The renal type H+/peptide symporter PEPT2: structure-affinity relationships.

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.

Analogues of arginine vasopressin modified in the N-terminal part of the molecule with enantiomers of N-methylphenylalanine.

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.

A novel inhibitor of the mammalian peptide transporter PEPT1.

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.

New analogues of bradykinin containing a conformationally restricted dipeptide fragment in their molecules.

The present paper describes the synthesis and some pharmacological properties of two new bradykinin analogues containing the ethylene-bridged dipeptide Phe-Phe in their molecules. In a further two peptides this modification was combined with acylation of the N-terminus with 1-adamantaneacetic acid. Finally, we synthesized four analogues by removing the Ser6 residue from the four peptides mentioned above. The activity of the new analogues was assayed on isolated rat uterus (RUT) and in rat blood pressure tests (BPT). The results clearly indicate that the proposed modification, alone or in combination with other changes, resulted in either a drop in antiuterotonic activity or even in conversion to an agonism. Although this tendency is not so distinct in blood pressure assays, the antagonistic potency of the new analogues is also diminished. Nevertheless, it was demonstrated that the D-amino acid in position 7 which, until recently, was considered necessary for antagonism, may be replaced, together with the amino acid occupying position 8, by a suitable, sterically restricted L,L-dipeptide unit.

Decisive structural determinants for the interaction of proline derivatives with the intestinal H+/peptide symporter.

To elucidate the decisive structural factors relevant for dipeptide-carrier interaction, the affinity of short amide and imide derivatives for the intestinal H+/peptide symporter (PEPT1) was investigated by measuring their ability to inhibit Gly-Sar transport in Caco-2 cells. Dipeptides with proline or alanine in the C-terminal position displayed affinity constants (Ki) of 0.15-1.2 mM and 0.08-9.5 mM, respectively. There was no clear relationship between hydrophobicity, size or ionization status of the N-terminal amino acid and the affinity of the dipeptides. However, analyzing the individual peptide bond conformations of Xaa-Pro dipeptides, a striking correlation between the cis/trans ratios (trans contents 24-70%) and the affinity constants was observed. After correcting the Ki values for the incompetent cis isomers, the Ki corr values of most dipeptides were in a small range of 0.1-0.16 mM. This result revealed the decisive role of peptide bond conformation even for a transport protein that is quite promiscuous in substrate translocation. When measuring affinity constants of Xaa-Pro and Xaa-Sar dipeptides, the cis/trans ratios cannot be ignored. Lower affinities of Lys-Pro, Arg-Pro and Pro-Pro indicate that additional molecular factors affect their binding at PEPT1. The Ki values obtained for the corresponding Xaa-Ala dipeptides support this conclusion. Potential substrates or inhibitors of peptide transport were found among Xaa-piperidides and Xaa-thiazolidides. Dipeptides with N-terminal proline displayed a very diverse affinity profile. However, in contrast to current knowledge, several Pro-Xaa dipeptides such as Pro-Leu, Pro-Tyr and Pro-Pro are recognized by PEPT1 with appreciable affinities. Binding seems mainly determined by the hydrophobicity of the C-terminal amino acid and the rigidity of the structure.

Transport of amino acid aryl amides by the intestinal H+/peptide cotransport system, PEPT1.

Transport of amino acid aryl amides by the intestinal H+/peptide symporter (PEPT1) was studied in Caco-2 cells and in Xenopus laevis oocytes expressing human PEPT1. Several amino acid amides were able to inhibit the uptake of [14C]glycylsarcosine in Caco-2 cells. Ala-4-nitroanilide (Ki = 0.08 mM), Phe-4-nitroanilide (Ki = 0.09 mM) and Ala-4-phenylanilide (Ki = 0.03 mM) were accepted as substrates with equal or higher affinity than natural Ala-Xaa dipeptides. Ala-anilide (Ki = 2.9 mM), Ala-7-amido-4-methylcoumarin (Ki = 0.2 mM), Ala-4-chloroanilide (Ki = 0.3 mM) and Ala-4-methylanilide (Ki = 0.3 mM) were also recognized by PEPT1 as substrates. In contrast, alanine, Ala-amide, Phe-amide, Ala-methyl ester, Ala-4-nitrobenzyl ester and Ala-methylamide were not recognized (Ki > 20 mM). In X. laevis oocytes, transport of Ala-4-nitroanilide, Ala-7-amido-4-methylcoumarin, Ala-4-methylanilide and Ala-anilide was associated with transfer of positive charge and the currents were saturable with respect to substrate concentration (K0.5 values: 0.1, 0.2, 0.8 and 3.1 mM, respectively). The currents induced by Ala-4-methylanilide were saturable with respect to the substrate concentration and influenced by the membrane potential. The affinity of the transporter for Ala-4-methylanilide was also found to be influenced by the membrane potential. We conclude that the intestinal H+/peptide cotransport system PEPT1 accepts amino acid aryl amides as substrates.

High-performance liquid chromatographic separation of cis-trans isomers of proline-containing peptides. II. Fractionation in different cyclodextrin systems.

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.

Conformational analysis of the tetrapeptide Pro-D-Phe-Pro-Gly in aqueous solution.

Conformational investigations of the tetrapeptide Pro-D-Phe-Pro-Gly in water solution were carried out by 1H and 13C NMR spectroscopy. The internal proline residue allows for the possibility of cis/trans isomerization about the D-Phe-Pro peptide bond resulting in two conformational isomers. The major isomer was identified as the trans isomer. The pH-dependence of the cis/trans equilibrium supports an additional stabilisation of the trans isomer by an intramolecular ionic interaction between the amino- and carboxy-terminus in the zwitterionic state. Based on 13C spin-lattice relaxation times (T1), different pyrrolidine ring conformations of Pro1 and Pro3 could be determined. By combination of several NMR data (vicinal coupling constants 3JN alpha, temperature dependence of the NH chemical shifts, differences in the chemical shifts between the beta and gamma carbons of the proline residues) and energy minimization calculations, a type II' beta-turn should contribute considerably to the overall structure of the trans isomer.

Structure-activity studies of novel casomorphin analogues: binding profiles towards mu 1-, mu 2- and delta -opioid receptors.

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.

Spectroscopic analysis of [Trp3]-beta-casomorphin analogs. Comparative structure conformation-activity studies.

A series of [3-tryptophan]-beta-casomorphin-5([Trp3]-beta-CM-5) analogs were investigated by circular dichroism (CD) and fluorescence spectroscopy to explore their structure-conformation properties in solution. In addition, the comparative opioid activities of these compounds were evaluated using the in vitro guinea pig ileum (GPI) and mouse vas deferens (MVD) assays. Specifically, the pentapeptide sequence of [Trp3]-beta-CM-5, H-Tyr-Pro-Trp-Pro-Gly-OH (I) was modified at Pro-2 and Pro-4 by D-Pro substitutions to provide two diastereometric analogs, [Trp3-D-Pro-4]-beta-CM-5 (II) and [D-Pro2,4,Trp3]-beta-CM-5 (III). In the GPI and MVD assays, beta-CM-5 effected IC50 values of 1.3 microM and 8.9 microM, respectively, which confirmed its known mu/delta-selectivity on these two peripheral opioid receptor subtypes. The potencies of compounds I, II, and III were 0.2, 2.0, and less than 0.005 relative to beta-CM-5 on the GPI assay. Compounds I and II exhibited pronounced mu/delta-selectivities (greater than 18.9- and 12.4-fold respectively), whereas compound III was essentially inactive in both the GPI and MVD assays. CD studies of beta-CM-5 and its [Trp3]-beta-CM-5 analogs showed striking differences in their near-UV and far-UV spectra in aqueous or organic solvents. In the far UV CD spectra, weak (20%) alpha-helicity (maximum at 193 nm and minima at 208 and 222 nm) for beta-CM-5 was obtained in trifluoroethanol (TFE); however, none of the [Trp3]-beta-CM-5 analogs showed such CD bands. Of potential relevance to gamma-turn or C7 secondary structure was the observation of a strong negative band at 245 nm for compounds II and III which was not solvent-dependent in H2O or TFE, whereas compound I showed this CD band exclusively in TFE. In the near-UV CD at 275 nm (Trp electronic transition), the relative order of intensities of this band were determined for the [Trp3]-beta-CM-5 compounds to be II greater than I greater than III, which was identical to their relative biological potencies in both the GPI and MVD assays. Fluorescence energy transfer (FET) experiments of compounds I-III provided the intramolecular distances (r) between their Tyr (donor) to Trp (acceptor) side-chains, by the Förster method, and were as follows: [Trp3]-beta-CM-5, r = 10.6 A; [Trp3, D-Pro4]-beta-CM-5, r = 9.6 A; and [D-Pro2,4,Trp3]-beta-CM-5, r = 11.0 A.(ABSTRACT TRUNCATED AT 400 WORDS)

Are diprotin A (Ile-Pro-Ile) and diprotin B (Val-Pro-Leu) inhibitors or substrates of dipeptidyl peptidase IV?

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.

Mechanism of proline-specific proteinases: (I) Substrate specificity of dipeptidyl peptidase IV from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum.

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.

Conformational analysis of morphiceptin by NMR spectroscopy.

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.

Metabolism of D-proline beta-casomorphin derivatives in the rat brain.

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.

Opiate receptor binding affinities of some D-amino acid substituted beta-casomorphin analogs.

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.

[Action of [3,14-L-selenocysteine, 8-D-tryptophan]-somatostatin on insulin and glucagon secretion of the isolated perfused pancrease of the Wistar rat]. / Zur Wirkung von [3,14-L-Selenozystein, 8-D-Tryptophan]-Somatostatin auf die Insulin- und Glukagonsekretion des isoliert perfundierten Pankreas der Wistar-Ratte.

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.

Is the proline-specific aminopeptidase P of the intestinal brush border an integral membrane enzyme?

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.

[Synthesis of cyclic and non-cyclic tachykinin partial sequences. 2. Synthesis of the homocyclic substance P(6-11) hexapeptide]. / Synthese cyclischer und cyclisch-verzweigter Tachykinin-Teilsequenzen. Teil 2: Darstellung des homodet-cyclischen Substanz P(6-11)-Hexapeptides.

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.

[Synthesis of cyclic and cyclically branched tachykinin partial sequences. 1. Synthesis of the homodet-cyclic eledoisin(6-11)-hexapeptide]. / Synthese cyclischer und cyclisch-verzweigter Tachykinin-Teilsequenzen. Teil 1: Darstellung des homodet-cyclischen Eledoisin(6-11)-Hexapeptides.

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.

Derivatives of beta-casomorphins with high analgesic potency.

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.