RESUMO
Diarrhea is one of the most important causes of mortality in the developing world, being responsible for 2.5 million deaths each year. Many of these deaths are caused by enterotoxigenic strains of bacteria, like Escherichia coli, that produce enterotoxins that cause acute watery diarrhea, commonly defined as secretory diarrhea. Studies on symptomatic patients indicate a high prevalence of enterotoxigenic E. coli strains producing the heat-stable toxin, STa. STa is a small, cysteine-rich peptide that binds to the extracellular receptor domain of guanylyl cyclase C (GCC), located at the luminal membrane of intestinal epithelial cells. GCC and its endogenous peptide ligands, guanylin and uroguanylin, play a key role in balancing water absorption and hydration of the intestinal lumen, as exemplified by the finding that loss of GCC function causes severe dehydration of the intestinal lumen, culminating in intestinal obstruction. From a mechanistic viewpoint, reduction of GCC activity offers an efficient approach to limit enterotoxigenic E. coli- provoked secretory diarrhea. Inhibition of GCC-mediated cGMP production would not only reduce anion secretion, but would also restore NHE3 activity, resulting in a comprehensive antidiarrheal action. In the present study, two novel phenylpyrimidinone derivatives were simultaneously synthesized and tested for their ability to block STa-induced CFTR activity in T84 cells.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Inibidores Enzimáticos/farmacologia , Pirimidinonas/farmacologia , Receptores de Enterotoxina/antagonistas & inibidores , Toxinas Bacterianas/metabolismo , Linhagem Celular , Diarreia/microbiologia , Diarreia/prevenção & controle , Enterotoxinas/metabolismo , Enterotoxinas/toxicidade , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Proteínas de Escherichia coli/metabolismo , Hormônios Gastrointestinais/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Peptídeos Natriuréticos/metabolismo , Pirimidinonas/síntese química , Pirimidinonas/química , Receptores de Enterotoxina/metabolismoRESUMO
A series of 4-substituted piperazine derivatives bearing a norbornene nucleus have been prepared and their affinity for serotonin 5-HT1A, 5-HT2A and 5-HT2C receptors has been evaluated. Compounds showing the highest affinity have been selected and evaluated on dopaminergic (D1 and D2) and adrenergic (alpha1 and alpha2) receptors. The combination of structural elements (heterocyclic nucleus, oxyalkyl chain and 4-substituted piperazine) known to be critical in order to have affinity on serotonin receptors and the proper selection of substituents led to compounds with higher receptor specificity and affinity. In binding studies, several molecules showed affinity in nanomolar range towards 5-HT1A, 5-HT2A and 5-HT2C receptors and moderate to no affinity for other relevant receptors (D1, D2, alpha1 and alpha2). Compound 2q 4-[2-[4-(3,4-dichlorophenyl)piperazin-1-yl]ethoxy]-4-aza-tricyclo[5.2.1.02,6]dec-8-ene-3,5-dione (Ki = 1.13 nM), was the most active and selective derivative for the 5-HT2C receptor with respect to other serotonin, dopaminergic and adrenergic receptors. Moreover, compound 3p showed mixed 5-HT2A/5-HT2C activity with affinity values in nanomolar range.
Assuntos
Norbornanos/síntese química , Norbornanos/farmacologia , Receptor 5-HT1A de Serotonina/efeitos dos fármacos , Receptor 5-HT2A de Serotonina/efeitos dos fármacos , Receptor 5-HT2C de Serotonina/efeitos dos fármacos , Serotoninérgicos/síntese química , Serotoninérgicos/farmacologia , Animais , Química Encefálica/efeitos dos fármacos , Ligantes , Espectroscopia de Ressonância Magnética , Masculino , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptor 5-HT1A de Serotonina/química , Receptor 5-HT2A de Serotonina/química , Receptor 5-HT2C de Serotonina/química , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/metabolismo , Receptores de Dopamina D1/química , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismoRESUMO
Several studies have been published on discovering involvement of PARs receptors in a number of disease states, including cancer and inflammation of the cardiovascular, respiratory, musculoskeletal, gastrointestinal and nervous systems. This mini-review will focus on recent advances in the synthesis of PAR ligands highlighting their therapeutic potential in the treatment of various inflammatory diseases.
Assuntos
Inflamação/tratamento farmacológico , Inflamação/metabolismo , Compostos Orgânicos/síntese química , Compostos Orgânicos/farmacologia , Receptores Ativados por Proteinase/agonistas , Receptores Ativados por Proteinase/antagonistas & inibidores , Animais , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Materiais Biomiméticos/uso terapêutico , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Compostos Heterocíclicos/uso terapêutico , Humanos , Ligantes , Compostos Orgânicos/química , Compostos Orgânicos/uso terapêutico , Receptores Ativados por Proteinase/metabolismoRESUMO
The interest in the microwave assisted organic synthesis has been growing during the recent years. It results from an increasing knowledge of fundamentals of the dielectric heating theory, availability of an equipment designed especially for the laboratory use as well as the discovery of the special techniques of the microwave syntheses. The efficiency of microwave flash-heating chemistry in dramatically reducing reaction times (reduced from days and hours to minutes and seconds) has recently been proven in several different fields of organic chemistry and this aspect is of great importance in high-speed combinatorial and medicinal chemistry. In this contribution, the current state of the art is summarized providing examples of the most recent applications in the field of microwave assisted synthesis of biologically active compounds both in heterocyclic and in peptide and peptidomimetic optimization.
Assuntos
Descoberta de Drogas/métodos , Micro-Ondas , Química Farmacêutica/métodos , Técnicas de Química Combinatória/métodos , Descoberta de Drogas/instrumentação , Compostos Heterocíclicos/síntese química , Compostos Heterocíclicos/química , Modelos Químicos , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/químicaRESUMO
Galanthamine is an alkaloid approved for the treatment of Alzheimer's disease. In this paper the syntheses and the anticholinesterase activities of new glucosyl and nitroxy derivatives substituted on position 6 are reported. Compounds 2, 3 and 5 presented a percentage of inhibition of 35.22%, 47.48% and 67.89% respectively.
Assuntos
Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/farmacologia , Galantamina/análogos & derivados , Galantamina/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Cromatografia Líquida de Alta Pressão , Galantamina/síntese química , Técnicas In Vitro , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Masculino , Conformação Molecular , Ratos , Ratos Sprague-Dawley , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND AND PURPOSE: Mesalamine is the first-line therapy for colitis, but it lacks potency and is only effective for mild-to-moderate forms of this disease. Hydrogen sulphide has been shown to be a potent, endogenous anti-inflammatory substance, modulating leukocyte-endothelial adhesion and leukocyte migration. The purpose of this study was to determine if an H(2)S-releasing derivative of mesalamine (ATB-429) would exhibit increased potency and effectiveness in a mouse model of colitis. EXPERIMENTAL APPROACH: Colitis was induced in mice with trinitrobenzene sulphonic acid and the effects of ATB-429 and mesalamine were compared in several treatment regimens. The severity of colitis was determined using several indices, including a disease activity score (comprised of scores for diarrhea, weight loss and fecal blood), colonic myeloperoxidase activity and macroscopic/microscopic scoring of tissue injury. KEY RESULTS: Irrespective of the treatment regiment, ATB-429 was more effective than mesalamine in reducing the severity of colitis. ATB-429 was particularly effective in reducing granulocyte infiltration into the colonic tissue (by approximately 70%), as well as reducing the expression of mRNA for several key proinflammatory cytokines/chemokines (e.g., TNFalpha, IFNgamma). Treatment with ADT-OH, the H(2)S-releasing moiety of ATB-429, did not affect severity of colitis. CONCLUSIONS AND IMPLICATIONS: ATB-429 exhibits a marked increase in anti-inflammatory activity and potency in a murine model of colitis, as compared to mesalamine. These results are consistent with recently described anti-inflammatory effects of H(2)S. ATB-429 may represent an attractive alternative to mesalamine for the treatment of inflammatory bowel disease.
Assuntos
Anti-Inflamatórios/farmacologia , Colite/prevenção & controle , Colo/efeitos dos fármacos , Dissulfetos/farmacologia , Fármacos Gastrointestinais/farmacologia , Sulfeto de Hidrogênio/metabolismo , Mesalamina/farmacologia , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/uso terapêutico , Quimiocinas/genética , Quimiocinas/metabolismo , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/metabolismo , Colite/patologia , Colo/metabolismo , Colo/patologia , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Dissulfetos/metabolismo , Dissulfetos/uso terapêutico , Relação Dose-Resposta a Droga , Feminino , Fármacos Gastrointestinais/metabolismo , Fármacos Gastrointestinais/uso terapêutico , Expressão Gênica/efeitos dos fármacos , Granulócitos/efeitos dos fármacos , Granulócitos/patologia , Mesalamina/metabolismo , Mesalamina/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/metabolismo , Índice de Gravidade de Doença , Fatores de Tempo , Ácido TrinitrobenzenossulfônicoRESUMO
A recombinant dengue 2 virus NS2B-NS3 protease (NS means non-structural virus protein) was compared with human furin for the capacity to process short peptide substrates corresponding to seven native substrate cleavage sites in the dengue viral polyprotein. Using fluorescence resonance energy transfer peptides to measure kinetics, the processing of these substrates was found to be selective for the Dengue protease. Substrates containing two or three basic amino acids (Arg or Lys) in tandem were found to be the best, with Abz-AKRRSQ-EDDnp being the most efficiently cleaved. The hydrolysis of dipeptide substrates Bz-X-Arg-MCA where X is a non-natural basic amino acid were also kinetically examined, the best substrates containing aliphatic basic amino acids. Our results indicated that proteolytic processing by dengue NS3 protease, tethered to its activating NS2B co-factor, was strongly inhibited by Ca2+ and kosmotropic salts of the Hofmeister's series, and significantly influenced by substrate modifications between S4 and S6'. Incorporation of basic non-natural amino acids in short peptide substrates had significant but differential effects on Km and k(cat), suggesting that further dissection of their influences on substrate affinity might enable the development of effective dengue protease inhibitors.
Assuntos
Aminoácidos Básicos/química , Corantes Fluorescentes/química , Serina Endopeptidases/química , Proteínas não Estruturais Virais/química , Sítios de Ligação , Ativação Enzimática , Transferência Ressonante de Energia de Fluorescência , Furina/química , Concentração de Íons de Hidrogênio , Hidrólise , Modelos Moleculares , Oligopeptídeos/química , Proteínas Recombinantes/química , Sais/química , Especificidade por SubstratoRESUMO
OBJECTIVE: To compare the bioavailability of two potassic losartan immediate release tablet (50 mg) formulations (Losartan from Laboratórios Cristália Ltd., Brazil, as a test formulation and Cozaar from Merck Sharp & Dohme Farmacêutica Ltd., Brazil, as a reference formulation) in 25 volunteers of both sexes. MATERIAL AND METHODS: The study was conducted in an open, randomized, 2-period crossover design and a 1-week washout period. Plasma samples were obtained over a 24-hour interval. The concentrations of losartan and its active metabolite losartan acid were analyzed by combined reversed phase liquid chromatography and tandem mass spectrometry (LC-MS-MS) with negative ion electrospray ionization using a selected ion monitoring method. From the losartan and losartan acid plasma concentrations vs. time curves the following pharmacokinetic parameters were obtained: AUClast, AUC0-inf and Cmax. RESULTS: The geometric mean and respective 90% confidence interval (CI) of Losartan/Cozaar losartan percent ratios were 92.9% (82.2-105.0%) for Cmax, 99.0% (92.5-105.9%) for AUClast, and 99.1% (92.7-105.8%) for AUC0-inf. Furthermore, the geometric mean and respective 90% CI of Losartan/Cozaar losartan acid percent ratios were 98.5% (91.5-106.0%) for Cmax, 97.9% (93.3 102.7%) for AUClast, and 98.1% (93.6-102.9%) for AUC0-inf. CONCLUSION: Since the 90% CI for Cmax, AUClast and AUC0-inf were within the 80-125% interval proposed by the US Food and Drug Administration, it was concluded that the potassic losartan immediate release 50 mg tablet was bioequivalent to the Cozaar immediate release 50 mg tablet, according to both the rate and extent of absorption. While there were no significant differences in the bioequivalence assessed by either losartan or losartan acid, future bioequivalence studies on losartan may be performed by quantifying losartan alone as the parent compounds are more discriminative.
Assuntos
Anti-Hipertensivos/farmacocinética , Losartan/farmacocinética , Adulto , Anti-Hipertensivos/sangue , Área Sob a Curva , Disponibilidade Biológica , Química Farmacêutica , Cromatografia Líquida de Alta Pressão , Estudos Cross-Over , Feminino , Meia-Vida , Humanos , Losartan/sangue , Masculino , Espectrometria de Massas , Equivalência TerapêuticaRESUMO
In this study, the mutagenicity of the anti-inflammatory parsalmide [5-amino-N-butyl-2-(2-propynyloxy)-benzamide] analogues PA7 [5-amino-N-butyl-2-cyclohexyloxy-benzamide], PA10 [5-amino-N-butyl-2-phenoxy-benzamide] and PA31 [5-amino-N-butyl-2-(p-tolyloxy)-benzamide] was determined by an Ames Salmonella assay. The experiments were performed by preincubating the compounds in the absence and presence of a post-mitochondrial fraction (S9) of rat liver homogenate from phenobarbital/beta-naphtoflavone treated rats. No mutagenic effect was observed after direct testing (no S9 added) in Salmonella typhymurium strains TA98, TA100, TA102, TA1535 and TA1537. However, in the presence of S9, the test substances triggered mutagenic responses in strains TA100 and TA98. PA31 presented the strongest mutagenic potential. The reversion rates in the presence of PA31 were about 2-19 fold higher than spontaneous mutation rates. In the presence of PA7, the reversion increased 2-14-fold over spontaneous rates. While PA10 showed a relatively mild mutagenic potential, as the number of revertants did not exceed 2.5 times the number of spontaneous mutations. Mass spectrometric analysis of the in vitro biotransformation showed that S9 converted (%), regioselectively, PA7 (19%), PA10 (7%) and PA31 (12%) into hydroxy-derivatives.
Assuntos
Benzamidas/química , Benzamidas/farmacologia , Mutagênicos/química , Mutagênicos/farmacologia , Animais , Anti-Inflamatórios , Benzamidas/síntese química , Biotransformação , Hidroxilação , Mitocôndrias Hepáticas/enzimologia , Estrutura Molecular , Testes de Mutagenicidade , Mutagênicos/síntese química , RatosRESUMO
Serotonin is a neuromediator, well-know for its implication in mood regulation, anxiety, depression and, insomnia as well as in normal human function such as sleep, sexual activity and appetite. In this way, serotonin (5-hydroxytryptamine, 5-HT) is one of the most attractive targets for medicinal chemists and pharmaceutical companies. Among 5-HTRs, the 5-HT1A subtype is the best studied, and it is generally accepted that it is involved in psychiatric disorders such as anxiety and depression. Several structurally different compounds are known to bind 5-HT1A receptor sites such as aminotetralins, ergolines, arylpiperazines, indolylalkylamines, aporphines and aryloxyalkyl-amines. In this review, we report an overview of the 5-HT1A receptor ligands, belonging to different chemical classes.
Assuntos
Receptor 5-HT1A de Serotonina/metabolismo , Serotoninérgicos/metabolismo , Ligantes , Receptor 5-HT1A de Serotonina/efeitos dos fármacos , Serotoninérgicos/química , Serotoninérgicos/classificação , Serotoninérgicos/farmacologiaAssuntos
Isquemia Encefálica/fisiopatologia , Cálcio/metabolismo , Hipóxia Encefálica/fisiopatologia , Fármacos Neuroprotetores , Oligomicinas/farmacologia , Trocador de Sódio e Cálcio/fisiologia , Sódio/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Bepridil/farmacologia , Transporte Biológico/efeitos dos fármacos , Desoxiglucose/farmacologia , Glioma , Glicólise/efeitos dos fármacos , Glicosilação , Humanos , L-Lactato Desidrogenase/análise , Modelos Neurológicos , Dados de Sequência Molecular , Peptídeos/farmacologia , Fosforilação , Trocador de Sódio e Cálcio/química , Células Tumorais CultivadasRESUMO
Heterocyclic compounds hold a special place among pharmaceutically important natural and synthetic materials. The remarkable ability of heterocyclic nuclei to serve both as biomimetics and reactive pharmacophores has largely contributed to their unique value as traditional key elements of numerous drugs. In both lead identification and lead optimization processes there is an acute need for new organic small molecules. Traditional methods of organic synthesis are orders of magnitude too slow to satisfy the demand for these compounds. The fields of combinatorial and automated medicinal chemistry have been developed to meet the increasing requirement of new compounds for drug discovery, within these fields, speed is of the essence. The efficiency of microwave flash-heating chemistry in dramatically reducing reaction times (reduced from days and hours to minutes and seconds) has recently been proven in several different fields of organic chemistry. We believe that the time saved by using focused microwaves is potentially important in traditional organic synthesis but could be of even greater importance in high-speed combinatorial and medicinal chemistry. In this review, it is impossible to cover all significant developments in the area of microwave-assisted organic synthesis (MAOS). Rather, outlines the basic principles behind the technology and summarizes the areas in which microwave technology has made an impact, to date. Specific attention is given to application of microwave irradiation in liquid systems, and in the solid state as well of several representative biologically interesting nuclei. In addition we report some of the most recently disclosed applications in combinatorial chemistry.
Assuntos
Técnicas de Química Combinatória/métodos , Compostos Heterocíclicos/síntese química , Micro-Ondas , Calefação/métodosRESUMO
Parsalmide (5-amino-N-butyl-2-(2-propynyloxy) benzamide) (5a), is a non-steroidal anti-inflammatory drug (NSAID), commercialised in Italy until 1985 with the brand name of Synovial(R), that has been widely used to treat arthritic patient. In addition, it was shown to spare gastric mucosa. Here we have synthesised a series of novel substituted benzamides, related to Parsalmide, and have evaluated their activity in vitro on COX-1 and COX-2 as well as in vivo in the carrageenin-induced rat paw edema, a classical in vivo anti-inflammatory assay. Compounds 5b, 11a and 11b, which showed a favourable profile in vitro and in vivo, were screened in comparison with Parsalmide for gastrointestinal (GI) tolerability in vivo in the rat. Results obtained showed that Parsalmide and compound 11b inhibited both COX-1 and COX-2 in vitro as well as they were active in vivo. Both compounds were devoid of gastric effect at the efficacious dose. In addition, both prevented indomethacin-induced gastric damage. Thus, these compounds may guide the definition of a new leading structure with anti-inflammatory activity that may allow designing new safer NSAIDs.
Assuntos
Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacologia , Benzamidas/síntese química , Benzamidas/farmacologia , Inibidores de Ciclo-Oxigenase/síntese química , Isoenzimas/antagonistas & inibidores , Estômago/efeitos dos fármacos , Animais , Anti-Inflamatórios/efeitos adversos , Anti-Inflamatórios/química , Benzamidas/efeitos adversos , Benzamidas/química , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/efeitos adversos , Inibidores de Ciclo-Oxigenase/química , Inibidores de Ciclo-Oxigenase/farmacologia , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Edema/induzido quimicamente , Edema/tratamento farmacológico , Privação de Alimentos , Concentração Inibidora 50 , Isoenzimas/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Proteínas de Membrana , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Ratos Wistar , Ovinos , Estômago/patologia , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The change of selectivity and the induction of antagonism by the insertion of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) in the second position of several opioid peptides have led to the interpretation of Tyr-Tic as a specific message domain for delta-opioid antagonists and to the discovery of dipeptides with substantial opioid activity. Selectivity and activity increase enormously when Tyr is substituted by 2',6'-dimethyl tyrosine (Dmt), hinting that the side chain of Dmt fits a hydrophobic cavity of the receptor very tightly and precisely. We have investigated the specificity of this fit by systematic changes of the substituents on the aromatic ring of ryr. Mono- and disubstitutions different from 2',6'- invariably lead to catastrophic decreases of activity. The only substitution compatible with retention of substantial antagonism is 2-methyl. An analysis of the conformational properties of all analogues reveals that substitutions do not affect the global shape of the molecule significantly. Accordingly, it is possible to use the shape of the different side chains to map the hydrophobic cavity of the receptor. The resulting complementary image is funnel shaped.
Assuntos
Encéfalo/metabolismo , Dipeptídeos/metabolismo , Isoquinolinas/química , Naltrexona/análogos & derivados , Antagonistas de Entorpecentes/química , Antagonistas de Entorpecentes/metabolismo , Tetra-Hidroisoquinolinas , Tirosina/análogos & derivados , Tirosina/metabolismo , Substituição de Aminoácidos/fisiologia , Animais , Sítios de Ligação/fisiologia , Ligação Competitiva/fisiologia , Conformação Molecular , Naltrexona/metabolismo , Antagonistas de Entorpecentes/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Opioides delta/antagonistas & inibidores , Termodinâmica , Tirosina/químicaAssuntos
Pressão Sanguínea/efeitos dos fármacos , Oligopeptídeos/farmacologia , Receptores de Trombina/metabolismo , Animais , Clorisondamina/farmacologia , Bloqueadores Ganglionares/farmacologia , Hipotensão/induzido quimicamente , Modelos Biológicos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/farmacologia , Fragmentos de Peptídeos/farmacologia , Ratos , Receptor PAR-1 , Receptor PAR-2RESUMO
SV-IV is a basic, thermostable, secretory protein of low Mr (9758) that is synthesized by rat seminal vesicle (SV) epithelium under strict androgen transcriptional control. This protein is of obvious pharmacological interest because it has potent nonspecies-specific immunomodulatory, anti-inflammatory, and pro-coagulant activities. In evaluating the clinical relevance and the possible use in medicine of SV-IV, we became interested in the study of its structure-function relationships and aimed to identify in its polypeptide chain specific peptide fragments possessing the marked anti-inflammatory properties of the protein not associated with other biological activities (pro-coagulation and immunomodulation) typical of this molecule. By using two different experimental approaches (the fragmentation of the protein into peptide derivatives by chemical methods and the organic synthesis on solid phase of selected peptide fragments), data were obtained showing that in this protein: (a) the immunomodulatory activity is related to the structural integrity of the whole molecule; (b) the anti-inflammatory activity is located in the N-terminal region of the molecule, the 8-16 peptide fragment being the most active; (c) the identified anti-inflammatory peptide derivatives do not seem to possess pro-coagulant activity, even though this particular function has been located in the 1-70 segment of the molecule.
Assuntos
Anti-Inflamatórios/síntese química , Fragmentos de Peptídeos/síntese química , Proteínas/química , Proteínas Secretadas pela Vesícula Seminal , Sequência de Aminoácidos , Animais , Anti-Inflamatórios/química , Coagulantes/síntese química , Coagulantes/química , Brometo de Cianogênio/química , Imunossupressores/síntese química , Imunossupressores/química , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Ratos , Ratos WistarRESUMO
We synthesized one series of fluorogenic substrates for cathepsin B derived from the peptide Bz-F-R-MCA (Bz=benzoyl, MCA=7-methyl-coumarin amide) substituting Phe at the P(2) position by non-natural basic amino acids that combine a positively charged group with aromatic or aliphatic radicals at the same side chain, namely, 4-aminomethyl-phenylalanine, 4-guanidine-phenylalanine, 4-aminomethyl-N-isopropyl-phenylalanine, 3-pyridyl-alanine, 4-piperidinyl-alanine, 4-aminomethyl-cyclohexyl-alanine, 4-aminocyclohexyl-alanine, and N(im)-dimethyl-histidine. Bz-F-R-MCA was the best substrate for cathepsin B but also hydrolyzed Bz-R-R-MCA with lower efficiency, since the protease accepts Arg at S(2) due to the presence of Glu(245) at the bottom of this subsite. The presence of the basic non-natural amino acids at the P(2) position of the substrate partially restored the catalytic efficiency of cathepsin B. All the kinetic parameters for hydrolysis of the peptides described in this paper are in accordance with the structures of the S(2) pocket previously described. In addition, the substrate with 4-aminocyclohexyl-alanine presented the highest affinity to cathepsin B although the peptide was obtained from a mixture of cis/trans isomers of the amino acid and we were not able to separate them. For comparison all the obtained substrates were assayed with cathepsin L and papain.
Assuntos
Diamino Aminoácidos/síntese química , Catepsina B/química , Endopeptidases , Corantes Fluorescentes/síntese química , Peptídeos/síntese química , Diamino Aminoácidos/química , Catepsina L , Catepsinas/química , Cisteína Endopeptidases , Desenho de Fármacos , Humanos , Hidrólise , Cinética , Estrutura Molecular , Papaína/química , Peptídeos/químicaRESUMO
Peptide T (ASTTTNYT), a fragment corresponding to residues 185-192 of gp120, the coat protein of HIV, is endowed with several biological properties in vitro, notably inhibition of the binding of both isolated gp120 and HIV-1 to the CD4 receptor, and chemotactic activity. Based on previous nuclear magnetic resonance (NMR) studies performed in our laboratory, which were consistent with a regular conformation of the C-terminal pentapeptide, and SAR studies showing that the C-terminal pentapeptide retains most of the biological properties, we designed eight hexapeptides containing in the central part either the TNYT or the TTNY sequence, and charged residues (D/E/R) at the two ends. Conformational analysis based on NMR and torsion angle dynamics showed that all peptides assume folded conformations. albeit with different geometries and stabilities. In particular, peptides carrying an acidic residue at the N-terminus and a basic residue at the C-terminus are characterized by stable helical structures and retain full chemotactic activity. The solution conformation of peptide ETNYTR displays strong structural similarity to the region 19-26 of both bovine pancreatic and bovine seminal ribonuclease, which are endowed with anti-HIV activity. Moreover, the frequent occurrence, in many viral proteins, of TNYT and TTNY, the two core sequences employed in the design of the hexapeptides studied in the present work, hints that the sequence of the C-terminal pentapeptide TTNYT is probably representative of a widespread viral recognition motif.
Assuntos
Fármacos Anti-HIV/química , Endorribonucleases/química , Epitopos/química , Oligopeptídeos/síntese química , Peptídeo T/química , Ribonuclease Pancreático/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Sítios de Ligação/fisiologia , Antígenos CD4/química , Antígenos CD4/metabolismo , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , Desenho de Fármacos , Estabilidade de Medicamentos , Endorribonucleases/farmacologia , HIV/efeitos dos fármacos , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Conformação Molecular , Mimetismo Molecular/fisiologia , Monócitos/citologia , Monócitos/metabolismo , Oligopeptídeos/farmacologia , Peptídeo T/análogos & derivados , Peptídeo T/farmacologia , Ribonuclease Pancreático/farmacologiaRESUMO
We explored the unique substrate specificity of the primary S(1) subsite of human urinary kallikrein (hK1), which accepts both Phe and Arg, using internally quenched fluorescent peptides Abz-F-X-S-R-Q-EDDnp and Abz-G-F-S-P-F-X-S-S-R-P-Q-EDDnp [Abz is o-aminobenzoic acid; EDDnp is N-(2,4-dinitrophenyl)ethylenediamine], which were based on the human kininogen sequence at the C-terminal region of bradykinin. Position X, which in natural sequence stands for Arg, received the following synthetic basic non-natural amino acids: 4-(aminomethyl)phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4-(aminomethyl)-N-isopropylphenylalanine (Iaf), N(im)-(dimethyl)histidine [H(2Me)], 3-pyridylalanine (Pya), 4-piperidinylalanine (Ppa), 4-(aminomethyl)cyclohexylalanine (Ama), and 4-(aminocyclohexyl)alanine (Aca). Only Abz-F-Amf-S-R-Q-EDDnp and Abz-F-H(2Me)]-S-R-Q-EDDnp were efficiently hydrolyzed, and all others were resistant to hydrolysis. However, Abz-F-Ama-S-R-Q-EDDnp inhibited hK1 with a K(i) of 50 nM with high specificity compared to human plasma kallikrein, thrombin, plasmin, and trypsin. The Abz-G-F-S-P-F-X-S-S-R-P-Q-EDDnp series were more susceptible to hK1, although the peptides with Gnf, Pya, and Ama were resistant to it. Unexpectedly, the peptides in which X is His, Lys, H(2Me), Amf, Iaf, Ppa, and Aca were cleaved at amino or at carboxyl sites of these amino acids, indicating that the S(1)' subsite has significant preference for basic residues. Human plasma kallikrein did not hydrolyze any peptide of this series except the natural sequence where X is Arg. In conclusion, the S(1) subsite of hK1 accepts amino acids with combined basic and aromatic side chain, although for the S(1)-P(1) interaction the preference is for aliphatic and basic side chains.
Assuntos
Substituição de Aminoácidos , Aminoácidos/síntese química , Aminoácidos/metabolismo , Calicreínas Teciduais/metabolismo , Sequência de Aminoácidos , Arginina/análogos & derivados , Arginina/síntese química , Arginina/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Histidina/análogos & derivados , Histidina/síntese química , Histidina/metabolismo , Humanos , Hidrólise , Calicreínas/antagonistas & inibidores , Calicreínas/sangue , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/síntese química , Fenilalanina/metabolismo , Especificidade por Substrato , Tripsina/metabolismoRESUMO
We have explored the substrate specificity of a recombinant cysteine proteinase of Leishmania mexicana (CPB2.8 Delta CTE) in order to obtain data that will enable us to design specific inhibitors of the enzyme. Previously we have shown that the enzyme has high activity towards substrates with a basic group at the P1 position [Hilaire, P.M.S., Alves, L.C., Sanderson, S.J., Mottram, J.C., Juliano, M.A., Juliano, L., Coombs, G.H. & Meldal M. (2000) Chem. Biochem. 1, 115--122], but we have also observed high affinity for peptides with hydrophobic residues at this position. In order to have substrates containing both features, we synthesized one series of internally quenched fluorogenic peptides derived from the sequence ortho-amino-benzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine, and substituted the Arg at the P1 position with the following non-natural basic amino acids: 4-aminomethyl-phenylalanine (Amf), 4-guanidine-phenylalanine (Gnf), 4-aminomethyl-N-isopropyl-phenylalanine (Iaf), 3-pyridyl-alanine (Pya), 4-piperidinyl-alanine (Ppa), 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine (Aca). For comparison, the series derived from ortho-amino-benzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine was also assayed with cruzain (the major cysteine proteinase of Trypanosoma cruzi), human cathepsin L and papain. The peptides ortho-amino-benzoyl-FAmfSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (k(cat)/K(m) = 12,000 mM(-1) x s(-1)) and ortho-amino-benzoyl-FIafSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (k(cat)/K(m) = 27,000 mM(-1) x s(-1)) were the best substrates for CPB2.8 Delta CTE. In contrast, ortho-amino-benzoyl-FAmaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine and ortho-amino-benzoyl-FAcaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine were very resistant and inhibited this enzyme with K(i) values of 23 nM and 30 nM, respectively. Cruzain hydrolyzed quite well the substrates in this series with Amf, Ppa and Aca, whereas the peptide with Ama was resistant and inhibited cruzain with a K(i) of 40 nM. Human cathepsin L presented an activity on these peptides very similar to that of CPB2.8 Delta CTE and papain hydrolyzed all the peptides with high efficiency. In conclusion, we have demonstrated that CPB2.8 Delta CTE has more restricted specificity at the S1 subsite and it seems possible to design efficient inhibitors with amino acids such as Ama or Aca at the P(1) position.