DISCRIMINATION OF A NOVEL TYPE OF RAT BRAIN 80PIOID RECEPTORS BY ENKEPHALIN ANALOG CONTAINING STRUCTURALLY CONSTRAINED CYCLOPROPYLPHENYLALANINE (VPhe)

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1 Vol. 42, No. 6, September 1997 Pages DISCRIMINATION OF A NOVEL TYPE OF RAT BRAIN 80PIOID RECEPTORS BY ENKEPHALIN ANALOG CONTAINING STRUCTURALLY CONSTRAINED CYCLOPROPYLPHENYLALANINE (VPhe) Yoshiro Chuman, Teruo Yasunaga,* Tommaso Costa,** and Yasuyuki Shimohigashi 1 Laboratory of Biochemistry, Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka , Japan, *Manufacturing Process Development Division, Otsuka Pharmaceutical Co., Ltd., Saga Factory, Kanzaki, Saga , Japan, and ** Laboratorio di Farmacologi~t, I~tituto Superiore di Sanitgt, Roma, Italy Received May 9, 1997 Received after revision May 28, 1997 Summary: Four different stereoisomers of cyclopropylphenylalanine (VPhe) were incorporated into [D-Ala2,LeuS]enkephalin at the position 4. These conforrnationally restricted enkephalin analogs were evaluated for their binding characteristics to ~t and 8 opioid receptors in rat brain. A striking finding is that the E-(2R,3S)-isomer binds to a novel class of 8 receptors and discriminates this receptor from the ordinary 8 receptor. This new type of 8 receptor suspected to be a receptor which suppresses the thermal analgesia mediated through ~t receptor. The Z-(2R,3R)-isomer was very potent with several times more enhanced affinity to i5 receptors than to ~t receptors, but could not differentiate the 8 receptors. The Z-(2S,3S)- isomer was weak, and E-(2S,3R)-isomer was almost inactive. Key words: Opioid receptors, receptor-subtypes, enkephalin analogs, cyclopropylphenylalanine, bioactive conformation, confornaational restriction. Introduetion The bioactive conlormation of a peptide ligand is a certain steric arrangement that the peptide adopts for occupying the binding site of receptor molecules. In general, the stabilization or fixation of bioactive conformation reinforces the ligand-receptor interaction, resulting in enhancement of biological activity. For peptidic ligands, incorporation of structural constraints is an effective way to restrict the conformation and to assess the stereochemical influence on the resulting biological profiles (1,2). For instance, simple ct,l~-dehydrogenation of an amino acid residue, creating c~,[3-dehydroamino acid, often produces unique effects on the peptide confoimation; i.e., an increase in rigidity owing to limited bond angles, a restriction 2Abbreviations: VAA, cyclopropylamino acids (the inverted triangle, V, is used to designate 'cyclopropyl'); VPhe, cyclopropylphenylalanine; DADLE, [D-AtaZ,D-Leu5]enkephalin; DAGO, [D-Ala2,MePhe4,Gty-olS]enkephalin; DEL, deltorphin II; DSLET, [D- Ser 2,Leu 5 ]enkephaly 1 -Thrt; E-( 2 R,3S)-isomer, [D-Ala 2,E-(2R,3S)-VPhe 4,Leu- 5 ]enkephalin; GPI, guinea pig ileum; MVD, mouse vas deferens; NAL, naloxone. 1To whom correspondence should be addressed /97/ /0 Copyright by Academic Press Australia. All rights of reproduction in any form reserved.

2 of side chain orientations, and an induction of backbone folding. The usefulness of such local conformatjonal restriction has been well demonstrated by us with the synthesis of a series of dehydro-enkephalins (3, 4). Another interesting structurally constrained series of amino acids is the 2-substituted 1- amino cyclopropane carboxylic acids, namely cyclopropylamino acids (VAA). 2 The orientation of the 13-substituents in VAA is restricted rigidly to only two possible positions, ;(1 = 0~ and 120 ~ corresponding to the Z- and E-configurations, respectively (Fig, 1). The incorporation of VAA into a peptide chain is expected to constrain effectively its conformation due to the small d? and (9 angles at VAA conformational energy minima. The structure-activity study using conformationaly restricted analogs appears to be very effective to design receptor specific and selective neuropeptides especially in a receptor-ligand multiplicity. In the opioid peptide-receptor system, the presence of ~ and ~ opioid receptors in brain was demonstrated by examining the binding affinities of radio-labeled ligands specific for each receptor subtype (5, 6). The heterogeneity of opioid receptors was also found in the peripheral tissues such as mouse vas deferens (MVD, cs-prototype) and guinea pig ileum (GPI, ~t-prototype) (7). These have been confirmed by recent isolation of edna coding respective receptors (8, 9). When we assayed enkephalin analog containing E-(2R,3S)-cyclopropyl- phenylalanine (VPhe), this analog exhibited a high affinity for ~5 receptors and a very weak affinity for ~t receptors in rat brain (10). However, it was completely inactive not only in GPI but also in MVD with no antagonist activity. Extremely weak affinity to MVD receptors was also evidenced by the binding assay (11). Although these data suggested that E-(2R,3S)-V Phe-containing enkephalin (hereafter designated as E-(2R,3S)-isomer) distinguishes the ~5 receptors in the central and peripheral nervous systems, it is not clear yet whether or not a novel type of 6 receptors exist either in the central or peripheral tissue. Cyclopropylphenylalanine possesses four stereoisomers in the E-(2R,3S)-, E-(2S,3R)-, Z-(2R,3R)-, and Z-(2S,3S)-configurafions (Fig. 1). Enkephalin analog containing E-(2S, 3R)- VPhe was completely inactive in any bioassay systems (10). Although the synthesis of enkephalin analogs containing the Z-isomers has been reported (12), no systemic assays have been done for a full set of VPhe-containing enkephalin isomers. In the present study, using highly receptor specific radio-labeled ligands, we have evaluated all of VPhe-containing enkephalin analogs for their ability to bind to 6 and g opioid receptors in rat brain and found that the E-(2R,3S)-isomer is able to discriminate a novel i5 receptor from the ordinary 6 receptors. Materials and Methods Peptide syntheses--all four stereoisomers of VPhe-containing enkephalin analogs were synthesized as reported previously (12, 13). The E- and Z-isomers were separately synthesized and purification was performed by HPLC. The purity was verified by high performance thinlayer chromatography and analytical HPLC. 1228

3 Tyr--D-A la--g~y Tyr-- D-A i a--g~y N\,,// NH - y,j Z-(2R,3R)- and Z-(2S,3S)-isomers n Leu E-(2R,3S)- and E-(2S,3R)-isomers Fig. 1. Structures of eyelopropylphenylalanine (VPhe)-eontaining enkephalin analogs. Receptor Binding Assays--Radio-ligand receptor binding assays involving rat brain preparations were carried out essentially as described previously (14). [3H]-[D-Ala2,MePhe 4, Gly-olS]enkephalin ([3H]DAGO) (1.80 TBq/mmol; New England Nuclear, Boston, Mass. USA) and ffh]naloxone ([3H]NAL) (1.85 TBq/mmol; Amersham, Buckinghamshire, UK) were used as tracers selective for g opioid receptors. For 6 opioid receptors, [3H]-[D-AIa2,D- LeuS]enkephatin ([3H]DADLE) (1.65 TBq/mmol; New England Nuclear), [3H]-[D-Ser2,LeuS]- enkephalyl-thr 6 ([3H]DSLET) (1.51 TBq/mmol; New England Nuclear), and [3H]-[IleS' 6]_ deltorphin II ([3H]DEL) (2.66 TBq/mmol; Amersham) were used. Incubations were carried out at 25~ for 60 rain in 50 mm Tris-HC1 buffer (ph 7.5) containing 0.1% bovine serum albumin. Bacitracin (100 gg/ml) was added as an enzyme inhibitor. Dose-response curves were constructed utilizing seven to fifteen doses, and the results were analyzed with the computer program, ALLFIT (15). The data were used to construct least-square estimates of the logistic curves relating the binding of labeled ligands to the concentrations of the non-labeled ligands. Results and Discussion Four different stereoisomers of [D-Alaz,VPhe4,LeuS]enkephalins were assessed for opioid receptors in rat brain using various type of radio-ligands specific for IX or 6 opioid receptors. Since rat brain does not contain the K receptors, no binding assay was carried out for this subtype. When [3H]DAGO was used as a typical ligand for bt receptors, each isomer exhibited non-overlapped binding curves in a wide range of concentrations (10-1~ -4 M). Figure 2 shows the concentration-dependent displacement curves analyzed by the computer program. DAGO was most potent, and the isomers of Z-(2R,3R), Z-(2S,3S), E-(2R,3S), and E-(2S,3R) followed in this order. It should be noted that the Z-isomers are much more potent (60-1,000-fold) than E-isomers. A similar activity profile was obtained in the assay using [3H]NAL, a non-peptide ligand specific for It receptors, instead of [3H]DAGO. For 6 receptors, several binding characteristics distinct from those in the ix assays became prominent. First, the Z-(2R,3R)-isomer was found to be very potent and more active than the standard 6 ligands of DSLEF and DADLE. Table 1 shows the IC50 values calculated from the curves. When the 6/It selectivity ratio (SR) was calculated as the ratio of IC50 using 3H-labeled 0-1igands divided by the IC50 obtained using 3H-labeled ~t-ligands, SR was about 1229

4 BIOCHEMISTRY and MOLECULAR,00 BIOLOGY INTERNATIONAL O 0 I i I i I i I i I i Log [peptide] (M) Fig. 2. Dose-response eurves of eyclopropylphenylalanine (VPhe)-eontaining enkephalin analogs on direct binding assays with [3H]-[D-Aia2,MePhe4,Glyol5]enkephalin ([3H]DAGO) in rat brain membranes. DAGO (1r Z-(2R,3R)- isomer, (O); Z-(2S,3S)-isomer, ((3); E-(2R,3S)-isomer, (I); and E-(2S,3R)-isomer, ([5]). Table 1. Binding affinities of four different stereoisomers of [D-AIa2,E-(2R,3S)-VPhe4,LeuS] - enkephatins for ~t and 6 opioid receptors in rat brain. Isomers IC50 (nm) [3H]DAGO [3H]NAL [3H]DADLE [3H]DSLET [3H]DEL (~t) (~t) (8) (8) (8) Z- (2R,3R) O t 2 Z-(2S,3S) E-(2R,3S) 3,290 2, E-(2S,3R) 6,770 3,770 1,290 2,560 1,640 10, indicating that this isomer is 6-selective. Although the Z-(2S,3S)-isomer was about 15-fold less potent in the 6 assays than the Z-(2R,3R) isomer, it was still 6-selective with SR = 4. The E-(2S,3R)-isomer was extremely weak in all the binding assays for 6 and ~t receptors. The most striking feature in binding to i5 receptors is much increased affinity observed for the E-(2R,3S)-isomer. The E-(2R,3S)-isomer was as active as the Z-(2S,3S)-isomer (IC50 = ca. 10 nm) in the assays using [3H]DADLE, [3H]DSLET, and [3H]DEL (Table 1). It was found in the present study, however, that the binding curves of this E-(2R, 3S)-isomer are very 1230

5 Vol. 42, No. 6, "1997 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL gently-sloping. This was particular when assayed using [3H]DSLET (Fig. 3) and [3H]DEL. Since the curves apeeared to be biphasic, we evaluated further the E-(2R, 3S)-isomer and DSLET in a detailed binding assay using [3H]DEL in rat brain. DSLET and dettorphin II are higly specific and selective ligands for i5 receptors (16, 17), DSLEr exhibits a usual monophasic binding curve. Whilst, the E-(2R,3S)-isomer shows a distinct biphasic curve. For the high affinity binding site, the E-(2R,3S)-isomer binds very strongly with the ICs0 value of approximately 0.28 nm, whereas it binds much more weakly (ICs0 of approximately 19 nm) for the low affinity binding site. Scatchard analysis gave indeed a biphasic line, and the dissociation constants were estimated to be about 6.7 x M and 1.4 x M for the high (about 19% population) and low (about 81%) affinity binding sites, respectively. These results clearly indicate that two distinct g receptors exist in rat brain and the E-(2R,3S)-isomer can discriminate them. DSLEF was not able to differentiate these two subtypes, binding to both receptors equally well. We have previously demonstrated that the E-(2R,3S)-isomer does not bind to the receptors in MVD, to which ordinary ~-ligands bind very strongly (11). The present results indicate that the high affinity binding site for the E-(2R,3S)-isomer does not exist in MVD. We have also shown that the E-(2R,3S)-isomer suppresses ~t receptor-mediated thermal analgesia by morphine (18). It is now strongly suspected that a novel subtype of ~ receptors exists in rat barain and this is the receptor cross-talking to ~t receptors, perhaps allosterically. The fact that 10o[ I i I, I i I, I Log [peptide] (M) Fig. 3. Dose-response curves of cyelopropylphenylalanine (VPhe)-containing enkephalin analogs on direct binding assays with [3H]-[D-AlaZ,LeuS]- enkephalyl-thr 6 ([~H]DSLET) in rat brain membranes. DSLET (lg); Z-(2R,3R)- isomer, (0); Z-(2S,3S)-isomer, (O); E-(2R,3S)-isomer, (I); and E-(2S,3R)-isomer, (~). 1231

6 ,00[ I = I = I I I i I I Log [peptide] (M) Fig. 4. Biphasic dose-response curve of E-(2R,3S)-isomer in contrast to monophasic curve of [D-Aia2,LeuS]enkephalyl-Thr 6 (DSLET) on the assay with [3H]-[lleS,6]deltorphin II in rat brain membranes. DSLET (~r and E- (2R,3S)-isomer (1). the E-(2R,3S)-isomer does recognize the novel class of 6 receptors further substantiates the usefulness of incorporation of structurally constrained amino acids, especially cyclopropane amino acids, for eliciting a specific interaction with the receptors, References 1. Shimohigashi, Y., Stammer, C. H., and T. Costa (1988) in Advances in Biotechnological Processes (Mizrahi, A., and van Wezel, A. L., Eds.), Vol. 10, Synthetic Peptides in Biotechnology, pp , Alan R. Liss, New York. 2. Beck-Sickinger, A. G. (1997) in Method in Molecular Biology (Irvine, G. B., and Williams, C. H., Eds.), Vol. 73, Neuropeptide Protocols, pp , Humana Press, Totowa. 3. Shimohigashi, Y., English, M. L., Stammer, C. H., and Costa, T. (1982) Biochem. Biophys. Res. Commun. 104, Shimohigashi, Y., Costa, T., Nitz, T. J., Chen, H.-C., and Stammer, C. H.(1984) Biochem. Biophys. Res. Commun. 121, Robson, L. E., and Kosterlitz, H. W. (1979) Proc. R. Soc. Lond. (Biol) 205, Chang, K.-J., and Cuatrecasas, P. (1979) J. Biol. Chem. 254, Lord, J. A. H., Waterfield, A. A., Hughes, J., and Kosterlitz, H. W. (1977) Nature 267, Kieffer, B. L., Befort, K., Gaveriaux-Ruff, C., and Hirth, C. G. (1992) Proc. Natl. Acad. Sci. USA 89, Chen, Y., Mestek, A., Liu, J., Hurley, J. A., and Yu, L. (1993) Mol. Pharmacol. 44, Shimohigashi, Y., Costa, T., Pfeiffer, A., Herz, A., Kimura, H., and Stammer, C. H.(1987) FEBS Letters 222, Vaughn, L. K., Wire, W. S., Davis, P., Shimohigashi, Y., Toth, G., Knapp, R. J., Hruby, V. J., Burks, T. F., and Yamamura, H. I. (1990) Eur. J. Pharmacol. 177,

7 12. Mapelli, V. P., Kimura, H., and Stammer, C. H. (1986) Int. J. Pep. Protein Res. 28, Kimura, H., Stammer, C, H., Shimohigashi, Y., Ren-Lin, C., and Stewart, J. (1983) Biochem. Biophys, Res. Commun. 115, Shimohigashi, Y., Costa, T., Matsuura, S,, Chen, H.-C., and Rodbard, D. (1982) Mol. Pharmacol. 21, De Lean, A., Munson, P. J., and Rodbard, D. (1978) Am. J. Physiol. 235, E97-E Gacel, G., Fournie-Zaluski, M. C., Fellion, E., and Roques, B. P. (1980) FEBS Lett. 118, Erspamer, V., Melchiorri, P., Falconieri-Erspamer, G., Negri, L., Corsi, R., Severini, C., Barra, D., Simmaco, M., and G. Kreil (1989) Proc. Natl. Acad. Sci. USA 86, Shimohigashi, Y., Takano, Y., Kanaiya, H., Costa, T., Herz, A., and Stammer, C. H. (1988) FEBS Lett. 233,