RESUMO
Native rat atrial natriuretic peptide (NANP) was shown to bind with high affinity and to increase intracellular levels of cGMP in cultured rat Leydig tumor cells. A linear analog of NANP which lacks the disulfide-linked bridge structure also bound with high affinity but did not increase levels of intracellular cGMP or antagonize the increase of this cyclic nucleotide by NANP. These data are consistent with the existence of two functional subpopulations of ANP receptors on cultured rat Leydig tumor cells; one which is capable of activating guanylate cyclase and one which is not linked to this enzyme.
Assuntos
Tumor de Células de Leydig/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Fator Natriurético Atrial/análogos & derivados , Fator Natriurético Atrial/metabolismo , Fator Natriurético Atrial/farmacologia , GMP Cíclico/biossíntese , Dissulfetos , Ativação Enzimática , Guanilato Ciclase/metabolismo , Masculino , Dados de Sequência Molecular , Ratos , Receptores do Fator Natriurético Atrial , Células Tumorais CultivadasRESUMO
A series of highly potent, structurally novel, non-nucleoside RT inhibitors has been described. Low nanomolar concentrations of 5-chloro-3-(phenylsulfonyl)-indole-2-carboxamide (1) inhibit the HIV-1 RT enzyme in vitro and HTLVIIIb viral spread in MT-4 human T-lymphoid cells. Good oral bioavailability was observed in rhesus monkeys upon oral dosing of 1 as a suspension in methocel. When compared to other non-nucleoside inhibitors (e.g. 15-18), 1 possesses improved inhibitory potency with respect to the wild-type RT, as well as the K103N and Y181C mutant enzymes. Additional studies within this class of inhibitors are in progress.
Assuntos
Antivirais/farmacologia , HIV-1/enzimologia , Indóis/farmacologia , Inibidores da Transcriptase Reversa , Sulfóxidos/farmacologia , Animais , Antivirais/química , Sequência de Bases , Disponibilidade Biológica , HIV/efeitos dos fármacos , Transcriptase Reversa do HIV , Indóis/química , Indóis/farmacocinética , Macaca mulatta , Dados de Sequência Molecular , Estrutura Molecular , Sulfóxidos/química , Sulfóxidos/farmacocinéticaAssuntos
Alquil e Aril Transferases , Antineoplásicos/síntese química , Inibidores Enzimáticos/síntese química , Piperazinas/síntese química , Piperazinas/farmacologia , Transferases/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase , Camundongos , Camundongos Nus , Conformação Molecular , Estrutura Molecular , Transplante de Neoplasias , Fosfatos de Poli-Isoprenil/metabolismo , Prenilação de Proteína , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , Sarcoma Experimental/tratamento farmacológico , Sarcoma Experimental/patologia , Sesquiterpenos , Proteínas ras/metabolismoRESUMO
The actions of atrial natriuretic factor (ANF) on the vascular wall are diverse and show a profound regional heterogeneity. ANF is a potent relaxant of aortic smooth muscle, a response which is associated with activation of particulate guanylate cyclase and elevation in tissue levels of cyclic GMP. However, many large and small muscular arteries and most veins are unresponsive to the peptide. The regional vascular heterogeneity may be due to an altered distribution of high affinity receptors and (or) alterations in the coupling of receptor activation to elevations in cyclic 3',5'-guanosine monophosphate (cGMP). Species differences exist in the structural requirements for receptor activation as well as the effects of infused ANF on peripheral resistance. Although the relaxation to ANF in vitro does not require an intact endothelium, endothelial cells contain multiple receptor subtypes for ANF. Differences amongst tissues and (or) species in the receptor profile for ANF may, in part, explain some of heterogeneity in responsiveness to ANF.
Assuntos
Fator Natriurético Atrial/farmacologia , Endotélio Vascular/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Aorta/metabolismo , Fator Natriurético Atrial/metabolismo , Bovinos , Células Cultivadas , GMP Cíclico/metabolismo , Endotélio Vascular/efeitos dos fármacos , Cinética , Receptores do Fator Natriurético AtrialRESUMO
The binding of fibrinogen to its platelet receptor, the glycoprotein IIb-IIIa complex, is mediated, in part, by an Arg-Gly-Asp (RGD) sequence within the fibrinogen A alpha chain. PAC1 is an IgM-kappa murine monoclonal antibody that binds to the platelet fibrinogen receptor, and its binding is inhibited by both fibrinogen and RGD-containing peptides. To identify the regions of PAC1 that interact with the fibrinogen receptor, we determined the mRNA sequences of PAC1 immunoglobulin heavy and light chain variable regions. Five out of the six complementarity-determining regions (CDRs) of PAC1 had entirely germline sequences with no regions of similarity to fibrinogen. However, CDR3 of the PAC1 heavy chain (H-CDR3) was very large and unique due to the insertion of a novel D region segment. H-CDR3 contained a sequence, Arg-Tyr-Asp (RYD), that, if present in the proper conformation, might behave like the RGD sequence in fibrinogen. A 21-residue synthetic peptide encompassing the H-CDR3 region inhibited fibrinogen-dependent platelet aggregation as well as the binding of PAC1 (Ki = 10 microM) and fibrinogen (Ki = 5 microM) to activated platelets. The RYD region of H-CDR3 appeared to be central to its function, because substitution of the tyrosine with glycine increased the inhibitory potency of the peptide by 10-fold, while replacing the tyrosine with D-alanine or inverting the RYD sequence sharply reduced the inhibitory potency. Thus, the linear sequence, RYD, within H-CDR3 of PAC1 appears to mimic the RGD receptor recognition sequence in fibrinogen. This type of immunologic approach could be useful in studying the structural basis of other receptor-ligand interactions.
Assuntos
Anticorpos Monoclonais , Fibrinogênio/genética , Glicoproteínas da Membrana de Plaquetas/metabolismo , Sequência de Aminoácidos , Complexo Antígeno-Anticorpo , Sequência de Bases , Plaquetas/metabolismo , Fibrinogênio/imunologia , Fibrinogênio/metabolismo , Humanos , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Glicoproteínas da Membrana de Plaquetas/imunologiaRESUMO
The mature proteins of retroviruses originate as a result of proteolytic cleavages of polyprotein precursors. Retroviruses encode proteases responsible for several of these processing events, making them potential antiviral drug targets. A 99-amino acid HIV-1 protease, produced by chemical synthesis or by expression in bacteria, is shown here to hydrolyze peptides corresponding to all of the known cleavage sites in the HIV-1 gag and pol polyproteins. It does not hydrolyze peptides corresponding to an env cleavage site or a distantly related retroviral gag cleavage site.
Assuntos
HIV-1/enzimologia , Peptídeo Hidrolases/metabolismo , Proteínas dos Retroviridae/metabolismo , Sequência de Aminoácidos , Antígenos Virais , Produtos do Gene gag , HIV-1/genética , Hidrólise , Cinética , Peptídeo Hidrolases/síntese química , Peptídeo Hidrolases/genética , Especificidade por SubstratoRESUMO
Retroviral proteins, including those from the human immunodeficiency virus (HIV), are synthesized as polyprotein precursors that require proteolytic cleavage to yield the mature viral proteins. A 99-residue polypeptide, encoded by the 5' end of the pol gene, has been proposed as the processing protease of HIV. The chemical synthesis of the 99-residue peptide was carried out by the solid-phase method, and the isolated product was found to exhibit specific proteolytic activity upon folding under reducing conditions. Upon size-exclusion chromatography, enzymatic activity was eluted at a point consistent with a dimeric molecular size. Specificity was demonstrated by the cleavage of the natural substrate HIV gag p55 into gag p24 and gag p17, as well as cleavage of small peptide substrates representing processing sites of HIV fusion proteins. The proteolytic action of the synthetic product could be inhibited by pepstatin, an aspartic protease inhibitor.
Assuntos
Endopeptidases/síntese química , Sequência de Aminoácidos , Endopeptidases/análise , Protease de HIV , Dados de Sequência MolecularRESUMO
Imidazolemethyl diaryl ethers are potent inhibitors of farnesyl-protein transferase. The SNAr displacement reaction used to prepare these diaryl ethers was amenable to rapid parallel synthesis of FPTase inhibitors. The use of a broad range of commercially available phenols quickly identified compounds which proved active in cells.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Éteres Fenílicos/farmacologia , Alquil e Aril Transferases/metabolismo , Animais , Ligação Competitiva , Linhagem Celular , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Imidazóis/química , Concentração Inibidora 50 , Biblioteca de Peptídeos , Éteres Fenílicos/síntese química , Ratos , Relação Estrutura-AtividadeRESUMO
The design and syntheses of non-thiol inhibitors of farnesyl-protein transferase are described. Substitutions on an imidazolylmethyl-AMBA-methionine template gave a highly potent and cell-active inhibitor.