Moacyr Comar

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.

Identification of a vaccine against schistosomiasis using bioinformatics and molecular modeling tools.

Schistosomiasis is a serious public health problem in Brazil and worldwide. Although the drugs used to treatment schistosomiasis are effective, the disease continues to expand in all endemic countries due to constant reinfection, poor sanitation, and the lack of effective programs for disease control. However, advances generated through genome projects have provided important information that has improved the understanding of the biology of this parasite. These advances, associated with the advent of bioinformatic analysis, are becoming an important tool in reverse vaccinology. Through database access to the DNA and protein sequences of Schistosoma mansoni and the use of bioinformatics programs, fourteen epitopes were identified. Five epitopes were obtained from proteins whose immunogenic potential had already been assessed in other studies (KP), and nine whose immunogenic potential is unknown (UP). To improve stimulation of the host immune system, the selected epitopes were modeled with a sugar moiety. After this addition, all of the epitopes showed structures similar to those observed in the native proteins, but only eleven of the peptides presented thermodynamically stable structures. Prediction analysis and molecular modeling showed that the glycopeptides presented here are important targets in the search for a vaccine against schistosomiasis. Additionally, they suggest that these molecules may be used in immunological assays to evaluate the level of protection, the effect on pathology reduction and the profile of cytokines and antibodies induced by them.

Docking, QM/MM, and molecular dynamics simulations of the hexose transporter from Plasmodium falciparum (PfHT).

Malaria is the most prevalent parasitic disease in the world. Currently, an effective vaccine for malaria does not exist, and chemotherapy must be used to treat the disease. Because of increasing resistance to current antimalarial drugs, new treatments must be developed. Among the many potential molecular targets, the hexose transporter of Plasmodium falciparum (PfHT) is particularly promising because it plays a vital role in glucose transport for the parasite. Thus, this study aims to determine the three-dimensional structure of PfHT and to describe the intermolecular interactions between active glycoside derivatives and PfHT. Such information should aid in the development of new antimalarial drugs. The receptor PfHT was constructed from primary sequences deposited in the SWISS MODEL database. Next, molecular docking simulations between O-(undec-10-en)-l-D-glucose and the constructed active site models were performed using Autodock Vina. The glycoside derivative-PfHT complexes were then refined using the hybrid QM/MM (PM3/ff03) method within the AMBER package. The models were then evaluated using Ramachandran plots, which indicated that 93.2% of the residues in the refined PfHT models (P5) were present in favorable regions. Furthermore, graphical plots using ANOLEA showed that the potential energies of interaction for atoms unbonded to P5 were negative. Finally, the O-(undec-10-en)-l-D-glucose-PfHT complex was evaluated using 20-ns Molecular Dynamics simulations with an ff03 force field. Docking and QM/MM studies revealed the amino acids essential for molecular recognition of and activity on glycosides. Inhibition of glucose transporters may prevent the development and metabolism of P. falciparum, so a description of the receptors structure is a critical step towards rational drug design.

Primary Structure of a Trypsin Inhibitor (Copaifera langsdorffii Trypsin Inhibitor-1) Obtained from C. langsdorffii Seeds.

In this study, the aim was to determine the complete sequence of the Copaifera langsdorffii trypsin inhibitor (CTI)-1 using 2-dimensional (2D)-PAGE, matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF), and quadrupole time-of-flight (QTOF) spectrometry. Spots A (CTI-1) and F (CTI-2) were submitted to enzymatic digestions with trypsin, SV8, and clostripain. The accurate mass of the peptide obtained from each digest was determined by mass spectrometry (MS) using MALDI-TOF. The most abundant peptides were purified and sequenced in a liquid chromatograph connected to an electrospray ionization-QTOF MS. When the purified trypsin inhibitor was submitted to 2D electrophoresis, different spots were observed, suggesting that the protein is composed of 2 subunits with microheterogeneity. Isoelectric points of 8.0, 8.5, and 9.0 were determined for the 11 kDa subunit and of 4.7, 4.6, and 4.3 for the 9 kDa subunit. The primary structure of CTI-1, determined from the mass of the peptide of the enzymatic digestions and the sequence obtained by MS, indicated 180 shared amino acid residues and a high degree of similarity with other Kunitz (KTI)-type inhibitors. The peptide also contained an Arg residue at the reactive site position. Its 3-dimensional structure revealed that this is because the structural discrepancies do not affect the canonical conformation of the reactive loop of the peptide. Results demonstrate that a detailed investigation of the structural particularities of CTI-1 could provide a better understanding of the mechanism of action of these proteins, as well as clarify its biologic function in the seeds. CTI-1 belongs to the KTI family and is composed of 2 polypeptide chains and only 1 disulfide bridge.

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.

Accurate Gaussian basis sets for atomic and molecular calculations obtained from the generator coordinate method with polynomial discretization.

Accurate Gaussian basis sets for atoms from H to Ba were obtained by means of the generator coordinate Hartree-Fock (GCHF) method based on a polynomial expansion to discretize the Griffin-Wheeler-Hartree-Fock equations (GWHF). The discretization of the GWHF equations in this procedure is based on a mesh of points not equally distributed in contrast with the original GCHF method. The results of atomic Hartree-Fock energies demonstrate the capability of these polynomial expansions in designing compact and accurate basis sets to be used in molecular calculations and the maximum error found when compared to numerical values is only 0.788 mHartree for indium. Some test calculations with the B3LYP exchange-correlation functional for N2, F2, CO, NO, HF, and HCN show that total energies within 1.0 to 2.4 mHartree compared to the cc-pV5Z basis sets are attained with our contracted bases with a much smaller number of polarization functions (2p1d and 2d1f for hydrogen and heavier atoms, respectively). Other molecular calculations performed here are also in very good accordance with experimental and cc-pV5Z results. The most important point to be mentioned here is that our generator coordinate basis sets required only a tiny fraction of the computational time when compared to B3LYP/cc-pV5Z calculations.

Molecular dynamics simulation of dopamine and ascorbic acid amid tetrafluoroborate 1-butyl-3-methylimidazolium compared to conventional solvents

Dopamine (DA) is an important neurotransmitter in the Central Nervous System (CNS) of mammals being found in significant quantities in the brain. In a low concentration is associated with Parkinsons disease [1], which makes it important detection. However, a major problem in determining DA is its joint resolution with coexisting species such as ascorbic acid (AA). In traditional solid electrodes, AA is oxidized at a potential close to that of DA, resulting in an overlapping voltammetric response [2]. Therefore, improving the selectivity of the monitoring techniques of AD has been the focus of much research. [3] Thus, ionic liquids at ambient temperatures (RTILs) tetrafluoroborate and 1-butyl-3-methylimidazolium (BMI.BF4) can be used as electrolytes for different electrochemical reactions, which have important properties, the selectivity of them, making the technique voltammetric more efficient. Our focus as this work is to analyze the behavior of these molecules (DA and AA) amid BMI.BF4 compared to conventional solvents, using a method known as Molecular Dynamics (MD), being a powerful tool for obtaining properties structural and thermodynamic properties of these substances. MD simulations were carried out for systems in DA [BMI.BF4] and AA [BMI.BF4]. For comparison, simulations were also carried out in water. All simulations were performed using the AMBER 9 package. We also used PTRAJ to perform analyses on the data we collected, such as calculating density, total energy, RDFs and self-diffusion coefficients, and we used xmgrace and gnuplot to aid us in visualizing the data. As part of evaluating the interference between molecules (AA and DA), we show the results of analyzes of AA. In this case the average density is 1.0768 cc / mol, while the water density is 1.027 cc / mole. As the total energy of the two systems have different values and constants (-19.5 kcal / mol in the midst of water and -12.5 kcal / mol among LI) along the dynamics. This difference in energy is given by unfavorable interactions that AA has with the molecules of the LI. For a better understanding of the systems were calculated the radial distribution functions for each of statepoints. The results presented in both solvent were similar, demonstrating that the AA is added in both LI well as for water, ie they have good solvation. Regarding the results of diffusivity, the 298K and 1 atm the coefficient of self-diffusion (MSD) showed lower values compared to the water in the same conditions. This result reveals the MSD in a higher viscosity of the LI and so may be an important property for separation of molecules (AA and DA) and therefore important in electrochemical process.