European Journal of Pharmacology - Molecular Pharmacology Section, 266 (1994) 175-180 175 © 1994 Elsevier Science B.V. All rights reserved 0922-4106/94/$07.00

EJPMOL 90563

Mechanism of 6-opioid receptor selection by the address domain of dermenkephalin

St6phane C ha rpen t i e r , Sandr ine Sagan, M o h a m m e d Naim, A n t o i n e D e l f o u r and P i e r r e Nicolas *

Laboratoire de Bioactivation des Peptides, lnstitut Jacques Monod, Universitd Paris 7, 2 Place Jussieu, 75251 Paris Cedex 05, France

Received 2 June 1993; revised MS received 28 September 1993; accepted 5 October 1993

Dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-AspNH 2) is a highly potent and selective 6-opioid peptide isolated from frog skin. It was recently recognized that the C-terminus His4-LeuS-Met6-Asp7NH2 of dermenkephalin was responsible for the addressing of the peptide towards the 6-opioid receptor. In order to investigate the role played by residues 4, 5 and 6 in this '6 address', we synthesized and evaluated 20 new analogues for their ability to displace tritiated ligands from/x- and 6-opioid sites. Results showed that position 4 of dermenkephalin contributes to 6 selectivity independently of 3-opioid receptor binding by preventing a high affinity p~ binding. Position 5 requires a hydrophobic side chain to enhance 6 affinity. A high 6 affinity was obtained with any amino acids introduced in position 6 suggesting that residue 6 serves as a neutral spacer. Thus, the main features responsible for the high 6-opioid selectivity of dermenkephalin are electrostatic repulsions with the/z-opioid receptor, additional hydrophobic interactions with the 6-opioid receptor and folding of the C-terminal domain.

Opioid; Dermenkephalin (binding); Structure-activity study; 6 Address

I. Introduction

As soon as the existence of at least three major types of opioid receptors (/~, 6, K) was admitted (for review, Paterson et al., 1984), the mechanisms of opi- oid receptor selection for the ligands were investigated. Unfortunately, the mammalian opioid peptides (en- kephalins, /3-endorphin, dynorphin) did not provide a thorough answer to this problem because of the low selectivities of these peptides towards the different types of opioid receptors (for review, Evans et al., 1988).

In this regard, the discovery of a new family of highly selective opioid peptides from the skin of South American frogs of the genus P h y l l o m e d u s a e , provides exceptional tools for structure-activity relationship studies. Indeed, despite the high homology in their primary structure, these peptides exhibit enormous dif- ferences in receptor selectivity. Thus, dermorphin (Tyr- D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), is the most potent and selective natural ~-opioid peptide known to date (Montecucchi et al., 1981; Amiche et al., 1987). Der- menkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH 2) (Amiche et al., 1989; Kreil et al., 1989; Lazarus et al.,

* Corresponding author. Tel.: (33-1) 44 27 69 52; Fax: (33-1) 44 27 59 94.

1989), deltorphin I (Tyr-o-Ala-Phe-Asp-Val-Val-Gly- NH 2) and deltorphin II (Tyr-o-Ala-Phe-Glu-Val-Val- Gly-NH z) are highly specific ~-opioid receptor agonists (Erspamer et al., 1989).

Previous structure-activity relationship studies demonstrated that although the N-terminal of both dermenkephalin and dermorphin display high affinity and selectivity for the ~-opioid receptor, these frag- ments can be reoriented towards the 6-opioid recep- tors by the addition of the C-terminal part of der- menkephalin (Sagan et al., 1989a,b). These observa- tions suggested that these opioid peptides conformed to the 'message-address' concept proposed by Schwyzer (Schwyzer, 1977). Accordingly, peptide ligands contain a 'message' domain responsible for receptor transduc- tion and an 'address' segment which determines recep- tor subtype selection. In this perspective we have un- dertaken an evaluation of the structural requirements of the 6 address of dermenkephalin, i.e. Hisa-Leu 5- Met6-Asp7NH2. For instance, the negative charge of the Asp 7 residue in dermenkephalin was shown to enhance 6 selectivity by preventing/~ binding (Lazarus et al., 1991; Sagan et al., 1992).

In this paper, we examine the hydropathic and structural requirements for the positions 4, 5 and 6 of dermenkephalin by the synthesis of 20 single amino acid substituted analogues, and evaluate their ability to displace either [tyrosyl-(3,5-3H)][o-Ala2,N-Me-Phe 4,

SSDI 0922-4106(93)E0159-H

176

Gly-olS]enkephalin ([3H]DAMGO, /~ probe) (Koster- litz and Paterson, 1981) or [tyrosyl-(3,5-aH)][o - Ser2,LeuS]enkephalyl-Thr6 ([3H]DSLET, ~ probe) (Gacel et al., 1980). A brief account of a part of this work appeared in the proceedings of the 22nd Euro- pean Peptide Symposium (Sagan et al., 1993).

2. Materials and methods

2.1. Radiochemicals

[tyrosyl-(3,5- 3 H)][D-S er 2,Leu5 ] E n k e p h a l y l - T h r 6 ([3H]DSLET, 52 C i / m m o l ) and [tyrosyl-(3,5-3H)][o - Ala2,N-Me-Phen,Gly-ol5]enkephalin ([3H]DAMGO, 52 C i / m m o l ) were purchased from Dupont de N e m o u r s / New England Nuclear.

2.2. Peptide synthesis

All the peptides were synthesized with a Mi l l iGen/ Biosearch 9050 PepSynthesizer by solid-phase method on A M (p[R,S-a-l(9H-fluoren-9-yl)-methoxyfor- mamido-2,4-dimethoxybenzyl]-phenoxyacetic acid)-lin- ked polyamide/k iese lguhr resin (PepSyn K). Coupling reactions were carried out using fluoren-9-ylmethoxy- carbonyl (Fmoc)-amino acids (Mil l iGen/Biosearch, USA) esterified with 2-(1H-benzotri-azol-l-yl)-l,l ,3,3- tetramethyluronium tetrafluoroborate (TBTU). Protec- tions for reactive side chains were: t-butyloxycarbonyl for Lys, tert-butyl for Tyr, Thr, Asp, Glu and triphenyl- methyl for His. Peptides were deprotected and cleaved from the resin with trifluoroacetic acid (82.5%) in the presence of phenol (5.0%), thioanisole (5.0%), water (5.0%) and ethyl-methyl-sulfide (2.5%). After ether extraction, the peptides were purified by a preparative high performance liquid chromatography (HPLC). Ho- mogeneity of the synthetic peptides was assessed by analytical HPLC, amino acid analysis and fast atomic bombarding mass spectrometry (FAB-MS) (Table 1).

2.3. Opioid binding assays

Brain membrane (minus cerebellum) from 180 g Sprague-Dawley rats (R. Janvier, France) were pre- pared as described elsewhere (Amiche et al., 1988). Binding assays were performed at 24°C in 50 mM Tris-HCl, pH %4, plus 0.1% bovine serum albumin and 0.01% bacitracin. Each assay contained, in a final volume of 2 ml, the membrane preparat ion (1.4 mg of membrane proteins) and the tritiated ligand at the desired concentration with or without the unlabeled ligand. The non-specific binding was determined in the presence of 1 ~ M of dermorphin for [3H]DAMGO and 1 /xM of dermenkephalin for [3H]DSLET. The tubes were incubated until the reaction had reached

equilibrium (1 h for [3H]DAMGO and 2 h for [3H]DSLET). The binding reaction was terminated by rapid vacuum filtration through 0.1% polyethylenimine coated Whatman glass fiber filters (GF /B) . The filters were washed twice with 5 ml of cold 50 mM Tris-HCl, pH 7.4, O.1% bovine serum albumin and transferred to vials containing 4 ml of Biofluor scintillation fluid (Dupont de N e m o u r s / N e w England Nuclear, USA). All measurements were performed in duplicates and each experiment repeated 2-3 times. The inhibitory constant (Ki, nM) of the various unlabeled ligands was calculated from the relation g i = IC50/[1 + (L/Ko)] (Cheng and Prusoff, 1973) where L is the concentra- tion of the labeled ligand, K d its equilibrium dissocia- tion constant determined by saturation binding analysis and IC50 the concentration for 50% inhibition of spe- cific binding.

3. Results

3.1. Position 4

Replacement of the imidazole side chain of His 4 in dermenkephalin with the hydrogen of Gly or with the aromatic side chain of Phe, generated peptides exhibit-

TABLE 1

Analytical data of new dermenkephalin analogues a.

Peptide analogues HPLC Retention Molecular weight time (min) b Calculated Found ¢

[Asn5 ]Dermenkephalin 22.60 956.1 956.3 [Thr 5]Dermenkephalin 22.76 943.1 943.4 lGly 5]Dermenkephalin 22.60 899.0 899.3 [AlaS]Dermenkephalin 23.13 913.1 913.3 [Met 5]Dermenkephalin 24.76 973.2 973.4 [PheS]Dermenkephalin 26.43 989.2 989.4 [Val5]Dermenkephalin 24.32 941.1 941.2 [Ile5]Dermenkephalin 21.86 955.1 955.4 [ProS]Dermenkephalin 23.27 939.1 939.4 [LysS]Dermenkephalin 21.45 970.1 970.5 [GluS]Dermenkephalin 22.79 971.1 971.3 [Asn6]Dermenkephalin 19.70 938.1 938.4 [Thr 6]Dermenkephalin 19.95 925.1 925.3 [Phe6]Dermenkephalin 25.88 971.1 971.4 [Val6]Dermenkephalin 21.54 923.1 923.4 [Pro6]Dermenkephalin 22.12 921.1 921.4 [Lys6]Dermenkephalin 17.32 952.1 952.4 [Glu6]Dermenkephalin 19.78 953.1 953.4

a Amino acid analysis was performed on a Pico-Tag system (Waters) (Nicolas et al., 1986) and showed correct amino acid composition for all the peptides, b HPLC elution was carried out with a Waters Delta Pak C18-300 .~ column, using a linear gradient from 0% to 60% (or 20% to 60% for position 6 substituted analogues) of acetonitrile in water, both containing 0.1% trifluoracetic acid, in 30 min (in 40 min for position 6 substituted analogues), at a flow rate of 1 ml/min. c Fast atomic bombarding mass spectrometry was performed as de- scribed (Nicolas et al., 1986).

TABLE 2

Opioid binding potencies (K i, nM) at the ~ (versus 1 nM [3H]DAMGO) and t~ sites (versus 2 nM [3H]DSLET) of derm- enkephalin (DRE) and related analogues with substitutions at posi- tion 4.

Peptide Ki(/x) Ki(a) Selectivity nM nM Ki(l~)/Ki(~)

DRE: Tyr-D-Met-Phe- His-Leu-Met-AspNH 2 380-+ 24 6.0_+ 0.2 63

[D-AIa2]DRE " 131 _+ 8 5.1 _+ 0.7 26 [D_AIa 2, GIy4]DRE b 25_+ 3.1 4.8_+ 0.3 5 [o-Ala 2, Phe4]DRE 20_+ 4.5 6.0_+ 0.5 3

[o-Ala 2, Pro4]DRE 1262 _+ 137 148 ± 17 8.5

[Lys4]DRE c 279_+ 24 304_+30 0.9 [Asp4]DRE c 38094- 172 82_+ 5.0 46

a Taken from Sagan et al., 1989a. b Taken from Sagan et al., 1989b. CTaken from Sagan et al., 1992. The values are the mean_+S.E, of 2-5 observations carried out in duplicates. The inhibitory constant K i (nM) was calculated from ICs0 values using the Cheng-Prusoff equation (Cheng and Prusoff, 1973).

ing a 5-9-fold lower 8 selectivity than the parent peptide (Table 2). This decrease in 8 selectivity was mainly due to an increase in /X affinity. The 8 affinity remained practically the same with His 4, Gly 4 or Phe 4 indicating that the side chain of the residue in position 4 is not involved in 8-opioid receptor binding.

In contrast, substitution of His 4 with Pro 4, which exerts high constraints on the peptide backbone, re- duced 8 affinity by more than 25-fold and/x affinity by only 3-fold, and caused a 7-fold loss in 8 selectivity relative to dermenkephalin (Table 2).

The introduction of a positive charge in position 4 by substituting His for Lys resulted in a dramatic drop of 8 affinity (50-fold) without modifying /x affinity (Table 2).

Finally, increase in the net negative charge of der- menkephalin by one unit through substitution of His 4 for Asp markedly reduced binding potencies at the/x- and 8-opioid receptors.

3.2. Position 5

Mono-substitutions of Leu 5 in dermenkephalin with hydrophobic amino acids, such as lie, Val, Phe, Met, Ala, yielded compounds which maintained a high affin- ity and a pronounced selectivity for ~-opioid receptors although they were slightly less potent than der- menkephalin (Table 3). The data obtained with re- placement of Leu 5 by neutral or hydrophilic amino acid residues were rather different. Indeed, substitu- tion of the Leu 5 residue by Gly or Thr led to peptides respectively 9 and 12 times less potent than der- menkephalin at the ~ sites. Substitution by Asn, which provided an isosteric but totally hydrophilic replace-

177

TABLE 3

Opioid binding potencies (Ki, nM) at the Iz (versus 1 nM [3H]DAMGO) and t5 sites (versus 2 nM [3H]DSLET) of derm- enkephalin (DRE) and related analogues with substitutions at posi- tion 5.

Peptide Ki(iz) Ki(a) Selectivity nM nM Ki(tz)/Ki(~)

DRE: Tyr-D-Met-Phe- His-Leu-Met-AspNH 2 380_+ 24 6.0_+ 0.2 63

[AsnS]DRE 335_+ 11 110 5 : 1 9 3.0 [ThrSIDRE 237_+ 15 72 + 17 3.3 [GIySIDRE 1424-_ 21 54 + 16 2.6 [AIaS]DRE 160-+ 13 13.4_+ 1.6 12 [MetS]DRE 258_+ 31 12.8_+ 5.2 20 [PheS]DRE 446_+ 54 10.3+_ 0.9 43 [ValS]DRE 422_+ 37 14.8_+ 3.4 28 [IIeS]DRE 421+_ 69 14.0-+ 3.0 30 [ProS]DRE 2168-+250 1400 _+348 1.5 [LysS]DRE 149-+ 15 182 _+ 54 0.8 [GIuS]DRE 1280_+163 216 _+ 3.5 5.9

The values are the mean + S.E. of 2-5 observations carried out in duplicates. The inhibitory constant K i (nM) was calculated from IC50 values using the Cheng-Prusoff equation (Cheng and Prusoff, 1973).

ment of Leu 5, was also detrimental to t~ binding po- tency. Interestingly, there is a nice correlation between the affinity towards the ~ sites and the hydrophobicity of the side chain in position 5 (Fig. 1).

Replacement of Leu 5 with Pro caused a dramatic loss in 8-opioid receptor affinity (264-fold; Ki(8) = 1400 nM) which far exceeded that observed with other sub- stitutes (Table 3). By comparison, the /x affinity was only slightly reduced (5.7-fold), resulting in a peptide analogue exhibiting a 46-fold lower selectivity than that of dermenkephalin.

The introduction of the positively charged residue Lys in place of Leu 5 led to peptide with no more 6 selectivity.

120 i hydrophilic hydrophobic

%

I O0 N T N ~ T T ] i ~ 80

60

40

M V 20 N F 0

- 4 - 3 - 2 -1 0 1 2 3 4 Hydropathy

Fig. 1. Effect of the hydropathic character of the substituting amino acid in position 5 (o) and position 6 (11]) of dermenkephalin on ~-opioid affinity (Ki(t~) , nM). Values± S.E.M. were taken from Ta- bles 3 and 4. Hydropathic scale was from Kyte and Doolittle (1982).

178

TABLE 4

Opioid binding potencies (Ki , nM) at the tz (versus 1 nM [3H]DAMGO) and 8 sites (versus 2 nM [3H]DSLET) of der- menkephalin (DRE) and related analogues with substitutions at position 6.

Peptide Ki(/z) Ki(6) Selectivity nM nM Ki(Ix)/Ki(6)

DRE: Tyr-o-Met-Phe- His-Leu-Met-AspNH2 380_+24 6.0+0.2 63

[Asn 6]DRE 323 _+ 35 9.6 5:0.9 35 [Thr 6]DRE 334 + 26 5.4 _+ 0.3 62

[Phe6]DRE 215+28 4.2+0.6 51 [VaI6]DRE 2645:12 5.7+ 1.2 46

[Pro 6]DRE 334 5:29 8.0 5:0.5 42

[Lys6]DRE 545:4.0 4.4_+0.2 12 [GIu6]DRE 378-1-33 17.85:0.8 21

The values are the mean + S.E. of 2-5 observations carried out in duplicates. The inhibitory cons tan t g i (nM) was calculated from IC50 values using the Cheng-Prusoff equation (Cheng and Prusoff, 1973).

Likewise, replacement of Leu 5 by the negatively charged amino acid, Glu, caused a marked decrease in

affinity and to a less extend in/~ affinity.

3.3. Position 6

The influence of the nature of the side chain of the sixth residue of dermenkephalin on 3 selectivity was explored through systematic substitutions of. Met 6. Whatever the substitutions tested in position 6 (Table 4), i.e. Ile 6, Val 6, Phe 6, Thr 6, Asn 6, Lys 6, Pro 6, none was found to affect significantly ~ affinity. All of the jz affinities were in the same order (200 n M < Ki(iz) < 380 nM) except that of [Lys6]dermenkephalin which was greatly improved (gi(tz) = 54 nM).

4. Discussion

These results highlighted the great difference be- tween positions 4, 5 and 6 of dermenkephalin, each amino acid residue playing a particular role in 6 ad- dressing.

Replacement of His 4 in dermenkephalin with either Gly or Phe was prejudicial to 6-opioid receptor selec- tivity. In contrast, these analogues retained a 3 binding affinity similar to that exhibited by the parent peptide, confirming results obtained by others (Salvadori et al., 1991). Thus, the 6-opioid receptor tolerates the ab- sence of side chain or the presence of an aromatic or imidazol side chain at position 4 of dermenkephalin. These results provide evidence that position 4 in der- menkephalin contributes to selectivity independently of

~-opioid receptor binding, mostly by preventing high affinity binding to/x-opioid receptor.

The insertion of Pro 4 in place of His 4 greatly re- duced ~ affinity. Since the side chain of His 4 was shown not to participate in a binding interaction with ~-opioid receptors, this result has to be interpreted in terms of alterations of the bioactive conformation ei- ther of the '6 address' domain 4-7 or of the opioid 'message' domain 1-3. Moreover it was previously shown that Tyr-D-Ala-Phe-ProNH 2 exhibited a high/~ potency (Ki( /z)= 7.3, Ki (~)= 3000) (Charpentier et al., 1991) similar to that of dermenkephalin-(1-4)NH 2 (gi(/~) = 8.0, Ki(~) = 1289) (Sagan et al., 1989a). Con- sequently, the weak ~ affinity of [D-Ala2,Pro4]dermen - kephalin may reflect a disturbed conformation of the address domain 4-7 due to the geometric constraints imposed by Pro.

The decline in /z- and ~-opioid affinities that fol- lowed the insertion of Lys in position 4 of der- menkephalin probably reflects the presence of a posi- tive charge at or near the binding site of opioid recep- tors. This hypothesis is supported by previous results obtained with the tetrapeptide Tyr-D-Ala-Phe-LysNH 2 (Ki(/~) = 13.2, gi(~) = 2790) (Charpentier et al., 1991).

Data obtained after mono-substitutions of Leu 5 by Gly, Asn, Thr, Phe, Met, Val, lie or Ala in der- menkephalin indicate that a hydrophobic side chain is mandatory at position 5 of dermenkephalin in order to confer, to this peptide, a high avidity towards the ~-opioid receptor. Similar results were recently ob- tained with position 5 in deltorphin (Sasaki et al., 1991). A hydrophobic side chain in position 5 caused an increase in ~ binding either through hydrophobic interactions with the ~-opioid receptor or through the stabilization of a ~ bioactive conformation of the N- terminal part of the molecule as suggested by Niki- forovich and Hruby (1990). To be effective, the side chain of residue 5 had to be precisely orientated since [ProS]dermenkephalin was found to be virtually inac- tive at the ~ site. The residue Pro probably hindered access to a region of the conformational space that is required for optimal interactions of the side chain of residue 5. These results are in agreement with a recent report showing that reorientation of the Leu 5 side chain through introduction of D-Leu in place of Leu has a drastic effect on ~ affinity (Lazarus et al., 1992). The importance of the topography of the lipophilic C-terminal tail of dermenkephalin and deltorphins has been recently pointed out by Misicka et al. (1992) who proposed a common three-dimensional organisation for these ~-opioid selective peptides.

An interesting observation was that all of the posi- tion-6 substituted analogues were found to be nearly as potent at the ~ sites as native dermenkephalin. The 8-opioid receptor tolerates the presence of a hydropho- bic, neutral, hydrophylic as well as aromatic group at

position 6. These data demonstrate that the nature of the side chain at position 6 is immaterial to the 8-opioid receptor recognition.

Moreover [Pro6]dermenkephalin still maintained a good 8 affinity and selectivity, in contrast to the Pro s analogue. This result has to be compared with those obtained by incorporation of D-Met 6 in lieu of L-Met 6 (Lazarus et al., 1992). In the latter case the 8 affinity of the resulting compound was equal to that of der- menkephalin. These overall data suggested that residue 6 in dermenkephalin serves as a neutral spacer posi- tioning different elements of the address domain such as the side chain of residue 5 or the negative charge of A s p 7.

The inclusion of Lys at position 6 greatly improved /x affinity compared to that of the parent peptide. This analogue was of utmost interest since side chains at position 6 have been previously shown to be neutral with respect to 8 and /z activities. Thus, alteration in affinities following substitution of Met 6 by Lys should reflect alteration in the net charge of the peptide. This result suggests that /z-opioid receptors possess an an- ionic group which facilitates binding of positively charged peptides and repulses negatively charged ones. This hypothesis is in agreement with conclusions drawn from studies of the roles of residues Asp 4 in deltorphin I and Asp 7 in dermenkephalin (Lazarus et al., 1991; Sagan et al., 1992).

In conclusion, the present study accounts for the mechanisms involved in ~-opioid addressing of der- menkephalin. Results showed that these mechanisms are of two types. The first one results in an overall increase in 8 binding affinity via hydrophobic interac- tions of the Leu s side chain of dermenkephalin. The second mechanism promotes rejection of der- menkephalin by the/z site. This rejection involves the side chain of His 4 which maintains a /z incompatible conformation and Asp 7 which prevents /z binding through electrostatic repulsions.

Acknowledgements

This work was supported in part by funds from the CNRS, the Fondation pour la Recherche M6dicale Fran(jaise, the Institut Na- tional pour la Sant6 et la Recherche M6dicale (CRE 90012). The authors wish to thank Dr. G. Ricart and Dr. B. Fournet (Lille, France) for performing mass spectrometry analysis. The expert assis- tance of J.J. Montagne was greatly appreciated.

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