The defensive functions of plant inhibitors are not restricted to insect enzyme inhibition

Three plant proteinase inhibitors BbKI (kallikrein inhibitor) and BbCI (cruzipain inhibitor) from Bauhinia bouhinioides, and a BrTI (trypsin inhibitor) from B. rufa, were examined for other effects in Callosobruchus maculatus development; of these only BrTI affected bruchid emergence. BrTI and BbKI share 81% identities in their primary sequences and the major differences between them are the regions comprising the RGD and RGE motifs in BrTI. These sequences were shown to be essential for BrTI insecticidal activity, since a modified BbKI [that is a recombinant form (BbKIm) with some amino acid residues replaced by those found in BrTI sequence] also strongly inhibited insect development. By using synthetic peptides related to the BrTI sequence, YLEAPVARGDGGLA-NH(2) (RGE) and IVYYPDRGETGL-NH(2) (RGE), it was found that the peptide with an RGE sequence was able to block normal development of C. maculatus larvae (ED(50) 0.16% and LD(50) 0.09%), this being even more effective than the native protein. (C) 2009 Elsevier Ltd. All rights reserved.

Studies towards the identification of putative bioactive conformation of potent vasodilator arylidene N-acylhydrazone derivatives

In this report we disclose the synthesis, vasodilatory activity, and identification of bioactive conformation of new N-acylhydrazone and N-methyl-N-acylhydrazone derivatives, structurally designed by bioisosteric replacements of previously described cardioactive compounds LASSBio-294 and its N-methyl derivative LASSBio-785. Some of these novel derivatives presented improved vasorelaxant properties, being new cardiovascular drug candidates. (C) 2009 Elsevier Masson SAS. All rights reserved.

Pharmacophore Model of Cruzain Inhibitors

Chagas disease, caused by the protozoan Trypanosoma cruzi, is one of the most serious amongst the so-called neglected diseases in Latin America, specially in Brazil. So far there has been no effective treatment for the chronic phase of this disease. Cruzain is a major cysteine protease of T cruzi and it is recognized as a valid target for Chagas disease chemotherapy. The mechanism of cruzain action is associated with the nucleophilic attack of an activated sulfur atom towards electrophilic groups. In this report, features of a putative pharmacophore model of the enzyme, developed as a virtual screening tool for the selection of potential cruzain inhibitors, are described. The final proposed model was applied to the ZINC v.7 database and afterwards experimentally validated by an enzymatic inhibition assay. One of the compounds selected by the model showed cruzain inhibition in the low micromolar range.

Flexibility and inhibitor binding in Cdc25 phosphatases

Cdc25 phosphatases involved in cell cycle checkpoints are now active targets for the development of anti-cancer therapies. Rational drug design would certainly benefit from detailed structural information for Cdc25s. However, only apo- or sulfate-bound crystal structures of the Cdc25 catalytic domain have been described so far. Together with previously available crystalographic data, results from molecular dynamics simulations, bioinformatic analysis, and computer-generated conformational ensembles shown here indicate that the last 30-40 residues in the C-terminus of Cdc25B are partially unfolded or disordered in solution. The effect of C-terminal flexibility upon binding of two potent small molecule inhibitors to Cdc25B is then analyzed by using three structural models with variable levels of flexibility, including an equilibrium distributed ensemble of Cdc25B backbone conformations. The three Cdc25B structural models are used in combination with flexible docking, clustering, and calculation of binding free energies by the linear interaction energy approximation to construct and validate Cdc25B-inhibitor complexes. Two binding sites are identified on top and beside the Cdc25B active site. The diversity of interaction modes found increases with receptor flexibility. Backbone flexibility allows the formation of transient cavities or compact hydrophobic units on the surface of the stable, folded protein core that are unexposed or unavailable for ligand binding in rigid and densely packed crystal structures. The present results may help to speculate on the mechanisms of small molecule complexation to partially unfolded or locally disordered proteins.

Three-Dimensional Quantitative Structure-Activity Relationship Study of Antitumor 2-Formylpyridine Thiosemicarbazones Derivatives as Inhibitors of Ribonucleotide Reductase

In order to extend previous SAR and QSAR studies, 3D-QSAR analysis has been performed using CoMFA and CoMSIA approaches applied to a set of 39 alpha-(N)-heterocyclic carboxaldehydes thiosemicarbazones with their inhibitory activity values (IC(50)) evaluated against ribonucleotide reductase (RNR) of H.Ep.-2 cells (human epidermoid carcinoma), taken from selected literature. Both rigid and field alignment methods, taking the unsubstituted 2-formylpyridine thiosemicarbazone in its syn conformation as template, have been used to generate multiple predictive CoMFA and CoMSIA models derived from training sets and validated with the corresponding test sets. Acceptable predictive correlation coefficients (Q(cv)(2) from 0.360 to 0.609 for CoMFA and Q(cv)(2) from 0.394 to 0.580 for CoMSIA models) with high fitted correlation coefficients (r` from 0.881 to 0.981 for CoMFA and r(2) from 0.938 to 0.993 for CoMSIA models) and low standard errors (s from 0.135 to 0.383 for CoMFA and s from 0.098 to 0.240 for CoMSIA models) were obtained. More precise CoMFA and CoMSIA models have been derived considering the subset of thiosemicarbazones (TSC) substituted only at 5-position of the pyridine ring (n=22). Reasonable predictive correlation coefficients (Q(cv)(2) from 0.486 to 0.683 for CoMFA and Q(cv)(2) from 0.565 to 0.791 for CoMSIA models) with high fitted correlation coefficients (r(2) from 0.896 to 0.997 for CoMFA and r(2) from 0.991 to 0.998 for CoMSIA models) and very low standard errors (s from 0.040 to 0.179 for CoMFA and s from 0.029 to 0.068 for CoMSIA models) were obtained. The stability of each CoMFA and CoMSIA models was further assessed by performing bootstrapping analysis. For the two sets the generated CoMSIA models showed, in general, better statistics than the corresponding CoMFA models. The analysis of CoMFA and CoMSIA contour maps suggest that a hydrogen bond acceptor near the nitrogen of the pyridine ring can enhance inhibitory activity values. This observation agrees with literature data, which suggests that the nitrogen pyridine lone pairs can complex with the iron ion leading to species that inhibits RNR. The derived CoMFA and CoMSIA models contribute to understand the structural features of this class of TSC as antitumor agents in terms of steric, electrostatic, hydrophobic and hydrogen bond donor and hydrogen bond acceptor fields as well as to the rational design of this key enzyme inhibitors.

Two New Ternary Complexes of Copper(II) with Tetracycline or Doxycycline and 1,10-Phenanthroline and Their Potential as Antitumoral: Cytotoxicity and DNA Cleavage

This paper reports on the synthesis and characterization of two new ternary copper(II) complexes: [Cu(doxy-cycline)(1,10-phenanthroline)(H(2)O)(ClO(4))](ClO(4)) (1) and [Cu(tetracycline)(1,10-phenanthroline)(H(2)O)(ClO(4))](ClO(4)) (2). These compounds exhibit a distorted tetragonal geometry around copper, which is coordinated to two bidentate ligands, 1,10-phenanthroline and tetracycline or doxycyline, a water molecule, and a perchlorate ion weakly bonded in the axial positions. In both compounds, copper(II) binds to tetracyclines`. via the oxygen of the hydroxyl group and oxygen of the amide group at ring A and to 1,10-phenanthroline via its two heterocyclic nitrogens. We have evaluated the binding of the new complexes to DNA, their capacity to cleave it, their cytotoxic activity, and uptake in tumoral cells. The complexes bind to DNA preferentially by the major groove, and then cleave its strands by an oxidative mechanism involving the generation of ROS. The cleavage of DNA was inhibited by radical inhibitors and/or trappers such as superoxide dismutase, DMSO, and the copper(I) chelator bathocuproine. The enzyme T4 DNA ligase was not able to relegate the products of DNA cleavage, which indicates that the cleavage does not occur via a hydrolytic mechanism. Both complexes present an expressive plasmid DNA cleavage activity generating single- and double-strand breaks, under mild reaction conditions, and even in the absence of any additional oxidant or reducing agent. In the same experimental conditions, [Cu(phen)(2)](2+) is approximately 100-fold less active than our complexes. These complexes are among the most potent DNA cleavage agents reported so far. Both complexes inhibit the growth of K562 cells With the IC(50) values of 1.93 and 2.59 mu mol L(-1) for compounds I and 2, respectively. The complexes are more active than the free ligands, and their cytotoxic activity correlates with intracellular copper concentration and the number of Cu-DNA adducts formed inside cells.

Uncovering false positives on a virtual screening search for cruzain inhibitors

Some unexpected promiscuous inhibitors were observed in a virtual screening protocol applied to select cruzain inhibitors from the ZINC database. Physical-chemical and pharmacophore model filters were used to reduce the database size. The selected compounds were docked into the cruzain active site. Six hit compounds were tested as inhibitors. Although the compounds were designed to be nucleophilically attacked by the catalytic cysteine of cruzain, three of them showed typical promiscuous behavior, revealing that false positives are a prevalent concern in VS programs. (C) 2007 Elsevier Ltd. All rights reserved.

Structure-activity relationships of hypervalent organochalcogenanes as inhibitors of cysteine cathepsins V and S

A new series of organotelluranes were synthesized and investigated, and the structure-activity relationships in cysteine proteases inhibition were determinated. It was possible to identify the relevance of structural components linked to the reactivity of these compounds as inhibitors. For example, dibromo-organotelluranes showed to be more reactive than dichloro-organotelluranes towards cysteine cathepsins V and S. Besides, no remarkable enantio-selectivity was verified. In general the achiral organotelluranes were more reactive than the chiral congeners against cysteine cathepsins V and S. A reactivity order for organochalcogenanes and cysteine cathepsins was proposed after the comparison of the inhibitory potencies of organotelluranes with the related organoselenanes. (C) 2011 Elsevier Ltd. All rights reserved.

Proteomic analysis reveals suppression of bark chitinases and proteinase inhibitors in citrus plants affected by the citrus sudden death disease

Citrus sudden death (CSD) is a disease of unknown etiology that greatly affects sweet oranges grafted on Rangpur lime rootstock, the most important rootstock in Brazilian citriculture. We performed a proteomic analysis to generate information related to this plant pathogen interaction. Protein profiles from healthy, CSD-affected and CSD-tolerant stem barks, were generated using two-dimensional gel electrophoresis. The protein spots were well distributed over a pI range of 3.26 to 9.97 and a molecular weight (MW) range from 7.1 to 120 kDa. The patterns of expressed proteins on 2-DE gels made it possible to distinguish healthy barks from CSD-affected barks. Protein spots with MW around 30 kDa and pI values ranging from 4.5 to 5.2 were down-regulated in the CSD-affected rootstock bark. This set of protein spots was identified as chitinases. Another set of proteins, ranging in pI from 6.1 to 9.6 with an MW of about 20 kDa, were also suppressed in CSD-affected rootstock bark; these were identified as miraculin-like proteins, potential trypsin inhibitors. Downregulation of chitinases and proteinase inhibitors in CSD-affected plants is relevant since chitinases are well-known pathogenesis-related protein, and their activity against plant pathogens is largely accepted.

Integration of Ligand- and Target-Based Virtual Screening for the Discovery of Cruzain Inhibitors

A myriad of methods are available for virtual screening of small organic compound databases. In this study we have successfully applied a quantitative model of consensus measurements, using a combination of 3D similarity searches (ROCS and EON), Hologram Quantitative Structure Activity Relationships (HQSAR) and docking (FRED, FlexX, Glide and AutoDock Vina), to retrieve cruzain inhibitors from collected databases. All methods were assessed individually and then combined in a Ligand-Based Virtual Screening (LBVS) and Target-Based Virtual Screening (TBVS) consensus scoring, using Receiving Operating Characteristic (ROC) curves to evaluate their performance. Three consensus strategies were used: scaled-rank-by-number, rank-by-rank and rank-by-vote, with the most thriving the scaled-rank-by-number strategy, considering that the stiff ROC curve appeared to be satisfactory in every way to indicate a higher enrichment power at early retrieval of active compounds from the database. The ligand-based method provided access to a robust and predictive HQSAR model that was developed to show superior discrimination between active and inactive compounds, which was also better than ROCS and EON procedures. Overall, the integration of fast computational techniques based on ligand and target structures resulted in a more efficient retrieval of cruzain inhibitors with desired pharmacological profiles that may be useful to advance the discovery of new trypanocidal agents.

Multimilligram enantioresolution of sulfoxide proton pump inhibitors by liquid chromatography on polysaccharide-based chiral stationary phase

The enantiomers of sulfoxide proton pump inhibitors - omeprazole, lansoprazole, rabeprazole and Ro 18-5364 - were enantiomerically separated by liquid chromatography at multimilligram scale on a poly saccharide-based chiral stationary phase using normal and polar organic conditions as mobile phase. The values of the recovery and production rate were significant for each enantiomer; better results were achieved using a solid-phase injection system. However, this system was applied just for the enantionteric separation of omeprazole to demonstrate the applicability of this injection mode at milligram scale. The chiroptical characterization of the compounds was performed using a polarimeter and a circular dichroism detector. The higher enantiomeric purity obtained for the isolated enantiomers suggests that the methods here described should be considered as a simple and rapid way to obtain enantiomeric pure standards for analytical purpose. (C) 2007 Elsevier B.V. All rights reserved.

2D QSAR and similarity studies on cruzain inhibitors aimed at improving selectivity over cathepsin L

Hologram quantitative structure-activity relationships (HQSAR) were applied to a data set of 41 cruzain inhibitors. The best HQSAR model (Q(2) = 0.77; R-2 = 0.90) employing Surflex-Sim, as training and test sets generator, was obtained using atoms, bonds, and connections as fragment distinctions and 4-7 as fragment size. This model was then used to predict the potencies of 12 test set compounds, giving satisfactory predictive R-2 value of 0,88. The contribution maps obtained from the best HQSAR model are in agreement with the biological activities of the study compounds. The Trypanosoma cruzi cruzain shares high similarity with the mammalian homolog cathepsin L. The selectivity toward cruzam was checked by a database of 123 compounds, which corresponds to the 41 cruzain inhibitors used in the HQSAR model development plus 82 cathepsin L inhibitors. We screened these compounds by ROCS (Rapid Overlay of Chemical Structures), a Gaussian-shape volume overlap filter that can rapidly identify shapes that match the query molecule. Remarkably, ROCS was able to rank the first 37 hits as being only cruzain inhibitors. In addition, the area under the curve (AUC) obtained with ROCS was 0.96, indicating that the method was very efficient to distinguishing between cruzain and cathepsin L inhibitors. (c) 2007 Elsevier Ltd. All rights reserved.

Fast separation of selective serotonin reuptake inhibitors antidepressants in plasma sample by nonaqueous capillary electrophoresis

A simple, fast, and sensitive liquid-liquid extraction method followed by nonaqueous capillary electrophoresis (LLE/NACE) was developed and validated for Simultaneous determination of four antidepressants (fluoxetine, sertraline, citalopram and paroxetine) in human plasma. Several experimental separation conditions using aqueous and nonaqueous media separation were tested by varying the electrolyte pH value (for aqueous medium) and the ionic strength concentration considering the similar mobility of the compounds. High-resolution separation was achieved with a mixture of 1.25 mol L(-1) of phosphoric acid in acetonitrile. The quantification limits of the LLE/CE method varied between 15 and 30 ng mL(-1), with a relative standard deviation (RSD) lower than 10.3%. The method was successfully applied in therapeutic drug monitoring and should be employed in the evaluation of plasma levels in urgent toxicological analysis. (C) 2009 Elsevier B.V. All rights reserved.