RESUMO
Introduction: The human umbilical artery (HUA), rat-isolated right atrium, and rat-isolated vas deferens present a basal release of 6-nitrodopamine (6-ND). The basal release of 6-ND from these tissues was significantly decreased (but not abolished) when the tissues were pre-incubated with Nω-nitro-L-arginine methyl ester (L-NAME). Methods: In this study, the effect of the pharmacological modulation of the redox environment on the basal release of 6-ND was investigated. The basal release of 6-ND was measured using Liquid chromatography with tandem mass spectrometry (LC-MS/MS). Results and Discussion: Pre-incubation (30 min) of the tissues with GKT137831 (1 µM) caused a significant increase in the basal release of 6-ND from all tissues. In the HUA, pre-incubation with diphenyleneiodonium (DPI) (100 µM) also caused significant increases in the basal release of 6-ND. Preincubation of the HUA with hydrogen peroxide (H2O2) (100 µM) increased 6-ND basal release, whereas pre-incubation with catalase (1,000 U/mL) significantly decreased it. Pre-incubation of the HUA with superoxide dismutase (SOD) (250 U/mL; 30 min) also significantly increased the basal release of 6-ND. Preincubation of the HUA with either allopurinol (100 µM) or uric acid (1 mM) had no effect on the basal release of 6-ND. Pre-treatment of the HUA with L-NAME (100 µM) prevented the increase in the basal release of 6-ND induced by GKT137831, diphenyleneiodonium, and H2O2. The results obtained indicate a major role of endogenous H2O2 and peroxidases as modulators of 6- ND biosynthesis/release and a lack of peroxynitrite contribution.
RESUMO
Hydrogen sulfide (H2S) is particularly produced in the skin, where it participates in the regulation of inflammation, pruritus, cytoprotection, scarring, and angiogenesis. In this study, we compared the effects of dexamethasone (Dex) with two H2S-releasing Dex derivatives in a murine model of atopic dermatitis (AD) induced by topical application of 2,4-dinitrochlorobenzene (DNCB). After sensitization with DNCB, the animals were topically treated for five consecutive days with either the H2S-releasing compounds 4-hydroxy-thiobenzamide (TBZ) and 5-(p-hydroxyphenyl)-1,2-dithione-3-thione (ADT-OH), Dex, or the derivatives Dex-TBZ or Dex-ADT. Topical treatment with equimolar doses of either Dex, Dex-TBZ, or Dex-ADT resulted in similar reductions in dermatitis score, scratching behavior, edema, eosinophilia, splenomegaly, and histological changes. In contrast with Dex, the H2S-releasing derivatives prevented IL-4 elevation and oxidative modification of skin proteins. On an equimolar dose basis, Dex-TBZ, but not Dex-ADT, promoted the elevation of endogenous H2S production and GPx activity. Neither Dex-TBZ nor Dex-ADT decreased GR activity or caused hyperglycemia, as observed with Dex treatment. We conclude that the presence of H2S-releasing moieties in the Dex structure does not interfere with the anti-inflammatory effects of this corticosteroid and adds beneficial therapeutical actions to the parent compound.
RESUMO
Human kallikrein 6 (KLK6) is highly expressed in the central nervous system and with elevated level in demyelinating disease. KLK6 has a very restricted specificity for arginine (R) and hydrolyses myelin basic protein, protein activator receptors and human ionotropic glutamate receptor subunits. Here we report a previously unreported activity of KLK6 on peptides containing clusters of basic amino acids, as in synthetic fluorogenic peptidyl-Arg-7-amino-4-carbamoylmethylcoumarin (peptidyl-ACC) peptides and FRET peptides in the format of Abz-peptidyl-Q-EDDnp (where Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-(2,4-dinitrophenyl) ethylenediamine), in which pairs or sequences of basic amino acids (R or K) were introduced. Surprisingly, KLK6 hydrolyzed the fluorogenic peptides Bz-A-R↓R-ACC and Z-R↓R-MCA between the two R groups, resulting in non-fluorescent products. FRET peptides containing furin processing sequences of human MMP-14, nerve growth factor (NGF), Neurotrophin-3 (NT-3) and Neurotrophin-4 (NT-4) were cleaved by KLK6 at the same position expected by furin. Finally, KLK6 cleaved FRET peptides derived from human proenkephalin after the KR, the more frequent basic residues flanking enkephalins in human proenkephalin sequence. This result suggests the ability of KLK6 to release enkephalin from proenkephalin precursors and resembles furin a canonical processing proteolytic enzyme. Molecular models of peptides were built into the KLK6 structure and the marked preference of the cut between the two R of the examined peptides was related to the extended conformation of the substrates.
Assuntos
Calicreínas/metabolismo , Cinética , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Aminoácidos Básicos/química , Aminoácidos Básicos/genética , Encefalinas/química , Encefalinas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Furina/química , Furina/metabolismo , Humanos , Hidrólise , Calicreínas/química , Calicreínas/genética , Metaloproteinase 14 da Matriz/química , Metaloproteinase 14 da Matriz/metabolismo , Modelos Moleculares , Fator de Crescimento Neural/química , Fator de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/metabolismo , Neurotrofina 3 , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Peptídeos/metabolismo , Conformação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Proteólise , Especificidade por SubstratoRESUMO
KLK7 substrate specificity was evaluated by families of fluorescence resonance energy transfer (FRET) peptides derived from Abz-KLFSSK-Q-EDDnp (Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-[2,4-dinitrophenyl] ethylenediamine), by one bead-one peptide FRET peptide library in PEGA resin, and by the FRET peptide libraries Abz-GXX-Z-XX-Q-EDDnp (Z and X are fixed and random natural amino acids, respectively). KLK7 hydrolyzed preferentially F, Y or M, and its S1' and S2' subsites showed selectivity for hydrophilic amino acids, particularly R and K. This set of specificities was confirmed by the efficient kininogenase activity of KLK7 on Abz-MISLM(↓)KRPPGFSPF(↓)RSSRI-NH2 ((↓)indicates cleavage), hydrolysis of somatostatin and substance P and inhibition by kallistatin. The peptide Abz-NLY(↓)RVE-Q-EDDnp is the best synthetic substrate so far described for KLK7 [kcat/Km=455 (mMs)(-1)] that was designed from the KLK7 substrate specificity analysis. It is noteworthy that the NLYRVE sequence is present in human semaphorin 6B. KLK7 is activated by GAGs, inhibited by neutral salts, and activated by high concentration of kosmotropic salt. Pyroglutamic acid inhibited KLK7 (Ki=33mM) and is present in skin moisturizing factor (124mM). The KLK7 specificity described here and elsewhere reflects its participation in patho-physiological events in skin, the gastrointestinal tract and central nervous system, where KLK7 is significantly expressed.
Assuntos
Glicosaminoglicanos/farmacologia , Calicreínas/metabolismo , Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Humanos , Hidrólise/efeitos dos fármacos , Cinética , Cininogênios/metabolismo , Dados de Sequência Molecular , Concentração Osmolar , Ácido Pirrolidonocarboxílico/farmacologia , Semaforinas/metabolismo , Serpinas/metabolismo , Somatostatina/metabolismo , Substância P/metabolismo , Especificidade por Substrato , Fatores de TempoRESUMO
The 5-[2-ethoxy-5-(4-methylpiperazin-1-ylsulfonyl)phenyl]-1-methyl-3-propyl-1,6-dihydro-7 H-pyrazolo[4,3-d]pyrimidin-7-one, sildenafil, is a cGMP-specific phosphodiesterase-5 (PDE5) inhibitor used for penile erectile dysfunction. In the search for more potent and selective PDE5 inhibitors, new sildenafil analogues (6a-v), characterized by the presence on the sulfonyl group in the 5' position of novel N-4-substituted piperazines or ethylenediamine moiety, were prepared by traditional and microwave-assisted synthesis and tested in rabbit isolated aorta and corpus cavernosum. Similarly to sildenafil, several analogues showed IC50 values in the nanomolar range. In the in vitro studies, all the tested compounds caused concentration-dependent relaxations in both rabbit isolated aorta and corpus cavernosum. All sildenafil analogues potentiated the nitric oxide-dependent vasodilation in endothelium-intact rabbit aorta. Compound 6f exhibited great pEC50 value in corpus cavernosum, and compounds 6r and 6u in isolated aorta were found as potent as sildenafil for inhibiting PDE5. Because several analogues were significantly more lipophilic than sildenafil, these compounds may offer a new lead for development of new sildenafil analogues.
Assuntos
Disfunção Erétil/tratamento farmacológico , Inibidores da Fosfodiesterase 5 , Piperazinas/síntese química , Pirimidinas/síntese química , Sulfonas/síntese química , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Endotélio Vascular/fisiologia , Humanos , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Masculino , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Pênis/efeitos dos fármacos , Pênis/fisiologia , Piperazinas/química , Piperazinas/farmacologia , Purinas/síntese química , Purinas/química , Purinas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Coelhos , Ratos , Citrato de Sildenafila , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade , Sulfonas/química , Sulfonas/farmacologiaRESUMO
We report microwave-assisted synthetic routes, the pharmacokinetic profile along with results from ulcerogenicity and mutagenicity studies of atenolol aspirinate, and an already described derivative, in which acetyl salicylic acid (aspirin) was connected to atenolol by an ester linkage. Atenolol aspirinate was stable towards aqueous hydrolysis but rapidly hydrolyzed in plasma (t(1/2) = 7.6 min). The results showed that the rapid and complete hydrolysis generates atenolol salicylate, which assumes a conformation stabilized by two intramolecular H-bonds, avoiding its further hydrolysis to salicylic acid and atenolol.
Assuntos
Anti-Hipertensivos/farmacocinética , Aspirina/química , Atenolol/farmacocinética , Animais , Anti-Hipertensivos/química , Anti-Hipertensivos/metabolismo , Área Sob a Curva , Atenolol/química , Atenolol/metabolismo , Mucosa Gástrica/efeitos dos fármacos , Meia-Vida , Humanos , Micro-Ondas , Modelos Moleculares , Testes de Mutagenicidade , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacocinéticaRESUMO
We explored the unique substrate specificity of the primary S, subsite of human urinary kallikrein (hK1), which accepts both Phe or Arg synthesizing and assaying peptides derived from Phenylacetyl-Phe-Ser-Arg-EDDnp, a previously described inhibitor with analgesic and anti-inflammatory activities [Emim et al., Br. J. Pharmacol. 130 (2000), 1099-1107]. Phe was substituted by amino acids containing larger aliphatic or aromatic side chains as well as by non-natural basic amino acids, which were designed to combine a large hydrophobic and/or aromatic group with a positively-charged group at their side chains. In general, all peptides with basic amino acids represented better inhibitors than those with hydrophobic amino acids. Furthermore, the S1 subsite specificity proved to be much more selective than the mere distinction between Phe and Arg, for minor differences in the side chains of the non-natural amino acids resulted in major differences in the Ki values. Finally, we present a series of peptides that were assayed as competitive inhibitors for human tissue kallikrein that may lead to the development of novel peptides, which are both more potent and selective.