RESUMO
Leu- and Met-enkephalin were the first endogenous opioid peptides identified in different mammalian species including the human. Comparative biochemical and bioinformatic evidence indicates that enkephalins are not limited to mammals. Various prodynorphin (PDYN) sequences in lower vertebrates revealed the presence of other enkephalin fingerprints in these precursor polypeptides. Among the novel enkephalins Ile-enkephalin (Tyr-Gly-Gly-Phe-Ile) was primarily observed in the African clawed frog (Xenopus laevis) PDYNs, while the structure of Phe-enkephalin (Tyr-Gly-Gly-Phe-Phe) was predicted by analyzing brain cDNA sequences encoding a PDYN of the African lungfish (Protopterus annectens). Ile-enkephalin can also be found in the PDYNs of four other fish species including the eel, bichir, zebrafish and tilapia, but no further occurrence for the Phe-enkephalin motif is available as yet. Based on sequencing data, the biological relevance of Phe- and Ile-enkephalin is suggested, because both of them can arise by regular posttranslational enzymatic processing of the respective neuropeptide precursors. In various receptor binding assays performed on rat brain membrane preparations both of the new peptides turned out to be moderate affinity opioids with a weak preference for the delta-opioid receptor (DOP) sites. Phe-enkephalin of the lungfish displayed rather unexpectedly low affinities toward the mu-opioid receptor (MOP) and DOP, while exhibiting moderate affinity toward the kappa-opioid receptor (KOP). In receptor-mediated G-protein activation assays measured by the stimulation of [(35)S]GTPgammaS binding, Met-enkephalin produced the highest stimulation followed by Leu-enkephalin, Ile-enkephalin and Phe-enkephalin, whereas the least efficacious among these endogenous peptides was still more effective than the prototype opiate agonist morphine in these functional tests.
Assuntos
Anuros/genética , Encéfalo/metabolismo , Encefalinas/genética , Encefalinas/metabolismo , Peixes/genética , Analgésicos Opioides/farmacologia , Animais , Sequência de Bases , DNA Complementar/genética , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Encefalinas/química , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Peptídeos Opioides/farmacologia , Ligação Proteica/efeitos dos fármacos , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Ratos , Ratos Wistar , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Trítio/farmacologiaRESUMO
The effects of novel nontoxic tripeptide aldehyde inhibitors of proteolytic enzymes were examined in order to investigate the possibility that serine-thiol protease(s) may be involved in PRL and GH secretion. Rat anterior pituitary cells maintained in culture for 7-8 days or freshly taken pituitary quarters were treated with BOC-DPhe-Pro-Arg-H (BOC-dPPA), DPhe-Pro-Arg-H (dPPA), BOC-DPhe-Leu-Lys-H (BOC-dPLL), or BOC-DPhe-Phe-Lys (BOC-dPPL). Newly synthetized [3H]PRL and [3H]GH as well as immunoreactive (i) hormones (iPRL, iGH) were measured in the incubation media and cell homogenates. Four hours of incubation in the presence of 0.1 mM dPPA resulted in a 30% decrease of [3H]PRL and iPRL release by cell cultures; the inhibition by BOC-dPPA was 60% and 48%, respectively. [3H]PRL biosynthesis was unchanged or slightly decreased. The effect of these tripeptide aldehydes on [3H]GH and iGH release was less pronounced but statistically significant. Pituitary quarters treated with 1.0 or 3.0 mM BOC-dPPA release 20% and 57% less [3H]PRL than the controls. In the same system BOC-dPPA in a 1.0 mM concentration did not effect GH secretion, and 3.0 mM BOC-dPPA inhibited [3H]GH output by 27%. Forty micromolars of BOC-dPPL decreased by 47%, 0.2 mM by 79%, and 1.0 mM by 94% [3H]PRL release from pituitary quarters. GH secretion was not influenced. A similar selectivity was observed when BOC-dPLL was used. It is clear that by serine-thiol protease inhibitors whose effects are sequence and dose dependent, PRL and GH release are decreased. The relative inhibiting potency on PRL release was BOC-dPPL greater than BOC-dPLL greater than BOC-dPPA greater than dPPA. The biosynthesis of [3H]PRL was reduced only in the presence of the highest tripeptide aldehyde concentrations or long (8 h) exposure, and only 1.0 mM Boc-dPPL reduced [3H]GH biosynthesis by 30%. The data suggest that proteolysis may be involved in the process of PRL and GH release and the enzyme(s) in question may be serine-thiol protease(s).
Assuntos
Hormônio do Crescimento/metabolismo , Oligopeptídeos/farmacologia , Prolactina/metabolismo , Inibidores de Proteases/farmacologia , Aldeídos/farmacologia , Animais , Células Cultivadas , Técnicas In Vitro , Adeno-Hipófise/metabolismo , Ratos , Ratos EndogâmicosRESUMO
A sensitive multipoint assay capable of measuring receptors for LHRH and its analogs using 5-10 micrograms membrane protein/incubation tube was used to determine binding characteristics of different agonists and antagonists of LHRH in membranes of male rat pituitary and human breast cancer specimens. This method also permitted Scatchard analysis of the receptor binding in pellet fractions of human breast cancer biopsies remaining from estrogen and progesterone receptor assays. The potent agonist [D-Trp6]LHRH bound to at least two classes of receptor sites, one with high affinity and one with low affinity in both rat pituitary and human breast cancer samples. The analysis of displacement curves of LHRH by agonists and antagonists showed that LHRH also bound to two classes of receptor sites in pituitary and one receptor site with lower affinity in human breast cancer membranes. Among the antagonists synthesized in our laboratory, SB-030, SB-077, SB-088, and SB-090 appeared to be the most potent in displacing labeled [D-Trp6]LHRH and showed the highest binding affinity to the pituitary and breast cancer membranes. Labeled antagonists showed somewhat less affinity to membranes of pituitaries and human cancers than the agonists and bound to only a single class of receptor population. Both agonists and antagonists were able to bind to membranes of human breast cancer samples, and some antagonists were very potent in this respect. Certain LHRH agonists or antagonists could be capable of exerting direct inhibitory effects on breast cancers depending upon the presence and characteristics of LHRH receptors.
Assuntos
Neoplasias da Mama/metabolismo , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/metabolismo , Hipófise/metabolismo , Neoplasias Hipofisárias/metabolismo , Receptores LHRH/metabolismo , Animais , Ligação Competitiva , Membrana Celular/metabolismo , Feminino , Humanos , Cinética , Masculino , Ratos , Ratos Endogâmicos , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Relação Estrutura-AtividadeRESUMO
D-Phe-Pro-Arg-H sulfate (GYKI-14166) is a highly active and selective inhibitor of thrombin both in vitro and in vivo. Recent studies on the stability of D-Phe-Pro-Arg-H in neutral aqueous solution at higher temperature have revealed that it is transformed into inactive 5,6,8,9,10,10a-hexahydro-2-(3'- guanidinopropyl)-5-benzyl-6-oxo- imidazo[1,2-a]pyrrolo[2,1-c]pyrazine. No such inactivation could be observed with Boc-D-Phe-Pro-Arg-H (GYKI-14451), but this compound was far less specific than the free peptide as it inhibited thrombin and, for instance, plasmin equally well. Assuming that the transformation of free tripeptide aldehyde, mentioned above, can only be initiated by a primary amino terminus, the N-alkyl derivatives of D-Phe-Pro-Arg-H were prepared. Of the new analogues, D-MePhe-Pro-Arg-H (GYKI-14766) proved to be as highly active and selective anticoagulant as its parent compound and was not inactivated by transformation into a heterocyclic compound.
Assuntos
Anticoagulantes/síntese química , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Animais , Anticoagulantes/farmacologia , Testes de Coagulação Sanguínea , Bovinos , Cães , Fibrinólise/efeitos dos fármacos , Técnicas In Vitro , CoelhosRESUMO
A series of peptide aldehydes synthetized in our institute during the last 15 years were screened to detect their inhibitory effect on blood coagulation. Simple conventional clotting assays, platelet function tests and fibrinolytic methods were used to evaluate the inhibitory potency of the compounds in complex clotting systems as well as their supposed antifibrinolytic effect in vitro. Special attention was paid to the possible interactions with blood cells and plasma proteins, and to the functional stability of the inhibitors in several tissue homogenates. D-Phe-Pro-Arg-H (GYKI-14166, RGH-2958), Boc-D-Phe-Pro-Arg-H (GYKI-14451) and D-MePhe-Pro-Arg-H (GYKI-14766) were found to be the most potent inhibitors. The peptide aldehydes via formation of reversible complexes with thrombin impede the enzyme to react with the coagulation factors, platelet membrane and vessel wall. The compounds inhibit platelet aggregation induced by thrombin specifically without changing the sensitivity of platelets to other inducers. D-Phe-Pro-Arg-H and D-MePhe-Pro-Arg-H showed no antifibrinolytic effect. D-MePhe-Pro-Arg-H and Boc-D-Phe-Pro-Arg-H proved to be stable in dry state for years and in solution at room temperature for several days. The anticoagulant activity of the compounds was declared in NIH antithrombin units.
Assuntos
Anticoagulantes/farmacologia , Oligopeptídeos/farmacologia , Animais , Bioensaio , Testes de Coagulação Sanguínea , Cães , Avaliação Pré-Clínica de Medicamentos , Estabilidade de Medicamentos , Humanos , Inibidores da Agregação Plaquetária/farmacologia , Contagem de Plaquetas , Coelhos , Estudos Retrospectivos , Trombina/antagonistas & inibidoresRESUMO
The antithrombotic action of the highly effective synthetic thrombin inhibitor D-MePhe-Pro-Arg-H (GYKI-14766) was studied in various models of experimental thrombosis. The compound administered to rats and rabbits by i.v. bolus injections, continuous i.v. infusions, subcutaneously and orally, respectively, induced significant decrease in thrombus weight (i) in a quantitative venous thrombosis model with stasis based on vascular lesion in rats, (ii) in an extracorporeal arterio-venous shunt model in rabbits, and (iii) prevented the occlusion of the vessel in arterial thrombosis induced by mechanical damage in rats. By using the arterio-venous shunt model in rabbits the inhibitory effect on thrombus growth could be demonstrated as a function of dose and time in self-controlled experiments. Blood level of the inhibitor determined by a bioassay varied between 0.09-0.67 microgram/ml whole blood when doses of 15 and 20 mg/kg were administered orally. A correlation was found between thrombin time, platelet aggregation induced by thrombin ex vivo and the weight of thrombi formed.
Assuntos
Anticoagulantes/farmacologia , Oligopeptídeos/farmacologia , Trombose/tratamento farmacológico , Sequência de Aminoácidos , Animais , Anticoagulantes/química , Derivação Arteriovenosa Cirúrgica , Modelos Animais de Doenças , Masculino , Dados de Sequência Molecular , Oligopeptídeos/química , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Coelhos , Ratos , Tromboflebite/sangue , Tromboflebite/tratamento farmacológico , Tromboflebite/patologia , Trombose/sangue , Trombose/patologiaRESUMO
D-Phe-Pro-Arg-H and D-MePhe-Pro-Arg-H synthetized in our institute were administered to mice, rats, rabbits and beagle dogs. The kinetics of the anticoagulant and antiplatelet effect was recorded by measuring various clotting parameters, platelet count and aggregation, and evaluated as proposed by Verstraete and Verwilghen. The minimum effective doses were found to be 0.25-0.5 mg kg-1h-1 by intravenous continuous infusions and 0.5-1.0 mg/kg by single injections. The dose-dependent prolongation of clotting times appeared after application within minutes and returned to baseline values as a function of dose. Blood level of the inhibitors was determined by a bioassay. Unlike heparin, no higher starting dose was required to reach the anticoagulant threshold level, i.e. 0.03-0.1 microgram/ml whole blood. The peptides did not cause significant changes in platelet count and function or in hemodynamic parameters (blood pressure, heart rate and ECG) and in respiration. They blocked platelet aggregation induced by thrombin ex vivo specifically. No rebound effect or bleeding could be demonstrated even after subtoxic doses of the compounds. The onset of the anticoagulant and antithrombotic effect appeared within 60 min after single oral doses and lasted for 3-6 h. In close correlation with the anticoagulant effect a complete or significant inhibition of platelet aggregation induced by thrombin ex vivo could also be recorded by using 5-10 mg/kg doses.
Assuntos
Anticoagulantes/farmacologia , Oligopeptídeos/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Trombina/antagonistas & inibidores , Animais , Tempo de Sangramento , Coagulação Sanguínea/efeitos dos fármacos , Sistema Cardiovascular/efeitos dos fármacos , Cães , Infusões Intravenosas , Injeções Intramusculares , Injeções Intravenosas , Injeções Subcutâneas , Masculino , Oligopeptídeos/sangue , Contagem de Plaquetas , Coelhos , Ratos , Respiração/efeitos dos fármacosRESUMO
A number of oligopeptides, protected at their N termini and possessing an aldehyde residue at their C terminal amino acids, are able to inhibit 45Ca2+ influx into anterior pituitary cells grown in monolayer culture and depolarized with high extracellular potassium concentration. In addition, the same oligopeptides interfere with hormone release, especially with that produced by lactotrophs. Our findings imply that oligopeptides may represent a new class of calcium channel ligands, and the pituitary cells are sensitive targets for them.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Hormônio do Crescimento/metabolismo , Oligopeptídeos/farmacologia , Adeno-Hipófise/efeitos dos fármacos , Prolactina/metabolismo , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Feminino , Adeno-Hipófise/metabolismo , RatosRESUMO
The analgesic ED50 values of some classical morphine congeners (morphine, methadone, fentanyl, azidomorphine) in the rat and mouse tail-flick tests were found to be similar. However, several synthetic derivatives of the natural enkephalins were more potent in mice than in rats. (These analogs contain D-amino acid in position 2 and D- or L-sulfonic (or phosphonic) acid residue in position 5). beta-Endorphin, D-Met2, Pro5-enkephalinamide and two partial agonists showed intermediate interspecies relative potencies. According to the data obtained, similar opiate receptors might mediate the analgesic action of classical opiates in rats and in mice. However, the opiate receptors responsible for the antinociceptive effects of the above mentioned enkephalin analogues must be dissimilar in the two species examined. The results are discussed in terms of the role of mu- and delta-receptors in mediation of the analgesic effect induced by different types of opioids.
Assuntos
Analgésicos , Encefalinas/farmacologia , Entorpecentes/farmacologia , Animais , Masculino , Camundongos , Ratos , Tempo de Reação/efeitos dos fármacos , Receptores Opioides/efeitos dos fármacos , Especificidade da EspécieRESUMO
The effect of Boc-DPhe-Phe-Lysinal (Boc-DPPL) on the 45Ca2+ uptake of rat anterior pituitary monolayer cultures was investigated. The compound decreased the basal Ca2+ uptake at 3 x 10(-4) mol/l. The 45Ca2+ uptake stimulated by potassium-induced depolarization was more sensitive to Boc-DPPL inhibition, a slight decrease was seen with 3 x 10(-6) mol/l and there was a half maximal inhibition at 3 x 10(-5) mol/l. Boc-DPPL is known to inhibit pituitary hormone release in similar concentrations, an effect might also be due to its calcium antagonist property.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Oligopeptídeos/farmacologia , Hipófise/metabolismo , Animais , Cálcio/metabolismo , Radioisótopos de Cálcio , Células Cultivadas , Masculino , Hipófise/citologia , Hipófise/efeitos dos fármacos , RatosRESUMO
The opioid activities of enkephalin analogues bearing D- or L-aminopentane-sulfonic/phosphonic acid at position 5 were studied in vitro, in electrically stimulated longitudinal muscle strip of guinea-pig ileum and mouse vas deferens preparations and in vivo in the rat tail-flick test. Using their in vitro effects Met-enkephalin-like, beta-endorphin-like, (nor)morphine-like and derivatives of intermediate character could be differentiated. Correlating the in vitro activities with the analgesic activity in vivo it is concluded that the enkephalin-like character in a pentapetide may hinder the expression of analgesic activity, when the compounds are given into the cerebroventricular system.
Assuntos
Analgésicos , Endorfinas/farmacologia , Encefalinas/farmacologia , Animais , Encefalinas/administração & dosagem , Cobaias , Íleo/efeitos dos fármacos , Técnicas In Vitro , Injeções Intravenosas , Injeções Intraventriculares , Masculino , Camundongos , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Ratos , Tempo de Reação/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Comparative studies on the anticoagulant effect of D-Phe-Pro-Arg-H (ALD) and D-Phe-Pro-Arg-CH2Cl (CMK) were carried out in order to estimate whether the reversible or the irreversible tripeptide inhibitor of thrombin would be more suitable to develop as a novel anticoagulant. Conventional screening assay methods in vitro were focused on the functional stability of the compounds in whole blood and blood components while ex vivo the changes in whole blood clotting time under parenteral application of the inhibitors were investigated. The efficacy of ALD relative to that of CMK was found to depend on the complexity of the test systems. Thus CMK was the more inhibitory in citrated plasma, but ALD showed the higher potency in whole blood. When incubated in various systems such as human whole blood, serum, solutions of isolated plasma proteins, digestive juices and tissue homogenates, respectively, the inhibitory activity of ALD showed only slight decreases for several hours while marked or substantial loss of activity was observed with CMK under identical conditions. ALD administered parenterally to rabbits proved to be powerful anticoagulant; CMK exhibited only a weak and transient anticoagulant effect presumably due to its ability to bind irreversibly to various plasma and tissue proteins. Accordingly, the reversible inhibitor ALD should be more suitable to develop as an anti-coagulant than CMK, its irreversibly acting analogue.
Assuntos
Anticoagulantes/farmacologia , Oligopeptídeos/farmacologia , Trombina/antagonistas & inibidores , Animais , Bile/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Suco Gástrico/metabolismo , Humanos , Pulmão/metabolismo , Masculino , CoelhosRESUMO
The pharmacologic activities of two thrombin inhibitors (D-MePhe-Pro-Arg-H, recombinant-hirudin) were compared in two animal models. The antithrombotic effect was investigated in vivo in rabbits using a modified Wessler stasis thrombosis model. During these experiments, blood was drawn for ex vivo testing to determine the coagulation profile and to determine plasma concentrations using pre-constructed calibration curves. A dose-dependent antithrombotic effect was observed for both agents. On an equigravimetric basis (100 micrograms/kg i.v.), r-hirudin showed a stronger antithrombotic effect than the tripeptide, which correlated well with the ex vivo anticoagulant effect. No adverse reactions were observed during this study. In a rabbit ear bleeding model, a dose and time dependent hemorrhagic effect was observed for both agents. Only slight bleeding effects were observed at 1.0 mg/kg dosages. These studies show that the tripeptide D-MePhe-Pro-Arg-H and r-hirudin are specific thrombin inhibitors with potent antithrombotic effects and a high therapeutic (antithrombotic/hemorrhagic) index. Furthermore, the results of these two animal models and ex vivo analyses can be used to determine the therapeutic index of thrombin inhibitors.
Assuntos
Anticoagulantes/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Fibrinolíticos/uso terapêutico , Hirudinas/análogos & derivados , Oligopeptídeos/uso terapêutico , Trombose/tratamento farmacológico , Sequência de Aminoácidos , Animais , Anticoagulantes/farmacologia , Relação Dose-Resposta a Droga , Fibrinolíticos/farmacologia , Terapia com Hirudina , Veias Jugulares , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Coelhos , Proteínas Recombinantes/uso terapêuticoRESUMO
The effects of new Agmatine (Agm) analogs of human growth hormone-releasing hormone (GH-RH) were compared to GH-RH (1-29)NH2 and to (D-Ala2)GH-RH(1-29)NH2 after intravenous (IV) and subcutaneous (SC) administration to pentobarbital-anesthetised male rats and in vitro using superfused rat pituitary cell system. After IV administration, the analogs: (D-MeAla2,Nle27)GH-RH(1-28)Agm(JG-75), (desamino-Tyr1,D-Ala2,Nle27)GH-RH(1-28)Agm(JG-77), (D-Ala2,Nle27)GH-RH(1-28)Agm(JG-73) and (D-Ala2)GH-RH(1-29)NH2 showed a potency 2.6-3.9 times greater than GH-RH(1-29)NH2 at 5 min and 1.6-2.7 times higher at 15 min. After SC administration these analogs were 30-74 times more potent than GH-RH(1-29)NH2. The ratio between the IV and SC GH-releasing activity of the analogs ranged from 2 to 5, while GH-RH(1-29)NH2 was about 50 times more active IV than SC. This indicates that 20-50% of the analogs can be absorbed from SC tissues, but only 2% of GH-RH(1-29)NH2. The in vitro activity of the agmatine analogs on GH release closely paralleled their IV potency and was 2.8-3.9 times greater than that of GH-RH(1-29)NH2. No significant difference in potency was found between (D-Ala2)GH-RH(1-29)NH2 and JG-75 after IV administration and in vitro, although JG-75 contained only 28 amino acids. We conclude that the reason for the large discrepancies between the previously reported activities of (D-Ala2)GH-RH(1-29)NH2 was simply due to the different ways of administration of this analog, SC vs IV, and not to species specificity. The replacement of Arg29 by Agmatine in (D-Ala2,Nle27)GH-RH(1-29)NH2 causes a 3 fold increase in SC potency, but the replacement of D-Ala2 with D-MeAla2 reduces the SC, but not the IV and in vitro activity in half.
Assuntos
Agmatina/farmacologia , Hormônio Liberador de Hormônio do Crescimento/análogos & derivados , Hormônio do Crescimento/sangue , Guanidinas/farmacologia , Fragmentos de Peptídeos/farmacologia , Agmatina/análogos & derivados , Animais , Arginina , Relação Dose-Resposta a Droga , Hormônio Liberador de Hormônio do Crescimento/administração & dosagem , Hormônio Liberador de Hormônio do Crescimento/farmacologia , Técnicas In Vitro , Masculino , Fragmentos de Peptídeos/administração & dosagem , Hipófise/efeitos dos fármacos , Ratos , Ratos Endogâmicos , SermorelinaRESUMO
Proteinase inhibitors were tested for their ability to inhibit prolactin (PRL) and growth hormone (GH) release by cultured anterior pituitary cells of the rat. Inhibitors of microbial origin (chymostatin, elastatinal, leupeptin) had either no or a moderate effect on hormone release while some tripeptide aldehydes, especially those with lysine at their C terminus, inhibited markedly PRL and to a lesser extent GH release. Boc-DPhe-Phe-lysinal was the most effective on lactotrophs inhibiting PRL release more than 50% at 10(-4) M. The site(s) of action of tripeptide aldehydes remain to be elucidated.
Assuntos
Hormônio do Crescimento/metabolismo , Adeno-Hipófise/metabolismo , Prolactina/metabolismo , Inibidores de Proteases/farmacologia , Animais , Aprotinina/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Leupeptinas/farmacologia , Oligopeptídeos/farmacologia , Fluoreto de Fenilmetilsulfonil/farmacologia , Adeno-Hipófise/efeitos dos fármacos , Ratos , Inibidor da Tripsina de Soja de Kunitz/farmacologiaRESUMO
Fibrin plate assay (FPA) and thrombelastography (TEG) were used to assess the antifibrinolytic effects of D-Phe-Pro-Arg-H (1), the prototype of peptide aldehyde inhibitors of thrombin, and two of its more stable derivatives, D-MePhe-Pro-Arg-H (2) and Boc-D-Phe-Pro-Arg-H (3). Inhibition of plasmin generation by tissue plasminogen activator, urokinase and streptokinase were studied by both FPA and TEG while that of plasmin could only be examined by FPA. TEG was more sensitive than FPA in general and for the detection of streptokinase inhibition in particular. Derivative (3) was 2-50 times more inhibitory than (1) or (2) depending on the enzyme studied and the assay system used. The thrombin selectivities of (1)-(3) were defined as the thrombin to fibrinolytic enzyme potency ratios. Data obtained by the FPA and thrombin time assay indicated (1) and (2) to be 2-80 times more selective for thrombin than (3). On the other hand, the values determined by TEG and recalcification assay showed the thrombin selectivity of (2) to be two to three times higher than that of (1), and (3) to have no such selectivity. According to TEG studies, (1) and (2) assisted rather than inhibited fibrinolysis by reducing the elasticity of human plasma clots.
Assuntos
Fibrinolisina/efeitos dos fármacos , Fibrinólise/efeitos dos fármacos , Oligopeptídeos/farmacologia , Trombina/antagonistas & inibidores , Estudos de Avaliação como Assunto , Estreptoquinase/antagonistas & inibidores , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidoresRESUMO
Imprinting was induced with synthetic oligopeptides in Chang liver cell cultures to test these molecules for signal molecule value. Investigations into imprinting overlaps (cross-imprinting) have shown that all oligopeptides (di-, tetra- and pentapeptides) carrying a terminal proline group were able to imprint the cells for the pentapeptide Tyr-D-Met-Gly-Phe-Pro-NH2, which displayed an outstanding imprinting potential for itself and an extraordinary opioid activity as well. The fact that exclusively the proline-deficient oligopeptide (a tetrapeptide) failed to imprint for the pentapeptide in question, indicates a decisive role of proline in the transformation of molecules to signal carriers (hormones). The pentapeptide in question did imprint for the related molecules (except the dipeptide) but to a much lesser degree than for itself. The marked inferiority of the pentapeptide's cross-imprinting potential to its self-imprinting potential supports the hypothetical implication that a considerable difference between the specific and non-specific binding capacities of a molecule, if not the loss of non-specific binding was an essential prerequisite of transformation to a signal molecule, i.e. of hormone evolution.
Assuntos
Evolução Biológica , Hormônios/fisiologia , Oligopeptídeos/farmacologia , Animais , Linhagem Celular , Fígado/efeitos dos fármacos , Fígado/fisiologia , Oligopeptídeos/fisiologia , Sinais Direcionadores de Proteínas/fisiologia , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/fisiologiaRESUMO
Hormonal imprinting induced in Tetrahymena and in Chang liver cells with di-, tri-, tetra- and pentapeptides (synthetic opioids and their fragments) has shown that both cell types are able to differentiate the related molecules from one another. The dipeptide phenylalanine + proline induced a measurable imprinting in the liver cells, and chain length increase, especially terminal coupling with tyrosine enhanced the imprinting potential enormously. Intra-chain changes in the amino acid sequence had a measurable effect on the intensity of imprinting. The molecules showing the relatively strongest physiological action accounted for the most intensive imprinting in both cell types; this indicates that, in all probability, induction of binding site formation plays a key role in the development of signal molecules, and thereby in hormone evolution.
Assuntos
Hormônios/biossíntese , Oligopeptídeos/farmacologia , Receptores de Superfície Celular/biossíntese , Animais , Linhagem Celular , Fígado/efeitos dos fármacos , Fígado/metabolismo , Tetrahymena/efeitos dos fármacos , Tetrahymena/metabolismoRESUMO
Research on synthetic peptides at the Institute for Drug Research (IDR) is exemplified by an overview of the projects that resulted in significant results.
Assuntos
Peptídeos/farmacologia , Animais , Anticoagulantes/síntese química , Anticoagulantes/farmacologia , Hormônios/síntese química , Hormônios/farmacologia , Humanos , Hungria , Peptídeos Opioides/síntese química , Peptídeos Opioides/farmacologia , Peptídeos/síntese químicaRESUMO
Research on synthetic peptides at the Institute for Drug Research (IDR) is exemplified by an overview of the projects that resulted in significant results. The first synthesis of oxytocin, a pituitary hormone, in 1953 launched the research on synthetic peptides all over the world. This synthesis was reproduced by Bodanszky at the IDR in 1954, then, after some improvements, the process was presented to Richter to produce synthetic oxytocin for therapeutic purposes. Significant result was the first synthesis of the 39-member whole molecule of human ACTH, another pituitary hormone. A short SAR study on luteinizing hormone-releasing hormone (LHRH) led to an interesting analog, Cit-8-LHRH, and somewhat later, to the D-Cit-6-LHRH analogues, of which SB-75 become marketed under the name Cetrorelix. Studies on the brain peptides, enkephalins, resulted in GYKI-14,238, the first analog that showed analgesic activity upon systemic administration and whose human efficacy could also be proven during clinical examination. Significant results were also achieved in the research on anticoagulant peptides. The first highly potent peptide aldehyde inhibitor of thrombin, GYKI-14,166, was identified at the IDR as well as its stable analog, GYKI-14,766. This compound was selected for detailed preclinical study, licensed to Eli Lilly Company, got the generic name efegatran, and entered clinical trials. The first non-covalent peptide inhibitor of thrombin, GYKI-14,525, was also identified at the IDR. Thus IDR really provided the prototype of original thrombin inhibitors in the mid 70's, and analogues were prepared in many laboratories through two decades. IDR's current research program's objective includes a quest for peptide originals that can inhibit both thrombin and factor Xa in solution and also within plasma clots in which these enzymes are entrapped. Structures with such inhibitory profile were identified among the efegatran-related alpha-hydroxy acid and ethoxycarbonyl-amino acid derivatives. The follow-up molecules are even more promising as antithrombotics, and may also be useful for treatment of disseminated intravascular coagulation, an often fatal syndrome, so we continue working on this project.