RESUMO
The cross-linked pentapeptides (2R,7R)-diaminooctanedioyl-bis(Tyr-Arg-Leu-Arg-Tyr-amide) ((2R,7R)-BVD-74D, (2R,7R)-1) and octanedioyl-bis(Tyr-Arg-Leu-Arg-Tyr-amide) (2) as well as the pentapeptide Ac-Tyr-Arg-Leu-Arg-Tyr-amide (3) were previously described as neuropeptide Y Y4 receptor (Y4R) partial agonists. Here, we report on a series of analogues of (2R,7R)-1 and 2 in which Arg2, Leu3, or Arg4 were replaced by the respective aza-amino acids. The replacement of Arg2 in 3 with a carbamoylated arginine building block and the extension of the N-terminus by an additional arginine led to the high-affinity hexapeptide Ac-Arg-Tyr-Nω-[(4-aminobutyl)aminocarbonyl]Arg-Leu-Arg-Tyr-amide (35), which was used as a precursor for a d-amino acid scan. The target compounds were investigated for Y4R functional activity in assays with complementary readouts: aequorin Ca2+ and ß-arrestin 1 or ß-arrestin 2 assays. In contrast to the parent compounds, which are Y4R agonists, several ligands were able to suppress the effect elicited by the endogenous ligand pancreatic polypeptide and therefore represent a novel class of peptide Y4R antagonists.
RESUMO
Hyaluronan (HA) is the main component of the extracellular matrix (ECM). Depending on its chain size, it is generally accepted to exert diverse effects. High molecular weight HA is anti-angiogenic, immunosuppressive and anti-inflammatory, while lower fragments are angiogenic and inflammatory. Human hyaluronidase Hyal-1 (Hyal-1) is one of the main enzymes in the metabolism of HA. This makes Hyal-1 an interesting target. Not only for functional and mechanistic studies, but also for drug development. In this work, Hyal-1 was expressed on the surface of E. coli, by applying Autodisplay, to overcome formation of inactive "inclusion bodies". With the cells displaying Hyal-1 an activity assay was performed using "stains-all" dye. Subsequently, the inhibitory effects of four saponins and 14 plant extracts on the activity of surface displayed Hyal-1 were evaluated. The determined IC50 values were 177 µM for glycyrrhizic acid, 108 µM for gypsophila saponin 2, 371 µM for SA1657 and 296 µM for SA1641. Malvae sylvestris flos, Equiseti herba and Ononidis radix extracts showed IC50 values between 1.4 and 1.7 mg/mL. In summary, Autodisplay enabled the expression of functional human target protein Hyal-1 in E. coli and facilitated an accelerated testing of potential inhibitors.
Assuntos
Antígenos de Neoplasias/metabolismo , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Histona Acetiltransferases/metabolismo , Hialuronoglucosaminidase/metabolismo , Extratos Vegetais/farmacologia , Antígenos de Neoplasias/genética , Membrana Celular/metabolismo , Inibidores Enzimáticos/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ácido Glicirrízico/farmacologia , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/genética , Humanos , Hialuronoglucosaminidase/antagonistas & inibidores , Hialuronoglucosaminidase/genética , Extratos Vegetais/química , Saponinas/farmacologiaRESUMO
The bioisosteric replacement of the acylguanidine moieties in dimeric histamine H2 receptor (H2R) agonists by carbamoylguanidine groups resulted in compounds with retained potencies and intrinsic activities, but considerably improved stability against hydrolytic cleavage. These compounds achieved up to 2500 times the potency of histamine when studied in [(35)S]GTPγS assays on recombinant human and guinea pig H2R. Unlike 3-(imidazol-4-yl)propyl substituted carbamoylguanidines, the corresponding 2-amino-4-methylthiazoles revealed selectivity over histamine receptor subtypes H1R, H3R and H4R in radioligand competition binding studies. H2R binding studies with three fluorescent compounds and one tritium-labeled ligand, synthesized from a chain-branched precursor, failed due to pronounced cellular accumulation and high non-specific binding. However, the dimeric H2R agonists proved to be useful pharmacological tools for functional studies on native cells, as demonstrated for selected compounds by cAMP accumulation and inhibition of fMLP-stimulated generation of reactive oxygen species in human monocytes.
Assuntos
Agonistas dos Receptores Histamínicos/química , Agonistas dos Receptores Histamínicos/farmacologia , Relação Estrutura-Atividade , Animais , Ligação Competitiva , Técnicas de Química Sintética , Avaliação Pré-Clínica de Medicamentos/métodos , Estabilidade de Medicamentos , Fluorescência , Guanidinas/química , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Cobaias , Agonistas dos Receptores Histamínicos/síntese química , Humanos , Ligantes , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Receptores Histamínicos H2/genética , Receptores Histamínicos H2/metabolismo , TrítioRESUMO
N1-Aryl(heteroaryl)alkyl-N2-[3-(1H-imidazol-4-yl)propyl]guanidines are potent histamine H2-receptor (H2R) agonists, but their applicability is compromised by the lack of oral bioavailability and CNS penetration. To improve pharmacokinetics, we introduced carbonyl instead of methylene adjacent to the guanidine moiety, decreasing the basicity of the novel H2R agonists by 4-5 orders of magnitude. Some acylguanidines with one phenyl ring were even more potent than their diaryl analogues. As demonstrated by HPLC-MS, the acylguanidines (bioisosteres of the alkylguanidines) were absorbed from the gut of mice and detected in brain. In GTPase assays using recombinant receptors, acylguanidines were more potent at the guinea pig than at the human H2R. At the hH1R and hH3R, the compounds were weak to moderate antagonists or partial agonists. Moreover, potent partial hH4R agonists were identified. Receptor subtype selectivity depends on the imidazolylpropylguanidine moiety (privileged structure), opening an avenue to distinct pharmacological tools including potent H4R agonists.
Assuntos
Guanidinas/síntese química , Guanidinas/farmacologia , Agonistas dos Receptores Histamínicos/química , Agonistas dos Receptores Histamínicos/farmacologia , Imidazóis/síntese química , Imidazóis/farmacologia , Receptores Histamínicos H2/metabolismo , Acilação , Animais , Linhagem Celular , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Guanidinas/química , Cobaias , Agonistas dos Receptores Histamínicos/síntese química , Humanos , Imidazóis/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Fatores de TempoRESUMO
PURPOSE: The aim of this study was to investigate the role of platelet-derived growth factor (PDGF) and PDGF receptors (PDGFRs) in the proliferation of human glioblastoma cells as a prerequisite for a new therapeutic approach to the treatment of malignant brain tumors with selective tyrosine kinase inhibitors such as imatinib. METHODS AND RESULTS: In the human glioblastoma cell lines U-87 MG, U-118 MG and U-373 MG different PDGF and PDGFR mRNAs were detected by RT-PCR, and the expression of the receptor proteins was demonstrated by immunostaining and flow cytometry. Moreover, functional activity of PDGFRs was demonstrated in PDGFRbeta expressing glioblastoma cell variants by measuring the mobilization of intracellular Ca(2+) upon PDGF-BB stimulation. However, addition of PDGF-BB to the serum-free culture medium had no stimulatory effect on cell proliferation. Furthermore, cell growth in serum-supplemented and serum-free medium was not affected by imatinib, leflunomide and AG-1296 at therapeutically relevant concentrations. CONCLUSION: Our results suggest that clinical antitumor effects of imatinib on glioblastoma, if any, are not mediated by the PDGFR.
Assuntos
Glioblastoma/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Perfilação da Expressão Gênica , Glioblastoma/tratamento farmacológico , Humanos , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , RNA Mensageiro/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Potent and specific inhibitors of hyaluronidases, a group of enzymes preferentially catalysing the hydrolysis of hyaluronic acid, are not known so far. Such compounds could be useful as pharmacological tools for studying the physiological and pathophysiological role of both hyaluronan and hyaluronidases. The effects of sulphated and non-sulphated structurally different oligosaccharides on bovine testicular hyaluronidase, hyaluronidase from bee venom and hyaluronate lyase from Streptococcus agalactiae (hylB (4755)) were studied with the Morgan-Elson reaction. Several active compounds were identified within a series of sulphated beta-(1,4)-galacto-oligosaccharides. The determined IC (50) values of these sulphated oligosaccharides ranged from 4 microM to 630 microM for all hyaluronan-degrading enzymes. Sulphated oligosaccharides like verbascose, planteose and neomycin showed comparable inhibition on all hyaluronidases, thereby possessing 100 - 500 times the activity of the widely accepted hyaluronidase inhibitor apigenin.