José A. F. P. Villar

21-Benzylidene Digoxin: A Proapoptotic Cardenolide of Cancer Cells That Up-Regulates Na,K-ATPase and Epithelial Tight Junctions

Cardiotonic steroids are used to treat heart failure and arrhythmia and have promising anticancer effects. The prototypic cardiotonic steroid ouabain may also be a hormone that modulates epithelial cell adhesion. Cardiotonic steroids consist of a steroid nucleus and a lactone ring, and their biological effects depend on the binding to their receptor, Na,K-ATPase, through which, they inhibit Na+ and K+ ion transport and activate of several intracellular signaling pathways. In this study, we added a styrene group to the lactone ring of the cardiotonic steroid digoxin, to obtain 21-benzylidene digoxin (21-BD), and investigated the effects of this synthetic cardiotonic steroid in different cell models. Molecular modeling indicates that 21-BD binds to its target Na,K-ATPase with low affinity, adopting a different pharmacophoric conformation when bound to its receptor than digoxin. Accordingly, 21-DB, at relatively high µM amounts inhibits the activity of Na,K-ATPase α1, but not α2 and α3 isoforms. In addition, 21-BD targets other proteins outside the Na,K-ATPase, inhibiting the multidrug exporter Pdr5p. When used on whole cells at low µM concentrations, 21-BD produces several effects, including: 1) up-regulation of Na,K-ATPase expression and activity in HeLa and RKO cancer cells, which is not found for digoxin, 2) cell specific changes in cell viability, reducing it in HeLa and RKO cancer cells, but increasing it in normal epithelial MDCK cells, which is different from the response to digoxin, and 3) changes in cell-cell interaction, altering the molecular composition of tight junctions and elevating transepithelial electrical resistance of MDCK monolayers, an effect previously found for ouabain. These results indicate that modification of the lactone ring of digoxin provides new properties to the compound, and shows that the structural change introduced could be used for the design of cardiotonic steroid with novel functions.

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.

Design and Synthesis of New Chacones Substituted with Azide/Triazole Groups and Analysis of Their Cytotoxicity Towards HeLa Cells

A series of new chalcones substituted with azide/triazole groups were designed and synthesized, and their cytotoxic activity was evaluated in vitro against the HeLa cell line. O-Alkylation, Claisen-Schmidt condensation and Cu(I)-catalyzed cycloaddition of azides with terminal alkynes were applied in key steps. Fifteen compounds were tested against HeLa cells. Compound 8c was the most active molecule, with an IC50 value of 13.03 µM, similar to the value of cisplatin (7.37 µM).

Differences of lipid membrane modulation and oxidative stress by digoxin and 21-benzylidene digoxin

&NA; Cardiotonic steroids (CTS) are compounds which bind to the Na,K‐ATPase, leading to its inhibition and in some cases initiating signaling cascades. Long utilized as a treatment for congestive heart disease, CTS have more recently been observed to inhibit proliferation and cause apoptosis in several cancer cell lines. A synthetic derivative of the CTS digoxin, called 21‐benzylidene digoxin (21‐BD), activates the Na,K‐ATPase rather than cause its inhibition, as its parent compound does. Here, the mechanism behind the unique effects of 21‐BD are further explored. In HeLa cancer cells, low (5 &mgr;M) and high (50 &mgr;M) doses of 21‐BD activated and inhibited the Na,K‐ATPase, respectively, without altering the membrane expression of the Na,K‐ATPase. While digoxin did not affect HeLa membrane cholesterol or phospholipid content, 50 &mgr;M 21‐BD increased both lipids via a mechanism reliant on an intact cell. Afterwards, the direct action of 21‐BD was evaluated on erythrocyte membranes; however, no effect was observed. As CTS may generate reactive oxygen species (ROS) which can affect plasma membrane fluidity and therefore Na,K‐ATPase activity, several markers involved in ROS generation were analyzed such as, lipid peroxidation (TBARS), reduced glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD). GSH content and catalase activity were unaffected by digoxin or 21‐BD. Surprisingly, TBARS and SOD activity was decreased with digoxin and with 50 &mgr;M 21‐BD. Thus, 21‐BD and digoxin altered components involved in ROS generation and inhibition in a similar fashion. This study suggests alterations to the Na,K‐ATPase and membrane lipids by 21‐BD is not reliant on ROS generation.

Synthesis and in vitro evaluation of novel triazole/azide chalcones

A series of 30 novel triazole/azide chalcone derivatives were synthesized by Claisen-Schmidt and Cu(I)-catalyzed cycloaddition reactions. The antiproliferative activity of each compound was evaluated against HeLa, RKO-AS45-1 and Wi-26VA4 cell lines. Terminal deoxynucleotidyl transferase dUTP nick end labeling assays indicated that compounds 4j and 5j significantly reduced the HeLa and RKO-AS45-1cell populations compared to the controls. The relative expression of the TP53 gene revealed changes in both cell lines after exposure to compounds 5j and 4j. The increased expression of the TP53 gene suggests a cellular attempt to repair DNA damage and indicates these triazole/azide chalcone derivatives as promising anticancer agents.

γ-Benzylidene digoxin derivatives synthesis and molecular modeling: Evaluation of anticancer and the Na,K-ATPase activity effect.

Cardiotonic steroids (CS), natural compounds with traditional use in cardiology, have been recently suggested to exert potent anticancer effects. However, the repertoire of molecules with Na,K-ATPase activity and anticancer properties is limited. This paper describes the synthesis of 6 new digoxin derivatives substituted (on the C17-butenolide) with γ-benzylidene group and their cytotoxic effect on human fibroblast (WI-26 VA4) and cancer (HeLa and RKO) cell lines as well as their effect on Na,K-ATPase activity and expression. As digoxin, compound BD-4 was almost 100-fold more potent than the other derivatives for cytotoxicity with the three types of cells used and was also the only one able to fully inhibit the Na,K-ATPase of HeLa cells after 24h treatment. No change in the Na,K-ATPase α1 isoform protein expression was detected. On the other hand it was 30-40 fold less potent for direct Na,K-ATPase inhibition, when compared to the most potent derivatives, BD-1 and BD-3, and digoxin. The data presented here demonstrated that the anticancer effect of digoxin derivatives substituted with γ-benzylidene were not related with their inhibition of Na,K-ATPase activity or alteration of its expression, suggesting that this classical molecular mechanism of CS is not involved in the cytotoxic effect of our derivatives.

Epitopes Rationally Selected through Computational Analyses Induce T-Cell Proliferation in Mice and Are Recognized by Serum from Individuals Infected with Schistosoma mansoni

Schistosomiasis is the second leading cause of death due to parasitic diseases in the world. Seeking an alternative for the control of disease, the World Health Organization funded the genome sequencing of the major species related to schistosomiasis to identify potential vaccines and therapeutic targets. Therefore, the aim of this work was to select T and B‐cell epitopes from Schistosoma mansoni through computational analyses and evaluate the immunological potential of epitopes in vitro. Extracellular regions of membrane proteins from the Schistosoma mansoni were used to predict promiscuous epitopes with affinity to different human Major Histocompatibility Class II (MHCII) molecules by bioinformatics analysis. The three‐dimensional structure of selected epitopes was constructed and used in molecular docking to verify the interaction with murine MHCII H2‐IAb. In this process, four epitopes were selected and synthesized to assess their ability to stimulate proliferation of CD4+ T lymphocytes in mice splenocyte cultures. The results showed that Sm041370 and Sm168240 epitopes induced significant cell proliferation. Additionally, the four epitopes were used as antigens in the Indirect Enzyme‐Linked Immunosorbent Assay (ELISA) to assess the recognition by serum from individuals infected with Schistosoma mansoni. Sm140560, Sm168240, and Sm041370 epitopes were recognized by infected individuals IgG antibodies. Therefore, Sm041370 and Sm168240 epitopes that stood out in in silico and in vitro analyses could be promising antigens in schistosomiasis vaccine development or diagnostic kits.

Design, synthesis, biological activity and structure-activity relationship studies of chalcone derivatives as potential anti- Candida agents

Vulvovaginal candidiasis (VVC) affects millions of women around the world every year. Candida albicans is the most frequently isolated pathogen in women and its rapid ability to develop resistance to first and second line therapies has boosted the search for new and effective antifungal agents. In this study, we show the in vitro anti-Candida activity of fifteen synthetic chalcone analogs and their antifungal potential in an in vivo model of VVC. Chalcone 12 showed potent antifungal effects, being able to inhibit the growth of Candida spp. at a concentration of 15.6 µg mL−1. In addition, mechanism of action studies have indicated the ergosterol fungal membrane as the target of this compound. Despite a considerable antifungal activity, the chalcone 12 showed high cytotoxicity in kidney cells lineages. Moreover, this compound was able to reduce Candida-associated virulence, impairing yeast–hyphal transition in C. albicans. An in vivo model of VVC showed that chalcone 12 significantly reduces the fungal load. Taken together, these findings showed that the chalcone 12 is a potent anti-Candida agent in vitro beyond of contribute to improve the fungal infection in a model of CVV. However, it showed low selectivity and high toxicity, suggesting molecular modifications to minimize these proprieties.

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

HighlightsUse of computational tools in the development of vaccines against human diseases.Rational development and structural verification of chimeric proteins.The importance of combining methodologies in the virtual screening process. Abstract 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.

Selectivity analyses of γ-benzylidene digoxin derivatives to different Na,K-ATPase α isoforms: a molecular docking approach

Abstract Digoxin and other cardiotonic steroids (CTS) exert their effect by inhibiting Na,K-ATPase (NKA) activity. CTS bind to the various NKA isoforms that are expressed in different cell types, which gives CTS their narrow therapeutic index. We have synthesised a series of digoxin derivatives (γ-Benzylidene digoxin derivatives) with substitutions in the lactone ring (including non-oxygen and ether groups), to obtain CTS with better NKA isoform specificity. Some of these derivatives show some NKA isoform selective effects, with BD-3, BD-8, and BD-13 increasing NKA α2 activity, BD-5 inhibiting NKA α1 and NKA α3, BD-10 reducing NKA α1, but stimulating NKA α2 and α3; and BD-14, BD-15, and BD-16 enhancing NKA α3 activity. A molecular-docking approach favoured NKA isoform specific interactions for the compounds that supported their observed activity. These results show that BD compounds are a new type of CTS with the capacity to target NKA activity in an isoform-specific manner. Graphical Abstract