Synthesis, biological evaluation and molecular docking studies of 3-(triazolyl)coumarin derivatives: Effect on inducible nitric oxide synthase

A series of 3-(triazolyl)-coumarins were synthesized and tested as anti-inflammatory agents. It was possible to infer that these compounds do not alter the interaction of LPS with TLR-4 or TLR-2, as the intracellular pathways involved in the TNF-alpha secretion and COX-2 activity were not affected. Nevertheless, the compounds inhibited iNOS-derived NO production, without affecting the eNOS activity. The outcome of the docking studies showed that it pi center dot center dot center dot pi interactions with the heme group are important for the iNOS inhibition, thus making compound 3c a promising lead. Moreover, the efficacy of this compound was visualized by the reduced number of neutrophils in the LPS-inflamed subcutaneous tissue. Together, biological and docking data show that triazolyl-substituted coumarins, that can act on iNOS, are a good scaffold to be explored. (C) 2012 Elsevier Masson SAS. All rights reserved.

Docking, synthesis and pharmacological activity of novel urea-derivatives designed as p38 MAPK inhibitors

p38 mitogen-activated protein kinase (p38 MAPK) is an important signal transducing enzyme involved in many cellular regulations, including signaling pathways, pain and inflammation. Several p38 MAPK inhibitors have been developed as drug candidates to treatment of autoimmune disorders, such as rheumatoid arthritis. In this paper we reported the docking, synthesis and pharmacological activity of novel urea-derivatives (4a-e) designed as p38 MAPK inhibitors. These derivatives presented good theoretical affinity to the target p38 MAPK, standing out compound 4e (LASSBio-998), which showed a better score value compared to the prototype GK-00687. This compound was able to reduce in vitro TNF-alpha production and was orally active in a hypernociceptive murine model sensible to p38 MAPK inhibitors. Otherwise, compound 4e presented a dose-dependent analgesic effect in a model of antigen (mBSA)-induced arthritis and anti-inflammatory profile in carrageenan induced paw edema, indicating its potential as a new antiarthritis prototype. (c) 2012 Elsevier Masson SAS. All rights reserved.

Design and synthesis of new (E)-cinnamic N-acylhydrazones as potent antitrypanosomal agents

We report herein the synthesis and trypanocidal profile of new (E)-cinnamic N-acylhydrazones (NAHs) designed by exploiting molecular hybridization between the potent cruzain inhibitors (E)-1-(benzo[d] 11,3)dioxol-5-yl)-3-(4-bromophenyl)prop-2-en-1-one and (E)-3-hydroxy-N'-((2-hydroxynaphthalen-1-yl)methylene)-7-methoxy-2-naphthohydrazide. These derivatives were evaluated against both amastigote and trypomastigote forms of Trypanosoma cruzi and lead us to identify two compounds that were approximately two times more active than the reference drug, benznidazole, and with good cytotoxic index. Although designed as cruzain inhibitors, the weak potency displayed by the best cinnamyl NAH derivatives indicated that another mechanism of action was likely responsible for their trypanocide action. (C) 2012 Elsevier Masson SAS. All rights reserved.

Biomimetic in vitro oxidation of lapachol: A model to predict and analyse the in vivo phase I metabolism of bioactive compounds

The bioactive naphtoquinone lapachol was studied in vitro by a biomimetic model with Jacobsen catalyst (manganese(III) salen) and iodosylbenzene as oxidizing agent. Eleven oxidation derivatives were thus identified and two competitive oxidation pathways postulated. Similar to Mn(III) porphyrins, Jacobsen catalyst mainly induced the formation of para-naphtoquinone derivatives of lapachol, but also of two ortho-derivatives. The oxidation products were used to develop a GC MS (SIM mode) method for the identification of potential phase I metabolites in vivo. Plasma analysis of Wistar rats orally administered with lapachol revealed two metabolites, alpha-lapachone and dehydro-alpha-lapachone. Hence, the biomimetic model with a manganese salen complex has evidenced its use as a valuable tool to predict and elucidate the in vivo phase I metabolism of lapachol and possibly also of other bioactive natural compounds. (C) 2012 Elsevier Masson SAS. All rights reserved.

Biochemical characterization and comparative analysis of two distinct serine proteases from Bothrops pirajai snake venom

This study reports the isolation and biochemical characterization of two different serine proteases from Bothrops pirajai snake venom, thus providing a comparative analysis of the enzymes. The isolation process consisted of three consecutive chromatographic steps (Sephacryl S-200, Benzamidine Sepharose and C2/C18), resulting in two serine proteases, named BpirSP27 and BpirSP41 after their molecular masses by mass spectrometry (27,121 and 40,639 Da, respectively). Estimation by SDS-PAGE under denaturing conditions showed that, when deglycosylated with PNGase F, BpirSP27 and BpirSP41 had their molecular masses reduced by approximately 15 and 42%, respectively. Both are acidic enzymes, with pI of approximately 4.7 for BpirSP27 and 3.7 for BpirSP41, and their N-terminal amino acid sequences showed 57% identity to each other, with high similarity to the sequences of other snake venom serine proteases (SVSPs). The enzymes showed different actions on bovine fibrinogen, with BpirSP27 acting preferentially on the B beta chain and BpirSP41 on both A alpha and B beta chains. The two serine proteases were also able to degrade fibrin and blood clots in vitro depending on the doses and incubation periods, with higher results for BpirSP41. Both enzymes coagulated the human plasma in a dose-dependent manner, and BpirSP41 showed a higher coagulant potential, with minimum coagulant dose (MCD) of similar to 3.5 mu g versus 20 mu g for BpirSP27. The enzymes were capable of hydrolyzing different chromogenic substrates, including S-2238 for thrombin-like enzymes, but only BpirSP27 acted on the substrate S-2251 for plasmin. They also showed high stability against variations of temperature and pH, but their activities were significantly reduced after preincubation with Cu2+ ion and specific serine protease inhibitors. In addition. BpirSP27 induced aggregation of washed platelets to a greater extent than BpirSP41. The results showed significant structural and functional differences between B. pirajai serine proteases, providing interesting insights into the structure-function relationship of SVSPs. (C) 2012 Elsevier Masson SAS. All rights reserved.

Aminoacylase 1-catalysed deacetylation of bioactives epoxides mycotoxin-derived mercapturates; 3,4-epoxyprecocenes as models of cytotoxic epoxides

The mycotoxin aflatoxin B1 (AFB1) is a carcinogenic food contaminant which is metabolically activated by epoxydation. The metabolism of mycotoxins via the mercapturate metabolic pathway was shown, in general, to lead to their detoxication. Mercapturic acids thus formed (S-substitued-N-acetyl-L-cysteines) may be accumulated in the kidney and either excreted in the urine or desacetylated by Acylase 1 (ACY1) to yield cysteine S-conjugates. To be toxic, the N-acetyl-L-cysteine-S-conjugates first have to undergo deacetylation by ACY 1. The specificity and rate of mercapturic acid deacetylation may determine the toxicity, however the exact deacetylation processes involved are not well known. The aim of this study was to investigate the role of ACY1 in the toxicity of some bioactive epoxides from Aflatoxin B1. We characterized the kinetic parameters of porcine kidney and human recombinant aminoacylase-1 towards some aromatic and aliphatic-derived mercapturates analogue of mycotoxin mercapturic acids and 3,4-epoxyprecocene, a bioactive epoxide derivated from aflatoxin. The deacetylation of mercapturated substrates was followed both by reverse phase HPLC and by TNBS method. Catalytic activity was discussed in a structure function relationship. Ours results indicate for the first time that aminoacylase-1 could play an important role in deacetylating mercapturate metabolites of aflatoxin analogues and this process may be in relation with their cyto- and nephrotoxicity in human. (C) 2012 Published by Elsevier Masson SAS.

Crystal structure of dihydroorotate dehydrogenase from Leishmania major

Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and has been exploited as the target for therapy against proliferative and parasitic diseases. In this study, we report the crystal structures of DHODH from Leishmania major, the species of Leishmania associated with zoonotic cutaneous leishmaniasis, in its apo form and in complex with orotate and fumarate molecules. Both orotate and fumarate were found to bind to the same active site and exploit similar interactions, consistent with a ping-pong mechanism described for class 1A DHODHs. Analysis of LmDHODH structures reveals that rearrangements in the conformation of the catalytic loop have direct influence on the dimeric interface. This is the first structural evidence of a relationship between the dimeric form and the catalytic mechanism. According to our analysis, the high sequence and structural similarity observed among trypanosomatid DHODH suggest that a single strategy of structure-based inhibitor design can be used to validate DHODH as a druggable target against multiple neglected tropical diseases such as Leishmaniasis, Sleeping sickness and Chagas' diseases. (C) 2012 Elsevier Masson SAS. All rights reserved.

The bactericidal effect of human secreted group IID phospholipase A(2) results from both hydrolytic and non-hydrolytic activities

The Human Secreted Group IID Phospholipase A(2) (hsPLA2GIID) may be involved in the human acute immune response. Here we have demonstrated that the hsPLA2GIID presents bactericidal and Ca2+-independent liposome membrane-damaging activities and we have compared these effects with the catalytic activity of active-site mutants of the protein. All mutants showed reduced hydrolytic activity against DOPC:DOPG liposome membranes, however bactericidal effects against Escherichia coli and Micrococcus luteus were less affected, with the D49K mutant retaining 30% killing of the Gram-negative bacteria at a concentration of 10 mu g/mL despite the absence of catalytic activity. The H48Q mutant maintained Ca2+-independent membrane-damaging activity whereas the G30S and D49K mutants were approximately 50% of the wild-type protein, demonstrating that phospholipid bilayer permeabilization by the hsPLA2GIID is independent of catalytic activity. We suggest that this Ca2+-independent damaging activity may play a role in the bactericidal function of the protein. (C) 2012 Elsevier Masson SAS. All rights reserved.

Isolation, enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom

A hyaluronidase (CdtHya1) from Crotalus durissus terrificus snake venom (CdtV) was isolated and showed to exhibit a high activity on hyaluronan cleavage. However, surveys on this enzyme are still limited. This study aimed at its isolation, functional/structural characterization and the evaluation of its effect on the spreading of crotoxin and phospholipase A(2) (PLA(2)). The enzyme was purified through cation exchange, gel filtration and hydrophobic chromatography. After that, it was submitted to a reverse-phase fast protein liquid chromatography (RP-FPLC) and Edman degradation sequencing, which showed the first N-terminal 44 amino acid residues whose sequence evidenced identity with other snake venom hyaluronidases. CdtHya1 is a monomeric glycoprotein of 64.5 kDa estimated by SDS-PAGE under reducing conditions. It exhibited maximum activity in the presence of 0.2 M NaCl, at 37 degrees C, pH 5.5 and a specificity to hyaluronan higher than that to chondroitin-4-sulphate, chondroitin-6-sulphate or dermatan. Divalent cations (Ca2+ and Mg2+) and 1 M NaCl significantly reduced the enzyme activity. The specific activity of CdtHya1 was 5066 turbidity reducing units (TRU)/mg, against 145 TRU/mg for the soluble venom, representing a 34.9-fold purification. The pure enzyme increased the diffusion of crotoxin and PLA (2) through mice tissues. CdtHya1 (32 TRU/40 mu L) potentiated crotoxin action, as evidenced by mice death, and it decreased the oedema caused by subplantar injections of buffer, crotoxin or PLA(2), thus evidencing the relevance of hyaluronidase in the crotalic envenoming. This work yielded a highly active antiedematogenic hyaluronidase from CdtV, the first one isolated from rattlesnake venoms. (C) 2012 Elsevier Masson SAS. All rights reserved.