Sandor Bajusz

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 First, Gly* has been replaced by a D-amino acid, the side-chain of which has been supposed to provide an extra binding-site for the receptor [l ] .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 To protect the fourth peptide bond of enkephalins, i.e., Phe4-x5, against proteolysis Met/I_eu’ has been replaced by Pro, resulting in a further increase of the in vivo biological activity [ 1 ] .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.

Comparative study on analgesic effect of Met5-enkephalin and related lipotropin fragments

HUGHES et al.1 have reported the isolation and structure of two pentapeptides from porcine brain with opiate agonist activity in isolated systems. The structure of one of these peptides, Met5-enkephalin, is identical with the sequence of pituitary β-lipotropin (β-LPH) between residues 61–65 (refs 2–4). To prove the biological correlation of brain enkephalin and pituitary β-LPH, a series of lipotropin fragments, LPH-(61–69)-5, LPH-(61–76)-6 and LPH-(61–91)-peptides7–10, have been isolated and shown to have opiate agonist activity in vitro. Only few and controversial data have been available so far on the analgesic effect in vivo of the above or similar lipotropin fragments. Enkephalins have recently been reported to induce analgesia in vivo11,12. Our preliminary data5,13, however, seemed to contradict these observations, rather suggesting that some larger fragment(s) of β-LPH may have analgesic properties. We have therefore compared the analgesic effects of Met5-enkephalin and some lipotropin fragments containing the complete structure of Met5-enkephalin at their NH2-terminus. The results show that the in vivo effect is a function of the length of the peptide chain, Met5-enkephalin being the least and LPH-(61–91)-peptide the most potent. During the preparation of this paper we have become aware of the recent observation of Bradbury and coworkers10,14 on a strong analgesic activity of LPH-(61–91)-peptide.

Attenuation of morphine tolerance and dependence by α-melanocyte stimulating hormone (α-MSH)

Abstract Repeated administration of morphine resulted in significant reduction of its analgesic potency. If 0.1 mg/kg α-MSH was coadministered, the tolerance development was attenuated, 1 mg/kg MIF (MSH release inhibiting factor), given simultaneously with morphine, did not affect tolerance. Injecting, however, MIF 1 hour prior to the daily opiate treatment resulted in accelerated development of tolerance supposedly by lowering the plasma α-MSH level at the time of morphine administration. Of the morphine abstinence symptoms the naloxone-induced jumping in morphine pretreated mice could not be modified either by α-MSH coadministration or by MIF pretreament, but the withdrawal body weight loss was found to be diminished by the former and increased by the latter peptide. The possible role of α-MSH in preventing the development of tolerance to the analgesic effect of endogenous opioid peptides is discussed.

Opioid agonist activity of β-lipotropin fragments: a possible biological source of morphine like substances in the pituitary

An endogenous peptide with opioid agonist activity, termed enkephalin, has been isolated from the porcine brain [ 1 ] , and more recently the primary structure of this pentapeptide has been elucidated: H-Tyr-GlyGly-Phe-Fe: -OH [2]. Hughes et al. [2] pointed out that the complete sequence of the major form of enkephalin, Met-enkephalin, is contained in the primary structure of pituitary /3-lipotropin between amino acid residues 61-65 [3-51. This sequence overlap raises the possibility that /I-lipotropin may be the biological precursor of enkephalin and/or other pituitary peptides with morphine-like activity. As an approach to this question the possible microheterogeneity of porcine /3-lipotropin at sequence position 65, and the opiate agonist activities of /3-lipotropin and some fragments of the hormone obtained by enzymic cleavages were investigated. The results of these studies are reported in this paper.

Effect of bacitracin on the biodegradation of β-endorphin

Abstract β-endorphin was incubated with rat brain homogenate, and the amino acids released were measured by amino acid analysis. Phe, Leu, Tyr, and Lys were liberated in the greatest amount indicating that the cleavage of Leu77-Phe78 and some Lys-X peptide bonds with endopeptidases followed by the removal of the terminal residues by exopeptidases are the main routes of β-endorphin degradation in the brain. Bacitracin considerably reduced the amino acid release from β-endorphin incubated with rat brain homogenate, and its action is suggested to be due to the inhibition of brain amino- and carboxypeptidases. Bacitracin also potentiated and prolonged the in vivo analgesic activity of β-endorphin.

Cross tolerance between morphine and β-endorphin in vivo

Experiments were performed to establish the development of cross tolerance between morphine and the C-terminal fragment of porcine β-lipotropin /LPH61–91, β-endorphin/ in rats. Repeated intracerebroventricular /ICV/ or peripheral administration of morphine induced tolerance both to morphine and β-endorphin. The tolerance induced by ICV administration of morphine was more pronounced than in the case of peripheral application. The results give support to the theory that at least in part similar sites and mechanisms are involved in the analgesic activity of morphine and the endorphins.

Enkephalin-like character and analgesia

The opioid activities of enkephalin analogues bearing D- or L-aminopentane-sulfonic/phosphonic acid at position 5 were studied in vitro, in electrically stimulated longitudinal muscle strip of guinea-pig ileum and mouse vas deferens preparations and in vivo in the rat tail-flick test. Using their in vitro effects Met-enkephalin-like, beta-endorphin-like, (nor)morphine-like and derivatives of intermediate character could be differentiated. Correlating the in vitro activities with the analgesic activity in vivo it is concluded that the enkephalin-like character in a pentapetide may hinder the expression of analgesic activity, when the compounds are given into the cerebroventricular system.

STRUCTURE-FUNCTION RELATIONSHIPS IN LIPOTROPINS

Twelve years ago Li and co-workers’, 2 reported the discovery of a new lipolytic polypeptide of adenohypophysis. Because of the considerable fatmobilizing effect of this polypeptide it was designated as lipotropic hormone (LPH) or 1ipotropin.l The lipolytic activity however, has not been a specific property of lipotropin; on the contrary almost all the adenohypophyseal hormones have lipolytic side-effect in addition to their main biologic function. This fact may, at least in part, explain why comparatively less attention has been paid to lipotropin in the last ten years. During this period of time only Dr. C. H. Li’s laboratory and few additional ones, namely the authors’, Dr. M. Chretien’s, Dr. Y. Pankov’s and more recently Dr. P. J. Lowry’s group, made efforts to elucidate the structure and to understand the physiologic role of these curious polypeptides. After the discovery of enkephalins-two brain pentapeptides structurally related to lipotropin-at the end of 1975,s lipotropin, this so far “useless” polypeptide, suddenly became the focus of interest and excitement, and in turn, the knowledge of the chemistry and biology of lipotropins acquired a special value. This paper reviews the most important results achieved in our institute during the last few years, but mostly in 1976, as regards the relationships between the structure and different biologic functions of lipotropins.

Tetrapeptide-amide analogues of enkephalin: the role of C-terminus in determining the character of opioid activity.

Abstract The opioid activities of tetrapeptide-amide analogues of enkephalin /H-Tyr-D-Met-Gly-Phe-NH2; H-Tyr-D-Nle-Gly-Phe-NH2/ were studied in isolated, electrically stimulated longitudinal muscle strip of guinea-pig ileum and mouse vas deferens preparations in vitro and in vivo in the rat tail-flick test. Their effects were compared to those of the parent L-Pro5-NH2 containing analogues, and to other enkephalin derivatives substituted with D-Met in position 2 and L-amino/imino acids of different character in position 5. It was found that whilst the opioid receptor in mouse vas deferens preferred aliphatic residues of acidic character at the C-terminus of pentapeptides and was highly sensitive to the removal of C-terminal amino acid, the other systems were either much less responsive to these changes, or the effects were opposite to those found in mouse vas deferens.

Enkephalin analogs containing amino sulfonic acid and amino phosphonic acid residues at position 5

Enkephalins, the endogenous opioid peptides: Tyr-Gly-Gly-Phe-Met and -Leu [ 11, have been known to show much higher agonist potency in the mouse vas deferens preparation (MVD) than in the guinea-pig ileum assay system (GPI) [2-61. The high MVD/GPI potency ratio is characteristic of enkephalins and of their synthetic analogs unless the pentapeptides are terminated by an amide function. For instance, potency ratios calculated for Met-enkephalin, its D-Ala2 analog and [D-Ala2, Met’]-enkeph~~ide were 9.0,8.4 and 2.0, respectively [2]. The MVDlGPI ratio for &endorphin, the 3 l-residue opioid peptide of the pituitary, is -1.2 and for nonpeptide narcotics, e.g., normorphine and morphine even smaller [2,4-61. Accordingly, the acidic COOH group at the C-terminus is essential for the ‘enkephalinoid’ character of pentapeptides. Starting from this consideration we synthesized some analogs of norleucine (Nle)-enkephahn in which the terminal COOH group is replaced by SOaH and POsH, group, respectively, i.e., Me’ is ~bstituted by a-amino~nt~e-s~fonic acid (NleS) and ff-amino-pentanephosphonic acid (MeP), respectively. The dissociation constant of a sulfonic acid or a phosphonic acid is known to be higher than that of the corresponding carboxylic acid. For instance, pKL values of taurine and p&nine are 1.5 and 3.6, respectively [7]. Similarly, pKH of glycine is 2.34 while the two pKH values of amino-methane-phosphonic acid are 1.85 and 5.35, respectively [S]. The more acidic terminal groups were expected to improve the enkephalin-like activity of the peptides. To study this question the opiate agonist potencies of the new compounds were