l-Hypaphorine and d-hypaphorine: Specific antiacetylcholinesterase activity in rat brain tissue.

Acetylcholinesterase (AChEis) inhibitors are used to treat neurodegenerative diseases like Alzheimer's disease (AD). l-Hypaphorine (l-HYP) is a natural indole alkaloid that has been shown to have effects on the central nervous system (CNS). The goal of this research was to synthesize l-HYP and d-HYP and test their anticholinesterasic properties in rat brain regions. l-HYP suppressed acetylcholinesterase (AChE) activity only in the cerebellum, whereas d-HYP inhibited AChE activity in all CNS regions studied. No cytotoxic effect on normal human cells (HaCaT) was observed in the case of l-HYP and d-HYP although an increase in cell proliferation. Molecular modeling studies revealed that d-HYP and l-HYP have significant differences in their binding mode positions and interact stereospecifically with AChE's amino acid residues.

Molecular modeling of a series of dehydroquinate dehydratase type II inhibitors of Mycobacterium tuberculosis and design of new binders.

Tuberculosis, caused by Mycobacterium tuberculosis (M. tuberculosis), is still responsible for a large number of fatal cases, especially in developing countries with alarming rates of incidence and prevalence worldwide. Mycobacterium tuberculosis has a remarkable ability to develop new resistance mechanisms to the conventional antimicrobials treatment. Because of this, there is an urgent need for novel bioactive compounds for its treatment. The dehydroquinate dehydratase II (DHQase II) is considered a key enzyme of shikimate pathway, and it can be used as a promising target for the design of new bioactive compounds with antibacterial action. The aim of this work was the construction of QSAR models to aid the design of new potential DHQase II inhibitors. For that purpose, various molecular modeling approaches, such as activity cliff, QSAR models and computer-aided ligand design were utilized. A predictive in silico 4D-QSAR model was built using a database comprising 86 inhibitors of DHQase II, and the model was used to predict the activity of the designed ligands. The obtained model proved to predict well the DHQase II inhibition for an external validation dataset ([Formula: see text] = 0.72). Also, the Activity Cliff analysis shed light on important structural features applied to the ligand design.

In vitro and in silico studies of 8(17),12E,14-labdatrien-18-oic acid in airways smooth muscle relaxation: new molecular insights about its mechanism of action.

In the field of experimental pharmacology, researchers continuously investigate new relaxant agents of the airway smooth muscle cells (ASMCs), since the pathophysiology of respiratory illnesses, such as asthma, involves hyperresponsiveness and changes in ASMC homeostasis. In this scenario, labdane-type diterpenes, like forskolin (FSK), are a class of compounds known for their relaxing action on smooth muscle cells (SMCs), being this phenomenon related to the direct activation of AC-cAMP-PKA pathway. Considering the continuous effort of our group to study the mechanism of action and prospecting for compounds isolated from natural sources, in this paper, we presented how the diterpene 8(17),12E,14-labdatrien-18-oic acid (LBD) promotes relaxant effect on ASMC, performing in vitro experiments using isolated guinea pig trachea and in silico molecular docking/dynamics simulations. In vitro experiments showed that in the presence of aminophylline, FSK and LBD had their relaxant effect potentiated (EC50 from 1.4 ± 0.2 × 10-5 M to 1.5 ± 0.3 × 10-6 M for LBD and from 2.0 ± 0.2 × 10-7 M to 6.4 ± 0.4 × 10-8 M for FSK) while in the presence of Rp-cAMPS this effect was attenuated (EC50 from 1.4 ± 0.2 × 10-5 M to 3 × 10-4 M for LBD and from 2.0 ± 0.2 × 10-7 to 3.1 ± 1.0 × 10-6 M for FSK). Additionally, in silico simulations evidenced that the lipophilic character of LBD is probably responsible for its stability on AC binding site. LBD presented two preferential orientations, where the double bonds of the isoprene moiety as well as the unique polar group (carboxylic acid) in this compound form important anchoring points. In this sense, we consider that the LBD can interact stabilizing the catalytic dimmer of AC as the FSK, although less efficiently.

Antiviral Potential of Spondias mombin L. Leaves Extract Against Herpes Simplex Virus Type-1 Replication Using In Vitro and In Silico Approaches.

Spondias mobin leaves have been traditionally used for treating cold sores. The study investigated the mechanism of antiherpes action of S. mombin extract, fractions, and geraniin. Different concentrations of samples were used to evaluate the in vitro antiherpes activity (anti-HSV-1) in virucidal, post-infection, attachment, and penetration assays. The mechanism of action of geraniin was investigated considering the glycoproteins gB and gD of HSV-1 surface as potential molecular targets. Molecular docking simulations were carried out for both in order to determine the possible binding mode position of geraniin at the activity sites. The binding mode position was posteriorly optimized considering the flexibility of the glycoproteins. The chemical analysis of samples was performed by LC-MS and revealed the presence of 22 substances, which are hydrolysable tannins, O-glycosylated flavonoids, phenolic acids, and a carbohydrate. The extract, tannin-rich fraction and geraniin showed important in vitro virucidal activity through blocking viral attachment but showed no relevant inhibition of viral penetration. The in silico approaches demonstrated a high number of potential strong intermolecular interactions as hydrogen bonds between geraniin and the activity site of the glycoproteins, particularly the glycoprotein gB. In silico experiments indicated that geraniin is at least partially responsible for the anti-herpes activity through interaction with the viral surface glycoprotein gB, which is responsible for viral adsorption. These results highlight the therapeutic potential of S. mombin anti-herpes treatment and provides support for its traditional purposes. However, further studies are required to validate the antiviral activities in vivo, as well as efficacy in humans.

Identification of a Selective PDE4B Inhibitor From Bryophyllum pinnatum by Target Fishing Study and In Vitro Evaluation of Quercetin 3-O-α-L-Arabinopyranosyl-(1→2)-O-α-L-Rhamnopyranoside.

Natural products are considered an important source of bioactive compounds especially in biodiversity-rich countries like Brazil. The identification of potential targets is crucial to the development of drugs from natural sources. In this context, in silico methodologies, such as inverse virtual screening (target fishing), are interesting tools as they are a rational and direct method that reduces costs and experimental time. Among the species of Brazilian biomes, Bryophyllum pinnatum (Lam.) Oken, native to Madagascar, is widely used by the population to treat inflammation conditions. It has a remarkable presence of flavonoids, including quercetin 3-O-α-L-arabinopyranosyl-(1→2)-O-α-L-rhamnopyranoside (1), considered one of its major compounds. However, until now there were no studies addressing its putative mechanism of action and explaining its pharmacological action. The enzyme PDE4B, known as an antiinflammatory protein, was indicated as a promising target by target fishing methods. This activity was confirmed by in vitro enzymatic inhibition, and an expressive selectivity of PDE4B over PDE4A was demonstrated. The interactions were investigated through molecular dynamics simulations. The results were pioneering, representing an advance in the investigation of the antiinflammatory action of B. pinnatum and confirm the potential of the flavonoid as a chemical extract marker. Also, the flavonoid was shown to be a promising lead for the design of other selective PDE4B blockers to treat inflammatory diseases.

Analogs of the Scorpion Venom Peptide Stigmurin: Structural Assessment, Toxicity, and Increased Antimicrobial Activity.

Scorpion venom is a rich source of biologically active components and various peptides with high-potential therapeutic use that have been characterized for their antimicrobial and antiproliferative activities. Stigmurin is a peptide identified from the Tityus stigmurus venom gland with high antibacterial and antiproliferative activities and low toxicity. Amino acid substitutions in peptides without a disulfide bridge sequence have been made with the aim of reducing their toxicity and increasing their biological activities. The purpose of this study was to evaluate the structural conformation and structural stability, as well as antimicrobial, antiproliferative, and hemolytic activities of two peptide analogs to Stigmurin, denominated StigA6 and StigA16. In silico analysis revealed the α-helix structure for both analog peptides, which was confirmed by circular dichroism. Data showed that the net charge and hydrophobic moment of the analog peptides were higher than those for Stigmurin, which can explain the increase in antimicrobial activity presented by them. Both analog peptides exhibited activity on cancerous cells similar to the native peptide; however, they were less toxic when tested on the normal cell line. These results reveal a potential biotechnological application of the analog peptides StigA6 and StigA16 as prototypes to new therapeutic agents.

Structural insights of dipeptidyl peptidase-IV inhibitors through molecular dynamics-guided receptor-dependent 4D-QSAR studies.

Dipeptidyl peptidase-IV (DPP-IV) inhibitors are promising antidiabetic agents. Currently, several DPP-IV inhibitors have been approved for therapeutic use in diabetes mellitus. Receptor-dependent 4D-QSAR is comparatively a new approach which uses molecular dynamics simulations to generate conformational ensemble profiles of compounds representing a dynamic state of compounds at a target's binding site. This work describes a receptor-dependent 4D-QSAR study on triazolopiperazine derivatives. QSARINS multiple linear regression method was adopted to generate 4D-QSAR models. A model with 9 variables was found to have better predictive accuracy with [Formula: see text], [Formula: see text] (leave-one-out) = 0.592 and [Formula: see text] predicted = 0.597. The location of these 9 variables at the binding site of DPP-IV revealed the importance of the residues Val711, Tyr662, Tyr666, Val202, Asp200 and Thr199 in making critical interactions with DPP-IV inhibitors. The study of these critical interactions revealed the structural features required in DPP-IV inhibitors. Thus, in this study the importance of a halogen substituent on a phenyl ring, the extent of substitution on the triazolopiperazine ring, the presence of an ionizable amino group and the presence of a hydrophobic substituent that can bind deeper in binding pocket of DPP-IV were revealed.

3D-QSAR with R: A new 3D-QSAR methodology applied to a set of DGAT1 inhibitors [corrected].

The rapid advances in computational methods for the drug design have resulted in the accurate predictions of biological activities of ligands with or without the availability of enzyme structures. 3D-QSAR is one of the computational methods used for such purpose. Currently, freely available 3D-QSAR methods suffer the limitations like complex methodologies, difficulty in the analysis of results, applying the statistical methods and validations of models built. Present work describes simple and novel 3D-QSAR methodology, which uses bash scripts LQTA_R_LJ, LQTA_R_QQ and LQTA_R_HB using freely available R statistical program. These scripts then generate Leenard-Jones, Coulomb and Hydrogen bond descriptors. These descriptors provide the steric 3D property, electrostatic property and hydrogen bond formation capacity respectively. These scripts have been tested for the set of DGAT1 inhibitors and results showed that the 3D-QSAR models built have better predictive abilities in terms of R2 0.735, Q2loo 0.635 and R2ext 0.715. The 3D-QSAR model suggested that the substitutions of the alkyl group at the oxadiazolyl ring at the 6th position of the pyrrolo-pyridazine ring is undesirable, on the contrary, substituted phenyl ring at 7th position is responsible for the improved DGAT1 inhibitory activity. The analysis also suggested that 6th position could be substituted with the oxadiazolyl ring or analogous heterocyclic rings, where the 3rd position of such heterocyclic rings substituted with rigid hydrophobic substitute can improve DGAT1 activity.

Analogs of the scorpion venom peptide stigmurin: structural assessment, toxicity, and increased antimicrobial activity

Scorpion venom is a rich source of biologically active components and various peptides with high-potential therapeutic use that have been characterized for their antimicrobial and antiproliferative activities. Stigmurin is a peptide identified from the Tityus stigmurus venom gland with high antibacterial and antiproliferative activities and low toxicity. Amino acid substitutions in peptides without a disulfide bridge sequence have been made with the aim of reducing their toxicity and increasing their biological activities. The purpose of this study was to evaluate the structural conformation and structural stability, as well as antimicrobial, antiproliferative, and hemolytic activities of two peptide analogs to Stigmurin, denominated StigA6 and StigA16. In silico analysis revealed the -helix structure for both analog peptides, which was confirmed by circular dichroism. Data showed that the net charge and hydrophobic moment of the analog peptides were higher than those for Stigmurin, which can explain the increase in antimicrobial activity presented by them. Both analog peptides exhibited activity on cancerous cells similar to the native peptide; however, they were less toxic when tested on the normal cell line. These results reveal a potential biotechnological application of the analog peptides StigA6 and StigA16 as prototypes to new therapeutic agents.

Synthesis, Molecular Modeling, and Evaluation of Novel Sulfonylhydrazones as Acetylcholinesterase Inhibitors for Alzheimer's Disease.

Alzheimer's disease (AD) is the most common type of dementia and related to the degeneration of hippocampal cholinergic neurons, which dramatically affects cognitive ability. Acetylcholinesterase (AChE) inhibitors are employed as drugs for AD therapy. Three series of sulfonylhydrazone compounds were designed, and their ability to inhibit AChE was evaluated. Fifteen compounds were synthesized and twelve of them had IC50 values of 0.64-51.09 µM. The preliminary structure-activity relationships indicated that the methylcatechol moiety and arylsulfonyl substituents generated better compounds than both the benzodioxole and alkylsulfonyl chains. Molecular dynamics studies of compound 6d showed that the interaction with the peripheral binding site of AChE was similar to donepezil, which may explain its low IC50 (0.64 µM). Furthermore, the drug-likeness of 6d suggests that the compound may have appropriate oral absorption and brain penetration. Compound 6d also presented antiradical activity and was not cytotoxic to LL24 cells, suggesting that this compound might be considered safe. Our findings indicate that arylsulfonylhydrazones may be a promising scaffold for the design of new drug candidates for the treatment of AD.

Structure and in vitro activities of a Copper II-chelating anionic peptide from the venom of the scorpion Tityus stigmurus.

Anionic Peptides are molecules rich in aspartic acid (Asp) and/or glutamic acid (Glu) residues in the primary structure. This work presents, for the first time, structural characterization and biological activity assays of an anionic peptide from the venom of the scorpion Tityus stigmurus, named TanP. The three-dimensional structure of TanP was obtained by computational modeling and refined by molecular dynamic (MD) simulations. Furthermore, we have performed circular dichroism (CD) analysis to predict TanP secondary structure, and UV-vis spectroscopy to evaluate its chelating activity. CD indicated predominance of random coil conformation in aqueous medium, as well as changes in structure depending on pH and temperature. TanP has chelating activity on copper ions, which modified the peptide's secondary structure. These results were corroborated by MD data. The molar ratio of binding (TanP:copper) depends on the concentration of peptide: at lower TanP concentration, the molar ratio was 1:5 (TanP:Cu2+), whereas in concentrated TanP solution, the molar ratio was 1:3 (TanP:Cu2+). TanP was not cytotoxic to non-neoplastic or cancer cell lines, and showed an ability to inhibit the in vitro release of nitric oxide by LPS-stimulated macrophages. Altogether, the results suggest TanP is a promising peptide for therapeutic application as a chelating agent.

Structure and in vitro activities of a Copper II-chelating anionic peptide from the venom of the scorpion Tityus stigmurus

An ionic Peptides are molecules rich in aspartic acid (Asp) and/or glutamic acid (Glu) residues in the primary structure. This work presents, for the first time, structural characterization and biological activity assays of an anionic peptide from the venom of the scorpion Tityus stigmurus, named TanP. The three-dimensional structure of TanP was obtained by computational modeling and refined by molecular dynamic (MD) simulations. Furthermore, we have performed circular dichroism (CD) analysis to predict TanP secondary structure, and UV-vis spectroscopy to evaluate its chelating activity. CD indicated predominance of random coil conformation in aqueous medium, as well as changes in structure depending on pH and temperature. TanP has chelating activity on copper ions, which modified the peptide's secondary structure. These results were corroborated by MD data. The molar ratio of binding (TanP: copper) depends on the concentration of peptide: at lower TanP concentration, the molar ratio was 1:5 (TanP: Cu2+), whereas in concentrated TanP solution, the molar ratio was 1:3 (TanP: Cu2+). TanP was not cytotoxic to non-neoplastic or cancer cell lines, and showed an ability to inhibit the in vitro release of nitric oxide by LPS-stimulated macrophages. Altogether, the results suggest TanP is a promising peptide for therapeutic application as a chelating agent.

Supramolecular aggregates of oligosaccharides with co-solvents in ternary systems for the solubilizing approach of triamcinolone.

A second compound is generally associated with oligosaccharides as a strategy to maximize the solubilizing effect for nonpolar compounds. This study elucidated the role and the mechanism whereby liquid compounds interact in these supramolecular aggregates in the solubilization of triamcinolone. Three different oligosaccharides (beta-cyclodextrin, 2-hydroxipropil-beta-cyclodextrin, and randomly methylated beta-cyclodextrin) and two potent co-solvents (triethanolamine and N-methyl pyrrolidone) were carefully evaluated by using three distinct experimental approaches. Incredibly stable complexes were formed with cyclodextrins (CDs). The structure of the complexes was elucidated by magnetic resonance spectra 2D-ROESY. The interactions of the protons of ring "A" of the drug with H(3) and H(5) protons of the CD cavity observed in the binary complexes remained in both ternary complexes. Unlike the observed ternary associations with triethanolamine, N-methyl pyrrolidone competed with the triamcinolone CD cavity and considerably decreased the stability of the complex and the solubility of the drug. The molecular dynamics (MD) and quantum mechanics:molecular mechanics (QM:MM) calculations supported that triethanolamine stabilized the drug-CD interactions for the conformer identified in the 2D-ROESY experiments, improving the quality and uniformity of the formed complex. The role played by the co-solvent in the ternary complexes depends on its specific ability to interact with the CD cavity in the presence of the drug, which can be predicted in theoretical studies to select the best candidate.

Homology modeling, vasorelaxant and bradykinin-potentiating activities of a novel hypotensin found in the scorpion venom from Tityus stigmurus.

In a recent work by our group involving a transcriptomics approach applied to the venom glands from Tityus stigmurus we identified a new family of peptides called Hypotensins (TSTI0006C) (Almeida et al., 2012). The cluster TSTI0006C was analyzed in the main 25 amino acid residues and named T. stigmurus Hypotensin (TistH), showing a molecular mass of 2.7 kDa, an absence of cysteines and the presence of two C-terminal proline residues, which are a bradykinin-potentiating peptide (BPP) signature. Here, we describe the homology modeling of the three-dimensional structure of TistH. In addition, we evaluated the cardiovascular effects elicited by TistH in normotensive rats. Firstly, TistH showed no cytotoxic effect on horse erythrocyte. Furthermore, in normotensive rats TistH was able to potentiate the hypotensive action of bradykinin (BK) and induced a vasorelaxant effect in mesenteric artery rings by endothelium-dependent release of nitric oxide (NO) and demonstrated independent inhibition of angiotensin converting enzyme (ACE). Our data can contribute to a better understanding of the structural and functional characteristics of TistH and suggest its potential use in cardiovascular diseases.

The receptor-dependent LQTA-QSAR: application to a set of trypanothione reductase inhibitors.

A new Receptor-Dependent LQTA-QSAR approach, RD-LQTA-QSAR, is proposed as a new 4D-QSAR method. It is an evolution of receptor independent LQTA-QSAR. This approach uses the free GROMACS package to carry out molecular dynamics simulations and generates a conformational ensemble profile for each compound. Such an ensemble is used to build molecular interaction field-based QSAR models, as in CoMFA. To show the potential of this methodology, a set of 38 phenothiazine derivatives that are specific competitive T. cruzi trypanothione reductase inhibitors, was chosen. Using a combination of molecular docking and molecular dynamics simulations, the binding mode of the phenotiazine derivatives was evaluated in a simulated induced fit approach. The ligands alignments were performed using both ligand and binding site atoms, enabling unbiased alignment. The models obtained were extensively validated by leave-N-out cross-validation and y-randomization techniques to test for their robustness and absence of chance correlation. The final model presented Q(2) LOO of 0.87 and R² of 0.92 and a suitable external prediction of [Formula: see text]= 0.78. The adapted binding site obtained is useful to perform virtual screening and ligand structure-based design and the descriptors in the final model can aid in the design new inhibitors.

4D-LQTA-QSAR and docking study on potent Gram-negative specific LpxC inhibitors: a comparison to CoMFA modeling.

A quasi 4D-QSAR has been carried out on a series of potent Gram-negative LpxC inhibitors. This approach makes use of the molecular dynamics (MD) trajectories and topology information retrieved from the GROMACS package. This new methodology is based on the generation of a conformational ensemble profile, CEP, for each compound instead of only one conformation, followed by the calculation intermolecular interaction energies at each grid point considering probes and all aligned conformations resulting from MD simulations. These interaction energies are independent variables employed in a QSAR analysis. The comparison of the proposed methodology to comparative molecular field analysis (CoMFA) formalism was performed. This methodology explores jointly the main features of CoMFA and 4D-QSAR models. Step-wise multiple linear regression was used for the selection of the most informative variables. After variable selection, multiple linear regression (MLR) and partial least squares (PLS) methods used for building the regression models. Leave-N-out cross-validation (LNO), and Y-randomization were performed in order to confirm the robustness of the model in addition to analysis of the independent test set. Best models provided the following statistics: [Formula in text] (PLS) and [Formula in text] (MLR). Docking study was applied to investigate the major interactions in protein-ligand complex with CDOCKER algorithm. Visualization of the descriptors of the best model helps us to interpret the model from the chemical point of view, supporting the applicability of this new approach in rational drug design.

LQTA-QSAR: a new 4D-QSAR methodology.

A novel 4D-QSAR approach which makes use of the molecular dynamics (MD) trajectories and topology information retrieved from the GROMACS package is presented in this study. This new methodology, named LQTA-QSAR (LQTA, Laboratório de Quimiometria Teórica e Aplicada), has a module (LQTAgrid) that calculates intermolecular interaction energies at each grid point considering probes and all aligned conformations resulting from MD simulations. These interaction energies are the independent variables or descriptors employed in a QSAR analysis. The comparison of the proposed methodology to other 4D-QSAR and CoMFA formalisms was performed using a set of forty-seven glycogen phosphorylase b inhibitors (data set 1) and a set of forty-four MAP p38 kinase inhibitors (data set 2). The QSAR models for both data sets were built using the ordered predictor selection (OPS) algorithm for variable selection. Model validation was carried out applying y-randomization and leave-N-out cross-validation in addition to the external validation. PLS models for data set 1 and 2 provided the following statistics: q(2) = 0.72, r(2) = 0.81 for 12 variables selected and 2 latent variables and q(2) = 0.82, r(2) = 0.90 for 10 variables selected and 5 latent variables, respectively. Visualization of the descriptors in 3D space was successfully interpreted from the chemical point of view, supporting the applicability of this new approach in rational drug design.

A diaryl sulfide, sulfoxide, and sulfone bearing structural similarities to combretastatin A-4.

Studies examining various spacer groups that link the two aromatic rings of combretastatin A-4 (CA4) have shown that the biological activity of analogs does not require the cis-stilbene configuration of CA4. Oxygen or nitrogen, carbonyl, methylene and ethylene spacers, for example, are present in CA4 analogs that show good activity. Up to now sulfur was not tested for this purpose. In this article we describe the synthesis of sulfide, sulfoxide and sulfone spacers between two aromatic rings comparable to those of CA4. We also compared them with CA4 for inhibitory effects on cell growth, tubulin polymerization, and the binding of [(3)H]colchicine to tubulin. We found that the sulfide is highly active and may be a lead compound for the preparation of antitumor compounds.