A.Z. Rónai

A superactive antinociceptive pentapeptide, (D-Met2,Pro5)-enkephalinamide

Our recent study [ 1 ] on synthetic analogs of enkephalins (Tyr-Gly-Gly-Phe-X, X = Met and Leu [2] ) has revealed some structural requirements for a pentapeptide to exert antinociceptive property by intravenous application. First, Gly* has been replaced by a D-amino acid, the side-chain of which has been supposed to provide an extra binding-site for the receptor [l ] . To support this view the D-Met*’ analog was more active both in vitro and in vivo than the D-Ala* and even the D-Nle* ones [ I] . Based on other considerations the replacement of Gly* by different D-amino acids has also been accomplished by other workers [3-51. To protect the fourth peptide bond of enkephalins, i.e., Phe4-x5, against proteolysis Met/I_eu’ has been replaced by Pro, resulting in a further increase of the in vivo biological activity [ 1 ] . Finally, an alkyl amide group has been introduced into the COOH-terminus of the molecule with the purpose that it should furnish a further receptor binding-site. Ethyl amide appeared to be superior to amyl amide indicating the size of the alkyl amide-group to be crucial. As the most significant result of the above attempts, ethyl amide of (D-Met’, Pro’)-enkephalin has been reported to have 55% of the analgesic activity of morphine by intravenous injection [ I] . In this paper we report the synthesis of an even more potent analgesic: (D-Met*, Pro’)-enkephalinamide.

Dissociation of presynaptic α2-adrenoceptors following prazosin administration: Presynaptic effect of prazosin

Evidence has been obtained that prazosin possesses a presynaptic alpha 2-adrenoceptor blocking action in the rat was deferens preparation stimulated at 1 Hz. In the presence of prazosin yohimbine failed to affect the presynaptic alpha 2-adrenoceptor stimulatory effect of 1-noradrenaline while the effect of xylazine was almost completely abolished. It is therefore suggested that there are two kinetically different prejunctional alpha 2-adrenoceptor sites.

Further enhancement of analgesic activity: enkephalin analogs with terminal guanidino group

There is a great body of evidence that a basic amino-terminal is essential for the opioid activity of enkephalins, Tyr-Gly-Gly-Phe-Met and -Leu [ 1 I, and their analogs. Removal of the amino-group of Tyr results in a practically inactive peptide [2], as does its acetylation [3]. Introduction of two methylgroups into the terminal amino-moiety brings about a substantial loss in the biological activity [4], while mono-methylation decreases the potency in the mouse vas deferens preparation but enhances activity in the guinea-pig ileum [S]. It was tempting to speculate that interaction between the peptide N-terminus and its counterpart on the receptor (presumably a carboxyl-group) might be favourably influenced by replacing the terminal amino-group with a guanidino function. The resonance stabilized guanidinium cation has the capacity to interact.with some bidentate structure in the receptor site establishing hydrogen bridges in addition to the electrostatic interaction. One could anticipate that the improved binding properties of the peptide N-terminus would result in enhanced biological activity. It has also to be taken into account, however, that this replacement leads to a distal shift of the positive charge (~1.2 A). Such modification at the e-amino group of the lysine side chain in certain peptides, i.e., conversion of lysine into homoarginine residue, makes the lysine bond resistant to trypsin [6]. Thus trypsin seems to be unable to combine simultaneously with both the carbonyl-group and the positive charge of the basic residue when the distance between these is greater than occurs in a lysine or arginine residue. In this context one may recall that papain, which is less specific than trypsin, can hydrolyse homoarginine

Correlation between circular dichroism data and biological activities of 2, 5 substituted enkephalin analogues

Abstract The relative structural rigidity of enkephalin analogues characterized by the molar ellipticity data obtained from the circular dichroism spectra of peptides was correlated with the opioid agonist activities of compounds displayed in isolated, electrically stimulated longitudinal muscle strip of guinea-pig ileum and mouse vas deferens preparations. It was found that the so called μ receptors modelled by guinea-pig ileum preferred the analogues with high capacity to exist in folded form, whilst the so called δ receptors (mouse vas deferens) accepted flexible ligands as readily as rigid ones.

The conversion of met-enkephalin-Arg6, Phe7 to met-enkephalin in rabbit ear artery

We studied the opioid agonist activities of met-enkephalin-Arg6, Phe7 heptapeptide (ME-A,P) and its D-Ala2-analogue in several isolated organs including rabbit ear artery (ART), guinea-pig ileum (GPI) mouse-, rat and rabbit vas deferens preparation. Both heptapeptides followed the in vitro activity pattern of met-enkephalin (ME). There was a significant parallelism in the sensitivity of ART and mouse vas deferens to opioid peptides. In ART, ME-A,P must be converted by a captopril sensitive enzyme to exert opioid agonist action. Incubation of heptapeptide with membranes prepared from rabbit ear artery revealed the captopril-sensitive enzymic conversion of ME-AP to ME as detected by high voltage paper electrophoresis.

Opioid binding profile of morphiceptin, Tyr-MIF-1 and dynorphin-related peptides in rat brain membranes

Opioid properties of several morphiceptin- (Tyr-Pro-Phe-Pro-NH2), Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) and dynorphin-derivatives were characterized in rat brain in vitro receptor binding assay and in electrically stimulated longitudinal muscle strip preparation of guinea pig ileum. In the case of morphiceptin-related peptides, an excellent correlation was found between the [3H]-naloxone binding displacement data and the agonist potencies determined in the bioassay. The turning point was the C-terminal amidation in the tri- and tetrapeptide pairs in both series. Tyr-MIF-1 derivatives showed weak affinity in the opioid receptor binding assay and none of them had any remarkable effect in the bioassay either as agonist or antagonist. The dynorphin A(1-10)-peptides modified at positions 5 and 8 retained their affinity with Pro5-, Pro8-, and Ala8-substituents, whereas some loss of affinity was observed in the case of Gly8-Dyn A(1-10).

Irreversible labelling of the opioid receptors by a melphalan-substituted [Met5]enkephalin-Arg-Phe derivative

[Met5]enkephalin-Arg-Phe (Tyr-Gly-Gly-Phe-Met-Arg-Phe) was modified with the methyl esther of melphalan (Mel; 4-bis(2-chloroethyl)amino-L-phenylalanine) and the resulting compounds were studied for their opioid binding properties in guinea pig and rat brain membranes. Three new peptides, with a substitution of a single amino acid, were synthesized (Mel-Gly-Gly-Phe-Met-Arg-Phe, Tyr-Gly-Gly-Mel-Met-Arg-Phe and Tyr-Gly-Gly-Phe-Met-Arg-Mel). In the rat brain, none of these ligands displayed any type specificity, whereas in guinea pig brain membranes the C-terminally modified peptide, Tyr-Gly-Gly-Phe-Met-Arg-Mel ([Mel7]peptide), displayed a kappa-binding profile and was a weak kappa-opioid-receptor agonist in isolated guinea pig ileum. The effect of sodium ions on [Mel7]peptide competition against [3H]naloxone binding indicated a weak agonist nature of the compound. When guinea pig brain membranes were preincubated with 1-10 microM of [Mel7]peptide, an apparently irreversible inhibition of [3H]naloxone ligand binding was observed. These results suggest that the heptapeptide containing melphalan at the C-terminus can be used as a relatively high-affinity irreversible label for the kappa-opioid receptor.

Agonist-based, receptor-type selective opioid peptides containing an alkylating moiety

Our team has a long-held tradition of synthesizing agonist-based opioid peptides containing an alkylating moiety (e.g. 1-3) to facilitate, among other projects, the isolation of opioid receptor(s) (4). The presented data represent a kind of summary of these synthetic and pharmacological activities, producing Tyr-D-Ala-Gly-MePhe-chloromeihyl ketone (DAMCK), Tyr-D-Ala-Gly-PheAsp-β-chloromethyl ketone (DAACK) and Melphalan 1 -D-Ala-Gly-Phe-Leu-OH (Mel 1 -DALE) and their nearest non-alkylating structural relatives, with a more detailed description of the latter pair which represent the most recent developments

Inhibition by p-nitrophenylphosphate of acetylcholine release induced by Na+-deprivation

The effect of p-nitrophenylphosphate (p-NPP) on the release of acetylcholine evoked by drugs and ionic environments known to inhibit Na+, K+-ATPase was studied in isolated cortical slices of rat brain and longitudinal muscle strip of guinea-pig ileum. p-NPP inhibited the release of acetylcholine induced by sodium deprivation provided that the circumstances were in favour of the function of the K+-activated part of ATPase. However, it failed to antagonize the increase in the acetylcholine release elicited by omission of K+ or by administration of ouabain. Therefore it is concluded that the K+-stimulated phosphatase moiety of the Na+, K+-ATPase might be involved in the release of acetylcholine.

Novel, receptor-type selective tetra- and pentapeptide opioid antagonists, related to a natural sequence found in snake venom

The BOC-protected Tyr-Lys-Lys-Trp-Trp-NH 2 peptide, the original pentapeptide sequence found in the venom of Philippine cobra, and its partially modified derivatives showed significant μ opioid receptor antagonist activity in both the mouse vas deferens and guinea-pig ileum bioassays. Neither the N-protected, nor the de-blocked peptides had any appreciable agonist activity. No antagonist activity was detected after the removal of the N-terminal protecting group. The Tyr can be replaced by Phe with minor loss of activity. Omitting one lysine from the sequence (BOC-Tyr-Lys-Trp-Trp-NH 2 ) gave an antagonist structure with increased κ selectivity