European Journal of Pharmacology - Molecular Pharmacology Section, 172 (1989) 417-423 417 Elsevier

EJPMOL 90040

Biologically active conformation of [Met s 1- and [Leu s ]enkephalin on 8 opioid receptor

Jfin Ga~par ik

Institute of Health, 821 01 Bratislava, Czechoslovakia

Received 20 April 1989, revised MS received 4 July 1989, accepted 21 July 1989

Investigations of the conformation of endogenous enkephalins are generally based on structural comparisons of enkephalins with other opiates and on experimental pharmacological studies. Based on such investigations, we now propose a novel model of the biologically active (receptor-bound) conformation of [MetS] - and [LeuS]enkephalin on the 8 opioid receptor. The model helps with the design of new opioid analgesics.

Opiates; [MetS]enkephalin; [LeuS]enkephalin; (Biologically active conformation)

1. Introduction

In 1975 oligopeptides with morphine-like activ- ity (Hughes et al., 1975), i.e. [MetS] - and [LeuS]en - kephalin, that react specifically with opioid recep- tors and are regarded as endogenous ligands of the opioid receptor system were found to exist in humans and experimental animals. The sequence of [MetS]enkephalin is identical to that of residue 61-65 amino acids contained in the pituitary C-fragment or to fl-endorphin (residue 61-91) that also has potent opioid activity (Bradbury et al., 1976b; Cox et al., 1976). Both these peptides, [MetS]enkephalin and fl-endorphin, are part of the structure of the pituitary hormone, fl-lipo- tropin, first isolated by C.H. Li et al. in 1965 (Li et al., 1965).

Several studies on, the conformation of en- kephalin in solution and in the crystalline state, together with theoretical energy calculations have yielded information about the conformational behavior and the various peptide conformations of

Correspondence to: Dr. Jan Gasparik, 1252 Pine Hill Drive, Annapolis, MD 21401, U.S.A.

enkephalin (for review see Schiller, 1984). It is supposed that the flexible molecule of the penta- peptide can take on various conformations in aqueous solution, and there is an equilibrium be- tween these conformations. According to one as- sumption only one of the various conformations already present might be recognized by the opioid receptor. Another alternative supposes that one or several of the conformers present in solution might undergo conformational changes when binding to the receptor. However, the receptor-bound confor- mation has not yet been optimized. Further, determination of the receptor-bound conforma- tion of endogenous enkephalins is complicated by the existence of/~, x (Gilbert and Martin, 1976; Martin et al., 1976) and 8 (Lord et al., 1977) opioid receptor subclasses that have different structural requirements. However, since the natu- ral enkephalins bind preferentially to ~ opioid receptors and have a reduced but still significant affinity to /~ receptors, it can be supposed that there are some similarities in the conformations.

The three-point model for opiate-receptor inter- action (Feinberg et al., 1976; Gorin and Marshall, 1977) requires the terminal aromatic amino acid, tyrosine, with its basic amine nitrogen in position

0922-4106/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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1 of the enkephalin and an additional hydro- phobic group within the peptide for stabilizing the receptor-active conformation. This second hydro- phobic group is represented by the phenylalanine residue in position 4 of endogenous enkephalins. The peptide backbone spacing between the two aromatic groups is responsible for secondary inter- actions with opioid receptor.

The common presence of a tyrosine residue in both the enkephalin and morphine suggested that the NH2-terminal tyrosine residue of enkephalin might represent the structural correlate of the tyramine part contained in morphine. The possi- ble correspondence of a common tyrosine residue emphasized the relevance of these findings to opioid structure-activity relationships and has formed the basis for structural comparisons be- tween enkephalin and opioid analgesics (Bradbury et al., 1976a; Feinberg et al., 1976). o-Methylation of the tyrosine hydroxy group of enkephalin re- suited in a dramatic decrease of morphine-like activity (Moritoki et al., 1981) to a level very close to that of derivatives of morphine, e.g. codeine. Acetylation (Ling and Guillemin, 1976) or the absence (Biischer et al., 1976) of the NH2-terminal amino group of enkephalin, which is essential for opioid activity, led to a nearly complete loss of activity. The N-allyl derivative of enkephalin (Pert et al., 1977) has partial antagonist properties remi- niscent of those of the N-allyl derivative of morphine, nalorphine, and the N,N-diallyl analog (Shaw et al., 1982) is a pure antagonist with activity similar to that of naloxone.

The second binding site in endogenous penta- peptide enkephalins is represented by the phenyl- alanine residue in position 4. Bradbury et al. (1976a) were first to propose that the phenyl- alanine side-chain of enkephalin and the phenethyl substituent of PEO (7a[1-(R-hydroxy-l-methyl-3- phenylpropyl)]- 6, 14-endo-ethanotetrahydrooripa- vine, fig. 1) might interact with the same receptor subsite. Feinberg et al. (1976) proposed that the uniquely high potency of phenazocine (fig. 1) and PET, an oripavine derivative similar to PEO with a methoxy instead of a hydroxy group on the benzene ring, is associated with a common locali- zation of ring P for both these drugs. Morphine and other opioid agonists lacking an appropriate

PEO

, o \ 1 % 1 c " ~ [ -

CH30 OH

Phenozocine

CH 3

Morphine

~ N-CH 3

OH

Methionine enkephalin 0 0 0 0 II II II PI

H2N~HC-NHCH2C-NHCH2C-N HCHC-NHCHCOOH I I

cH2

OH CH3

Demorphin

0 0 0 0 0 0 0 II II II II II H II II

H2NCHC-NHCH-C-NHCHC-NHCH2C-NHCHC-N--C-C- NHCHC-NH2 I I I I I I I

OH OH

Fig. 1. Structures of opioid analgesics. PEO is the abbreviation for 7a-[1-(R-hydroxy-l-methyl-3-phenylpropyl)]-6,14-endoeth-

enotetrahydrooripavine.

ring P generally have a weaker opioid activity. Binding of the benzene ring T of morphine and the amine nitrogen transforms the receptor to the 'agonist conformation'. The enhancement in potency of PEO, PET and phenazocine is however completed by stabilization of the agonist confor- mation through binding of ring P to the specific agonist binding site (fig. 1).

The 'minimal active fragment' of enkephalin could be represented by the structure Tyr-Gly- Gly-phenethylamide (Morley, 1980) which is the decarboxylated analog of Tyr-Gly-Gly-Phe, the smallest sequential analog of enkephalin to exhibit full opioid activity (Terenius et al., 1976). Like- wise, Tyr-D-Ala-Gly-phenethylamide has an in vitro activity similar to that of the tetrapeptide Tyr-D-Ala-Gly-Phe (Kosterlitz et al., 1980). The aromatic amino acids of both these parent en-

TABLE 1

Potencies of enkephalin analogs on the guinea pig ileum. Potencies are relative to [MetS]enkephalin (Tyr-Gly-Gly-Phe- Met) = 100 (from Vavrek et al., 1981).

Peptide Peptide structure Relative no. potency

Tyr-Gly-Gly-Phe-Met 100 Tyr-D-Ala-benzylamide 8 Tyr-D-Ala-phenethylamide 6 Tyr-D-Ala-phenytpropylamide 77 Tyr-D-Ala-phenylbutylamide 13

kephalin peptides - - Tyr-Gly-Gly-Phe and Tyr- D-Ala-Gly-Phe - - are separated from each other by a peptide backbone consisting of two amino acids. A potent opioid peptide agonist, der- morphin, isolated from frog skin was discovered in 1981 (Montecucchi et al., 1981). Although hav- ing less than a two-amino acid equivalent distance between the aromatic amino acids, tyrosine and phenylalanine, and therefore being different from endogenous opioid peptides, dermorphin has strong opioid activity. It becomes necessary to re-evaluate the requirement for the dipeptide 'con- necting chain'. In the course of elegant work on the structure-activity relationships of opiates, Vavrek et al. (1981) also investigated some di- and tripeptide analogs of the enkephalins with L- glycine in position 2 replaced by D-alanine, which however could be considered as dermorphin ana- logs. A series of these dermorphin-like aral- kylamides of dipeptide Tyr-D-Ala show interest- ing opioid activity (table 1).

2. Materials and methods

The biological activity of enkephalin analogs presented in table 1 was investigated on the stimulated guinea pig ileum, essentially as described by Chipkin et al. (1981).

3. Results

3.1. Localization of the Phe 4 binding site of endoge- nous enkephafins

The availability of these simple aralkylamido- peptides allows the localization of the phenyl-

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alanine binding site of endogenous enkephalins to be defined by direct drug-receptor interaction. The Tyr-D-Ala-phenethylamide, which can be considered as the decarboxylated analog of the dermorphin derivative, Tyr-D-Ala-Phe, has only weak opioid potency. It should, however, be the most potent analog of these four dermorphin-re- lated aralkylamidopeptides, if its benzene ring (necessary for potent biological activity) binds to the Phe 4 opioid receptor subsite as in the case of endogenous enkephalins. The phenylalanine re- sidue of dermorphin thus does not seem to act at the same binding site of the opioid receptor as does the phenylalanine residue of endogenous opiates. The pressure of only one amino acid 'chain' between Tyr I and Phe 3 of dermorphin does not allow the phenylalanine of dermorphin to occupy the Phe 4 binding site, as it does in the case of endogenous enkephalins. This suggestion, that there exist different phenylalanine binding sites for dermorphin is supported by the difference between the effect of nitrated enkephalins and of dermorphins. Nitration of the phenylalanine ben- zene ring of dermorphin leads to decreased activ- ity, while the nitrated benzene ring of phenyl- alanine provides an increase in potency of [LeuS]enkephalin (Schiller et al., 1983). Extending the alkyl chain and increasing the flexibility of the alkylamide chain yielded the most potent analog, Tyr-D-Ala-phenylpropylamide, of these four aral-

Tyr-D-Ala - benzy lamide Tyr - D-Ata- phenethyta m ide

0

Tyr-D-Atct- phenylpropylarn ide

0

Tyr- D-AIa- phenytbutyla mide

0

Fig. 2. Modified formulas of various aralkylamidopeptides. Localization of the benzene ring of phenylpropylamide is closely reminiscent of the analogous position of ring P of PEO

and phenazocine.

4 2 0

Methionine enkephalin

" ~ ~'~4~N ~ ~ Phe4

Gly 3 1~ ~ 4 Met5

/

PEO

Phenazocine

Leucine enkepholin

Gly 3 " ~ Leu , ¢ - -

Fig. 3. Biologically active conformation of [Met 5 ]enkephalin, phenazocine, PEO and [Leu 5 ]enkephalin (Dreiding models).

kylamido-peptides. The almost 10-fold increase in activity of Tyr-D-Ala-phenylpropylamide com- pared to the three others could be explained by the fact that the aromatic benzene ring, ring P, can reach a fixed position (benzene ring of Phe 4 of endogenous enkephalin) (fig. 2). Based on the above it can be shown that only the flexible di- peptide backbone spacing between the two aromatic amino acids Tyr I and Phe 4 is necessary, because it allows the phenylalanine of endogenous enkephalin to occupy its binding site on the opioid receptor and, thus, to achieve the biologically ac- tive conformation (fig. 3).

3.2. A model for stepwise binding of endogenous enkephalins to the 8opioid receptor

The considerations just discussed enable us to assume that one of the flexible peptide conformers of [Met5] - and [LeuS]enkephalin binds initially to the 8 opioid receptor by the tyrosine part of the molecule, and that the rest of the molecule then undergoes the conformational changes in several steps. First, the flexible peptide backbone takes up

its position between the two benzene rings of Tyr 1 and Phe 4, respectively, simultaneously giving rise to an intramolecular hydrogen bond between Phe 4 and Gly 2. The benzene ring of phenylalanine then takes its fixed position and, finally, methionine or leucine takes its position, giving rise to the second hydrogen bond between Met 5 or Leu 5 and Gly 3 (fig. 3). These hydrogen bonds strengthen the bio- logically active conformation which was created as a result of energy-requiring conformational changes. These active binding processes result in a stepwise formation of the peptide-receptor com- plex in accordance with a 'zipper' type mechanism (Burgen et al., 1975). The slow association and dissociation rate constants that had been found in binding studies with [MetS]enkephalin (Simantov et al., 1978) could then be determined from the conformational changes now found. On the other hand, the biologically active conformation of rigid opiates already present in solution permits a quick drug-receptor interaction without conformational changes, according to Fischer's lock-and-key model, e.g. morphine.

Each of many possible conformations of en- kephalin present in aqueous solution - - the en- vironment most similar to physiological condi- tions -- requires some amount of energy from active subcellular processes to effect the change from minimal energetic conformational forms in solution to biologically active forms with high conformational energy when bound to the recep- tor. Theoretically, the zipper type of ligand bind- ing could allow all flexible enkephalin molecules to bind to the opioid receptor, however, with various amounts of energy needed for the binding process. The conformer requiring the smallest amount of energy to overcome the energetic bar- rier for binding might be the first to occupy the opioid receptor. Thus the enkephalin will only take biologically active conformational form on its true, after it has bound actively to the opioid receptor.

Likewise, the biologically active conformation of the strong opioid analgesics, PEO and phenazocine, is reminiscent of that of [MetS]en - kephalin. Thus, PEO and phenazocine, with their benzene rings T and P as well as the amine nitrogen, all crucial for biological activity, can take positions similar to those in endogenous peptide ligands, [MetS]- and [LeuS]enkephalin (fig. 3).

4. Discussion

Since endogenous enkephalins have an affinity for the 3 opioid receptor only 10 times greater than that for the # subtype (Lord et al., 1977), there could be strong similarities in the active site of both binding sites. The fact that there is the same potency increase on introduction of a nitro substituent in the para position of the Phe 4 aromatic ring of cyclic enkephalin analogs such as H-Tyr-cyclo[NV-D-Dbu-Gly-Phe-Leu] as in linear enkephalins leads to the assumption that the cyclic analogs and the linear enkephalins share a com- mon mode of binding that involves interaction with identical receptor subsites (Schiller and Di- Maio, 1983). However, comparison of this cyclic analog with the corresponding open-chain analog, [D-Abu2,LeuS]enkephalinamide, showed that the

421

/~ receptor selectivity of H-Tyr-cyclo-[NV-D-Dbu - Gly-Phe-Leu] is a direct consequence of the con- formational constraint introduced through ring closure (Schiller and DiMaio, 1982). The high affinity and relatively good selectivity of this cyclic enkephalin peptide for the/~ receptor led Maigret et al. (1986) to propose the/x-active conformation for this cyclic enkephalin analog. A two-step bind- ing mechanism was subsequently proposed for the interactions of this cyclic peptide with the ~ recep- tor. The mechanism could be an intermediate be- tween the zipper model proposed for flexible lin- ear peptides and the lock-and-key model proposed for rigid opiates. The fact that the potency in- crease found on introduction of a nitro substituent in the para- position of the Phe 4 aromatic ring in the cyclic enkephalin analog, H-Tyr-cyclo-[NV-D- Dbu-Gly-Phe-Leu], was the same as in linear en- kephalins but with differences in affinity and selectivity for/ t over 8 receptors due to cycliza- tion, suggests similar conformations of enkepha- lins at 8 and at/~ receptors. These conformations would differ slightly because of subtle changes in peptide backbone conformation.

Although natural enkephalins bind prefer- entially to 3 receptors, they show a reduced but still significant affinity for /~ receptors. It can therefore be supposed that the Phe 4 of endoge- nous enkephalins can take similar positions on 3 and /~ receptor. Next to tyrosine binding to the Tyr ~ binding'site on the 3 or/~ opioid receptor, occupation of Phe 4 binding site by phenylalanine is the crucial condition for biological activity. The Tyr-D-Ala portion of the molecule of enkephalin- like aralkylamidopeptides isosteric to the Tyr-Gly fragment of endogenous enkephalins binds to the Tyr ~ subsite on the opioid receptor, so that only a flexible carbon atom chain determines the binding of the benzene ring of Tyr-D-Ala-phenylpro- pylamide to the Phe 4 binding site, resulting in maximal pharmacological effect.

Dreiding models indicate that if the two flat benzene rings of the Tyr I and Phe 4 of endogenous enkephalins take on their fixed positions, the peptide backbone takes its position between the two aromatic groups. It is assumed that if the benzene ring of Phe 4 of endogenous enkephalins is to take up the position of benzene ring P, the

422

peptide backbone must contract its form, a pro- cess that requires energy. The entire molecule of enkephalin thus takes on a bioactive conformation with high conformational energy. The intramolec- ular hydrogen bonds that thus arise contribute to the creation of the receptor-bound conformation.

Finally it could be concluded that the confor- mation proposed for the natural flexible linear peptides, [MetS]- and [LeuS]enkephalin, can be achieved on the 8 opioid receptor. However, since similar positions of Tyr 1 and Phe 4 of natural enkephalins could be achieved on 8 and # recep- tors, two questions arise: whether the peptide backbone of endogenous enkephalins takes on another conformation on the/~ opioid receptor, or whether the peptide backbone can take on the same conformation as on the 8 opioid receptor. However because of different conformational re- quirements for the peptide backbone on # recep- tors with different conformations, decreased bio- logical activity follows from incomplete occupa- tion of the backbone binding site. The fact that the substitution of glycine for D-amino acids at position 2 in synthetic enkephalins leads to en- hanced /~ selectivity, primarily by increasing # potency while holding the ~ potency relatively stable, might favour the latter hypothesis. Further studies on the relationship between the 8- and g-active conformations in relation to their opioid receptors are now in progress.

References

Bradbury, A.F.D., D.G. Smyth and C.R. Snell, 1976a, Biosyn- thetic origin and receptor conformation of methionine en- kephalin, Nature 260, 165.

Bradbury, A.F.D., D.G. Smyth, C.R. Snell, N.J.M. Birdsall and E.C. Hulme, 1976b, C-fragment of lipotropin has a high affinity for brain opiate receptors, Nature 260, 793.

Burgen, A.S.V., G.C.K. Roberts and J. Feeney, 1975, Binding of flexible ligands to macromolecules, Nature 253, 753.

Biischer, H.H., R.C. Hill, D. RSmer, F. Cardinaux, A. Closse, D. Hauser and D. Pless, Jr., 1976, Evidence for analgesic activity of enkephalin in the mouse, Nature 261,423.

Chipkin, R.E., D.H. Morris, M.G. English, J.D. Rosamond, C.H. Stammer, E.J. York and J.M. Stewart, 1981, Potent tetrapeptide enkephalins, Life Sci. 28, 1517.

Cox, B.M., A. Goldstein and C.H. Li, 1976, Opioid activity of

a peptide, B-lipotropin-/61-91/, derived from fl-lipo- tropin, Proc. Natl. Acad. Sci. U.S.A. 73, 1821.

Feinberg, A.P., I. Creese and S.H. Snyder, 1976, The opiate receptor: a model explaining structure-activity relationships of opiate agonists and antagonists, Proe. Natl. Acad. Sci. U.S.A. 73, 4215.

Gilbert, P.E. and W.R. Martin, 1976, The effects of morphine- and nalorphine-like drugs in the nondependent, morphine- dependent cyclazocine-dependent chronic spinal dog, J. Pharmocol. Exp. Ther. 198, 66.

Gorin, F.A. and G.R. Marshall, 1977, Proposal for the biologi- cally active conformation of opiates and enkephalin, Proc. Natl. Acad. Sci. U.S.A. 74, 5179.

Hughes, J., T.W. Smith, H.W. Kosterlitz, L.A. Fothergill, B.A. Morgan and H.R. Morris, 1975, Identification of two re- lated pentapeptides from the brain with potent opiate agonist activity, Nature 258, 577.

Kosterlitz, H.W., J.A.H. Lord, S.J. Patterson and A.A. Water- field, 1980, Effect of changes in the structure of enkepha- lins and narcotic analgesic drugs on their interactions with #-receptors and 8-receptors, Br. J. Pharmacol. 68, 333.

Li, C.H., L. Barnafi, M. Chretien and D. Chung, 1965, Isola- tion and amino acid sequence of fl-LPH from sheep pitui- tary glands, Nature 208, 1093.

Ling, N. and R. Guillemin, 1976, Morphinomimetic activity of synthetic fragments of/3-1ipotropin and analogs, Proc. Natl. Acad. Sci. U.S.A. 73, 3308.

Lord, J.A.H., A.A. Waterfield, J. Hughes and H.W. Kosterlitz, 1977, Endogenous opioid peptides: multiple agonists and receptors, Nature 267, 495.

Maigret, B., M,C. Fournie-Zaluski, B. Roques and S. Premilat, 1986, Proposals for the #-active conformation of the en- kephalin analog Tyr-cyclo-[Nr-D-A2-bu-Gly-Phe-Leu-], Mol. Pharmacol. 29, 314.

Martin, W.R., C.G. Eades, J.A. Thompson, R.E. Huppler and P.E. Gilbert, 1976, The effects of morphine- and nalorphine-like drugs in the nondependent and morphine- dependent chronic spinal dog, J. Pharmacol, Exp. Ther. 197, 517.

Montecucchi, P.C., R. de Castiglione, S. Piani, L. Gazzini and V. Erspamer, 1981, Amino acid composition and sequence of dermorphin, a novel opiate-like peptide from the skin of Phillomedusa sauoage, Int. J. Pept. Protein Res. 17, 275.

Moritoki, H., Y. Kiso, K. Kageyama and K. Matsumoto, 1981, Morphine-like activities of synthetic enkephalin analogues, J. Pharm. Pharmacol. 33, 54.

Morley, J.S., 1980, Structure activity relationships of enkepha- lin-like peptides, Annu. Rev. Pharmacol. 20, 8l.

Pert, C.B., D.L. Bowie, A. Pert, J.L. Morell and E. Gross, 1977, Agonist-antagonist properties of N-allyl-D[D-Ala2]-Met - enkephalin, Nature 269, 73.

Schiller, P.W., 1984, Conformational analysis of enkephalin and conformation-activity relationships, in: The Peptides, eds. V.J. Hruby and J. Meienhofer (Academic Press, New York) p. 219.

Schiller, P.W. and J. DiMaio, 1982, Opiate receptor subclasses differ in their conformational requirements, Nature 297, 74.

Schiller, P.W. and J. DiMaio, 1983, in: Peptides: Structure and Function, eds. V.J. Hruby and D.H. Rich (Pierce Chemical Co., Rockford) p. 269.

Schiller, P.W., T.M. Nguyen, J. DiMaio and C. Lemieux, 1983, Composition of mu receptor, delta receptor and kappa receptor binding sites through pharmacologic evaluation of paranitrophenylalanine analogs of opiate peptides, Life Sci. 33, Suppl. 1, 319.

Shaw, J.S. and M.J. Turnbull, 1978, In vitro profile of some opioid pentapeptide analogues, European J. Pharmacol. 49, 313.

Shaw, J.S., L. Miller, M.J. Turnbull, J.J. Gormley and J.S.

423

Morley, 1982, Selective antagonists at the opiate delta-re- ceptor, Life Sci. 31, 1529.

Simantov, R., S.R. Childers and S.H. Snyder, 1978, The opiate receptor binding interactions of [3H]methionine enkepha- lin, an opioid peptide, European J. Pharmacol. 47, 319.

Terenius, L., A. Wahlstr~m, G. Lindeberg, S. Karlsson and U. Ragnarsson, 1976, Opiate receptor affinity of peptides re- lated to Leu-enkephalin, Biochet-n. Biophys. Res. Commun. 71, 175.

Vavrek, R.J., L.-H. Hsi, E.J. York, M.E. Hall and J.M. Stewart, 1981, Minimum structure opioids - - dipeptide and tri- peptide analogs of the enkephalins, Peptides 2, 303.