Structure-activity relationship of dibenzylideneacetone analogs against the neglected disease pathogen, Trypanosoma brucei.

Trypanosoma brucei is a protozoan parasite that causes Human African Trypanosomiasis (HAT), a neglected tropical disease (NTD) that is endemic in 36 countries in sub-Saharan Africa. Only a handful drugs are available for treatment, and these have limitations, including toxicity and drug resistance. Using the natural product, curcumin, as a starting point, several curcuminoids and related analogs were evaluated against bloodstream forms of T. b. brucei. A particular subset of dibenzylideneacetone (DBA) compounds exhibited potent in vitro antitrypanosomal activity with sub-micromolar EC50 values. A structure-activity relationship study including 26 DBA analogs was initiated, and several compounds exhibited EC50 values as low as 200 nM. Cytotoxicity counter screens in HEK293 cells identified several compounds having selectivity indices above 10. These data suggest that DBAs offer starting points for a new small molecule therapy of HAT.

Phenolic chalcones as agents against Trichomonas vaginalis.

Trichomonas vaginalis, a flagellated and anaerobic protozoan, is a causative agent of trichomoniasis. This disease is among the world's most common non-viral sexually transmitted infection. A single class drug, nitroimidazoles, is currently available for the trichomoniasis treatment. However, resistant isolates have been identified from unsuccessfully treated patients. Thus, there is a great challenge for a discovery of innovative anti-T. vaginalis agents. As part of our ongoing search for antiprotozoal chalcones, we designed and synthesized a series of 21 phenolic chalcones, which were evaluated against T. vaginalis trophozoites. Structure-activity relationship indicated hydroxyl group plays a role key in antiprotozoal activity. 4'-Hydroxychalcone (4HC) was the most active compound (IC50 = 27.5 µM) and selected for detailed bioassays. In vitro and in vivo evaluations demonstrated 4HC was not toxic against human erythrocytes and Galleria mellonella larvae. Trophozoites of T. vaginalis were treated with 4HC and did not present significant reactive oxygen species (ROS) accumulation. However, compound 4HC was able to increase ROS accumulation in neutrophils coincubated with T. vaginalis. qRT-PCR Experiments indicated that 4HC did not affect the expression of pyruvate:ferredoxin oxidoreductase (PFOR) and ß-tubulin genes. In silico simulations, using purine nucleoside phosphorylase of T. vaginalis (TvPNP), corroborated 4HC as a promising ligand. Compound 4HC was able to establish interactions with residues D21, G20, M180, R28, R87 and T90 through hydrophobic interactions, π-donor hydrogen bond and hydrogen bonds. Altogether, these results open new avenues for phenolic chalcones to combat trichomoniasis, a parasitic neglected infection.

Design, synthesis and antibacterial activity of chalcones against MSSA and MRSA planktonic cells and biofilms.

Staphylococcus aureus is the one of the most successful modern pathogens. The same bacterium that lives as a skin and mucosal commensal can be transmitted in health-care and community-settings and causes severe infections. Thus, there is a great challenge for a discovery of novel anti-Staphylococcus aureus compounds, which should act against resistant strains. Herein, we designed and synthesized a series of 17 chalcones, substituted by amino group on ring A, which were evaluated against methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus MRSA planktonic cells. The antibacterial potency was improved by substituents on ring B, which were designed according to Topliss' manual method. 4-bromo-3'-aminochalcone (5f) was the most active, demonstrating minimum inhibitory concentration (MIC) values of 1.9 µg mL-1 and 7.8 µg mL-1 against MSSA and MRSA, respectively. The association of 5f with vancomycin demonstrated synergistic effect against MSSA and MRSA, with Fractional Inhibitory Concentration Index (FICI) values of 0.4 and 0.3, respectively. Subinhibitory concentration of 5f inhibited the MSSA and MRSA adhesion to human keratinocytes. Chalcone 5f was able to reduce MSSA and MRSA biofilm formation, as well as acts on preformed biofilm in concentration-dependent mode. Scanning electron microscopy analyses confirmed severe perturbations caused by 5f on MSSA and MRSA biofilm architecture. The acute toxicity assay, using Galleria mellonella larvae, indicated a low toxic effect of 5f after 72 h, displaying lethality of 20% and 30% at 7.8 µg mL-1 and 78.0 µg mL-1, respectively. In addition, the antibacterial activity spectrum of 5f indicated action against planktonic cells of Enterococcus faecalis (MIC = 7.8 µg mL-1), Acinetobacter baumannii (MIC = 15.6 µg mL-1) and Mycobacterium tuberculosis (MIC = 5.7 µg mL-1). Altogether, these results open new avenues for 5f as an anti-Staphylococcus aureus agent, with potential applications as antibacterial drug, adjunct of antibiotics and medical devices coating.

Antibacterial activity of a new monocarbonyl analog of curcumin MAC 4 is associated with divisome disruption.

Curcumin (CUR) is a symmetrical dicarbonyl compound with antibacterial activity. On the other hand, pharmacokinetic and chemical stability limitations hinder its therapeutic application. Monocarbonyl analogs of curcumin (MACs) have been shown to overcome these barriers. We synthesized and investigated the antibacterial activity of a series of unsymmetrical MACs derived from acetone against Mycobacterium tuberculosis and Gram-negative and Gram-positive species. Phenolic MACs 4, 6 and 8 showed a broad spectrum and potent activity, mainly against M. tuberculosis, Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus (MRSA), with MIC (minimum inhibitory concentration) values ranging from 0.9 to 15.6 µg/mL. The investigation regarding toxicity on human lung cells (MRC-5 and A549 lines) revealed MAC 4 was more selective than MACs 6 and 8, with SI (selectivity index) values ranging from 5.4 to 15.6. In addition, MAC 4 did not demonstrate genotoxic effects on A549 cells and it was more stable than CUR in phosphate buffer (pH 7.4) for 24 h at 37 °C. Fluorescence and phase contrast microscopies indicated that MAC 4 has the ability to disrupt the divisome of Bacillus subtilis without damaging its cytoplasmic membrane. However, biochemical investigations demonstrated that MAC 4 did not affect the GTPase activity of B. subtilis FtsZ, which is the main constituent of the bacterial divisome. These results corroborated that MAC 4 is a promising antitubercular and antibacterial agent.

Identification of a Prenyl Chalcone as a Competitive Lipoxygenase Inhibitor: Screening, Biochemical Evaluation and Molecular Modeling Studies.

Cyclooxygenase (COX) and lipoxygenase (LOX) are key targets for the development of new anti-inflammatory agents. LOX, which is involved in the biosynthesis of mediators in inflammation and allergic reactions, was selected for a biochemical screening campaign to identify LOX inhibitors by employing the main natural product library of Brazilian biodiversity. Two prenyl chalcones were identified as potent inhibitors of LOX-1 in the screening. The most active compound, (E)-2-O-farnesyl chalcone, decreased the rate of oxygen consumption to an extent similar to that of the positive control, nordihydroguaiaretic acid. Additionally, studies on the mechanism of the action indicated that (E)-2-O-farnesyl chalcone is a competitive LOX-1 inhibitor. Molecular modeling studies indicated the importance of the prenyl moieties for the binding of the inhibitors to the LOX binding site, which is related to their pharmacological properties.

Investigating the Modes of Action of the Antimicrobial Chalcones BC1 and T9A.

Xanthomonas citri subsp. citri (X. citri) is an important phytopathogen and causes Asiatic Citrus Canker (ACC). To control ACC, copper sprays are commonly used. As copper is an environmentally damaging heavy metal, new antimicrobials are needed to combat citrus canker. Here, we explored the antimicrobial activity of chalcones, specifically the methoxychalcone BC1 and the hydroxychalcone T9A, against X. citri and the model organism Bacillus subtilis. BC1 and T9A prevented growth of X. citri and B. subtilis in concentrations varying from 20 µg/mL to 40 µg/mL. BC1 and T9A decreased incorporation of radiolabeled precursors of DNA, RNA, protein, and peptidoglycan in X. citri and B. subtilis. Both compounds mildly affected respiratory activity in X. citri, but T9A strongly decreased respiratory activity in B. subtilis. In line with that finding, intracellular ATP decreased strongly in B. subtilis upon T9A treatment, whereas BC1 increased intracellular ATP. In X. citri, both compounds resulted in a decrease in intracellular ATP. Cell division seems not to be affected in X. citri, and, although in B. subtilis the formation of FtsZ-rings is affected, a FtsZ GTPase activity assay suggests that this is an indirect effect. The chalcones studied here represent a sustainable alternative to copper for the control of ACC, and further studies are ongoing to elucidate their precise modes of action.

Short-term exposure to chrysin promotes proliferative responses in the ventral male prostate and female prostate of adult gerbils.

Chrysin (5,7-dihydroxyflavone) is a bioactive compound found in different fruits, vegetables, honey and propolis. This flavone has been suggested for the treatment of reproductive dysfunction, mainly because of its antioxidant and hormonal properties. However, the effects of this polyphenol on the prostate are still poorly understood. The purpose of this study was to evaluate the effects of short-term chrysin exposure on the ventral male and female prostates of adult gerbils. To evaluate the androgenic potential of chrysin, gerbils were also exposed to testosterone. Male and female gerbils were exposed to chrysin (50 mg/kg/day, orally) or testosterone cypionate (1 mg/kg/week, subcutaneously) for 3, 7 and 21 days. Prostates were dissected for morphological, stereological and immunohistochemical analyses. Serum levels of testosterone and 17ß-estradiol were measured by ELISA. Serum testosterone levels were not increased by chrysin supplementation in males or females. However, only females treated with chrysin for 21 days showed an increase in estradiol levels. Increased androgen receptor immunoreactivity, higher proliferation rates and glandular hyperplasia were observed in male and female prostates for all chrysin treatment times. Additionally, increased oestrogen receptor alpha immunoreactivity was observed in all chrysin-treated females. Although chrysin and testosterone promoted similar morphological changes in the gerbil prostate, chrysin supplementation was less deleterious to prostate health, since it resulted in lower incidence of hyperplasia and an absence of neoplastic foci.

Antibacterial activity of 3,3'-dihydroxycurcumin (DHC) is associated with membrane perturbation.

Curcumin is a plant diphenylheptanoid and has been investigated for its antibacterial activity. However, the therapeutic uses of this compound are limited due to its chemical instability. In this work, we evaluated the antimicrobial activity of diphenylheptanoids derived from curcumin against Gram-positive and Gram-negative bacteria, and also against Mycobacterium tuberculosis in terms of MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) values. 3,3'-Dihydroxycurcumin (DHC) displayed activity against Enterococcus faecalis, Staphylococcus aureus and M. tuberculosis, demonstrating MIC values of 78 and 156 µg/mL. In addition, DHC was more stable than curcumin in acetate buffer (pH 5.0) and phosphate buffer (pH 7.4) for 24 h at 37 °C. We proposed that membrane and the cell division protein FtsZ could be the targets for DHC due to that fact that curcumin exhibits this mode of antibacterial action. Fluorescence microscopy of Bacillus subtilis stained with SYTO9 and propidium iodide fluorophores indicated that DHC has the ability to perturb the bacterial membrane. On the other hand, DHC showed a weak inhibition of the GTPase activity of B. subtilis FtsZ. Toxicity assay using human cells indicated that DHC has moderate capacity to reduce viability of liver cells (HepG2 line) and lung cells (MRC-5 and A549 lines) when compared with doxorubicin. Alkaline comet assay indicated that DHC was not able to induce DNA damage in A549 cell line. These results indicated that DHC is promising compound with antibacterial and antitubercular activities.

Anabolic effects of chrysin on the ventral male prostate and female prostate of adult gerbils (Meriones unguiculatus).

Chrysin is a bioflavonoid found in fruits, flowers, tea, honey and wine, which has antioxidant, anti-inflammatory, antiallergic and anticarcinogenic properties. This flavone has also been considered as beneficial for reproduction due its testosterone-boosting potential. Thus, the aim of this study was to evaluate the effects of chrysin on the prostate and gonads of male and female adult gerbils. In addition, a comparative analysis of the effects of testosterone on these same organs was conducted. Ninety-day-old male and female gerbils were treated with chrysin (50mgkg-1day-1) or testosterone cypionate (1mgkg-1week-1) for 21 days. The ventral male prostate and female prostate were dissected out for morphological, morphometric-stereological and ultrastructural assays. Testes and ovaries were submitted to morphological and morphometric---stereological analyses. Chrysin treatment caused epithelial hyperplasia and stromal remodelling of the ventral male and female prostate. Ultrastructurally, male and female prostatic epithelial cells in the chrysin group presented marked development of the organelles involved in the biosynthetic-secretory pathway, whereas cellular toxicity was observed only in female glands. Chrysin preserved normal testicular morphology and increased the number of growing ovarian follicles. Comparatively, testosterone treatment was detrimental to the prostate and gonads, since foci of prostatic intraepithelial neoplasia and gonadal degeneration were observed in both sexes. Thus, under the experimental conditions of this study, chrysin was better tolerated than testosterone in the prostate and gonads.

Antibacterial and Antitubercular Activities of Cinnamylideneacetophenones.

Cinnamaldehyde is a natural product with broad spectrum of antibacterial activity. In this work, it was used as a template for design and synthesis of a series of 17 cinnamylideneacetophenones. Phenolic compounds 3 and 4 exhibited MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) values of 77.9 to 312 µM against Staphylococcus aureus, Streptococcus mutans, and Streptococcus sanguinis. Compounds 2, 7, 10, and 18 presented potent effects against Mycobacterium tuberculosis (57.2 µM ≤ MIC ≤ 70.9 µM). Hydrophilic effects caused by substituents on ring B increased antibacterial activity against Gram-positive species. Thus, log Po/w were calculated by using high-performance liquid chromatography-photodiode array detection (HPLC-PDA) analyses, and cinnamylideneacetophenones presented values ranging from 2.5 to 4.1. In addition, the effects of 3 and 4 were evaluated on pulmonary cells, indicating their moderate toxicity (46.3 µM ≤ IC50 ≤ 96.7 µM) when compared with doxorubicin. Bioactive compounds were subjected to in silico prediction of pharmacokinetic properties, and did not violate Lipinski's and Veber's rules, corroborating their potential bioavailability by an oral route.

The 2',4'-dihydroxychalcone could be explored to develop new inhibitors against the glycerol-3-phosphate dehydrogenase from Leishmania species.

The enzyme glycerol-3-phosphate dehydrogenase (G3PDH) from Leishmania species is considered as an attractive target to design new antileishmanial drugs and a previous in silico study reported on the importance of chalcones to achieve its inhibition. Here, we report the identification of a synthetic chalcone in our in vitro assays with promastigote cells from Leishmania amazonensis, its biological activity in animal models, and docking followed by molecular dynamics simulation to investigate the molecular interactions and structural patterns that are crucial to achieve the inhibition complex between this compound and G3PDH. A molecular fragment of this natural product derivative can provide new inhibitors with increased potency and selectivity.

Ethyl ferulate, a component with anti-inflammatory properties for emulsion-based creams.

Ethyl ferulate (FAEE) has been widely studied due to its beneficial heath properties and, when incorporated in creams, shows a high sun protection capacity. Here we aimed to compare FAEE and its precursor, ferulic acid (FA), as free radical scavengers, inhibitors of oxidants produced by leukocytes and the alterations in rheological properties when incorporated in emulsion based creams. The cell-free antiradical capacity of FAEE was decreased compared to FA. However, FAEE was more effective regarding the scavenging of reactive oxygen species produced by activated leukocytes. Stress and frequency sweep tests showed that the formulations are more elastic than viscous. The viscoelastic features of the formulations were confirmed in the creep and recovery assay and showed that the FAEE formulation was less susceptive to deformation. Liberation experiments showed that the rate of FAEE release from the emulsion was slower compared to FA. In conclusion, FAEE is more effective than FA as a potential inhibitor of oxidative damage produced by oxidants generated by leukocytes. The rheological alterations caused by the addition of FAEE are indicative of lower spreadability, which could be useful for formulations used in restricted areas of the skin.

Antiprotozoal activity of quinonemethide triterpenes from Maytenus ilicifolia (Celastraceae).

The present study describes the leishmanicidal and trypanocidal activities of two quinonemethide triterpenes, maytenin (1) and pristimerin (2), isolated from Maytenus ilicifolia root barks (Celastraceae). The compounds were effective against the Trypanosomatidae Leishmania amazonensis and Leishmania chagasi and Trypanosoma cruzi, etiologic agents of leishmaniasis and Chagas' disease, respectively. The quinonemethide triterpenes 1 and 2 exhibited a marked in vitro leishmanicidal activity against promastigotes and amastigotes with 50% inhibitory concentration (IC(50)) values of less than 0.88 nM. Both compounds showed IC(50) lower than 0.3 nM against Trypanosoma cruzi epimastigotes. The selectivity indexes (SI) based on BALB/c macrophages for L. amazonensis and L. chagasi were 243.65 and 46.61 for (1) and 193.63 and 23.85 for (2) indicating that both compounds presented high selectivity for Leishmania sp. The data here presented suggests that these compounds should be considered in the development of new and more potent drugs for the treatment of leishmaniasis and Chagas' disease.

Membranolytic Activity Profile of Nonyl 3,4-Dihydroxybenzoate: A New Anti-Biofilm Compound for the Treatment of Dermatophytosis.

The ability of dermatophytes to live in communities and resist antifungal drugs may explain treatment recurrence, especially in onychomycosis. Therefore, new molecules with reduced toxicity that target dermatophyte biofilms should be investigated. This study evaluated nonyl 3,4-dihydroxybenzoate (nonyl) susceptibility and mechanism of action on planktonic cells and biofilms of T. rubrum and T. mentagrophytes. Metabolic activities, ergosterol, and reactive oxygen species (ROS) were quantified, and the expression of genes encoding ergosterol was determined by real-time PCR. The effects on the biofilm structure were visualized using confocal electron microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). T. rubrum and T. mentagrophytes biofilms were susceptible to nonyl and resistant to fluconazole, griseofulvin (all strains), and terbinafine (two strains). The SEM results revealed that nonyl groups seriously damaged the biofilms, whereas synthetic drugs caused little or no damage and, in some cases, stimulated the development of resistance structures. Confocal microscopy showed a drastic reduction in biofilm thickness, and transmission electron microscopy results indicated that the compound promoted the derangement and formation of pores in the plasma membrane. Biochemical and molecular assays indicated that fungal membrane ergosterol is a nonyl target. These findings show that nonyl 3,4-dihydroxybenzoate is a promising antifungal compound.

Pyridine alkaloids from Senna multijuga as acetylcholinesterase inhibitors.

As part of an ongoing research project on Senna and Cassia species, five new pyridine alkaloids, namely, 12'-hydroxy-7'-multijuguinol (1), 12'-hydroxy-8'-multijuguinol (2), methyl multijuguinate (3), 7'-multijuguinol (4), and 8'-multijuguinol (5), were isolated from the leaves of Senna multijuga (syn. Cassiamultijuga). Their structures were elucidated on the basis of spectroscopic data analysis. Mass spectrometry was used for confirmation of the positions of the hydroxy groups in the side-chains of 1, 2, 4, and 5. All compounds exhibited weak in vitro acetylcholinesterase inhibitory activity as compared with the standard compound physostigmine.

Antiprotozoal sesquiterpene pyridine alkaloids from Maytenus ilicifolia.

As part of a bioprospecting program aimed at the discovery of antiprotozoal agents from the Brazilian flora, two new sesquiterpene pyridine alkaloids, ilicifoliunines A (1) and B (2), along with the known alkaloids aquifoliunine E-I (3) and mayteine (4), were isolated from the root bark of Maytenus ilicifolia. The structures of 1 and 2 were established on the basis of spectroscopic data interpretation. Alkaloid 3 displayed potent in vitro antiprotozoal activity against Leishmania chagasi and Trypanosoma cruzi, with IC(50) values of 1.4 and 41.9 µM, respectively, as well as low cytotoxicity against murine peritoneal macrophages (IC(50) of 1.8 mM).

Antifungal activity and toxicity of an octyl gallate-loaded nanostructured lipid system on cells and nonmammalian animals.

Aim: Octyl gallate (OG) loaded into a nanostructured lipid system (NLS) was tested for antifungal activity and in vitro and in vivo toxicity. Methods & Results: The features of NLS-OG were analyzed by dynamic light scattering and showed adequate size (132.1 nm) and homogeneity (polydispersity index = 0.200). OG was active against Paraccoccidioides spp., and NLS-OG did not affect antifungal activity. NLS-OG demonstrated reduced toxicity to lung cells and zebrafish embryos compared with OG, whereas NLS was toxic to hepatic cells. OG and NLS-OG did not show toxicity in a Galleria mellonella model at 20 mg/kg. All toxic concentrations were superior to MIC (antifungal activity). Conclusion: These results indicate good anti-Paracoccidioides activity and low toxicity of NLS-OG.

Plain language summary Drugs for the treatment of fungal diseases are limited in number and present side effects, drug interactions, risks for pregnant women and fungal resistance. The authors produced a derivative compound from plants called octyl gallate (OG) and then incorporated it into a nanoparticle lipid system (NLS) for better distribution in biological fluids. NLS-OG was tested against a fungus called Paracoccidioides, which causes lung infections. The toxicity profile of NLS-OG was also evaluated in lung and hepatic cells as well as novel animal models. NLS-OG presented good antifungal activity and low toxicity in lung cells and embryos.

Mitochondrial Imbalance of Trypanosoma cruzi Induced by the Marine Alkaloid 6-Bromo-2'-de-N-Methylaplysinopsin.

Chagas disease, caused by Trypanosoma cruzi, affects seven million people worldwide and lacks effective treatments. Using bioactivity-guided fractionation, NMR, and electrospray ionization-high resolution mass spectrometry (ESI-HRMS) spectral analysis, the indole alkaloid 6-bromo-2'-de-N-methylaplysinopsin (BMA) was isolated and chemically characterized from the marine coral Tubastraea tagusensis. BMA was tested against trypomastigotes and intracellular amastigotes of T. cruzi, resulting in IC50 values of 62 and 5.7 µM, respectively, with no mammalian cytotoxicity. The mechanism of action studies showed that BMA induced no alterations in the plasma membrane permeability but caused depolarization of the mitochondrial membrane potential, reducing ATP levels. Intracellular calcium levels were also reduced after the treatment, which was associated with pH alteration of acidocalcisomes. Using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)/MS analysis, alterations of mass spectral signals were observed after treatment with BMA, suggesting a different mechanism from benznidazole. In silico pharmacokinetic-pharmacodynamic (PKPD) parameters suggested a drug-likeness property, supporting the promising usefulness of this compound as a new hit for optimizations.

Flavonoid Derivatives Targeting BCR-ABL Kinase: Semisynthesis, Molecular Dynamic Simulations and Enzymatic Inhibition.

BACKGROUND: Natural products have been universally approached in the research of novel trends useful to detail the essential paths of the life sciences and as a strategy for pharmacotherapeutics. OBJECTIVE: This work focuses on further modification to the 6-hydroxy-flavanone building block aiming to obtain improved BCR-ABL kinase inhibitors. METHODS: Ether derivatives were obtained from Williamson synthesis and triazole from Microwave- assisted click reaction. Chemical structures were finely characterized through IR, 1H and 13C NMR and HRMS. They were tested for their inhibitory activity against BCR-ABL kinase. RESULTS: Two inhibitors bearing a triazole ring as a pharmacophoric bridge demonstrated the strongest kinase inhibition at IC50 value of 364 nM (compound 3j) and 275 nM (compound 3k). CONCLUSION: 6-hydroxy-flavanone skeleton can be considered as a promising core for BCR-ABL kinase inhibitors.