Int. J . Peptidekotein Res. 15, 1980, 122-129

M E T H I O N I N E ENKEPHALIN A N D I S O S T E R I C A N A L O G U E S Part 11: Receptor Conformation of Methionine Enkephalin

DEREK HUDSON, ROBERT SHARPE and MICHAEL SZELKE

Department of Chemical Pathology, Royal Postgraduate Medical School, London, England

Received I 2 February, accepted for publication 10 May 1979

Biological activities are reported for two different types of analogues of methionine enkephalin. Cyclic analogues, bridged between the amino- and carboxy- terminals of the parent peptide, are inactive. In contrast, significant levels of activity are displayed by linear isosterically modified analogues in which the Tyr' -Cly2 peptide bond is replaced by either -CH,NH- or -CH2CH2-. Similar replacements o f the GIy2-Gly3 peptide bond yield compounds with much reduced potency. These modifications serve as useful probes of the receptor conformation. Based on these findings, a model is proposed for interaction between enkephalin and its receptor.

Key words: methionine enkephalin; isosteric analogue; opiate receptor; receptor confor- mation

It has been stated recently that the elucidation of the conformation of the analgetic pharmaco- phore is one of the most challenging problems in medicinal chemistry (Clarke et af., 1978). A knowledge of how enkephalins bind t o the opiate receptor will lead t o the rational design of clinically useful opiates and antagonists, as well as giving a more general insight into the receptor interaction of other biologically active peptides.

Although certain features of the opiate receptor have been deduced from structure- activity studies on conformationally rigid alkaloid derivatives (Lewis e t aL, 1971), there are still many questions to be answered. Widely differing conclusions on the receptor confor- mation of enkephalin have been drawn from spectroscopic studies in solution, and from conformational energy calculations. Much of

Abbreviations: The abbreviations used are as in Part I with the addition of PEO to stand for 7-11-phenyl-3- hydroxybutyl-3-] pndoethenotetrahydrooripavine.

the speculation is based on structural resemblance between methionine enkephalin and nonpeptidic opiates. The picture is further complicated by the use of different test systems, by the existence of various types of opiate receptor with differing requirements, and by the possibility that different types of analgetic may bind to different conformational forms of the same receptor.

Solution studies, such as 'H n.m.r. (Roques etal., 1976;Joneset al., 1976) and fluorescence spectra (SchilIer, 1977; SchiIler et al., 1978), suggest a type 11' 0-bend centered on Gly3 -Phe4 stabilised by a hydrogen bond from the carbonyl of Gly' to the amino group of Met' (GP 0-bend). Conformational energy calculations point to a multiplicity of structures, as does circular dichroism (Spirtes e t aL, 1978). Most authors agree that the receptor conformation involves a GP 0-bend. Some (Isogai e t al., 1977; Clarke et al., 1978) conclude that the crucial tyrosine side-chain is orientated inward towards the &bend, perhaps accompanied by the

122 036 7-8377/80/020 122-08 $02.00/0 0 1980 Munksgaard, Copenhagen

RECEPTOR CONFORMATION OF MET-ENKEPHALIN

formation of a hydrogen bond between the phenolic hydroxy group and the peptide back- bone. Others (Bradbury et al., 197&; h e w & Burt, 1978; Smith & Griffin, 1978) suggest an opposite outward facing orientation for the tyrosine side-chain. Smith & Griffin (1978) proposed that Leuenkephalin binds to the receptor in a similar form to that found in the solid state by X-ray crystallography. This structure is described as a rigid framework based on a &bend centered on the Glyz-Gly3 sequence (GG &bend). it is stabilised by two intiamolecular hydrogen bonds, one between the carbonyl of Tyr’ and the -NH-group of Phe4, and another between the terminal amino group and the carbonyl of the Phe4. This shows considerable resemblance to the original proposal of Bradbury et al. (197642) for Met- enkephalin, except that in this earlier suggestion 4 -+ 1 hydrogen bonding only is implicated, and the C-terminal amino acid residue is positioned so that the methionine side-chain lies between the two aromatic rings. This brings the thiomethyl group into a similar orientation to that of the nonaromatic C6 hydroxy group of morphine and the corre- sponding methoxy group of the oripavine alkaloids. In the structure proposed by Smith & Griffin the carboxy terminus is thought to correspond to this important polar group. In an alternative proposal (Go& & Marshall, 1977) the Tyr’ and Phe4 side chains are brought into close proximity such that the meta and para positions of the Phe4 aromatic ring correspond to Cs and C6 of morphine. Other workers have suggested that the amino and carboxy groups are near enough to form a salt bridge (Garbay- Jaureguiberry et al., 1976; Jones et al., 1977; Balodis et al., 1978).

The study of conventional peptide analogues of the enkephalins, prepared by modification or replacement of amino acid residues, has thrown some light on the importance of side- chains and their configuration for productive receptor interaction. This paper reports the biological activity of two types of analogues:-

(a) cyclic molecules in which the amino and carboxy termini of enkephalin are linked by a bridging group, in order to test the importance of having the termini in close proximity and,

@)linear analogues in which some of the peptide bonds are replaced with CHz-NH- O r

-CHZ -CHz - groups in order to assess the contri- bution of the peptide backbone to receptor binding.

The synthesis o f these analogues is described in the preceding paper (Part I of this series).

The simplest example of the first type of andogues is H 2 13, cyclo(-Gly-Tyr-Gly-ely-Phe- Met-), in which a glycine residue connects the termini. This compound, however, is extremely insoluble and therefore impossible to assay. Additionally, in H 21 3 the crucial “tyramine” nitrogen atom is present as an arnide and is no longer capable of bearing a positive charge. H2 14, cyclo [ - (NHCH, CH,)TyrClyGly-Phe- Met-], was prepared to overcome both de- ficiencies. In this analogue bridging is by a 2- aminoethyl group; the tyrosine nitrogen being a basic secondary mine. As its hydrochloride H214 is water-soluble, but is totally inactive in both in vivo assays (rat tail flick assay after either intracerebroventricular or intravenous administration of large doses) and in v i m assays (guinea pig ileum, mouse vas deferens and brain membrane radioreceptor assays). In the design of these compounds it was assumed that they would adopt conformations like those of the long established intrarnolecularly hydrogen bonded cyclic hexapeptides (Schwyzer, 1959). Perhaps the inactivity of H 2 1 4 resides in incorrect orientation of the amino group as well as of the methionine side- chain, which is prevented from adopting either the conformation proposed by Bradbury et al. (197@), or indeed that describec later in this paper.

Table 1 shows the structures anld activities of linear analogues of methionine enkephalin modified by replacement of the Tyr’ -Gly2 and Gly2-Gly3 peptide bonds with either -CH2 -CH2- (“hydrocarbon” analogues) or -CH2 -NH- (“reduced” analogues). Such isosteric rnodifi- cation of widely differing classes of peptides gives rise to active analogues; for example, gonadoliberin (Szelke et al., 1077) and an inhibitory tetrapeptide of renin fParry et al., 1972). All the enkephalin analogues shown in Table 1 are definitely, if only weakly, active in both the H-naloxone brain membrane receptor assay and the guinea pig iIeum isolated tissue assay. The reduced isosteric peptides are more active than their hydrocarbon counterparts. Substitution of the Tyr’ S l y 2 bond gives rise

123

II 211

I 1 21s 1 x 10.6 50

3

56

I ('I I * I

I 1 216 NH, ~HCH,NtiCH:COCly-Phe-Metol 2.5 x lo-' 200 8 3

I I 2 1 2 11- ~ y r - N l l C l l , CH CH. CH CO-Phe-Met-NH 5 x 10-5 1 < 0.3

II 218 ti-Tyr-NIICH ,CH, NHCti ,CO-Phe-hlet-OH 1 x 10-5 5 < 1

I I 219 ll-T~r-NllCll~ Cll NIICH, CO-Phe-Metol 1.5 x 10-5

Ii 220 I I-Tyr-NHCII ( 7 1 NtICH , CO-Phe-Met-NH 1.7 x 10-5

3 0.3

3 0.3

to analogues with high activity. In both of the assay systems methionine enkephalin amide is known to have a similar activity to methionine enkephalin (Frederickson er al., 1976). Additional combination of C-terminal methion- inol (Roemer er al., 1977) with the Tyr' Gly' reduced isostere gives H216. which is more potent than the parent peptide in the radio- receptor assay.

The tyrosine carbonyl group is not important for activity. However, comparison of the isomeric peptides H 2 16 and H 2 19, as well as of H215 with its isomer H220, shows that reduction of the Gly' carbonyl rather than the Tyr' carbonyl decreases activity by a factor of 20 to 60 in the radioreceptor assay, and by a factor of 180 to 280 in the guinea pig ileum assay. Fig. 1 shows displacement curves in the radioreceptor assay for naloxone and the reduced isosteric analogues. Despite their low activity, H218, H219 and H220 still possess full efficacy and the displacement curves parallel closely that produced by methionine 124

enkephalin but not that of naloxone. The Gly2 carbonyl is important but not essential for activity.

If enzymic degradation occurred to a signifi- cant extent in the assay systems then the relative activities might not be attributed directly to differences in receptor affinity. Methionine enkephalin would be expected to be much more susceptible to both amino- and endopeptidase digestion than the isosteric analogues themselves. 5-Norleucine enkephalin was shown to be stable to the radioreceptor assay conditions, since exactly superimposable displacement curves were produced when incubations were performed for either 10 or 20min. Under similar conditions (Miller et aZ., 1977) 50%1OOfig/ml of bacitracin has been shown to be sufficient to prevent degradation for up to 40min at 25"; and other workers (Meunier & Moisand, 1977) have used 30pg/ml of bacitracin compared to the lOOfig/ml concentration used in this study (Experimental Section). In the guinea pig ileum assay some

RECEPTOR CONFORMATION or MLT-ENKEPHALIN - 1

! i I

4

!

i

I I )

i

I FIGURE I Displaccnient of specifi- cally bound ’[I-naloxonc from rat bran rnenibrmc

I I l 0

1 I preparations by reduced isosteric analogues of Met- enkcph;ilin.

degradation did occur. On addition of methionine enkephalin to the assay bath, inhibition of the electrically evoked twi:ih occurred and a plateau value, used for measure- ment, was reached quite rapidly. If no iurther sample was added, the twitch returned slowly to the baseline value, perhaps throb& the combined effects of tachyphylaxis and degradation. The half-life for this return was more than 10 times the period taken for each measurement. In the assay 2-D-Ala-5-methionine enkephalin was 6.6 times as active as the parent compound, in agreement with the value obtained by other workers (Kosterlitz & Hughes, 1978) who attributed this increase to enzymic stability. Since all the isosteric analogues should be more stable than methionine enkephdin, division by 6.6 should represent a maximum amount by which to compensate the 180-’_80-fold difference in activity of the pairs of isomeric peptides discussed above. Indeed, although it may well be pure coincidence because of different populations of receptors, making this allowance gives similar relative activities in the two assay Systems.

In neither of the assays can the results be explained by differential enzymic stability; rather, the observed values are a true reflection of receptor affinity. It is concluded, therefore, that the tyrosine carbonyl group is not impor-

tant in the stabilisation ofreceptor conformation by hydrogen bonding to the Phe4 -NH- group, nor is it involved in direct interaction with the receptor, such as by taking up a position similar to that proposed for the carbonyl group of several nonpeptidic opiates (Clark et al., 1978). Moreover, the Tyr’GIy’ peptide bond can be replaced by two methylene groups (H 2 1 1 ) with retention of significant activity. The conclusion that the carbonyl of Tyr‘ is not involved in an intramolecular hydrogen bond is supported by the finding that N-methyl-Phe4 analogues of enkephalin are highly active analgetics (Roemer et al., 1977). Conversely, the Gly2 carbonyl group is important but not essential for receptor interaction. It is more likely to be involved in binding to the receptor than in forming an intramolecular hydrogen bond with the -NH- of Mets since high activity is retained when this nitrogen atom is methylated, the Met residue is replaced by proline (Bajusz et al., 1977) or completely eliminated (Morgan wf al., 1977).

These conclusions have been incorporated into the receptor confirmation depicted in Fig. 2A. There is no intrachain hydrogen bonding but the Gly2Gly3 peptide bond is hydrogen bonded to a binding site on the receptor which may be an amino group. This interaction has no recognised counterpart in the binding of non- peptidic analgetics. The binding of the power-

125

D. HLIDSON ET AL

b

FIGURE 2A Perspective drawing of the proposed conformation of Met-enkephalin bound to the opiate receptor.

ful synthetic opiate PEO is shown in Fig. 2B. Recent findings (Kosterlitz er al., 1979; Gillan ef al., 1979) show that different types of opiate bind to different receptors, and that oripavine opiates rather than morphine or morphinan based derivatives show similar binding characteristics to the enkephdins, and therefore provide a sounder basis for comparison. Additional receptor binding sites for methionine enkephalin other than those shown in Fig. 2A may be inferred by the obvious similarities with Fig. 2B.

The conformation proposed bears some resemblance to other "F-ring" models, for example that suggested by Bradbury et ~ l . (197&), except that in this case the methionine sidechain projects below the plane of the peptide backbone and the C-terminal carboxy group rather than the thiomethyl group occupies the polar binding position corre- sponding to theCc hydroxy group of morphine. The methionine sidechain might occupy a hydrophobic cavity, which might also be occupied by the non-polar 6 , 14endoetheno bridge of PEO. This would provide additional stabilisation, and it is tempting to speculate that analogues such as D-Met' , Pro' enkephalin amide (Bajusz ef aZ., 1977) are highly active

because the D-Met' side cham can occupy the same hydrophobic binding site.

In the proposed receptor conformation amino and carboxy termini are well separated. The amino group is not invdwd in head to tail interaction but is available to bind to an aniomc site on the receptor. Since analogues of enkephalin extended at the N-terminus by a single amino acid residue are stdl highly active (Terenius ef al., 1976), the requirements for this interaction are not as exacting as might be expected by analogy with the opiate alkaloids (Lewis et al., 1971; Feinberg er al., 1976). Productive receptor interaction may occur in a series of consecutive steps progressively freezing the flexible solution conformation to one complementary to the receptor. The initial step is likely to be binding of the amino group to the anionic site, followed by hydrophobic binding of the tyrosine sidechain and hydrogen bonding of the Gly2Gly3 peptide bond to complementary sites on the receptor. This hydrogen bonding of the GlyZGly3 peptlde bond helps guide the Phe4 and Met4 sidecham to their appropriate hydrophobic binding sites.

The method of systematic isosteric modifi- cation of peptide bonds is a valuable tool in receptor mapping. The model suggested is

126

RECEPTOR CONFORMATION OF MET-ENKEPHALIN

FIGURE 2B BONDIhG Perspective drdwing of 8 'TE PEO bound to the opldte

recep'or

speculative but is consistent with known structure-activity relationships. it explains the inconsistencies of intramolecularly hydrogen bonded conformations as well as providing a basis for understanding the high activity of the hexapeptide H -Tyr -Gly -Gly -Gly-Phe - Leu-OH (Terenius e t al., 1976). Continuing studies of the Gly3 -Phe4 and Phe4 -Mets regions are likely to provide further important information and insight. For example in the model the Gly3 carbonyl group falls very close to the position occupied by the tertiary CI9 hydroxy group of PEO; and its reduction is predicted to lead to considerable loss of activity. With more distortion, however, the Gly3 carbonyl group can be aligned with the polar site corresponding to the C, hydroxy group of morphine, in which case even greater loss of activity would be expected. On the other hand, isosteric modifi- cation of the Phe4-Met5 peptide bond should result in retention of binding affinity and activity.

EXPERIMENTAL PROCEDURES

peptides were prepared by solid phase synthesis using a phenolic resin support as described in Part I and elsewhere (Hudson et al., 1979).

Dore-Smith '4tomunit kits (Capitai Bio- technic Developments, London, W3) were used to build molecular models of PEO apd Met- enkephalin. These are very suitable since they retain a rigid configura:ion without locking for ready comparison yet are easily manipulated. Photographs of the models in the desired orientations were used as a basis for the perspective drawings shown in Fig. 2A and B. Slight distortion of some bond angles and distances were made to reveal obscured atoms and bonds.

Guinea pig ileum assays were performed as described by Kosterlitz & Watt (1968) except that EDso values were calculated from cumulative dose-response curves. The time to reach a plateau value for each dose was less than 1 min. The mouse vas deferens assays were performed as described by Hughes et d'(1975); increasing doses of the analogues were injected into the Krebs stream perfusing a 5-ml bath at 29" at a flow rate of 3 ml/min.

Opiate binding assays were carried out using a modification of a method described elsewhere (Bradbury ef ab, 1976b). A hypotonically lysed (1OmM Tris-HCl, pH 7.4). extensively washed crude synaptosome preparation was made from whole supratentorial rat brain. The membranes were incubated with the peptides in 0.05M

127

D. HUDSON ET AL.

Tris-HCI buffer pH 7.6 with 0.1 51 NaCI, 0.1% bovine seruni albumin and 0.01% bacitracin containing tritiated naloxone (New England Nuclear. 2OCi mmol-' , 1 x M). After 20niin incubation at 25" the suspension was centrifuged ( 1 5 OOOg, 1.5 min) the pellets rapidly and superficially washed with 0.1 M NaCl in 0.05 M Tris-HC1 buffer pH 7.6, the pellets resuspended in water (0.5ml) and solubilised with scintillator (6 gllitre of PPO in toluene containing 20% (v/v) Triton X 100). counted at 30% efficiency to determine the bound counts. Specific binding was defined as that fraction of the bound radioactivity dis- placed by morphine (lo-' M). Incubations were carried out in triplicate, in a t least two separate experiments. Standards of naloxone, Metenkephalin and 5-norleucine enkephalin were used.

Rat tail flick assays were performed as described by Walker et al. (1977).

A C K N O W L E D G E M E N T S

The authors are indebted to their colleagues and friends for unselfish collaboration. Thanks are due to B.A. Morgan, A. Wilson and C.F.C. Smith, Reckitt and Colman, Hull, England for guinea pig ileum and mouse vas deferens assays, and to G. Fink, Department of Human Anatomy, Oxford University for in vivo assays. Special thanks are due to C.R. Snell, National Institute for Medical Research, Mill Hill, London NW7, for valuable discussions and for the brain membrane radio receptor assays; as well as to D. Simmonds of the Royal Postgraduate Medical School for the preparation of Fig. 2A and B.

REFERENCES

Bajusz, S., Rbnai, A.Z., Szkkely, J.I. , Grif, L., Dunai- Kovks, 2. & Berzktei, I. (1977) FEBS Lett. 76, 91-92

Balodis, Y.Y., Nikiforovich, G.V., Grinsteine, I.V., Vegner, R.E. & Chipens, G.I. (1978) FEBS Lett. 86,239-242

Bradbury, A.F., Smyth, D.G. & Snell, C.R. (1976a) Nature 260,165-166

Bradbury, A.F., Smyth, D.G., Snell, C.R., Birdsall, N.J.M. & Hulme, E.C. (19766) Nature 260, 793- 795

Clarke, F.H., Jaggi, H. & LoveU, R.A. (1978) J. Med. Chem. 21,600-606

Feinberg, A P., Creese, I. & Snyder, S.H. (1976)Proc. Natl. Acad. Sci. US 73.4215-4219

Frederickson, R.C.A., Nicklander, R.. Smithwick, E.L., Sharman, R. & Norris, F.H. (1976) in Opiates arid Opioid Peptides (Kosterlitz, H.w.. ed.). pp. 239-243, North Holland, Amsterdam

Garbay-Jaureguiberry, C.. Roques, B.P., Oberlin, R., Anteunis, M. & Lala, A.K. (1976) Biochem. Biophys. Res. Commun. 71,558-565

Gillan, M.G.C., Kosterlitz, H.W. & Paterson, S.J. (1979) Br. J. Pharmacol., in press.

Gorin, F.A. & Marshall, G.R. (1977) Proc. Natl. Acad. Sci. US 74 ,5 179-5 183

Hudson, D., Sharpe, R., Szelke, M., Maclntyre, I . & Fink, G. (1979) UK Patent Application GB 2 000 783 A

Hughes, J . , Kosterlitz, H.W. & Leslie, F.M. ( 1 9 7 5 ) B r . J. Phannacol. 53, 371-381

Isopai, Y., Nemethy, G. & Scheraga, H.A. (1977)proc. Natl. Acad. Sci. US 74, 4 14 -4 18

Jones, C.R., Gibbons, W.A. & Garsky, V. (1976) Nature 262,779-782

Jones, C.R., Garsky, V. & Gibbons, W.A. (1977) Biochem. Biophys. Res. Commun. 76,619-625

Kosterlitz, H.W. & Hughes, J. (1978) in Advances i , ~ Biochemical Psychopharmacology (Costa, E. & Trabucchi, M., eds.), vol. 18, pp. 31-44, Raven Press, New York

Kosterlitz, H.W. &Watt , A.J. (1968) Br. J. Pharmacol. 33,266-276

Kosterlitz, H.W., Lord, J.A.H., Paterson, S.J. and Waterfield, A.A. (1979) Br. J. Pharmacol., in press

Lewis, J.W., Bentley, K.W. & Cowan, A. (1971)Ann. Rev. Pharmacol. 11,241-270

Loew, G.H. & Burt, S.K. (1978)Proc. Natl. Acad. Sci. US 75,7-11

Meunier, J.C. & Moisand, C. (1977) FEBS Lett. 77, 209-213

Miller, R.J., Chang, K.J. & Cuatrecasas, P. (1977) Biochem. Biophys. Res. Commun. 74,13 1 1 - 1 3 17

Morgan, B.A., Bower, J.D., Guest, K.P., Handa, B.K., Metcalf, G . & Smith, C.F.C. (1977) in Peptides, Proceedings of the Fifth American Peptide Symposium (Goodman, M. & Meienhofer, I., eds.), pp. 111-113, John Wiley, New York

Parry, M.J., Russell, A.B. & Szelke, M. (1972) in Chemispy and Biology ofpeptides (Meienhofer, I., ed.), pp. 541-544, Ann Arbor Science, Michigan

Roemer, D., Buescher, H.H., Hill, R.C. Pless, J., Bauer, W., Cardinaux, F., Closse, A., Hauser, D. & Huguenin, R. (1977) Nature 268,547-549

Roques, B.P., Garbay-Jaureguiberry, C., Oberlin, R., Anteunis, M. & Lala, A.K. (1976) Nature 262, 778-779

Schiller, P.W. (1977) Biochem. Biophys. Res. Commun. 79,493-498

Schiller, P.W., Yam, C.F. & Prosmanne, J. (1978) J. Med. Chem. 21,1110-1116

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Schwyzer, R. (1959) Rec. Chem. Progr. 20,147-156 Smith, G.D. & Griffin, J.F. (1978) Science 199,

Spirtes, M.A., Schwartz, R.W., Mattice, W.L. & Coy, D.H. (1978) Biochem. Biophys. Res. Commun.

Szelke, M., Hudson, D., Sharpe, R., MacIntyre, I., Fink, G. & Pickering, A.J.M.C. (1977) inMolecular Endocrinology (Maclntyre, I. & Szelke, M., eds.), 57-70, ElsevierlNorth Holland, Amsterdam

Terenius, L., Wahlstrom, A., Lindeberg, G., Karlsson, S . & Ragnarsson, U. (1976) Biochem. Biophys. Res. Commun. 71,175-179

1214-1216

81,602-609

Walker, J.M., Berntson, G.G., Sandman, C.A., Coy, D.H., Schally, A.V. & Kastin, A.J. (1977) Science 196,85-87

Address: Dr. D. Hudson Department of Chemical Pathology Royal Postgraduate Medical School Duane Road London, W12 OHS England

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