A novel trypsin Kazal-type inhibitor from Aedes aegypti with thrombin coagulant inhibitory activity

Kazal-type inhibitors play several important roles in invertebrates, such as anticoagulant, vasodilator and antimicrobial activities. Putative Kazal-type inhibitors were described in several insect transcriptomes. In this paper we characterized for the first time a Kazal unique domain trypsin inhibitor from the Aedes aegypti mosquito. Previously, analyses of sialotranscriptome of A. aegypti showed the potential presence of a Kazal-type serine protease inhibitor, in female salivary glands, carcass and also in whole male, which we named AaTI (A. aegypti trypsin inhibitor). AaTI sequence showed amino acid sequence similarity with insect thrombin inhibitors, serine protease inhibitor from Litopenaeus vannamei hemocytes and tryptase inhibitor from leech Hirudo medicinalis (LDTI). In this work we expressed, purified and characterized the recombinant AaTI (rAaTI). Molecular weight of purified rAaTI was 7 kDa rAaTI presented dissociation constant (K(i)) of 0.15 and 3.8 nM toward trypsin and plasmin, respectively, and it weakly inhibited thrombin amidolytic activity. The rAaTI was also able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. AaTI transcription was confirmed in A. aegypti female salivary gland and gut 3 h and 24 h after blood feeding, suggesting that this molecule can act as anticoagulant during the feeding and digestive processes. Its transcription in larvae and pupae suggested that AaTI may also play other functions during the mosquito`s development. (C) 2010 Elsevier Masson SAS. All rights reserved.

Adhesion and protease activity in cell lines from human salivary gland tumors are regulated by the laminin-derived peptide AG73, syndecan-1 and beta 1 integrin

We studied the induction of protease activity by the laminin alpha 1-derived peptide AG73 in cells from adenoid cystic carcinoma (CAC2) and myoepithelioma (M1), respectively a malignant and a benign salivary gland tumors. Laminin alpha 1 chain and MMP9 were immunolocalized in adenoid cystic carcinoma and myoepithelioma in vivo and in vitro. Cells grown inside AG73-enriched laminin-111 exhibited large spaces in the extracellular matrix, suggestive of remodeling. The broad spectrum MMP inhibitor GM6001 decreased spaces induced by AG73 in CAC2 and M I cells. This result strongly suggests that AG73-mediated matrix remodeling involves matrix metalloproteinases. CAC2 and M1 cells cultured on AG73 showed a dose-dependent increase of MMP9 secretion, as detected by zymography. Furthermore, siRNA silencing of MMP9 decreased remodeling in 3D cultures. We searched for AG73 receptors regulating MMP9 activity in our cell lines. CAC2 and M1 cells grown on AG73 exhibited colocalization of syndecan-1 and beta 1 integrin. siRNA knockdown of syndecan-1 expression in these cells resulted in decreased adhesion to AG73 and reduced protease and remodeling activity. We investigated syndecan-1 co-receptors in both cell lines. Silencing beta 1 integrin inhibited adhesion to AG73, matrix remodeling and protease activity. Double-knockdown experiments were carried out to further explore syndecan-1 and beta 1 integrin cooperation. CAC2 cells transfected with both syndecan-1 and beta 1 integrin siRNA oligos showed significant decrease in adhesion to AG73. Simultaneous silencing of receptors also induced a decrease in protease activity. Our results suggest that syndecan-1 and beta 1 integrin signaling downstream of AG73 regulate adhesion and MMP production by CAC2 and M1 cells. (c) 2008 Elsevier B.V./International Society of Matrix Biology. All rights reserved.

Metallopeptidase inhibitors arrest vital biological processes in the fungal pathogen Scedosporium apiospermum

P>Scedosporium apiospermum is an emerging agent of opportunistic mycoses in humans. Previously, we showed that mycelia of S. apiospermum secreted metallopeptidases which were directly linked to the destruction of key host proteins. In this study, we analysed the effect of metallopeptidase inhibitors on S. apiospermum development. As germination of inhaled conidia is a crucial event in the infectious process of S. apiospermum, we studied the morphological transformation induced by the incubation of conidia in Sabouraud-dextrose medium at 37 degrees C. After 6 h, some conidia presented a small projection resembling a germ-tube. A significant increase, around sixfold, in the germ-tube length was found after 12 h, and hyphae were exclusively observed after 24 h. Three distinct metallopeptidase inhibitors were able to arrest the transformation of conidia into hyphae in different ways; for instance, 1,10-phenanthroline (PHEN) completely blocked this process at 10 mu mol l-1, while ethylenediamine tetraacetic acid (EDTA) and ethylene glycol-bis (beta-aminoethyl ether; EGTA) only partially inhibited the differentiation at up to 10 mmol l-1. EGTA did not promote any significant reduction in the conidial growth, while PHEN and EDTA, both at 10 mmol l-1, inhibited the proliferation around 100% and 65%, respectively. The secretion of polypeptides into the extracellular environment and the metallopeptidase activity secreted by mycelia were completely inhibited by PHEN. These findings suggest that metallo-type enzymes could be potential targets for future therapeutic interventions against S. apiospermum.

Non-steroidal anti-inflammatory drugs may modulate the protease activity of Candida albicans

The phenotypic pressure exerted by non-steroidal anti-inflammatory drugs (NSAIDs) on autochthonous and pathogenic microbiota remains sparsely known. In this study, we investigated if some NSAIDs increment or diminish the secretion of aspartyl-proteases (Sap) by Candida albicans grown under different phenotypes and oxygen availability using a set of SAP knock-out mutants and other set for genes (EFG1 and CPH1) that codify transcription factors involved in filamentation and protease secretion. Preconditioned cells were grown under planktonic and biofilm phenotypes, in normoxia and anoxia, in the presence of plasma concentrations of acetylsalicylic acid, diclofenac, indomethacin, nimesulide, piroxicam, ibuprofen, and acetaminophen. For diclofenac, indomethacin, nimesulide, and piroxicam the secretion rates of Sap by SAP1-6, EFG1. and CPH1 mutants were similar or, even, inferior to parental wildtype strain. This suggests that neither Sap 1-6 isoenzymes nor Efg1/Cph1 pathways may be entirely responsible for protease release when exposed to these NSAIDs. Ibuprofen and acetaminophen enhanced Sap secretion rates in three environmental conditions (normoxic biofilm, normoxic planktonic and anoxic planktonic). In other hand, aspirin seems to reduce the Sap-related pathogenic behavior of candidal biofilms. Modulation of Sap activity may occur according to candidal phenotypic state, oxygen availability, and type of NSAID to which the cells are exposed. (C) 2010 Elsevier Ltd. All rights reserved.

HOST-MEDIATED INDUCTION OF alpha-AMYLASES BY LARVAE OF THE MEXICAN BEAN WEEVIL Zabrotes subfasciatus (COLEOPTERA: CHRYSOMELIDAE: BRUCHINAE) IS IRREVERSIBLE AND OBSERVED FROM THE INITIATION OF THE FEEDING PERIOD

Larvae of Zabrotes subfasciatus secrete alpha-amylases that are insensitive to the alpha-amylase inhibitor found in seeds of Phaseolus vulgaris. By analyzing amylase activities during larval development on P. vulgaris, we detected activity of the constitutive amylase and the two inducible amylase isoforms at all stages. When larvae were transferred from the non alpha-amylase inhibitor containing seeds of Vigna unguiculata to P. vulgaris, the inducible alpha-amylases were expressed at the same level as in control larvae fed on P. vulgaris. Interestingly, when larvae were transferred from seeds of P. vulgaris to those of V. unguiculata, inducible alpha-amylases continued to be expressed at a level similar to that found in control larvae fed P. vulgaris continuously. When 10-day-old larvae were removed from seeds of V. unguiculata and transferred into capsules containing flour of P. vulgaris cotyledons, and thus maintained until completing 17 days ( age when the larvae stopped feeding), we could detect higher activity of the inducible alpha-amylases. However, when larvae of the same age were transferred from P. vulgaris into capsules containing flour of V. unguiculata, the inducible alpha-amylases remained up-regulated. These results suggest that the larvae of Z. subfasciatus have the ability to induce insensitive amylases early in their development. A short period of feeding on P. vulgaris cotyledon flour was sufficient to irreversibly induce the inducible alpha-amylase isoforms. Incubations of brush border membrane vesicles with the alpha-amylase inhibitor 1 from P. vulgaris suggest that the inhibitor is recognized by putative receptors found in the midgut microvillar membranes. (C) 2010 Wiley Periodicals, Inc.

Discovery of novel Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase inhibitors

Based on its essential role in the life cycle of Trypanosoma cruzi, the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has been considered a promising target for the development of novel chemotherapeutic agents for the treatment of Chagas` disease. In the course of our research program to discover novel inhibitors of this trypanosomatid enzyme, we have explored a combination of structure and ligand-based virtual screening techniques as a complementary approach to a biochemical screening of natural products using a standard biochemical assay. Seven natural products, including anacardic acids,. avonoid derivatives, and one glucosylxanthone were identified as novel inhibitors of T. cruzi GAPDH. Promiscuous inhibition induced by nonspecific aggregation has been discarded as specific inhibition was not reversed or affected in all cases in the presence of Triton X-100, demonstrating the ability of the assay to find authentic inhibitors of the enzyme. The structural diversity of this series of promising natural products is of special interest in drug design, and should therefore be useful in future medicinal chemistry efforts aimed at the development of new GAPDH inhibitors having increased potency. (C) 2009 Elsevier Ltd. All rights reserved.

Anacardic acid derivatives as inhibitors of glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi

Chagas` disease, a parasitic infection caused by the flagellate protozoan Trypanosoma cruzi, is a major public health problem affecting millions of individuals in Latin America. On the basis of the essential role in the life cycle of T. cruzi, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been considered an attractive target for the development of novel antitrypanosomatid agents. In the present work, we describe the inhibitory effects of a small library of natural and synthetic anacardic acid derivatives against the target enzyme. The most potent inhibitors, 6-n-pentadecyl-(1) and 6-n-dodecylsalicilic acids (10e), have IC(50) values of 28 and 55 mu M, respectively. The inhibition was not reversed or prevented by the addition of Triton X-100, indicating that aggregate-based inhibition did not occur. In addition, detailed mechanistic characterization of the effects of these compounds on the T. cruzi GAPDH-catalyzed reaction showed clear noncompetitive inhibition with respect to both substrate and cofactor. (C) 2008 Elsevier Ltd. All rights reserved.

Quantitative structure-activity studies on a series of migrastatin analogs as inhibitors of cancer cell metastasis

Migrastatin, a macrolide natural product, and its structurally related analogs are potent inhibitors of cancer cell metastasis, invasion and migration. In the present work, a specialized fragment-based method was employed to develop QSAR models for a series of migrastatin and isomigrastatin analogs. Significant correlation coefficients were obtained (best model, q(2) = 0.76 and r(2) = 0.91) indicating that the QSAR models possess high internal consistency. The best model was then used to predict the potency of an external test set, and the predicted values were in good agreement with the experimental results (R(2) (pred) = 0.85). The final model and the corresponding contribution maps, combined with molecular modeling studies, provided important insights into the key structural features for the anticancer activity of this family of synthetic compounds based on natural products.

Classical and hologram QSAR studies on a series of inhibitors of trypanosomatid Glyceraldehyde-3-Phosphate Dehydrogenase

Leishmaniasis and trypanosomiasis are major causes of morbidity and mortality in both tropical and subtropical regions of the world. The current available drugs are limited, ineffective, and require long treatment regimens. Due to the high dependence of trypanosomatids on glycolysis as a source of energy, some glycolytic enzymes have been identified as attractive targets for drug design. In the present work, classical Two-Dimensional Quantitative Structure -Activity Relationships (2D QSAR) and Hologram QSAR (HQSAR) studies were performed on a series of adenosine derivatives as inhibitors of Leishmania mexicana Glyceraldehyde-3-Phosphate Dehydrogenase (LmGAPDH). Significant correlation coefficients (classical QSAR, r(2)=0.83 and q(2) =0.81; HQSAR, r(2)=0.91 and q(2) =0.86) were obtained for the 56 training set compounds, indicating the potential of the models for untested compounds. The models were then externally validated using a test set of 14 structurally related compounds and the predicted values were in good agreement with the experimental results (classical QSAR, r(pred)(2) = 0.94; HQSAR, r(pred)(2) = 0.92).

Discovery of New Inhibitors of Schistosoma mansoni PNP by Pharmacophore-Based Virtual Screening

Schistosomiasis is considered the second most important tropical parasitic disease, with severe socioeconomic consequences for millions of people worldwide. Schistosoma monsoni, one of the causative agents of human schistosomiasis, is unable to synthesize purine nucleotides de novo, which makes the enzymes of the purine salvage pathway important targets for antischistosomal drug development. In the present work, we describe the development of a pharmacophore model for ligands of S. mansoni purine nucleoside phosphorylase (SmPNP) as well as a pharmacophore-based virtual screening approach, which resulted in the identification of three thioxothiazolidinones (1-3) with substantial in vitro inhibitory activity against SmPNP. Synthesis, biochemical evaluation, and structure activity relationship investigations led to the successful development of a small set of thioxothiazolidinone derivatives harboring a novel chemical scaffold as new competitive inhibitors of SmPNP at the low-micromolar range. Seven compounds were identified with IC(50) values below 100 mu M. The most potent inhibitors 7, 10, and 17 with 1050 of 2, 18, and 38 mu M, respectively, could represent new potential lead compounds for further development of the therapy of schistosomiasis.

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.

MPO Inhibitors Selected by Virtual Screening

The hemeprotein myeloperoxidase (MPO) participates in innate immune defense through its ability to generate potent microbicidal oxidants. However, these oxidants are also key mediators of the tissue damage associated with many inflammatory diseases. Thus, there is considerable interest in developing therapeutically useful MPO inhibitors. Here, we used structure-based drug design (SBDD) and ligand-based drug design (LBDD) to select for potentially new and selective MPO inhibitors. A pharmacophore model was developed based on the crystal structure of human MPO in complex with salicylhydroxamic acid (SHA), a known inhibitor of the enzyme. The pharmacophore model was used to screen the ZINC database for potential ligands, which were further filtered on the basis of their physical-chemical properties and docking score. The filtered compounds were visually inspected, and nine were purchased for experimental studies. Surprisingly, almost all of the selected compounds belonged to the aromatic hydrazide class, which had been previously described as MPO inhibitors. The compounds selected by virtual screening were shown to inhibit the chlorinating activity of MPO; the top four compounds displayed IC(50) values ranging from 1.0 to 2.8 mM. MPO inactivation by the most effective compound was shown to be irreversible. Overall, our results show that SBDD and LBDD may be useful for the rational development of new MPO inhibitors.

Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics

Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 +/- 0.2) x 10(7) M(-1) s(-1)) and peroxynitrite ((3.7 +/- 0.4) x 10(5) M(-1) s(-1)) at pH 7.4 and 25 degrees C. (C) 2011 Elsevier Inc. All rights reserved.

Digestive physiology and characterization of digestive cathepsin L-like proteinase from the sugarcane weevil Sphenophorus levis

Sugarcane is an important crop that has recently become subject to attacks from the weevil Sphenophorus levis, which is not efficiently controlled with chemical insecticides. This demands the development of new control devices for which digestive physiology data are needed. In the present study, ion-exchange chromatography of S. levis whole midgut homogenates, together with enzyme assays with natural and synthetic substrates and specific inhibitors, demonstrated that a cysteine proteinase is a major proteinase, trypsin is a minor one and chymotrypsin is probably negligible. Amylase, maltase and the cysteine proteinase occur in the gut contents and decrease throughout the midgut; trypsin is constant in the entire midgut, whereas a membrane-bound aminopeptidase predominates in the posterior midgut. The cysteine proteinase was purified to homogeneity through ion-exchange chromatography. The purified enzyme had a mass of 37 kDa and was able to hydrolyze Z-Phe-Arg-MCA and Z-Leu-Arg-MCA with k(cat)/K(m) values of 20.0 +/- 1.1 mu M(-1) s(-1) and 30.0 +/- 0.5 mu M(-1) s(-1), respectively, but not Z-Arg-Arg-MCA. The combined results suggest that protein digestion starts in the anterior midgut under the action of a cathepsin L-like proteinase and ends on the surface of posterior midgut cells. All starch digestion takes place in anterior midgut. These data will be instrumental to developing S. levis-resistant sugarcane. (C) 2011 Elsevier Ltd. All rights reserved.

Geranylation of benzoic acid derivatives by enzymatic extracts from Piper crassinervium (Piperaceae)

The ability to carry out geranylations on aromatic substrates using enzymatic extracts from the leaves of Piper crassinervium (Piperaceae) was evaluated. A literature analysis pointed out its importance as a source of prenylated bioactive molecules. The screening performed on aromatic acceptors (benzoic acids, phenols and phenylpropanoids) including geranyl diphosphate as prenyl donor, showed the biotransformation of the 3,4-dihydroxybenzoic acid by the crude extract, and the p-hydroxybenzoic acid by both the microsomal fraction and the crude extract, after treating leaves with glucose. The analysis of the products allowed the identification of C- and O-geranylated derivatives, and the protease (subtilisin and pepsin) inhibition performed on the O-geranylated compounds showed weak inhibition. Electrophoretic profiles indicated the presence of bands/spots among 56-58 kDa and pI 6-7, which are compatible with prenyltransferases. These findings show that P. crassinervium could be considered as a source of extracts with geranyltransferase activity to perform biotransformations on aromatic substrates. (C) 2010 Elsevier Ltd. All rights reserved.