Enkephalin Derivatives Research Articles

Photolabile opioid derivatives of D-Ala2-Leu5-enkephalin and their interactions with the opiate receptor.

Photolabile derivatives of D-Ala2-Leu5-enkephalin were prepared by synthetic procedures in which a 2-nitro-4-azidophenyl group is linked to the terminal carboxyl group of the enkephalin by means of an ethylenediamine or ethylenediamine beta-alanine spacer. These peptides bind to opiate receptors with nanomolar affinities and inhibit electrically stimulated contractions of the mouse vas deferens and adenylate cyclase activity of NG108-15 neuroblastoma x glioma hybrid cell membranes. Both inhibitions are reversed by the opiate antagonist naloxone. Photolysis of the ligands bound to rat brain membranes results in the loss of approximately 50% of the receptor sites. This decrease in receptor number is blocked by naloxone and requires light. A photolabile [3H]enkephalin derivative labels an equivalent number of sites under similar irradiation conditions.

Correlation of 15N and 13C relaxation data as a mobility probe for linear peptides. A theoretical and experimental study using enkephalins and related models

The potential of 15N NMR relaxation data for conformational studies of small linear peptides is discussed, using 15N enriched enkephalin derivatives as follows: Tyr- ∗Gly- ∗Gly- ∗Phe, I; Boc-Tyr- ∗Gly- ∗Gly- ∗Phe-OCH 3, II; and Tyr- ∗Gly- ∗Gly- ∗Phe- ∗Leu, III. 15N relaxation data ( T 1/NOE) are interpreted in terms of molecular motion using the simplified two-spin system assumption for the NH bonds. In that way, two different approaches are developed: (i) the comparison of the temperature variation of T 1 for 15N and for 13C α which shows the occurrence of concerted motion for linked 15N and 13C, nuclei along the peptide backbone; (ii) the correlation of 15N relaxation data pairs ( T 1/NOE) by means of different molecular motion models to check which best fits with the experimental results. The isotropic overall motion model with internal libration appears most appropriate. The 15N “antisymmetric” T 1 obtained with an INEPT-derived pulse sequence provide useful parameters to study the conformational preferences in free linear peptides, because of the sensitivity of these quantities to intramolecular exchange processes which are distance dependent. All the results support folded conformations for the free peptides I and III and a random one for the diprotected peptide II.

Studies on the release by somatic stimulation from rat and cat spinal cord of active materials which displace dihydromorphine in an opiate-binding assay

Using the spinal superfusion procedure, in anesthetized rats and cats, the presence of active factors which displace dihydromorphine in brain opiate binding studies, has been observed. Separation of this activity on a Sephadex G-10 column reveals the presence of two fractions which occur before (Fraction I) and after (Fraction II) the salt peak which account for over 70% of the observed dihydromorphine-displacing activity. The ratio of activity in Fraction II/Fraction I is 33 and 21, in the resting spinal perfusates of the rat and cat, respectively. High intensity, bilateral stimulation of the sciatic nerve in cats, results in a 30- and 5.4-fold increase in the levels of Fraction I and Fraction II, respectively, over pre-stimulation levels. In rat, bilateral stimulation of the hind paws, resulted in a frequency-dependent increase in the levels of Fraction I (1.9- and 3.2-fold at 5 and 50 Hz, respectively). Dynorphin 1–13 fragment elutes at least partly in Fraction I. With regard to Fraction II, the peak co-chromatographs with hexapeptide derivatives of enkephalin. Met- and Leu-enkephalin (Fraction III), elute off the column at a point where opiate receptor displacing activity is relatively small. Electrophoretic separation of Fraction I radioreceptor activity of alkaline and acid pH on agarose columns revealed two principle peaks which co-migrated with α-neoendorphin and dynorphin 1–13. Fraction II activity appeared primarily in a single peak which was isographic with enkephalin hexapeptides. Using radioimmunoassays, detectable levels of dynorphin and Met-enkephalin were observed and sciatic nerve stimulation resulted in significant increases. Neither column-coupled radioreceptor assays nor radioimmunoassays revealed the presence of ß-endorphin. The present experiments demonstrate the releasibility by high intensity somatic stimulation of a variety of opioid peptides present in spinal terminals. Significantly, however, the majority of this activity appears to be found in fractions different from those of the pentapeptide enkephalins.

Binding characteristics of a monoclonal beta-endorphin antibody recognizing the N-terminus of opioid peptides.

The present paper describes the isolation and characterization of a clone of hybrid myelomas (3-E7) secreting a mouse monoclonal antibody to beta-endorphin. An examination of its specificity against a series of human beta-lipotropin fragments and other opioid peptides revealed that the N-terminus portion of beta-endorphin is the determinant. Complete or almost complete cross-reactivity was obtained to methionine- and leucine-enkephalin, beta-lipotropin 60-65, and BAM 22; partial cross-reactivity was seen to dynorphin1-13 and alpha-neo-endorphin, whereas beta-lipotropin, alpha-N-acetyl-beta-endorphin, Des-Tyr1-beta-endorphin, in addition to a series of synthetic enkephalin derivatives, completely lacked cross-reactivity. The use of the monoclonal antibody in radioimmunoassay (RIA) for beta-endorphin resulted in a lower sensitivity related to respective polyclonal antibodies. An increase of 100% in tracer binding could, however, be obtained by use of beta-endorphin iodinated with its N-terminal tyrosine protected by coupling to an antibody. A solid-phase RIA was developed involving the internally 3H-labeled monoclonal antibody, which resulted in a 10-fold increase in sensitivity as compared with the homogenous RIA. These data indicate that for the binding to this antibody a tyrosine residue in position 61 is essential, and it thus recognizes a site that is of functional significance for many naturally occurring opioid peptides.

A quantitative structure-activity relationship study of the inhibitory action of a series of enkephalin-like peptides in the guinea pig ileum and mouse vas deferens bioassays.

The potencies of a series of enkephalin derivatives with general structure H . Tyr-D-Ala-Gly-X-Y . NH2 (X and Y are variable amino acid residues), to depress the contractions of electrically stimulated guinea pig ileum and mouse vas deferens preparations, were analyzed. The doses for half-maximal inhibition could be expressed as linear functions of three structural parameters--electronic, hydrophobic, and steric--which characterized the side chains of X and Y. A general methodology was devised for the quantitative estimation of these substituent parameters for amino acid side chains. The excellent statistics obtained for the equation of regression is an indication that no other parameters need to be considered to account for the opiate activity in this series. The relative importance of these factors and their intercorrelation were established, and the predictive value of the model was tested.

Characteristics of a monoclonal β-endorphin antibody recognizing the N-terminus of opioid peptides

The properties of a mouse monoclonal antibody to β-endorphin secreted by a clone of hybrid myelomas (3-E7) are described. The antibody displays virtually complete cross-reactivity to met-enkephalin and leu-enkephalin, but no cross-reactivity to β-lipotropin, α-N-acetyl-β-endorphin and des-Tyr 1-β-endorphin. Substantial cross-reactivity is seen with some other naturally occuring opioid peptides bearing the enkephalin sequence, such as dynorphin, α-neo-endorphin and BAM 22, but cross-reactivity is lacking in the case of certain synthetic enkephalin derivatives possessing a D-amino acid in position 2. The data indicate that for the binding of an antigen to the antibody the N-terminal tyrosine moiety is essential. The antibody recognizes, thus, a site which is of functional significance for the interaction of many naturally occuring opioid peptides with the opiate receptor.

Polymer-supported biopolymer synthesis: 1. High load solid (gel) phase strategy for peptide synthesis with a phenolic poly(acryloylmorpholine)-based support

An efficient, low-cost, reaction strategy for the solid (gel) phase synthesis of peptides and protected peptide segments has been developed. The strategy involves the use of a new poly(acryloylmorpholine)-based phenolic support matrix, Koch-Light Peptide Resin A. Illustrative syntheses of N-terminal Boc- and Z-protected[Leu]- enkephalin derivatives, including C-terminal acid hydrazides and esters, are described. The strategy, which is effective for the synthesis of peptides at high matrix loadings, is adopted readily for large-scale application.

Selectivity in the trimethylsilylation and acylation of peptide bonds, and its application to modification of the enkephalins

N.m.r. spectra of N-acylated peptides, formed by reaction of protected peptides with silylating agents followed by acylation, have provided a means for assessing selectivity in the acylation of amide bonds. Amino-acids such as valine and phenylalanine prevent significant acylation on neighbouring amide bonds while N-acylation occurs readily at glycyl amide bonds, one residue away from a hindered centre. Selective N-acylation of the Gly-Gly bond in enkephalin derivatives has been carried out using this methodology. Trimethylsilylation of Z-protected enkephalin derivatives provide a mild method for conversion into hydantoin (2,4-dioxoimidazoline) analogues of the enkephalins.

Further synthesis of enkephalinol analogs containing the dipeptide unit Tyr-Arg (kyotorphin).

In order to obtain enkephalin derivatives with high analgesic activity, four enkephalinol analogs having the Tyr-D-Arg unit in the N-terminal position were synthesized. Of these, H-Tyr-D-Arg-Gly-MePhe-Met (O)-ol exhibited analgesic activity comparable to that of morphine after intravenous administration to mice.

Electrophysiological interactions of enkephalins with neuronal circuitry in the rat hippocampus. I. Effects on pyramidal cell activity

Effects of enkephalins on hippocampal pyramidal cell activity were studied in situ and in the in vitro hippocampal slice. Active enkephalin derivatives produced a dose-dependent naloxone-reversible excitation in both preparations whereas inactive enkephalin derivatives had no effect. Several different types of experiments, carried out in the slice, strongly suggest that this excitation is due to blockade of inhibitory pathways. First, when the pyramidal cell population spike is increased during enkephalin administration, no change is seen in the simultaneously recorded EPSP. Second, the magnitude of the enkephalin effect is highly correlated with the amount of inhibition, as judged by paired-pulse stimulation, initially present in the slice. Third, if inhibitory pathways are depressed by a brief period of hypoxia, enkephalin has little effect. Finally, enkephalin responses are mimicked by picrotoxin, which selectively antagonizes inhibitory input to the pyramidal neuron. Since enkephalins do not block the effects of GABA, the putative inhibitory transmitter, these data suggest that opioid peptides depress the inhibitory interneurons and disinhibit the pyramidal cells.

Electrophysiological interactions of enkephalins with neuronal circuitry in the rat hippocampus. II. Effects on interneuron excitability

The effects of active and inactive enkephalin derivatives and naloxone on putative interneurons were studied in the in vitro hippocampal slice. Inhibitory interneurons were recorded from extracellularly, and identified electrophysiologically on the basis of their characteristic action potential shape and pattern of evoked firing in response to single and multiple electrical stimuli. Active enkephalin derivatives elicited a dose-dependent depression in excitability whereas inactive derivatives had no effect. Naloxone reliably and reproducibly antagonized the depressant action of active enkephalins. These data confirm the hypothesis outlined in the preceding communication, that the direct effect of enkephalins in the hippocampus is a depression of firing of inhibitory neurons, and support the hypothesis that enkephalin-induced excitations of pyramidal cells are brought about by disinhibition.

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

The opioid activities of tetrapeptide-amide analogues of enkephalin /H-Tyr-D-Met-Gly-Phe-NH 2; H-Tyr-D-Nle-Gly-Phe-NH 2/ 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-Pro 5-NH 2 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.

Photoaffinity inactivation of the enkephalin receptor.

An arylazide enkephalin derivative, [D-Ala2,Met5]enkephalin-Tyr-N-(2-nitro-4-azidophenyl) ethylenediamine (ETN), has been synthesized. In the dark, it inhibited the binding of [3H]enkephalinamide to enkephalin receptor-rich NG-108 cell membranes with an I50 = 2.2 X 10(-8) M or KI = 7 X 10(-9) M, assuming competitive inhibition. Photolysis of membranes in the presence of ETN caused irreversible inactivation of the enkephalin receptor, but inactivation was prevented by the addition of enkephalin, the half-effective concentration being 3 x 10(-9) M. ETN appears to be an effective photoaffinity label for the enkephalin receptor.

Enkephalin analogs. Introduction of stereochemical constraints, metal binding sites and fluorescent groups

There has been considerable speculation on the biologically active conformations of the enkephalins and their possible structural similarity to the opiates [1- 4]. A number of models have been proposed on the basis of theoretical calculations [2] and computer modelling [3]. Recent NMR studies suggest a high degree of conformational flexibility at ${Gly}^2$ and ${Gly}^3$ implying that a favoured conformation does not exist in aqueous solution [5]. The replacement of the Gly residues by Aib residues will greatly restrict the available conformations at positions 2 and 3 of the pentapeptides and may thereby allow a better definition of the steric requirements for biological activity. This approach appears particularly attractive in view of the extremely well defined conformations adopted by Aib containing peptides [6 -10]. An earlier report described the synthesis of ${Aib}^2$-${Met}^3$-enkephalin-amide [11]. Here we present the preparation and compare the biological properties of the ${Aib}^2$, ${Aib}^3$ and ${Aib}^2-{Aib}^3 {Met}^5$-enkephalin derivatives. The properties of 3-nitro-Tyr analogs, designed to bind paramagnetic NMR shift probes and a fluorescent ${Aib}^2$ analog developed for studies of receptor interactions, are also described.

Fluorescent and photo-affinity enkephalin derivatives: Preparation and interaction with opiate receptors

The fluorescent and photo-affinity derivatives of enkephalin, Tyr-D-Ala-Gly-Phe-Leu-Lys-N ε-Rhodamine (II) and Tyr-D-Ala-Gly-Phe-Leu-Lys-N ε-nitro-azidophenyl (III), were prepared by conventional methods followed by chemical modification. The two peptides inhibit the binding of 125I-labeled enkephalin to brain membrane preparations, with apparent IC 50 values of 5.9 nM and 5.5 nM for peptides II and III, respectively. The iodinated derivative of peptide III binds specifically to brain membrane preparations with an apparent K d of about 2.1 × 10 −9M.

Opiate activity of [DAla 2,Δ zPhe 4]-methionine-enkephalinamide

The opiate-like activity of [DAla 2,Δ zPhe 4, Met 5]-enkephalinamide (ΔFE) was evaluated in the stimulated guinea pig ileum assay and the mouse tailflick test. In vitro it caused a naloxone-reversible inhibition of the electrically induced twitch at a dose about one-fifth that of [DAla 2,Met 5]-enkephalinamide (DAE). The ED 50 for the analgesic effect of ΔFE after intracerebral injection was also approximately one-fifth that of DAE. However, ΔFE was not active after intraperitoneal injection. These data imply that some increases in the potency of enkephalin derivatives may not be entirely attributable to resistance to enzymatic degradation, but may be related to preferred structural conformation at the receptor.

Deprotection of O-methyltyrosine by a ‘push–pull’ mechanism using the thioanisole–trifluoromethanesulphonic acid system. Application to the convenient synthesis of a potent N-methylenkephalin derivative

The methyl group attached at the phenolic oxygen of tyrosine can be smoothly cleaved by a thioanisole–trifluoromethanesulphonic acid system; this deblocking method was successfully applied to the synthesis of a new potent enkephalin derivative, MeTyr-Gly-Gly-Phe-Metol (Metol = L-methioninol residue).

Fluorescent enkephalin derivatives with biological activity

Fluorescent dansylated derivatives of enkephalins were prepared, with the dansyl group either at the N- or C-terminal part of methionine-enkephalin (Met-E) or its peptidase-resistant analogue D-Ala 2-Met-E. An energy transfer, from the Tyr to the dansyl group, was found for all compounds and spectral properties were dependent upon the environment polarity. Met-E-(CH 2) 2 dansyl II and D-Ala 2-Met-E-(CH 2) 2 dansyl III exhibit biological activities on guinea-pig ileum and binding affinities on striatal membranes, similar to Met-E. III exhibits significant analgesic activity in mice after i.v. administration (about 35 % that of morphine). Fluorescent enkephalins potentially represent useful probes for the exploration of opiate receptors, studies of ligand-receptor interactions and evaluation of enkephalins cleaving peptidases in various tissues.

Comparison of the analgesic properties of lipotropin C-Fragment and stabilized enkephalins in the rat

The C-Fragment of lipotropin (LPH 61–91) was shown to produce strong and long-lasting analgesia on intraventricular infusion in the rat. Its analgesic properties were compared with those of three synthetic derivatives of methionine enkephalin (LPH 61–65) which had been stabilized against enzymic degradation by blocking one or both termini with N-methyl and C-amide groups. C-Fragment was approximately 50 times more potent on a molar basis than N-methyl methionine enkephalin amide. The singly blocked pentapeptides, like methionine enkephalin, produced little more than transient analgesia. It was concluded that the analgesic properties of C-Fragment depend on the length and nature of the peptide chain rather than on the resistance of its N-terminal pentapeptide to degradation.