to control the pathogen in stored seeds.

Evaluation of potential breath biomarkers for active smoking: assessment of smoking habits

Different compounds have been reported as biomarkers of a smoking habit, but, to date, there is no appropriate biomarker for tobacco-related exposure because the proposed chemicals seem to be nonspecific or they are only appropriate for short-term exposure. Moreover, conventional sampling methodologies require an invasive method because blood or urine samples are required. The use of a microtrap system coupled to gas chromatography–mass spectrometry analysis has been found to be very effective for the noninvasive analysis of volatile organic compounds in breath samples. The levels of benzene, 2,5-dimethylfuran, toluene, o-xylene, and m- p-xylene have been analyzed in breath samples obtained from 204 volunteers (100 smokers, 104 nonsmokers; 147 females, 57 males; ages 16 to 53 years). 2,5-Dimethylfuran was always below the limit of detection (0.005 ppbv) in the nonsmoker population and always detected in smokers independently of the smoking habits. Benzene was only an effective biomarker for medium and heavy smokers, and its level was affected by smoking habits. Regarding the levels of xylenes and toluene, they were only different in heavy smokers and after short-term exposure. The results obtained suggest that 2,5-dimethylfuran is a specific breath biomarker of smoking status independently of the smoking habits (e.g., short- and long-term exposure, light and heavy consumption), and so this compound might be useful as a biomarker of smoking exposure