Beta-endorphin: peripheral opioid activity of homologues from six species

The peripheral opioid activity of six homologous beta-endorphins (beta-EPs) were assayed on the guinea pig ileum and the vas deferens of the mouse, the rat and the rabbit. In the guinea pig ileum assay, human beta-EP (beta h-EP) was less potent than camel, turkey, and ostrich beta-EPs, of the same potency as equine beta-EP and more active than des-acetyl salmon beta-EP. In the rat vas deferens, mammalian beta-EPs showed higher activity than those from the bird and the fish, whereas in the mouse vas deferens assay, beta h-EP is more active than those from other species. In the rabbit vas deferens, however, all homologous beta-EPs show very weak activity. The relative potency of beta-EP homologues obtained from rat vas deferens assay is in good correlation with the analgesic potency, while the receptor binding activity does not correlate with any of the four bioassays, but appears to be related to the charge properties of the peptides.     

Beta-endorphin. Biological activity of synthetic analogs with analgesia inhibiting property in rat vas deferens and guinea pig ileum assays

Three synthetic analogs of human beta-endorphin (beta h-EP) (I, [Gln8, Gly31]-beta h-EP-Gly-Gly-NH2; II, [Arg9,12,24,28,29]-beta h-EP and III, [Cys11,26, Phe27, Gly31]-beta h-EP), which have been shown to possess potent inhibiting activity to beta h-EP-induced analgesia, were assayed in rat vas deferens and guinea pig ileum bioassay systems. In the rat vas deferens assay, relative potencies of these analogs were beta h-EP, 100; I, 30; II, 40; III, 1, whereas in the guinea pig ileum assay: beta h-EP, 100; I, 184; II, 81; III, 163. From previous studies on their analgesia potency in mice and opiate receptor-binding activity in rat brain membranes, their activity in rat vas deferens correlates well with the analgesic potency and the activity from guinea pig ileum assay shows good correlations with that from the opiate receptor-binding assay.     

Analgesic, receptor binding, and peripheral opioid activities of synthetic dermorphin-dynorphin hybrid peptides

The effects of substituting the enkephalin moiety of dynorphin with the dermorphin sequence were studied on the receptor preference, analgesic, and peripheral opioid potencies by using synthetic dermorphin-dynorphin hybrid peptides as the probe. Replacement of the enkephalin moiety of dynorphin with the dermorphin or dermorphin1-5 sequences caused a remarkable increase in analgesic potency, and a 3-6 fold increase in potency of binding against [3H]-dihydromorphine. The potency of receptor binding against [3H]-EKC was also increased by incorporation of the whole dermorphin sequence into the dynorphin molecule. In the presence of NaCl (100 mM), the effect of enhancing binding against [3H]-EKC due to dermorphin substitution disappeared, suggesting the contribution of opioid mu-receptor. Peripheral opioid activities assayed by various smooth muscle preparations showed that dermorphin incorporation caused a decreased in the potency of inhibition of the contractions of the guinea pig ileum and the rabbit vas deferens, no change in potency on the mouse vas deferens, and a marked increase in the inhibition of the rat vas deferens. Among the peripheral opioid activities only that assayed with the rat vas deferens appears to correlate approximately with the analgesic and the receptor binding activities. Judging from the relative potencies obtained from all assays, it is evident that the N-terminal dermorphin moiety, but not the C-terminal dynorphin fragment, dominates the opioid activity and receptor preference of the hybrid peptide.     

Binding characteristics of dermorphin, and [dermorphin1-7]-beta c-endorphin in rat brain membranes

Dermorphin and a camel beta-endorphin (beta c-EP) analog in which residues 1-7 correspond to the dermorphin sequence ([Dermorphin1-7]-beta c-EP) have been investigated with respect to their receptor binding characteristics using human and camel beta-EP as reference peptides. Tritiated dihydromorphine, [D-Ala2, D-Leu5]-enkephalin, ethylketocyclazocine and human beta-endorphin were used as primary ligands in the rat brain membrane preparation for radioreceptor assay. Camel beta-endorphin was the most potent peptide in all experiments. [Dermorphin1-7]-beta c-EP is significantly less potent towards 3H-ethylketocyclazocine and 3H-[D-Ala2, D-Leu5]-enkephalin but is as potent towards 3H-dihydromorphine and 3H-human beta-endorphin. Dermorphin itself weakly displaces tritiated dihydromorphine, [D-Ala2, D-Leu5]-enkephalin and ethylketocyclazocine (potency relative to camel beta-EP, 1-4%) but it is more potent (9%) in competition with tritiated human beta-endorphin. Dermorphin and the [Dermorphin-1-7]-beta c-EP appear to interact preferentially with mu opiate receptors.     

Synthesis and opioid activity of new fentanyl analogs

Three new fentanyl analogs (compounds 3-4-5) have been synthesized and evaluated for antinociceptive properties using the writhing test. The analgesic property of the active compound, N-[1-phenylpyrazol-3-yl]-N-[1-(2-phenethyl)-4-piperidyl)] propenamide (compound 4), was tested using the hot plate test in mice. Its opioid agonistic activity was characterized using three isolated tissues: guinea pig ileum, mouse vas deferens, and rabbit vas deferens. Compound 4 was as effective as fentanyl or morphine and it showed less antinociceptive potency than fentanyl but it was more potent than morphine. The duration of the antinociception was similar to that of fentanyl. This compound inhibited the electrically evoked contractions of myenteric plexus-longitudinal muscle strips of guinea pig ileum and of mouse vas deferens but not those of rabbit vas deferens. These effects could be reversed by micro selective antagonists (naloxone and/or CTOP) but not by the delta selective antagonist naltrindole, thus indicating that the compound acted as a micro opioid agonist. Finally, the binding data confirmed that compound 4 had high affinity and selectivity for the micro-receptor.     

Synthetic peptides related to the dermorphins. II. Synthesis and biological activities of new analogues

A new series of analogues of the potent opiate-like peptides dermorphins (mainly tetra- and pentapeptides) were synthesized in order to better evaluate the structure-activity relationships. Relative potencies were referred to dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂), the prototype of this class of frog skin peptides. Peripheral opioid activity (guinea pig ileum and mouse vas deferens) was determined for all the dermorphin analogues. For a selected number of them also central analgesic (hot plate and tail-flick tests) and cataleptic activities were assayed in the rat by intracerebroventricular administration.     

Opioid receptor binding profile of selected dermorphin-like peptides

The receptor binding profile of a selected group of dermorphin-like peptides was determined and correlated with the results of the guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays and with the currently used antinociception tests in the rat. For the peptides with the characteristic dermorphin D-Ala2-Phe3-Gly4 sequence, a linear negative correlation was found between the reciprocal of sodium shift and relative affinity for the mu-type opioid receptor. For the same peptides, a positive correlation was evidenced between relative potency on GPI and MVD and relative affinity for mu- and delta-type receptors, respectively.     

Examination of the requirement for an amphiphilic helical structure in beta-endorphin through the design, synthesis, and study of model peptides

In our approach to beta-endorphin modeling, we have proposed that the biological properties of the natural peptide are determined by the combination of three basic structural units: a highly specific opiate recognition sequence at the NH2 terminus (residues 1-5) connected via a hydrophilic peptide link (residues 6-12) to a potential amphiphilic helix in the COOH-terminal residues 13-31. In the alpha-helical conformation the hydrophobic domain twists around the length of the helix and covers almost one-half of its surface. The other distinctive features of the helix include its basicity and the two aromatic residues Phe18 and Tyr27. In contrast to previous models we have studied, peptide 4 is a "negative" model in the sense that it was designed and examined in order to determine how the lack of a well defined amphiphilic structure affects the biological properties of beta-endorphin. For this purpose, peptide 4 retains the three structural units previously postulated for beta-endorphin, but the amino acids of the 13-31 region are arranged in such a way that no definite continuous hydrophobic zone could be formed in an alpha- or pi-helical conformation of this region. In aqueous buffered solutions, peptide 4 showed almost the same amount of alpha-helical structure as beta-endorphin, with a slight tendency toward less helicity in 50% aqueous 2,2,2-trifluoroethanol. In rat brain homogenate, peptide 4 was degraded slightly slower than beta-endorphin, in contrast to the apparently much higher stability of previous models under the same conditions. With regard to opiate receptor binding, peptide 4 was twice as potent as beta-endorphin in mu-receptor assays but half as potent in delta-receptor assays. The opiate potency of peptide 4 on the guinea pig ileum was higher than that of beta-endorphin. In contrast, in the rat vas deferens assay, which is very specific for beta-endorphin, the potency of peptide 4 was very low and could be shown not to be mediated by the same opiate mechanism or by the same opiate receptor. A comparison of these results with those of previous model peptides provides further evidence for the importance of an amphiphilic helical structure in beta-endorphin residues 13-31, which determines the resistance to proteolysis of the natural molecule and contributes to the delta- and mu-opiate receptor interaction. The amphiphilicity of this helical structure must also be essential for high opiate activity on the rat vas deferens (epsilon-receptors), whereas no such structural requirement appears to be necessary for interaction with the opiate receptors on the guinea pig ileum.     

New analogues of oxotremorine and oxotremorine-M: estimation of their in vitro affinity and efficacy at muscarinic receptor subtypes

Two subsets of tertiary amines (1a-6a) and methiodides (1b-6b) with a structural resemblance to oxotremorine and oxotremorine-M were tested at rabbit vas deferens (M1), guinea pig left atrium (M2), guinea pig ileum and urinary bladder (M3) muscarinic receptor subtypes. The pharmacological profile of the derivatives under study has been discussed by evaluating their potency, affinity and efficacy as well as the regional differences in muscarinic receptor occupancy.     

Structure-activity studies of dermorphin. The role of side chains of amino acid residues on the biological activity of dermorphin

Seventeen analogues of dermorphin were synthesized and bio-assayed to determine the influence of side chains of the individual amino acid residues forming the sequence of dermorphin on the biological activity of this opioid peptide. Syntheses were carried out using solid-phase procedure, and the analogues obtained were purified by gel filtration on Sephadex G-10. Biological activities determined in guinea pig ileum (GPI) and mouse vas deferens (MVD) tests showed that the N-terminal tetrapeptide is responsible for the activity of dermorphin. Substitutions in the C-terminal fragment, particularly in position 5, for other amino acid residues results in substantial differentiation towards mu and delta receptors.     

14-membered cyclic opioids related to dermorphin and their partially retro-inverso modified analogues. I. Synthesis and biological activity.

As a continuation of our program to study structure-activity relationships of opiate peptides, we report the syntheses and biological activities of a series of 14-membered cyclic dermorphin analogues closely related to enkephalin analogue Tyr-c[D-A2bu-Gly-Phe-Leu] incorporating a phenylalanine at the third position in place of glycine. In addition to two parent dermorphin analogues Tyr-c[D-A2bu-Phe-Phe-(L and D)-Leu], four stereoisomeric retro-inverso modified analogues Tyr-c[D-A2bu-Phe-gPhe-(S and R)-mLeu] with a reversed amide bond between residues four and five, and Tyr-c[D-Glu-Phe-gPhe-(L and D)-rLeu] with two reversed amide bonds between residues four and five, and between residue five and the side chain of residue two have been synthesized. The results from the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays show that all analogues are superactive at either one or both opiate receptors and in general display higher activities as compared to the corresponding enkephalin analogues with a glycine at the third position. Results from the in vitro biological assays and conformational analysis using 1H-NMR spectroscopy (adjoining paper) will provide useful information to understand the role of the Phe3 aromatic side chain in dermorphin, and that of the Phe4 aromatic side chain in enkephalin, on opiate activity since these cyclic dermorphin analogues contain two Phe residues at both the third and fourth positions.     

Structure-activity relationship of rubiscolins as delta opioid peptides

To study the structure-activity relationship of rubiscolins (YPLDLF and YPLDL), delta opioid peptides derived from the spinach Rubisco, we substituted the amino acid residues and evaluated their activities by mouse vas deferens (MVD) and guinea pig ileum (GPI) assays as well as receptor affinity. Replacement of Leu(3) with Ile and Met in rubiscolin-6 potentiated the delta opioid activity by about four times in MVD assay. Asp(4) cannot be replaced by Ala, Glu or His. The original Leu(5) was optimal, while substitution of Phe(6) with Val potentiated its delta opioid activity by more than 10 times. The most potent derivative we obtained was YPMDLV, which was nearly 20 times more potent than rubiscolin-6 in MVD assay. The derivatives thus obtained showed higher delta receptor affinity and more potent antinociceptive activity than rubiscolins.     

In vitro pharmacological profile of peptide III-BTD: a novel ligand for nociceptin/orphanin FQ and opioid receptors

Peptide III-BTD has been recently identified as a non-selective nociceptin/orphanin FQ receptor ligand by screening of a synthetic peptide combinatorial library. In the present study we evaluated the pharmacological profile of peptide III-BTD in isolated tissues (mouse and rat vas deferens, guinea pig ileum) sensitive to both nociceptin and opioid peptides. In the mouse vas deferens and guinea pig ileum, III-BTD concentration dependently inhibited the electrically induced twitch (pEC50 5.91 and 6.18, respectively; Emax 94 +/- 1% and 94 +/- 2%) and this effect was blocked by naloxone (1 microM). In the rat vas deferens, III-BTD was inactive in most of the tissues, while in few others it elicited a slight inhibition only at the highest concentration tested (10 microM). In the presence of 1 microM naloxone, 1 microM III-BTD shifted to the right the concentration response curve of nociceptin in a parallel manner, showing pKB values in the range 6.6-6.9. These data confirm on native nociceptin receptors the pharmacological profile of peptide III-BTD which behaved as a mixed nociceptin receptor antagonist/opioid receptor agonist in the [35S]GTPyS binding assay performed on cells expressing the recombinant human receptors.     

A pituitary endorphin with novel properties.

We describe the further purification of an opioid peptide from a porcine pituitary concentrate. The peptide has typical naloxone-reversible opioid activity in the guinea pig ileum myenteric-plexus preparation and mouse vas deferens, and it inhibits stereospecific binding at opiate receptors. It is distinguished from β-endorphin and the enkephalins by its apparent molecular weight, its slow reversal with washing in the guinea pig ileum preparation, and the resistance of its biologic activity to cyanogen bromide treatment. In beef pituitary, slow-reversing, cyanogen bromide resistant activity is found principally in neurointermediate lobe.     

High and low affinity opioid binding sites: relationship to mu and delta sites

Binding and pharmacological studies suggest a common opiate and enkephalin binding site in addition to their previously reported selective sites. This common high affinity site has tentatively been named mu1, distinguishing it from the morphine-selective site (mu2) and enkephalin-selective site (delta). The existence of this additional common high affinity site and its association with opiate and opioid peptide analgesia may help explain some pharmacological observations, such as the cross tolerance between morphine and enkephalin analgesia and the lack of cross tolerance between them in the guinea pig ileum and mouse vas deferens bioassays.     

Opioid activities of beta-casomorphins

β-Casomorphin-7 (H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-OH) and its analogues: β-casomorphin-6, (-5) and (-4) (derived by sequential removal of respectively one, two or three amino acid residues from the C-terminus), were tested for their opioid activities in a variety of assay systems. Each of the four peptides displayed opioid activity in an opiate receptor binding assay, the isolated mouse vas deferens (MVD), the guinea-pig ileum longitudinal muscle myenteric plexus preparation (GPI) and produced naloxone-reversible analgesia after intracerebroventricular injection into rats. In contrast, none of the peptides displayed opioid activity in the isolated rat vas deferens preparation (RVD). β-Casomorphin-5 was the most potent compound in all the assays employed. Each β-casomorphin was more potent on the GPI than on the MVD. In view of the fact that the GPI, MVD and RVD are populated predominantly by μ-, δ- and ε-receptors, respectively, the β-casomorphins probably represent μ-type opiate receptor agonists.     

Humoral endorphin: A new endogenous factor with opiate-like activity

Humoral (H) endorphin, a novel endogenous opioid ligand detected in brain, blood and cerebrospinal fluid was tested in a series of opiate sensitive assays. H-endorphin displaced radiolabeled enkephalin from its specific bindings sites and inhibited the electrically evoked contraction of the guinea pig ileum and mouse vas deferens. When injected to unanesthesized animals, humoral endorphin induced analgesia in rats and mydriasis in mice. The activity of H-endorphin both $\text{in}\text{vitro}$ and $\text{in}\text{vivo}$ attests to its opioid nature. However, while its antinociceptive effect was blocked by naloxone, mydriasis induced by H-endorphin was resistant to the effect of the opiate antagonist. Similarly, intermediate concentrations of naloxone inhibited the effect of H-endorphin on the guinea pig ileum while its effect on the mouse vas deferens was completely refractory to naloxone. The physiological function of humoral endorphin in various naturally occuring states that show similar paradoxical interactions with naloxone is discussed.     

A novel opioid peptide, leumorphin, acts as an agonist at the kappa opiate receptor

The primary structure of the common precursor of porcine beta-neo-endorphin and dynorphin (preproenkephalin B) has shown the existence of a third leucine-enkephalin (leu-enkephalin) sequence with a C-terminal extension of 24 amino acids. This nonacosapeptide, named leumorphin, was approximately 70 times more potent than leu-enkephalin in inhibiting the contraction of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum. This action of leumorphin, like those of beta-neo-endorphin and dynorphin, was antagonized less effectively by naloxone than that of leu-enkephalin, but more effectively by Mr2266, an antagonist relatively specific for the kappa type opiate receptor. The inhibitory action of leumorphin or beta-neo-endorphin on the contraction of the guinea pig ileum muscle strip was reduced in a dose-dependent manner by pretreatment with dynorphin and vice versa. Leumorphin as well as beta-neo-endorphin and dynorphin inhibits the contraction of the rabbit vas deferens which is known to have only the kappa type opiate receptor. This action was also effectively antagonized by Mr2266. It is concluded that leumorphin has potent opioid activity and acts at the kappa receptor, like other opioid peptides derived from preproenkephalin B.     

Glycoconjugates of opioid peptides. Synthesis and biological activity of [Leu5]enkephalin related glycoconjugates with amide type of linkage.

Three N-glycoconjugates of the general formula H-Tyr-Gly-Gly-Phe-Leu-NH-R (R = carbohydrate residue) were synthesized in order to determine the influence of some carbohydrate molecules (6-amino-6-deoxy-D-glucopyranose, 2-amino-2-deoxy-D-glucopyranose, beta-D-glucopyranosylamine) on the biological activity, conformation, and stability of the opioid pentapeptide [Leu5]enkephalin. For the preparation of this compound different methods of peptide synthesis (active ester and mixed anhydride) were investigated. In comparison with [Leu5]enkephalin, all three N-glycoconjugates showed higher potency in the guinea pig ileum assay and lower potency in the mouse vas deferens assay, indicating a decrease in delta opioid receptor selectivity.     

Comparative study on the effect of morphine and the opioid-like peptides in the vas deferens of rodents: species and strain differences, evidence for multiple opiate receptors

The effect of an opiate alkaloid and an opioid-like peptide was studied on the electrically evoked twitching of the vas deferens of 3 common laboratory rodents. Normorphine and the synthetic opioid peptide D-Alanine₂ methionine enkephalinamide (D-Ala₂) produced dose dependent inhibitions of the twitching response in the mouse vas deferens. In the rat vas, while β-endorphin (β-EP) caused an inhibitory effect in three strains of rats to a similar degree, morphine produced a dose related enhancement of the twitching. In the guinea pig, both morphine and β-EP caused an increased in the muscular twitch. The results are interpreted in terms of an heterogenous mixture of opiate receptors present in the vas deferens from these rodents. The mouse appears to contain mainly δ receptors while the rat has mostly ε receptors characterized by their specificity and sensitivity to the action of β-EP. The guinea pig vas deferens has apparently lost the sensitivity to the inhibitory influence of the opioids, suggesting the absence of μ or δ opiate receptors in this tissue.