Jan W. van Nispen

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 one-pot N-protection of L-arginine

A facile, one-pot synthesis of Nα-t-Butyloxycarbonyl,Nδ,Nϖ-di-benzyloxycarbonyl-L-Arginine (3a) and Nα,Nδ,Nϖ-tri-benzyloxycarbonyl-L-Arginine (3b) is reported. Nα-t-Butyloxycarbonyl-L-Arginine (1b) is treated with trimethylsilylchloride and the tri-silylated intermediate 2c is subsequently allowed to react with benzyloxycarbonyl chloroformate to give 3a in 50% overall yield. Starting from 1a or 1c, 3b was prepared according to the same procedure in 72% and 60–85% yield, respectively.

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)

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.

Chapter 6. Formation and Degradation of Neuropeptides

Publisher Summary Synaptic communication was long thought to be mediated solely by classical neurotransmitters such as monoamines, amino acids and acetylcholine. In the last decade, however, an increasing number of peptides have been found in CIS neurons. Leuropeptides can be defined as those peptides who affect communication between neurons either by direct transmitter action or indirectly via neuro-modulation. The availability of newer diagnostic and analytical techniques has greatly facilitated the identification of such peptides. The situation is further complicated by the frequent coexistence of neuropeptides with each other or with classical neurotransmitters. Co-transmitters may greatly increase the number of chemical signals available for neuronal communication. Neuropeptides are involved in homeostatic systems, including the regulation of pain, blood pressure, temperature, thirst, feeding, learning, memory and tropic function. Neuropeptides have also been implicated in neurological diseases such as Alzheimers disease, schizophrenia, huntingtons chorea and epilepsy, as well as in the immune system. The amino acid sequences of the majority of characterized neuropeptides have been provided in this chapter that discusses biosynthesis and post-translational processing of peptides. The discussion is on neuropeptides in mammalian CIS, the human POWC precursor molecule and products of its cleavage at paired basic amino acid residues. The chapter also provides details on enzymatic degradation of neuropeptides in the CNS.

Chapter 6 Neuropeptides and Their Processing: Targets for Drug Design

Publisher Summary This chapter discusses the enzyme inhibitors and neuropeptides as enzymes, as drugs still present serious problems in the context of access to the central nervous system (CNS). The chapter explains a previous survey of the formation and degradation of neuropeptides, and describes progress in designing drugs that can influence these processes. In principle, this includes enzymes, enzyme inhibitors, and neuropeptide agonists or antagonists. Precursors for neuropeptides processed at paired basic amino acid residues appear to share a common β-turn secondary structure around the cleavage site, while monobasic processing may be directed by adjacent proline residues. New endopeptidases include a rat metallopeptidase cleaving NT at the Pro-Tyr bond and a bovine enzyme cleaving the occupational therapy (OT) precursor on the carboxyl side of the Lys-Arg doublet. Rat brain may contain a specific thyrotropin-releasing hormone (TRH)-degrading enzyme that is distinct from the more widely distributed pyroglutaniyl peptidase. A free radical, alternative mechanism has been suggested for peptide α-amidation on the basis of studies with an abiotic system. New exopeptidases include a serine carboxypeptidase of porcine origin that processes human adrenocorticotropic hormone (ACTH) and β-LPH, and cathepsin B (a preferential peptidyl dipeptidase) that cleaves pro-opioid oligopeptides.

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

An in vitro autoradiographic study was performed to characterize specific rat brain binding sites for non-opioid neuroleptic-like γ-type endorphins, using [ 35 S]Met-des-enkephalin-γ-endorphin ([ 35 S]Met-DEγE; [ 35 ]S-β-endorphin(5–17)) with high specific activity as radioligand. The binding sites appeared to be confined to rat forebrain region, 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 [ 35 S]Met-DEγE (500 pM) with DEγE yielded an IC 50 of 0.6 nM whereas DEαE (β-endorphin(6–16)) had an IC 50 of 210 nM. Various endophins, sharing the γ-endorphin C terminus, displaced [ 35 S]Met-DEγE to the same extent as non-labelled DEγE (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 (μ) and DPDPE (δ) displaced [ 35 S]Met-DEγE to some extent at 10 −6 M whereas U69,593 (κ) was inactive, suggesting that the binding sites for γ-type endorphins may resemble μ- and δ-opioid receptors in some aspects. Similarly, relationships with dopamine receptors were investigated. Haloperidol partially displaced [ 35 S]Met-DEγE whereas sulpiride, SKF38,393 and 3-PPP at 10 −6 M did not induce significant displacement. Thus, binding sites are distinct from dopamine receptors. Finally, the monoclonal anti-DEγE anti-idiotypic antibody CR14.1 appeared to be a potent competitor of [ 35 S]Met-DEγE. The results obtained indicate that [ 35 S]Met-DEγE labels a specific class of binding sites in the brain. These binding sites are selective for γ-type endorphins, and are distinct from opioid and dopamine receptors. In view of their topography and binding characteristics, the binding sites for γ-type endorphins may be of relevance for the neuroleptic-like activity of these peptides.

Des-enkephalin-γ-endorphin (DEγE): Biotransformation in rat, dog and human plasma

SummaryBiotransformation of [3H-Lys9] DEΓE was investigated afterin 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 thein vitro disappearance of DEΓE 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 β-endorphin (βE) sequences 7–17, 8–17, 9–17, 6–15, 7–15 and 8–15. The abundance of βE6–15, βE7–15 and βE8–15 in rat and human plasma suggests preferential, subsequent carboxypeptidase and aminopeptidase mechanisms, whereas in dog plasma DEΓE is predominantly degraded by aminopeptidase activities (major peptide metabolites: βE7–17 and βE8–17). In thein vivo studies with rats and dogs the same radioactive peptide fragments were detected in blood as found in thein 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 metabolitein vivo, exceeding 70% of total radioactivity in rat and dog blood 10 min after3H-DEΓE dosing.

Behavioral activity of met-enkephalin and ACTH4–10 and other peptides containing a phenylalanine and methionine residue

Abstract Met-enkephalin, injected i.p. 5 minutes before training, decreased acquisition of a step-up active avoidance response in rats. ACTH4–10 exerted a similar effect if given i.p. within 15 minutes of training. ACTH4–10 and Met-enkephalin share a phenylalanine and a methionine residue. The possibility was studied that these amino acid residues are a structural requirement for this effect. Accordingly, some linear and cyclic peptides sharing the sequence Phe-Met were synthesized and tested. The cyclic peptide cyclo (-Phe-Met-e-Ahx-) decreased acquisition but with a longer time course of efficacy than for ACTH4–10 or Met-enkephalin and also after oral administration.

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.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.