Peptide-derived transition state analogue inhibitors of thrombin; synthesis, activity and selectivity.

In a study to combine the transition state analogue concept with the principle of catalytic site spanning, a series of peptide-derived transition state analogue (TSA) inhibitors of thrombin has been synthesized and tested. In the sequence H-D-Phe-Pro-Arg-Gly-OH (2) the Arg-Gly amide bond has been replaced by three classes of transition state analogues, being the ketomethylene, the hydroxyethylene and the hydroxymethylene amide bond replacements. Compound 12a, in which the amide bond has been replaced by the ketomethylene group, was found to be the most potent thrombin inhibitor of the series studied. Subsequently, penta- and hexapeptide sequences with good affinity for thrombin were developed, i.e. H-D-Phe-Pro-Arg-Gly-Phe-OH (16) and H-D-Phe-Pro-Arg-Gly-Phe-Lys-OH (26). In these sequences the Arg-Gly amide bond was then replaced by the ketomethylene group. The resulting compounds 43a and 47a, respectively, were evaluated in vitro as inhibitors of thrombin and factor Xa. Compound 47a was found to be the most potent thrombin inhibitor of the series studied (Ki = 29 nM). The combination of the transition state analogue concept and the principle of peptide elongation (tetrapeptide-->hexapeptide) yields thrombin inhibitors of high potency and selectivity. The effects of these two alterations reinforce each other indicating a synergistic effect. This might be rationalized by entropy factors.

Topography and characteristics of specific binding sites for non-opioid gamma-type endorphins in the rat brain as studied by autoradiography with [35S]Met-desenkephalin-gamma-endorphin.

An in vitro autoradiographic study was performed to characterize specific rat brain binding sites for non-opioid neuroleptic-like gamma-type endorphins, using [35S]Met-des-enkephalin-gamma-endorphin ([35S]Met-DE gamma E; [35]S-beta-endorphins(5-17)) with high specific activity as radioligand. The binding sites appeared to be confined to rat forebrain regions, e.g., orbital cortex, frontal cortex, cingulate cortex, piriform cortex, nucleus accumbens, amygdala, mediodorsal nucleus of the thalamus and arcuate and periventricular nuclei of the hypothalamus. These regions are part of the mesocorticolimbic feedback circuit. Densitometric analysis of the autoradiographs revealed that the density of the binding sites was highest in the mediodorsal nucleus of the thalamus and the amygdala. Concentration-dependent displacement of [35S]Met-DE gamma E (500 pM) with DE gamma E yielded an IC50 of 0.6 nM whereas DE alpha E (beta-endorphin(6-16)) had an IC50 of 210 nM. Various endorphins, sharing the gamma-endorphin C terminus, displaced [35S]Met-DE gamma E to the same extent as non-labelled DE gamma E (at 10(-6) M) whereas non-endorphin peptides did not show displacing capacity. Possible relationships of the binding sites with opioid receptors were investigated. DAMGO (mu) and DPDPE (delta) displaced [35S]Met-DE gamma E to some extent at 10(-6) M whereas U69,593 (kappa) was inactive, suggesting that the binding sites for gamma-type endorphins may resemble mu- and delta-opioid receptors in some aspects. Similarly, relationships with dopamine receptors were investigated. Haloperidol partially displaced [35S]Met-DE gamma E whereas sulpiride, SKF38,393 and 3-PPP at 10(-6) M did not induce significant displacement. Thus, binding sites are distinct from dopamine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Difficult couplings in stepwise solid phase peptide synthesis: predictable or just a guess?

Stepwise solid phase peptide synthesis, Fmoc-approach, of 88 peptides varying in length from 4 to 24 amino acid residues was performed using a uniform procedure for coupling, monitoring and deprotection steps. The data of 696 couplings have been incorporated into a computer programme in order to study whether the degree of coupling can be predicted. Parameters studied are the nature of the amino acid to be coupled, the amino acid to be acylated and the length of the growing peptide chain. Using this information, prediction of "good" or "difficult" sequences, that is, peptides that can be synthesized without appreciable repeated couplings or the opposite, seems possible.

Capillary electrophoresis of peptides. Analysis of adrenocorticotropic hormone-related fragments.

Capillary electrophoresis can be used successfully to analyse small peptides to give additional information to that obtained using high-performance liquid chromatography (HPLC). The separation of a modified adrenocorticotropic hormone (4-9) fragment (Org 2766) and several of its fragments was investigated using capillary zone electrophoresis. Prediction of migration in aqueous systems using pKa-related data and the migration behaviour using sodium dodecyl sulphate in the buffer are discussed, as is the choice of buffer systems. The electrophoretic patterns are compared with the HPLC separation.

ACTH/MSH-like peptides inhibit the binding of dopaminergic ligands to the dopamine D2 receptor in vitro.

ACTH-(1-24) decreased the binding of the dopamine D2 receptor agonist, [3H]N-propylnorapomorphine ([3H]NPA), to rat striatal membranes in a concentration-dependent manner, with a Ki of 5 x 10(-7) M. Saturation curves for [3H]NPA binding in the presence of increasing concentrations of ACTH-(1-24) were performed. Scatchard analysis in the presence of ACTH-(1-24) revealed an increased dissociation constant (Kd), while the binding capacity (Bmax) was not affected by the peptide, suggesting an apparent competitive interaction between ACTH-(1-24) and [3H]NPA. ACTH-(1-24) also reduced the binding of the dopamine D2 receptor antagonist [3H]spiperone to striatal membranes, with a Ki of 10(-6) M. Much higher concentrations of ACTH-(1-24), up to 10(-4) M, were needed for the displacement of appropriate radiolabelled ligands from dopamine D1 receptors, serotonin 5-HT1A, serotonin 5-HT1B, muscarinic M1 acetylcholine and histamine H1 receptors. ACTH-(1-24) also inhibited the binding of [3H]spiperone to dopamine D2 receptors in membranes of the pituitary gland, the septum and the substantia nigra. ACTH-(1-39) and most ACTH fragments and analogs were less potent than ACTH-(1-24) in displacing [3H]NPA from the dopamine D2 receptor in striatal membranes. In general there was a relationship between displacing potency and chain length. ACTH-(7-16)-NH2 and benzyloxycarbonyl-ACTH-(8-16)-NH2, however, were more potent than ACTH-(1-24) in reducing the binding of [3H]NPA to dopamine D2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of specific antisera against 9-desglycinamide-8-arginine vasopressin by site-specific immunization.

Four specific and sensitive antisera against 9-desglycinamide-8-arginine vasopressin (DGAVP) were obtained by means of site-specific immunization in rabbits, using a Keyhole Limpet haemocyanin-DGAVP conjugate synthesized by a specific reaction of a thiol function with a maleimide group. Compared with conventionally raised antisera, the cross-reactivity with endogenous arginine vasopressin (AVP) and vasopressin-derived fragments was substantially lower. Interassay variability was 18.1% at maximum. Possible applicability of the new antisera in pharmacokinetic studies of DGAVP with respect to blood-brain barrier transport was tested by means of a pilot study in the rat.

Structural modifications of the ACTH-(4-9) analog ORG 2766 yields peptides with high biological activity.

The behavioral effects of two peptides (HOE 427) and ORG 31433) related to the ACTH-(4-9) analog ORG 2766 were investigated in Wistar rats in a number of tests in which Org 2766 is active. Subcutaneous administration of HOE 427 in a dose of 0.5 ng/kg or ORG 31433 in doses of 0.5-5.0 ng/kg facilitated passive avoidance behavior whereas these peptides attenuated the avoidance response in doses of 25 ng/kg and 250 ng/kg respectively. ORG 31433 (0.1 - 1.0 microgram/kg) decreased motor activity of group housed rats tested under low light conditions. Furthermore subcutaneous (1.0- 10.0 ng/kg) or oral (10 microgram/kg) administration of ORG 31433 accelerated functional recovery from 6-hydroxydopamine (6-OHDA)-induced lesions in the nucleus accumbens which cause motor hypoactivity. The experiments show that as compared to ORG 2766 the peptides HOE 427 and ORG 31433 induce qualitatively similar responses but are approximately 10 to 100 times more potent. These data may imply that substitution of the C-terminal COOH group of ORG 2766 yields neuropeptides with increased potency.

Acid-catalyzed hydrolysis of peptide-amides in the solid state.

The hydrolysis of peptide-amides in the solid state at 4 degrees due to the presence of residual strong acid was investigated. It was found that even small molar excesses of these acids cause substantial deamidation in one day. The degree of amide hydrolysis depends not only upon the pKa and the volatility of the acid, but also upon the accessibility of the amide function; peptides with bulky C-terminal residues are more stable than the less-hindered ones.

Synthesis and 11C-labelling of the ACTH fragment analogue H-Met(O2)-Glu-His-Phe-D-Lys-Phe-OH (Org 2766) via its homocystine-containing precursor.

The hexapeptide dimer (H-Hcy-Glu-His-Phe-D-Lys-Phe-OH)2 was synthesized using solution methods and characterized. Its conversion into H-Met(O2)-Glu-His-Phe-D-Lys-Phe-OH, Org 2766, was studied on a small scale in as short a time as possible; reduction of the disulfide bond using Na/NH3, reaction with CH3I, oxidation with H2O2 and catalyst and purification by HPLC were carried out starting with 2 mg of the dimer in a total preparation time of approximately 22 min, starting with the addition of CH3I. The preparation of the 11C-labelled analogue was carried out by methylation with 11CH3I. Restrictions imposed by working with carbon-11 will be discussed.

Neuropeptide processing in regional brain slices: effect of conformation and sequence.

The central enzymatic stability of des-enkephalin-gamma-endorphin and its synthetic analogs [cycloN alpha 6, C delta 11]beta-endorphin-[6-17] and [Pro7, Lys(Ac)9]-beta-endorphin[6-17] was studied in vitro using a newly developed, regionally dissected rat brain slice, time course incubation procedure. Tissue slice viability was estimated as the ability of the brain slice to take up or release gamma-[3H]aminobutyric acid after high K+ stimulation. Results demonstrated stability of uptake/release up to 5 hr of incubation, suggesting tissue viability over this period. The estimated half-life of peptides based on the results obtained in our incubation protocol suggest that the peptides studied are metabolized at different rates in the individual brain regions tested. A good correlation exists between the high enzyme activity of neutral endopeptidase (EC 3.4.24.11) and the rapid degradation of des-enkephalin-gamma-endorphin and [cycloN alpha 6, C delata 11]beta-endorphin-[6-17] in caudate putamen. Proline substitution combined with lysine acetylation appears to improve resistance to enzymatic metabolism in caudate putamen and hypothalamus. However, cyclization of des-enkephalin-gamma-endorphin forming an amide bond between the alpha-NH2 of the N-terminal threonine and the gamma-COOH of glutamic acid did not improve peptide stability in any brain region tested. The present study has shown that the brain slice technique is a valid and unique approach to study neuropeptide metabolism in small, discrete regions of rat brain where peptides, peptidases and receptors are colocalized and that specific structural modifications can improve peptide stability.

Complementary information from isotachophoresis and high-performance liquid chromatography in peptide analysis.

Reversed-phase high-performance liquid chromatography is a valuable analytical technique to support the synthesis, isolation and purification of peptides, as is illustrated by some critical separations. In addition to this technique, capillary isotachophoresis can give useful information on the purity determination of peptides and on the presence of ionic compounds of a non-peptidic nature. With regard to the latter aspect, isotachophoresis proved to be a suitable technique as a check on the effective removal of salts after preparative high-performance liquid chromatography.

Des-enkephalin-gamma-endorphin (DE gamma E): biotransformation in rat, dog and human plasma.

Biotransformation of [3H-Lys9] DE gamma E was investigated after in vitro incubation of the tritiated peptide with rat, dog and human plasma. In addition, its metabolite profile in blood was studied following intravenous administration to rats and dogs. Half-lives for the in vitro disappearance of DE gamma E in plasma were 13.0 +/- 0.8 min (dog), 15.7 +/- 1.2 min (rat) and 19.2 +/- 0.9 min (human), indicating very rapid degradation of the peptide by proteolytic enzymes. Biotransformation products were identified on the basis of co-chromatography on HPLC with synthetic reference peptides. The six principal fragments appeared to be beta-endorphin (beta E) sequences 7-17, 8-17, 9-17, 6-15, 7-15 and 8-15. The abundance of beta E6-15, beta E7-15 and beta E8-15 in rat and human plasma suggests preferential, subsequent carboxypeptidase and aminopeptidase mechanisms, whereas in dog plasma DE gamma E is predominantly degraded by aminopeptidase activities (major peptide metabolites: beta E7-17 and beta E8-17). In the in vivo studies with rats and dogs the same radioactive peptide fragments were detected in blood as found in the in vitro experiments with plasma. In both species their blood levels were already maximal within a minute after intravenous administration of the parent peptide, thereafter they declined rapidly. 3H-Lysine was the main radioactive metabolite in vivo, exceeding 70% of total radioactivity in rat and dog blood 10 min after 3H-DE gamma E dosing.

[3H]9-desglycinamide,8-arginine vasopressin: metabolism and in-vivo fate.

Half-lives based on the disappearance of [3H]9-desglycinamide,8-arginine vasopressin ([3H]DGAVP) following in-vitro incubation in plasma were 1.7 h (dog), 5.8 h (rat) and greater than 12 h (man). For all three species, and particularly dogs, biotransformation of the peptide in plasma occurred predominantly through carboxypeptidase activities, leading to the accumulation of AVP-(1-7). Disappearance of [3H]DGAVP from rat blood after a single i.v. injection followed a biphasic decay with half-lives of 2.2 +/- 0.8 (S.D.) min (distribution phase) and 14.4 +/- 1.2 min (elimination phase). The central and peripheral volumes of distribution were high and of the same order of magnitude, being 0.21 and 0.25 litres/kg respectively. Blood clearance values ranged from 36 to 45 ml/min per kg. In addition to [3H]AVP-(1-7), [3H]tyrosine was also found to be a major radioactive metabolite in blood. Compared with i.v. dosing, the s.c. route of administration for [3H]DGAVP resulted in longer-lasting peptide levels in blood which persisted for up to 4-5 h after injection. Maximal concentrations were reached at 7.5 min, whereafter they declined bi-exponentially with terminal half-lives of 31.1 +/- 8.7 min. The mean bioavailability for DGAVP was almost 100%, demonstrating virtually complete absorption from the s.c. injection site.

A luminescent label for the immunoassay of oxytocin.

Chemiluminescent labels have been shown to be interesting alternatives to radioisotope labels. Disadvantages of the latter are preparation of e.g. labelled protein/peptides every four to six weeks, and problems with storage and disposal. Amino-Butyl-Ethyl-Isoluminol(ABEI) was attached to the alpha-amino function of the N-terminal amino acid residue of oxytocin; this complex was used in immunoassays for oxytocin. This non-isotopic label did not require heating at 60 degrees C for optimal light-signal development, a procedure usually required for chemiluminescent labels. Standard curves were set up employing the ABEI-label on the one hand and 125I-label on the other. Under identical conditions of final antibody concentration and amount of label, a comparison was made between the performance of the luminescent immunoassay (LIA) and that of the radioimmunoassay (RIA). We conclude that the LIA systems resulted in standard curves of high precision; in comparison with RIA, the sensitivity of the LIA curves is not yet sufficient for the determination of oxytocin concentrations in e.g. human biological fluids. Further improvements in sensitivity of the LIA systems are to be expected by selection of other luminescent labels or by the use of a more sensitive measuring device.

Inhibition of gamma-endorphin generating endopeptidase activity of rat brain by peptides: structure activity relationship.

gamma-Endorphin generating endopeptidase (gamma EGE) activity is an enzyme activity which converts beta-endorphin into gamma-endorphin and beta-endorphin-(18-31). The inhibitory potency on gamma EGE activity of neuropeptides and analogues or fragments of neuropeptides was tested. Dynorphin-(1-13) (IC50: 0.14 microM), human beta-endorphin-(1-31) (IC50: 15.5 microM), porcine ACTH-(1-39) (IC50: 6.3 microM), and substance P (IC50: 26 microM) had an inhibitory activity on gamma EGE activity. beta-Endorphin-(18-31) (IC50: 0.35 microM) but not gamma-endorphin potently inhibited gamma EGE activity. The IC50 of poly (Lys)40-60 was 0.8 microM. It is concluded that 1) gamma EGE activity is strongly inhibited by its product beta-endorphin-(18-31), 2) the enzyme is strongly inhibited by peptides with an aromatic amino acid at the NH2-terminal and/or basic amino acids in the COOH-terminal of the peptide chain.

Application of reversed-phase HPLC in some critical peptide separations.

The application of reversed-phase high performance liquid chromatography in peptide chemistry is illustrated. Its use is described for separation of closely related endorphins, as an optical purity criterion, for rapid determination of the species of origin of insulin preparations, and finally for analysis of small, basic (hydrophilic) peptides.

Oxytocin is a precursor of potent behaviourally active neuropeptides.

An oxytocin fragment which accumulated during the incubation of oxytocin with brain synaptic membranes was chemically characterized as the hexapeptide pGlu-Asn-Cys(Cys)-Pro-Leu-Gly-NH2 [( pGlu4, Cyt6]OXT-(4-9]. This peptide was approximately a hundred times more potent than oxytocin in attenuating memory consolidation as tested in a passive avoidance test situation; the dose-response relationship was bell-shaped. The des-glycinamide derivative [pGlu4, Cyt6]OXT-(4-8) was nearly as active, but showed a linear dose-response relationship. The data indicate that oxytoxin can act as precursor for potent behaviourally active neuropeptides.

A major metabolite of arginine vasopressin in the brain is a highly potent neuropeptide.

A peptide that accumulated as the major product during the proteolysis of arginine vasopressin by rat brain synaptic membranes was isolated and its structure was shown to be the hexapeptide pGlu-Asn-Cys(Cys)-Pro-Arg-Gly-NH2. When administered intracerebroventricularly in extremely low doses, this vasopressin fragment and its desglycinamide derivative facilitated memory consolidation in a passive avoidance situation. These vasopressin metabolites, which are devoid of pressor activity, constitute highly potent neuropeptides with selective effects on memory and related processes; they are activated via proteolytic processing of vasopressin by brain peptidases.

Analytical isotachophoresis in peptide chemistry. I. Analysis of anions in peptides.

The application of analytical capillary, carrier-free isotachophoresis as a rapid method (approx. 10-15 min per run) for the simultaneous determination of anions present in peptides (as counterion, contaminating ion or peptide anion) is described. Only small amounts of material, approx. 0.1-1.0 mg, are necessary to obtain information on the nature of the anion (qualitative) and of the amount of anion present (quantitative aspect).