Digoxin Derivatives Sensitize a Saccharomyces cerevisiae Mutant Strain to Fluconazole by Inhibiting Pdr5p.

The poor outcome of treatments for fungal infections is a consequence of the increasing incidence of resistance to antifungal agents, mainly due to the overexpression of efflux pumps. To surpass this mechanism of resistance, a substance able to inhibit these pumps could be administered in association with antifungals. Saccharomyces cerevisiae possesses an efflux pump (Pdr5p) homologue to those found in pathogenic yeast. Digoxin is a natural product that inhibits Na+, K+-ATPase. The aim of this study was to evaluate whether digoxin and its derivatives (i.e., DGB, digoxin benzylidene) can inhibit Pdr5p, reversing the resistance to fluconazole in yeasts. An S. cerevisiae mutant strain that overexpresses Pdr5p was used in the assays. The effects of the compounds on yeast growth, efflux activity, and Pdr5p ATPase activity were measured. All derivatives enhanced the antifungal activity of fluconazole against S. cerevisiae, in comparison to fluconazole alone, with FICI values ranging from 0.031 to 0.500. DGB 1 and DGB 3 presented combined effects with fluconazole against a Candida albicans strain, with fractional inhibitory concentration index (FICI) values of 0.625 and 0.281, respectively The compounds also inhibited the efflux of rhodamine 6G and Pdr5p ATPase activity, with IC50 values ranging from 0.41 µM to 3.72 µM. The results suggest that digoxin derivatives impair Pdr5p activity. Considering the homology between Pdr5p and efflux pumps from pathogenic fungi, these compounds are potential candidates to be used in association with fluconazole to treat resistant fungal infections.

Alkaloid and phenolic compounds of Xylopia aromatica inhibits tumor growth by down-regulating matrix metalloproteinase-2 (MMP-2) expression.

Annonacea species have been reported to possess antitumor properties. However, the in vitro and in vivo antitumor activities of Xylopia aromatica (Annonacea) have not yet been elucidated. This study aimed to investigate the effects of Xylopia aromatica leaves hexane fraction (XaHF) on Ehrlich ascites carcinoma cells lines (EAC), both in vitro and in vivo. In vitro assays revealed a significant cytotoxic effect with the two lower XaHF concentrations (62.5 and 32.3mg/mL). EAC (2.5x106 cells) were inoculated in the right flank of Swiss mice, and the animals were treated intraperitoneally with 32.3mg kg-1 of XaHF daily, for 20 days. Our findings indicate that XaHF suppressed the growth of EAC in vivo, with a significant decrease (46%) in tumor volume. There was also a decrease in the necrosis area (71%), inflammatory infiltrate, and MMP-2 expression. High-Performance Liquid Chromatography with Diode Array Detector (HPLC-DAD) identified secondary metabolites possibly related to phenolic acids, flavonoids, and alkaloids. Thus, the results confirmed the antitumoral activity that may be related to the presence of the identified metabolites in XaHF extract.

Implications of Synthetic Modifications of the Cardiotonic Steroid Lactone Ring on Cytotoxicity.

Na,K-ATPase (NKA) and cardiotonic steroids (CTS) have shown potent cytotoxic and anticancer effects. Here, we have synthesized a series of CTS digoxin derivatives (γ-benzylidene) with substitutions in the lactone ring and evaluated the cytotoxicity caused by digoxin derivatives in tumor and non-tumor cells lines, as well as their effects on NKA. The cytotoxicity assay was determined in HeLa, A549, and WI-26 VA4 after they were treated for 48 h with increased concentrations of CTS. The effects of CTS on NKA activity and immunoblotting of α1 and ß1 isoforms were evaluated at IC50 concentrations in A549 cell membrane. NKA activity from mouse brain cortex was also measured. The majority of CTS exhibited low cytotoxicity in tumor and non-tumor cells, presenting IC50 values at micromolar concentrations, while digoxin showed cytotoxicity at nanomolar concentrations. BD-15 presented the lowest IC50 value (8 µM) in A549 and reduced its NKA activity in 28%. In contrast, BD-7 was the compound that most inhibited NKA (56% inhibition) and presented high IC50 value for A549. In mouse cortex, only BD-15 modulated the enzyme activity in a concentration-dependent inhibition curve. These results demonstrate that the cytotoxicity of these compounds is not related to NKA inhibition. The substitutions in the lactone ring of digoxin led to an increase in the cytotoxic concentration in tumor cells, which may not be interesting for cancer, but it has the advantage of increasing the therapeutic margin of these molecules when compared to classic CTS, and can be used safely in research for other diseases.

The γ-Benzylidene Digoxin Derivative BD-15 Increases the α3-Na, K-ATPase Activity in Rat Hippocampus and Prefrontal Cortex and no Change on Heart.

Our study aimed to investigate the effects of the new cardiotonic steroid BD-15 (γ-benzylidene derivatives) in the behavioral parameters, oxidative stress and the Na, K-ATPase activity in the hippocampus, prefrontal cortex and heart from rats to verify the safety and possible utilization in brain disorders. For this study, groups of male Wistar rats were used after intraperitoneal injection of 20, 100 and 200 µg/Kg with BD-15. The groups were treated for three consecutive days and the control group received 0.9% saline. BD-15 did not alter behavior of rats treated with different doses. An increase in the specific α2,3-Na, K-ATPase activity was observed for all doses of BD-15 tested in the hippocampus. However, in the prefrontal cortex, only the dose of 100 µg/Kg increased the activity of all Na, K-ATPase isoforms. BD-15 did not cause alteration in the lipid peroxidation levels in the hippocampus, but in the prefrontal cortex, a decrease of lipid peroxidation (~ 25%) was observed. In the hippocampus, GSH levels increased with all doses tested, while in the prefrontal cortex no changes were found. Subsequently, when the effect of BD-15 on cardiac tissue was analyzed, no changes were observed in the tested parameters. BD-15 at a dosage of 100 µg/Kg proved to be promising because it is considered therapeutic for brain disorders, since it increases the activity of the α3-Na, K-ATPase in the hippocampus and prefrontal cortex, as well as decreasing the oxidative stress in these brain regions. In addition, this drug did not cause changes in the tissues of the heart and kidneys, preferentially demonstrating specificity for the brain.

Pharmacologic potential of new nitro-compounds as antimicrobial agents against nosocomial pathogens: design, synthesis, and in vitro effectiveness.

Nosocomial infections are an important cause of morbi-mortality worldwide. The increase in the rate of resistance to conventional drugs in these microorganisms has stimulated the search for new therapeutic options. The nitro moiety (NO2) is an important pharmacophore of molecules with high anti-infective activity. We aimed to synthesize new nitro-derivates and to evaluate their antibacterial and anti-Candida potential in vitro. Five compounds [3-nitro-2-phenylchroman-4-ol (3); 3-nitro-2-phenyl-2H-chromene (4a); 3-nitro-2-(4-chlorophenyl)-2H-chromene (4b); 3-nitro-2-(4-fluorophenyl)-2H-chromene (4c), and 3-Nitro-2-(2,3-dichlorophenyl)-2H-chromene (4d)] were efficiently synthesized by Michael-aldol reaction of 2-hydroxybenzaldehyde with nitrostyrene, resulting in one ß-nitro-alcohol (3) and four nitro-olefins (4a-4d). The antibacterial and anti-Candida potentials were evaluated by assaying minimal inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and minimum bactericidal concentration (MBC). Mono-halogenated nitro-compounds (4b and 4c) showed anti-staphylococcal activity with MIC values of 15.6-62.5 µg/mL and MBC of 62.5 µg/mL. However, the activity against Gram-negative strains was showed to be considerably lower and our data suggests that this effect was associated with the outer membrane. Furthermore, nitro-compounds 4c and 4d presented activity against Candida spp. with MIC values ranging from 7.8-31.25 µg/mL and MFC of 15.6-500 µg/mL. In addition, these compounds were able to induce damage in fungal cells increasing the release of intracellular material, which was associated with actions on the cell wall independent of quantitative changes in chitin and ß-glucan. Together, these findings show that nitro-compounds can be exploited as anti-staphylococcal and anti-Candida prototypes.

21­Benzylidene digoxin, a novel digoxin hemi-synthetic derivative, presents an anti-inflammatory activity through inhibition of edema, tumour necrosis factor alpha production, inducible nitric oxide synthase expression and leucocyte migration.

Recent findings have demonstrated new therapeutic functions of cardiotonic steroids, a process that is termed drug repositioning. Despite the confirmed anti-inflammatory effects of cardiotonic steroids, their clinical use has been discouraged due to toxicity related to inhibition of the Na+/K+ ATPase. A novel synthetic compound derived from digoxin, 21­benzylidene digoxin (21­BD), does not inhibit this enzyme. Herein, we evaluated the anti-inflammatory and antinociceptive effects and acute toxicity of 21­BD. Murine (Swiss mice) models of paw oedema induced by carrageenan, acetic acid-induced abdominal writhing, and formalin and acute toxicity tests were used. Oral administration of 21­BD (0.3 mg/kg) showed a significant and prolonged inhibition of paw oedema. Histological analysis demonstrated a reduction in inflammatory cells and expression of inducible nitric oxide synthase (iNOS) in footpads 6 h after administration of carrageenan. 21­BD (0.3 mg/kg) also reduced the levels of tumour necrosis factor (TNF)-α 2 and 4 h after carrageenan. 21­BD demonstrated antinociceptive activity, inhibiting abdominal writhes at all tested doses. However, in the formalin test, 21­BD did not present antinociceptive activity. In the acute toxicity test, 21­BD did not cause symptoms of toxicity or mortality. The present study demonstrated, for the first time, that 21­BD is safe and exhibits a marked anti-inflammatory activity in acute local inflammation. This effect might be a consequence of its ability to inhibit the release of the PMN leucocyte-derived mediators, including TNF-α, and iNOS expression as well as its inhibitory effect on oedema and PMN leucocyte infiltration.

Rational selection of immunodominant and preserved epitope Sm043300e from Schistosoma mansoni and design of a chimeric molecule for biotechnological purposes.

Human schistosomiasis is a neglected tropical disease of great importance in public health. A large number of people are infected with schistosomiasis, making vaccine development and effective diagnosis important control strategies. A rational epitope prediction workflow using Schistosoma mansoni hypothetical proteins was previously presented by our group, and an improvement to that approach is presented here. Briefly, immunodominant epitopes from parasite membrane proteins were predicted by reverse vaccinology strategy with additional in silico analysis. Furthermore, epitope recognition was evaluated using sera of individuals infected with S. mansoni. The epitope that stood out in both in silico and in vitro assays was used to compose a rational chimeric molecule to improve immune response activation. Out of 2185 transmembrane proteins, four epitopes with high binding affinities for human and mouse MHCII molecules were selected through computational screening. These epitopes were synthesized to evaluate their ability to induce TCD4+ lymphocyte proliferation in mice. Sm204830e and Sm043300e induced significant TCD4+ proliferation. Both epitopes were submitted to enzyme-linked immunosorbent assay to evaluate their recognition by IgG antibodies from the sera of infected individuals, and epitope Sm043300 was significantly recognized in most sera samples. Epitope Sm043300 also showed good affinity for human MHCII molecules in molecular docking, and its sequence is curiously highly conserved in four S. mansoni proteins, all of which are described as G-protein-coupled receptors. In addition, we have demonstrated the feasibility of incorporating this epitope, which showed low similarity to human sequences, into a chimeric molecule. The stability of the molecule was evaluated by molecular modeling aimed at future molecule production for use in diagnosis and vaccination trials.

Synthesis and biological evaluation of novel 3-alkylpyridine marine alkaloid analogs with promising anticancer activity.

Cancer continues to be one of the most important health problems worldwide, and the identification of novel drugs and treatments to address this disease is urgent. During recent years, marine organisms have proven to be a promising source of new compounds with action against tumoral cell lines. Here, we describe the synthesis and anticancer activity of eight new 3-alkylpyridine alkaloid (3-APA) analogs in four steps and with good yields. The key step for the synthesis of these compounds is a Williamson etherification under phase-transfer conditions. We investigated the influence of the length of the alkyl chain attached to position 3 of the pyridine ring on the cytotoxicity of these compounds. Biological assays demonstrated that compounds with an alkyl chain of ten carbon atoms (4c and 5c) were the most active against two tumoral cell lines: RKO-AS-45-1 and HeLa. Micronucleus and TUNEL assays showed that both compounds are mutagenic and induce apoptosis. In addition, Compound 5c altered the cellular actin cytoskeleton in RKO-AS-45-1 cells. The results suggest that Compounds 4c and 5c may be novel prototype anticancer agents.