RESUMEN
Dipeptidyl peptidase IV preferably hydrolyzes peptides and proteins with a penultimate proline residue. Umezawa and co-workers (Umezawa et al. (1984) J. Antibiotics 37, 422-425) reported that diprotin A (Ile-Pro-Ile) and diprotin B (Val-Pro-Leu) are inhibitors for dipeptidyl peptidase IV. We could show that both compounds as well as other tripeptides with a penultimate proline residue are substrates for dipeptidyl peptidase IV. An apparent competitive inhibition by those compounds is a kinetic artifact due to the substrate-like structure of such tripeptides.
Asunto(s)
Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Oligopéptidos/metabolismo , Secuencia de Aminoácidos , Dipeptidil Peptidasa 4 , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/antagonistas & inhibidores , Cinética , Datos de Secuencia Molecular , Oligopéptidos/farmacología , Especificidad por SustratoRESUMEN
The substrate specificity of dipeptidyl peptidase IV (dipeptidyl peptide hydrolase, EC 3.4.14.5) from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum, was investigated with a series of N-terminal unprotected (dipeptidyl peptidases IV) and succinylated dipeptidyl-p-nitroanilides (proline-specific endopeptidase). Both enzymes are specific for the S configuration of the amino-acid residue in P1 and P2 position if the penultimate residue is proline. In the case of alanine substrates (Ala in P1, dipeptidyl peptidase IV hydrolyzes such compounds where the configuration of the P2 residue is R. The penultimate residue with dipeptidyl peptidase IV can be, beside proline and alanine, dehydroproline, hydroxyproline and pipecolic acid. Proline substrates (Pro in P1) with an R configuration in P2 are inhibitors of the hydrolysis of proline substrates with an S,S configuration in an uncompetitive (dipeptidyl peptide IV) or mixed inhibition type (proline-specific endopeptidase). Derivatives of Gly-Pro-pNA where the N-terminal amino group is methylated are hydrolyzed by dipeptidyl peptidase IV.
Asunto(s)
Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Flavobacterium/enzimología , Riñón/enzimología , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Dipéptidos , Dipeptidil Peptidasa 4 , Cinética , Prolil Oligopeptidasas , Especificidad por Sustrato , PorcinosRESUMEN
beta-Casomorphin-(5) and some analogs modified by the introduction of some D-amino acids and D-pipecolic acid as well as by C-terminal amidation were tested for their affinities to mu- and delta-binding sites in rat brain membranes. The binding affinities of these compounds are compared with the known activities in the guinea pig ileum (GPI) and mouse vas deferens (MVD) test and their antinociceptive potencies in rats. The substitution of D-proline for proline in position 4 in beta-casomorphin-(5) and beta-casomorphin-(4)amide (morphiceptin) results in derivatives with very high mu-binding affinity and mu-selectivity. These affinities correspond to the respective analgesic potencies. Both binding to mu-receptors and analgesic potency are also enhanced by the introduction of D-Phe in position 3. Testing D-Ala2 substituted derivatives with respect to their ability to compete for 3H-naloxone, we observed apparent differences between the pentapeptide amides (biphasic displacement curves) and the tetrapeptide amides (monophasic displacement curves). The substitution of L-Pro2 by D-pipecolic acid yields an analog with preferential delta-receptor affinity in the organ preparations (MVD) but preferential mu-receptor affinity in brain membranes. This finding suggests a possible difference between peripheral and central mu-binding sites.
Asunto(s)
Encéfalo/metabolismo , Caseínas/metabolismo , Endorfinas/metabolismo , Receptores Opioides/metabolismo , Aminoácidos , Animales , Unión Competitiva , Femenino , Cinética , Masculino , Ratas , Ratas Endogámicas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Beta-casomorphin (5) Tyr-Pro-Phe-Pro-Gly, a partial sequence of bovine beta-casein with moderate opioid properties and mu-receptor affinity, was modified by substituting for the natural L-amino acids their D-analogs, and D-pipecolic acid, as well as by amidation of the C-terminal. Substitution of D-Pro or D-pipecolic acid for L-Pro4 considerably increased the analgesic action and the potency on guinea-pig ileum of beta-casomorphin (5) as well as of casomorphin [4] amide. The resulting D-Pro4 analogs Deprolorphin and Deproceptin which showed high analgesic potency after both intracerebroventricular and intravenous administrations. Also, the substitution of D-Phe for L-Phe3 enhanced, even though to a lesser degree, the antinociceptive action. Both naltrexone and naloxone completely blocked the effects in vivo and in vitro. The substitution of D-Pro for L-Pro2 abolished the opioid-like actions, while substituting D-pipecolic acid for L-Pro2 resulted in an increased analgesic effect of remarkably long duration. The correlation of analgesic action with the effects on isolated organs separates the L-Pro4-substituted derivatives and D-Phe3-CM(5) from the other modified casomorphins and morphine, indicating that the analgesic potency of the former was about ten times that of the latter group in the case of identical GPI-potency. This may involve different subpopulations of opiate mu-receptors.
Asunto(s)
Analgésicos , Caseínas/farmacología , Endorfinas/farmacología , Secuencia de Aminoácidos , Animales , Relación Dosis-Respuesta a Droga , Endorfinas/administración & dosificación , Semivida , Inyecciones Intraventriculares , Masculino , Naltrexona/farmacología , Ratas , Ratas Endogámicas , Relación Estructura-ActividadRESUMEN
beta-Cyclodextrin-bonded silica is demonstrated to be a suitable stationary phase for high-performance liquid chromatography of conformational isomers of proline-containing peptides. In contrast to reversed-phase chromatography, the principle of inclusion complexation shows significant selectivities in conformer resolution based on a variety of interactions. New results of inclusion HPLC of biologically active oligopeptides related to beta-casomorphin on stationary phases containing bonded cyclodextrins of different internal diameters indicate a steric discrimination process during the conformer separation. beta-Cyclodextrin used as a mobile phase additive in reversed-phase systems is shown to offer the opportunity to investigate conformational changes using commercially available reversed-phase columns.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Ciclodextrinas , Péptidos/aislamiento & purificación , Prolina/análisis , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Péptidos/químicaRESUMEN
The beta -casomorphin-5 sequence was systematically modified by substitution of the naturally occurring amino acids. The derivatives are compared on the basis of their affinities towards mu 1-, mu 2 and delta -opioid binding sites estimated by means of binding studies with [3H]dihydromorphine and [3H]D-Ala2-Leu5-enkephalin as labels and computer curve fitting. A C-terminal amide group which is known to increase mu-site affinity of beta -CM-4 and beta -CM-5 mainly enhances the mu 2-site affinity. Furthermore, the Pro4-amide structure, which seems to be responsible for the affinity enhancement can be substituted by the pyrrolidide ring structure. Modifications in position 2, 3 and 4 can lead to an increase in mu 1-, mu 2- or delta -site affinity and/or selectivity. The specificity of these effects might be dependent on the respective changes in the charge or hydrophobicity due to the introduction of other amino acids. The results suggest firstly that the alternating proline residues in the beta -CM-5 molecule are not absolutely necessary for its mu-site affinities, and secondly that both opioid receptor subtype affinity and selectivity may be modified by changing charge and/or hydrophobicity in the middle part of the beta -casomorphin molecule.
Asunto(s)
Receptores Opioides/metabolismo , Secuencia de Aminoácidos , Animales , Endorfinas , Femenino , Técnicas In Vitro , Cinética , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos , Ratas , Ratas Endogámicas , Receptores Opioides/efectos de los fármacos , Receptores Opioides delta , Receptores Opioides mu , Relación Estructura-ActividadRESUMEN
The synthesis of somatostatin by linking together Boc-Ala-Gly-Cys(Bzl)-Lys(Z)-Asn-Phe-NHNH2 and H-Phe-Trp-Lys[Z(pCl)]-Thr-Phe-Thr-Ser-Cys(Bzl)-OBzl is described. The two partial sequences were obtained from segments which in general are also used in preparing somatostatin analogues. The synthesis of the partial sequences was optimized. The intracerebroventricular application of the somatostatin thus obtained to rats produced an increase of the dopamine and serotonin contents in the striatum. The results achieved were identical with those realized with standard somatostatin (Serono).
Asunto(s)
Somatostatina/síntesis química , Animales , Fenómenos Químicos , Química , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Inyecciones Intraventriculares , Ratas , Serotonina/metabolismo , Somatostatina/farmacologíaRESUMEN
The authors describe the synthesis of a shorter-chained somatostatin analogue in which the ring size of the native molecule is largely preserved by incorporation of S-carboxymethyl-L-homocysteinamid and ring closure via its side-chain function. This synthesis involves the linkage of Boc-Phe-D-Trp-Lys[Z(pCl)]-Thr-Phe-Thr-Ser-OH to H-Hcy(CH2-CO-Lys[Z(oCl)]-Asn-Phe-OMe)-NH2 and subsequent azide cyclization. In contrast to somatostatin, the somatostatin analogue thus obtained produces, when applied intracerebroventricularly to rats, a decrease in the dopamine content of the striatum.
Asunto(s)
Somatostatina/análogos & derivados , Animales , Química Encefálica/efectos de los fármacos , Fenómenos Químicos , Química , Dopamina/metabolismo , Inyecciones Intraventriculares , Ratas , Somatostatina/administración & dosificación , Somatostatina/síntesis química , Somatostatina/farmacologíaRESUMEN
The authors describe the synthesis of beta-casomorphine-5 (Tyr-Pro-Phe-Pro-Gly) by segment condensation and stepwise building-up. Intracerebroventricular application of the pentapeptide to the rat produces a long-lasting analgesia against thermal stimuli (which can be inhibited by naloxone) and counteracts the extinction of a passive avoidance reaction for no less than 4 days following post-shock application. beta-Casomorphin-5 is very rapidly degraded by dipeptidylpeptidase IV; the degradation starts from the N-terminus giving dipeptides. The degradation products are competitive inhibitors of this enzyme.
Asunto(s)
Endorfinas/síntesis química , Fragmentos de Péptidos , Analgésicos , Animales , Reacción de Prevención/efectos de los fármacos , Biotransformación , Endorfinas/antagonistas & inhibidores , Endorfinas/metabolismo , Extinción Psicológica/efectos de los fármacos , Inyecciones Intraventriculares , Cinética , Naloxona/farmacología , RatasRESUMEN
Ala-Phe-Ile-Gly-Leu-Met has been disengaged by cyclization of H-Leu-Met-Ala-Phe-Ile-Gly-OH by means of dicyclohexylcarbodiimide/N-Hydroxysuccinimide or the adequate p-nitrophenylester. Acc. to various strategic variants, the design of the linear precursor has been performed by condensation of the segments of Boc-Leu-Met-OH and H-Ala-Phe-Ile-Gly-OH. The resulting cyclo-[Eledoisin(6-11)-Hexapeptide] has in a clearly separated range of dose dual agonistic and antagonistic effects at the guinea-pig ileum.
Asunto(s)
Eledoisina/síntesis química , Fragmentos de Péptidos/síntesis química , Acetilcolina/farmacología , Animales , Fenómenos Químicos , Química , Eledoisina/farmacología , Femenino , Cobayas , Íleon/efectos de los fármacos , Técnicas In Vitro , Contracción Muscular/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Sustancia P/farmacologíaRESUMEN
The authors describe the synthesis of Gln-Phe-Phe-Gly-Leu-Met by cyclization of H-Leu-Met-Gln-Phe-Phe-Gly using three different methods. The linear sequence was obtained by a (2+4)-segment condensation. The resulting cyclopeptide showed only a small kinin activity on isolated guinea pig ileum compared to substance P, but it is a full agonist.
Asunto(s)
Oligopéptidos/síntesis química , Fragmentos de Péptidos , Sustancia P/síntesis química , Secuencia de Aminoácidos , Animales , Ciclización , Cobayas , Íleon/efectos de los fármacos , Técnicas In Vitro , Contracción Muscular/efectos de los fármacos , Oligopéptidos/farmacología , Ácido Pirrolidona Carboxílico/análogos & derivados , Sustancia P/farmacologíaAsunto(s)
Caseínas/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Endopeptidasas/metabolismo , Endorfinas/metabolismo , Serina Endopeptidasas , Animales , Caseínas/farmacología , Endorfinas/farmacología , Cobayas , Hidrólisis , Técnicas In Vitro , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Prolil Oligopeptidasas , Especificidad por SustratoRESUMEN
The H(+)/peptide cotransporter PEPT2 is expressed in a variety of organs including kidney, lung, brain, mammary gland, and eye. PEPT2 substrates are di- and tripeptides as well as peptidomimetics, such as beta-lactam antibiotics. Due to the presence of PEPT2 at the bronchial epithelium, the aerosolic administration of peptide-like drugs might play a major role in future treatment of various pulmonary and systemic diseases. Moreover, PEPT2 has a significant influence on the in vivo disposition and half-life time of peptide-like drugs within the body, particularly in kidney and brain. PEPT2 is known to have similar but not identical structural requirements for substrate recognition and transport compared to PEPT1, its intestinal counterpart. In this review we compiled available affinity constants of 352 compounds, measured at different mammalian tissues and expression systems and compare the data whenever possible with those of PEPT1.
Asunto(s)
Riñón/metabolismo , Simportadores/metabolismo , Dipéptidos/farmacología , Humanos , Transportador de Péptidos 1 , Relación Estructura-Actividad , Simportadores/química , Simportadores/efectos de los fármacosRESUMEN
It has been found that the microvillous membrane of rat enterocytes contains an aminopeptidase P which is one of the few enzymes capable to hydrolyze the peptide imido bond on the N-terminal side of proline residues. The enzyme was enriched 9-fold in chromatographically purified brush border membrane vesicles of the small intestine. Various extraction procedures and proteinase treatments yielded strong evidence that it is an integral part of the membrane without a stalked hydrophilic head exposed to the outer surface. It was solubilized by detergent and further enriched by ion exchange chromatography up to 73-fold.
Asunto(s)
Aminopeptidasas/metabolismo , Intestino Delgado/enzimología , Microvellosidades/enzimología , Animales , Fraccionamiento Celular , Mucosa Intestinal/enzimología , Microscopía Electrónica , Microvellosidades/ultraestructura , Prolina , Ratas , Ratas EndogámicasRESUMEN
Three exorphins, beta-casomorphin-5, morphiceptin and its D-Pro4 analog, were studied in DMSO by means of 1H and 13C NMR spectroscopy, with the aim of detecting conformational features of potential biological significance for the mu opioid activity since the presence of two Pro residues restricts the accessible conformational space more than in all other peptides. It is found that the conformational mixtures present in solution contain relevant fractions of folded conformers, a feature that assures the observation of four different Tyr OH signals in the 500 MHz spectrum of morphiceptin. The conformer distribution of (very active) (D-Pro4)-morphiceptin is different from those of its (less active) congeners.
Asunto(s)
Endorfinas , Espectroscopía de Resonancia Magnética , Péptidos , Conformación Proteica , EstereoisomerismoRESUMEN
The aim of the present study was to evaluate the metabolic breakdown of two biologically active derivatives of beta-casomorphin (beta CM)-i.e. D-proline4-beta CM (D-Pro4-beta CM) and des-tyrosine 1-D-proline4-beta CM (DT-D-Pro4-beta CM)- in the rat brain. After intracerebroventricular (icv.) administration of 0.78 nmoles of both [3H]D-Pro4-beta CM and [3H]DT-D-Pro4-beta CM, the concentration of the intact peptides and their metabolites was estimated in brain stem and corpus striatum. Both peptides were degraded in brain tissue forming a common metabolite. Phe-D-Pro-Gly. The metabolic half-lives of DT-D-Pro4-beta CM in brain stem and c. striatum were 22.6 min and 28.6 min, whereas those of D-Pro4-beta CM were 7.8 min and 8.2 min, respectively. According to both half-lives of the intact peptide and the kinetics of the formation of the stable metabolite Phe-D-Pro-Gly. DT-D-Pro4-beta CM seemed to be more resistant to biological degradation in brain tissue than D-Pro4-beta CM. In the case of D-Pro4-beta CM, the dipeptidylpeptidase IV (DP IV) (EC 3.4.14.-) is presumed to be the cardinal enzyme for the breakdown. Since the degradation of these peptides in brain tissue results in the unique metabolite Phe-D-Pro-Gly, the differences in the pattern of biological activities between D-Pro4-beta CM and DT-D-Pro4-beta CM may be due to the action of the corresponding intact peptide.
Asunto(s)
Encéfalo/metabolismo , Endorfinas/metabolismo , Fragmentos de Péptidos , Animales , Tronco Encefálico/metabolismo , Ventrículos Cerebrales/metabolismo , Cuerpo Estriado/metabolismo , Cinética , Masculino , Ratas , Ratas EndogámicasRESUMEN
By isolated perfused pancreas of Wistar rats the glucose (11 mmol/l) and arginine (10 mmol/l) stimulated insulin (IRI) and glucagon (IRG) secretion was measured in order to investigate the inhibitory activities of somatostatin-14 (SS 14) and the somatostatin analogue [3,14-L-seleno-cysteine, 8-D-tryptophan]-somatostatin (SeSS). SS-14 or SeSS (152.8 nmol/l) inhibit the glucose stimulated IRI secretion by 75 and 65%, respectively. Only the second phase of the biphasic arginine stimulated insulin secretion pattern by 40%. SeSS has under these conditions no effect, whereas 58 nmol/l SS-14 or SeSS show a suppressing effect on the first (20 and 55%, respectively) and second phase (65 and 85%, respectively) of the insulin secretion. Using 5.8 nmol/l SS-14 or SeSS the arginine stimulated IRG secretion was inhibited only in the second phase of the biphasic glucagon secretion pattern by about 40%. 58 nmol/l SS-14 or SeSS show an inhibiting effect on the first and on the second phase of secretion, in both cases about 50%. It is concluded that in the SS-14 molecule the sulfur of cysteine in position 3 and 14 can be exchanged by selenium without modifying the biological activities measured in the glucose or arginine stimulated IRI and IRG secretion in vitro. The D-Trp8 in the SeSS analogue does not show the typical better inhibitory action of D-Trp8-SS-14 on insulin and glucagon secretion compared with SS-14. Possibly the selenium in the SeSS analogue abolishes this effect.
Asunto(s)
Glucagón/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Animales , Femenino , Glucosa/farmacología , Técnicas In Vitro , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Cinética , Perfusión , Ratas , Ratas Endogámicas , Somatostatina/análogos & derivados , Somatostatina/farmacología , Relación Estructura-ActividadRESUMEN
This study was initiated to develop inhibitors of the intestinal H(+)/peptide symporter. We provide evidence that the dipeptide derivative Lys[Z(NO(2))]-Pro is an effective competitive inhibitor of mammalian PEPT1 with an apparent binding affinity of 5-10 microM. Characterization of the interaction of Lys[Z(NO(2))]-Pro with the substrate binding domain of PEPT1 has been performed in (a) monolayer cultures of human Caco-2 cells expressing PEPT1, (b) transgenic Pichia pastoris cells expressing PEPT1, and (c) Xenopus laevis oocytes expressing PEPT1. By competitive uptake studies with radiolabeled dipeptides, HPLC analysis of Lys[Z(NO(2))]-Pro in cells, and electrophysiological techniques, we unequivocally show that Lys[Z(NO(2))]-Pro binds with high affinity to PEPT1, competes competitively with various dipeptides for uptake into cells, but is not transported itself. Lack of transport was substantiated by the absence of Lys[Z(NO(2))]-Pro in Caco-2 cell extracts as determined by HPLC analysis, and by the absence of any positive inward currents in oocytes when exposed to the inhibitor. The fact that Lys[Z(NO(2))]-Pro can bind to PEPT1 from the extracellular as well as the intracellular site was shown in the oocyte expression system by a strong inhibition of dipeptide-induced currents under voltage clamp conditions. Our findings serve as a starting point for the identification of the substrate binding domain in the PEPT1 protein as well as for studies on the physiological and pharmacological role of PEPT1.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Dipéptidos/metabolismo , Nitrobencenos/metabolismo , Simportadores , Animales , Unión Competitiva , Transporte Biológico Activo/efectos de los fármacos , Células CACO-2/metabolismo , Proteínas Portadoras/biosíntesis , Dipéptidos/antagonistas & inhibidores , Dipéptidos/farmacología , Humanos , Cinética , Nitrobencenos/farmacología , Oocitos/metabolismo , Técnicas de Placa-Clamp , Transportador de Péptidos 1 , Pichia/genética , Pichia/metabolismo , Xenopus laevisRESUMEN
After intracarotid injection of [3H]beta-casomorphin-5 (beta CM5) in rats, the accumulation of radioactivity was determined in 18 brain regions and the anterior pituitary. The relative accumulation in all regions significantly exceeded that of [3H]inulin by a factor of 2.5, indicating a low but measurable brain uptake of the peptide. In blood-brain barrier-free areas, the accumulation of radioactivity was 15-fold higher than in blood-brain barrier-protected areas. The relative accumulation was not dependent on the total beta CM5 concentration in the range of 0.3-1.1 microM, and was not depressed by 400 microM L-tyrosine. We conclude that beta CM5, like other peptides, is accumulated in the blood-brain barrier-free areas to a relatively high but differing degree, whereas in the areas with a tight endothelium the accumulation is relatively low and nearly uniform. A binding to endothelial cells may contribute to the low accumulation of beta CM5, especially in blood-brain barrier-protected areas.
Asunto(s)
Barrera Hematoencefálica , Encéfalo/metabolismo , Endorfinas/metabolismo , Fragmentos de Péptidos , Animales , Arterias Carótidas , Endorfinas/administración & dosificación , Inyecciones Intraarteriales , Masculino , Adenohipófisis/metabolismo , Ratas , Ratas Endogámicas , Distribución TisularRESUMEN
Four new analogues of arginine vasopressin (AVP) substituted in positions 2 and 3 with all possible combinations of enantiomers of N-methylphenylalanine were synthesized and studied to assess the influence of N-methylation of the peptide bonds between the first three amino acids on the pharmacological properties of the resulting peptides. The next three analogues were designed to learn how the shortening of the peptide chain, by removal of one of the N-methylphenylalanine residues, would affect pharmacological properties of the resulting compounds. The activity of the analogues was tested in the in vitro uterotonic, pressor and antidiuretic tests. None of the prepared analogues displayed significant biological activity with the exception of [Me-d-Phe(2), Me-Phe(3)]AVP and [Me-d-Phe(2,3)]AVP, which showed low antiuterotonic activity (pA(2) = 6.6 and pA(2) = 6.4, respectively). Our results, while not impressive in terms of biological activity, may be helpful for designing potent and selective oxytocin antagonists.