Cruzain inhibition by hydroxymethylnitrofurazone and nitrofurazone: investigation of a new target in Trypanosoma cruzi.

Nitrofurazone (NF) and its derivative, hydroxymethylnitrofurazone (NFOH), have presented antichagasic activity. NFOH has higher activity and lower mutagenicity. The aim of this work was to assess whether NF and its derivative NFOH would also be inhibitors of cruzain, besides their trypanothione reductase inhibitory activity. In vitro cruzain inhibition tests were performed for both compounds, and the 50% inhibitory concentration (IC50) for NF and NFOH presented values of 22.83 +/- 1.2 microM and 10.55 +/- 0.81 microM, respectively. AM1 semi-empirical molecular modeling studies were performed to understand the activity of the compounds, corroborating the observed cruzain inhibitory activity.

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.

Kininogenase activity of Thalassophryne nattereri fish venom.

Accidents caused by the venomous fish Thalassophryne nattereri are characterized by edema, intense pain and necrosis at the site of the sting. This study assessed the nociceptive and edematogenic activities of T. nattereri venom after injection into the mouse hindpaw and determination of the paw licking duration and weight. Subplantar injections of the venom (0.1-6 microg) induced a dose-related increase of the paw licking time and paw swelling with maximal values at 3 microg (209.5 +/- 57.5 s and 135.0 +/- 6.8 mg, respectively). Pretreatment of mice with either indomethacin (10 mg/kg, i.p.), a cyclooxygenase inhibitor, dexamethasone (1 mg/kg, s.c.), a steroid anti-inflammatory agent, cyproheptadine (1 mg/kg, i.p.), antagonist of serotonin receptors or L-NAME (100 mg/kg, s.c.), inhibitor of nitric oxide syntase, did not affect the venom-induced nociceptive and edematogenic responses. Injection of the opioid analgesic fentanyl (0.1 mg/kg, s.c.) reduced the paw licking time induced by 1 microg venom by 84% of control, without affecting the paw swelling. Both nociceptive and edematogenic responses were reduced after treatment with a specific tissue kallikrein inhibitor (TKI, 100 mg/kg, i.p.) by 78% and 24% from control values, respectively. Administration of a specific plasma kallikrein inhibitor (PKSI(527,) 100 mg/kg, s.c.) did not affect the venom-induced nociceptive response, but it decreased the paw edema by 15% from control. After injection of the angiotensin-converting enzyme inhibitor captopril (100 mg/kg, i.p.) the venom-induced nociceptive end edematogenic responses were increased by two-fold. The role of kallikreins possibly present in the venom was further assessed by hydrolysis of human kininogen and kininogen-derived synthetic peptides, showing the release of kallidin (Lys-bradykinin). The hydrolysis was inhibited by metal chelating agents but not by serino-, aspartyl- or cysteino-proteinase inhibitors. The data suggest that a protease with tissue-kallikrein-like activity plays a major role in nociception and edema induced by T. nattereri venom and this should be considered to achieve efficient treatments for human accidents with this venom.

Angiotensin I-converting enzyme inhibitor peptides derived from the endostatin-containing NC1 fragment of human collagen XVIII.

Extracellular matrix and soluble plasma proteins generate peptides that regulate biological activities such as cell growth, differentiation and migration. Bradykinin, a peptide released from kininogen by kallikreins, stimulates vasodilatation and endothelial cell proliferation. Various classes of substances can potentiate these biological actions of bradykinin. Among them, the best studied are bradykinin potentiating peptides (BPPs) derived from snake venom, which can also strongly inhibit angiotensin I-converting enzyme (ACE) activity. We identified and synthesized sequences resembling BPPs in the vicinity of potential proteolytic cleavage sites in the collagen XVIII molecule, close to endostatin. These peptides were screened as inhibitors of human recombinant wild-type ACE containing two intact functional domains; two full-length ACE mutants containing only a functional C- or N-domain catalytic site; and human testicular ACE, a natural form of the enzyme that only contains the C-domain. The BPP-like peptides inhibited ACE in the micromolar range and interacted preferentially with the C-domain. The proteolytic activity involved in the release of BPP-like peptides was studied in human serum and human umbilical-vein endothelial cells. The presence of enzymes able to release these peptides in blood led us to speculate on a physiological mechanism for the control of ACE activities.

Cathepsin B carboxydipeptidase specificity analysis using internally quenched fluorescent peptides.

We have examined in detail the specificity of the subsites S1, S2, S1' and S2' for the carboxydipeptidase activity of cathepsin B by synthesizing and assaying four series of internally quenched fluorescent peptides based on the sequence Dnp-GFRFW-OH, where Dnp (2,4-dinitrophenyl) is the quenching group of the fluorescence of the tryptophan residue. Each position, except the glycine, was substituted with 15 different naturally occurring amino acids. Based on the results we obtained, we also synthesized efficient and sensitive substrates that contained o -aminobenzoic acid and 3-Dnp-(2,3-diaminopropionic acid), or epsilon-amino-Dnp-Lys, as the fluorescence donor-receptor pair. The higher kinetic parameter values for the carboxydipeptidase compared with the endopeptidase activity of cathepsin B allowed an accurate analysis of its specificity. The subsite S1 accepted preferentially basic amino acids for hydrolysis; however, substrates with phenylalanine and aliphatic side-chain-containing amino acids at P1 had lower K m values. Despite the presence of Glu245 at S2, this subsite presented clear preference for aromatic amino acid residues, and the substrate with a lysine residue at P2 was hydrolysed better than that containing an arginine residue. S1' is essentially a hydrophobic subsite, and S2' has particular preference for phenylalanine or tryptophan residues.

Comparaçäo da especificidade da catepsina B para suas atividades endo e expopeptidásicas utilizando substratos peptídicos de fluorescência apagada internamente / Comparison of endo and exopeptidase activities od cathepsin B throught the use of internally quenched fluorogenic peptide substrates

Exploramos a especificidade da catepsina B recombinante humana expressa em levedura, Pichia pastoris. Sintetizamos e caracterizamos dois tipos de substratos de fluorescência interna apagada, isto é, um formado por Abz-Dnp e outro por Trp-Dnp (Abz= acido oito-aminobenzoico, Trp=triptofano, Dnp= 2,4-dinitrophenila). como pares doador-aceptor de fluorescência, todos com a carboxila C-terminal livre. Preparamos três séries de substratos, usando como referência os seguintes peptídeos: AbzFRDap(Dnp)[ ou K(Dnp)]P-OH, Abz-GFFRK(Dnp)W-OH e Dnp-GFRFW-OH. Fizemos o estudo da cinética de hidrólise destes peptídeos e determinamos as ligaçoes clivadas para cada substrato. Isto nos garantiu que estávamos acompanhando a atividade carboxidipeptidásica. As observaçoes mais relevantes, para a especificidade da atividade exopeptidásica da catepsina B, sao as seguintes: Phe na posiçao P2 é o aminoácido preferencial Arg ou Phe na posiçao P1 mostraram ser preferenciais na' dependência do aminoácido colocado em P2'. Assim, com Pro nesta posiçao o resíduo Arg é preferencial, já com Trp em P2' tanto Arg como Phe em P1 resultaram em bons substratos. K(Dnp) ou Dap(Dnp) no lado P' nao se diferenciam quanto à geraçao de substratos eficientes para catepsina B. A natureza do aminoácido na posiçao P2' tem um efeito muito determinante na susceptibilidade dos substratos. O Trp é o resíduo preferencial nesta posiçao, e a presença de aminoácidos hidrofílicos ou carregados resulta em péssimos substratos. - Aminoácidos hidrofóbicos sao preferidos na posiçao Pi'.,_(au