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Deltorphin, a novel amphibian skin peptide with high selectivity and affinity for δ opioid receptors
European Journal of Pharmacology, 162 (1989) 123-128 Elsevier
123
EJP 50688
Deltorphin, a novel amphibian skin peptide with high selectivity and affinity for 8 opioid receptors G u n t h e r Kreil t, D o n a t e l l a B a r r a 2, M a u r i z i o S i m m a c o 2, V i t t o r i o E r s p a m e r 3 G i u l i a n a F a l c o n i e r i E r s p a m e r 3, L u c i a N e g r i 3, Cinzia Severini 3, R i t a Corsi 3' a n d Pietro M e l c h i o r r i 3,. t Institute of Molecular Biology, Salzburg, Austria, : Department of Biochemical Sciences and Center for Molecular Biology of the National Research Council, and 3 Institute of Medical Pharmacology, University La Sapienza, Rome, Italy Received 28 July 1988, revised MS received 18 November 1988, accepted 13 December 1988
With a cDNA library prepared from skin of Phyllomedusa sauvagei, the sequence of the precursor of dermorphin was elucidated recently. The sequence suggested the existence of another peptide, distantly related to dermorphin. Two variants of this peptide have now been synthesized containing either L- or D-methionine as the second amino acid. The peptide containing the D-methionine exhibited high-affinity and selectivity for 8 opioid receptors in the mouse vas deferens and in rat brain homogenates. Moreover, using the synthetic peptide as marker, we could isolate small quantities of the corresponding natural peptide containing D-methionine as the second amino acid from skin extracts of Phyllomedusa sauvagei. The name deltorphin is proposed for this new peptide and its sequence is Tyr-D-Met-PheHis-Leu-Met-Asp-NH> Deltorphin; 80pioid receptor agonists; (Isolation, Sequencing)
1. Introduction
The heptapeptide dermorphin, isolated from the skin of the South American frog Phyllomedusa sauvagei, displays unusually high opioid activity. It binds selectively and with high affinity to the/~ binding site of opioid receptors (Rossi et al., 1986). The biological activity of dermorphin is dependent on the presence of a D-alanine residue in the second position; homologues with an L-amino acid in this position are inactive (Broccardo et al., 1981). Using recombinant D N A techniques, Richter et al. (1987) showed that dermorphin, like numerous other peptides, is derived in multiple
* To whom all correspondence should be addressed: Institute of Medical Pharmacology III, University of Rome 'La Sapienza', P.za A. Moro 5, 00185 Rome, Italy.
copies from larger precursors. In the cloned cDNAs of these precursors, a normal alanine codon was found at the position where D-alanine is present in the end product, pointing to a posttranslational conversion of specific alanine residues from the L to the D isomer. From the sequence of one of these cloned cDNAs, the existence of another heptapeptide, only distantly related to dermorphin, could be predicted, assuming that the same processing mechanism as that which liberates dermorphin from its precursors operated (fig. 1). Two variants of this peptide have now been synthesized containing either L- or Dmethionine as the second amino acid. We now report that the peptide with the D isomer selectively bound to 8 opioid receptors with an affinity that was an order of magnitude higher than that of the synthetic cyclic bis-penicillamine enkephalin analogues (Mosberg et al., 1983) and which
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
124 a.......
Lys-Arg-Tyr-Ala-Phe-Gly-Tyr-Pro-Ser-Gly-Glu-Ala-Lys-Lys...
b.......
Lys-Arg-Tyr-Met-Phe-His-Leu-Met-Asp-Gly-Glu-Ala-Lys-Lys...
Fig. 1. Segment of the amino acid sequence of prepro-dermorphin as predicted from the nucleotide sequence of clone D - l / 2 (for details see Richter et al., 1987). Amino acids present in the end product dermorphin (a) and the predicted peptide deltorphin (b) are in bold type.
was in the same range of affinity exhibited by the recently described, highly &selective linear hexapeptides D S T B U L E T and BUBU (Delay-Goyet et al., 1988). Moreover, the peptide could be isolated from skin extracts of Phyllomedusa sauvagei. This peptide is thus the first naturally occurring peptide with a high selectivity for 6 receptors. This peptide has accordingly been named deltorphin. 2. Materials and methods
The heptapeptides Tyr-Met-Phe-His-Leu-MetAsp-NH 2 with either L- or D-methionine as the second amino acid were custom synthesized by Biotec Nordhorn (Nordhorn, FRG). Samples of these peptides were also kindly supplied by Drs. Gozzini and De Castiglione (Farmitalia Carlo Erba, Milano, Italy). Dermorphin (DER), [DAlaZ,D-Leu 5]enkephalin (DADLE), [D-Ala 2,MePhe4,Gly-olS]enkephalin (DAGO) and [D-Pen2,D PenS]enkephalin (DPDPE) were purchased from Peninsula Labs (Belmont, CA, USA), [D-Thr 2, Leu 5]enkephalyl-Thr 6 (DTLET) and AllylZ-TyrAib-Aib-Phe-Leu-PH (ICI 174,864) from CRB (Cambridge, England). The peptides were purified by HPLC (see below) to a purity of more than 95%, and their claimed sequence was confirmed by automatic Edman degradation carried out on an Applied Biosystems peptide sequencer model 470A (see below) before they were used in the biological assays and as marker substances for the isolation of deltorphin from skin extracts. Radioactive peptides were purchased from Amersham (England).
2.1. Isolation and characterization of deltorphin from skin extracts of Phyllomedusa sauvagei Frog skin was minced with scissors and extracted at room temperature for 24 h with four volumes ( v / w ) of methanol. This procedure was
repeated three times and the supernatants were combined and evaporated under vacuum. The dried methanol extract was dissolved in 50% aqueous acetonitrile and fractionated (extract from 2 g skin per run) by reverse phase high performance liquid chromatography (RP-HPLC) (Aquapore RP 300, C 18, 7 × 250 mm, 7 t~m, Brownlee Labs, USA) with a linear gradient of solvent B (0.2% trifluoroacetic acid (TFA, Pierce Chemical Company, USA) in acetonitrile-isopropyl alcohol 4:1, v / v ) on solvent A (0.2% TFA) in water) at a flow rate of 2.8 m l / m i n (fig. 2a). The biological activity of the collected fractions was tested on in vitro preparations of electrically stimulated mouse vas deferens and expressed as nmol of synthetic deltorphin (fig. 2a: black bars). The fraction that eluted between 25 and 30% of solvent B, corresponding to the elution zone of the biological activity, was further purified by RP-HPLC (Spheri 5, C18, 4.6 × 250 mm, 5 /1, Brownlee Labs) at a flow rate of 1 ml/min, with a 40 rain gradient from 30 to 70% acetonitrile on 0.2% aqueous TFA (fig. 2b). Pooled bioactive fractions were submitted to a further RP-HPLC fractionation (Supelcosil LC18DB, 4.6 × 250 mm, 5 /~, 100 A °, Supelco, USA) with a different acetonitrile gradient in aqueous TFA at a flow rate of 1.5 m l / m i n (solvent A, 0.1% TFA in water, solvent B, 0.1% TFA in acetonitrile; 10-100% B in four steps: 10-15% over 10 min, 15-25% over 30 min, 25-50% over 25 min, and 50-100% over 20 min). The elution profiles of the two synthetic deltorphins in this system are shown by the dashed upper trace in fig. 2c. The retention times were 47.7 rain for [L-Met2]deltorphin and 51.4 min for deltorphin. The peptide co-eluting with synthetic deltorphin after the third purification step was used for the sequence analysis as well as for the biological assays. Sequence analysis of this peptide was performed on an Applied Biosystems Model 470A gas-phase protein sequencer equipped with an Ap-
125
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Fig. 2. Purification of deltorphin from skin extracts of Phyllomedusasauvagei. (a) HPLC on an Aquapore RP 300 column of the crude extract from 2 g of the amphibian skin. (b) Further HPLC of bioactive fractions collected in the previous chromatogram (see a black columns) on Spheri 5, C18, column. (c) Final chromatography of active fractions from b on Supelcosil LC18DB column. The elution profiles of the two synthetic deltorphins, [L-Met2]deltorphin (e) and deltorphin (11), used as reference standards in thL, system, are shown by dashed upper trace.
126 plied Biosystems Model 120A P T H Analyzer for the on-line detection of phenylthiohydantoins. The sample was dissolved in 30% acetonitrile and loaded onto a glass fiber filter coated with polybrene and pre-washed according to the manufacturer's instructions.
Non-specific binding was defined by 1 t~M D A G O and 1 /~M D T L E T in /~ and 8 binding assays, respectively.
2.2. Assays of biological activity
The synthetic peptides Tyr-Met-Phe-His-LeuM e t - A s p - N H 2 with either L- or D-methionine as the second amino acid were used as markers to search for their natural counterparts in amphibian skin. A methanol extract from the skin of PhylIomedusa sauvagei was fractionated by R P - H P L C as described in the Methods. Fractions were tested for biological activity on the mouse vas deferens (fig. 2). The fraction that co-eluted with deltorphin, the D-MetZ-containing peptide, after the third purification step (fig. 2c) was used for sequence analysis, The automated Edman degradation of the skin peptide gave the sequence: TyrMet-Phe-His-Leu-Met-Asp. The possible presence of a D-methionine and the amidation of the COOH-terminal aspartate could not be determined from this analysis. However, the peptide isolated from skin extracts not only co-eluted with synthetic Tyr-D-Met-Phe-His-Leu-Met-Asp-NH 2, but also had the same biological activity. We tested the inhibitory action of synthetic deltorphin, [L-MetZ]deltorphin and reference peptides on electrically evoked contractions of the myenteric plexus-longitudinal muscle of the guinea pig ileum and of the mouse, rat and rabbit vas deferens. The results obtained with these peptides in the bioassays are summarized in table 1. The high 8 receptor selectivity and affinity of del-
The peptides were tested for their inhibitory action on electrically evoked contractions in isolated preparations of myenteric plexus-longitudinal muscle of guinea-pig ileum and of mouse, rat~ and rabbit vas deferens. The synthetic reference peptides were DER, D A D L E and DPDPE. The assays were performed in the presence or absence of naloxone and the 6 opioid receptor antagonist ICI 174,864. The equilibrium constant K e for the antagonists naloxone and ICI 174,864 was calculated by using the formula Ke = Q / ( D R - 1 ) , where Q is the antagonist concentration (nM) and D R is the dose ratio in antagonist-treated versus antagonist-free preparations.
2.3. Receptor-binding assays The binding of the peptides to opioid receptors in rat brain homogenates was assayed as described by Mosberg et al. (1983). The /~ binding site was selectively labeled with [3H]DAGO (1 nM), the 8 binding site with [3H]DTLET (1 nM) in the presence of 100 nM D A G O to suppress ~t binding, and the k-binding site with [3H](-)-bremazocine (0.15 nM) in the presence of 100 nM each of D A G O and D T L E T to suppress t~ and ~ binding.
3. Results
TABLE 1 Inhibitory potencies (ICso) of dermorphin, DADLE, DPDPE, synthetic dehorphin, natural deltorphin and L[Meta]dehorphin in guinea pig ileum (GPI) and mouse vas deferens (MVD) asays. Peptides Dermorphin DADLE DPDPE Synthetic deltorphin Natural deltorphin [L-Met2]dehorphin
ICs0 (riM _+S.E.) MVD
GPI
MVD/GPI
23.16_+ 4.56 0.63_+ 0.066 4.74+_ 0.393 1.09 +_ 0.078 0.85_+ 0.047 1 186 +_282
1.07 + 0.08 9.12_+ 0.85 2698 _+645 2460 _+735 2500 ± 880 > 100.000
21.54 0.07 0.0018 0.0004 0.0004
127 TABLE 2 Receptor binding affinity (Ki) and selectivity of dermorphin, DADLE, DTLET. D P D P E and deltorphin for ~t, 6 and ~ opioid receptors. Peptides
Dermorphin DADLE DTLET DPDPE Deltorphin
K i (nM-+ S.E.)
0.35_+ 0.03 11.36_+ 0.89 28.45_+ 8.24 990 -+ 98 1 630 + 114
8
g
Receptor selectivity (Ki6/Ki'u')
1730 _+28 2.31_+ 0.33 2.22_+ 0.57 18.87-+ 0.64 2.41 -+ 0.03
18200_+ 350 10350_+ 895 22380_+1042 >25000 > 25 000
4943 0.20 0.08 0.02 0.0015
torphin was evident from these tests. On the mouse vas deferens, the ICs0 for deltorphin was about 1000, 20 and 6 times lower than that for [LMet2]deltorphin, D E R and DPDPE, respectively, and only about 25% higher than the ICs0 for DADLE. In the same assay, the K e values (nM) for naloxone were: 76.9 _+ 2.1 for deltorphin, 43.2 +_ 3.4 for D P D P E and 5.3 + 0.6 for DER. This confirms that naloxone is a weak antagonist of 8 receptor selective opioids. In the mouse vas deferens, ICI 174,864 was a slightly better antagonist of deltorphin and D P D P E than naloxone was, but was completely inactive with D E R as agonist. The K e values (riM) for ICI 174,864 were: 23.2 + 4.5 for deltorphin, 19.5 + 3.8 for D P D P E and 150 + 5.2 for DER. On the guinea pig ileum, deltorphin was 2 300 and 270 times less active than D E R and DADLE, respectively. In this assay, the D-Met2-containing peptide was again at least 100 times more potent than the L isomer. The ratio of the ICs0 value in the mouse vas deferens to the ICs0 value in the guinea pig ileum is considered to give an indication of the selectivity of a compound for 8 opioid receptors (Corbett et al., 1984). The very low ratios obtained for deltorphin and D P D P E (table 1) further demonstrate their high selectivity in this respect. Conversely, the selective/~ receptor ligand DER had the highest ICs0 ratio in these two assays. Both deltorphin and [L-Met2]deltorphin were completely inactive on the rat and rabbit vas deferens (data not shown). This confirms that these peptides do not bind to/~ receptors (rat vas deferens) and also indicates that they have no significant affinity for x receptors (rabbit vas deferens).
The deltorphin isolated from skin extracts was only tested on the mouse vas deferens and guinea pig ileum because of the low amount present. In these assays, the activity of the natural compound was indistinguishable from that of the synthetic peptide (table 1). Table 2 shows the apparent affinity (Ki, nM) of these peptides at /~, 8 and x sites in rat brain homogenates. Receptor selectivity was calculated by the ratio of the K i for 8 sites to the K i for/~ sites. It is evident that both the affinity and the selectivity of deltorphin for the 8 binding sites in brain homogenates were more than one order of magnitude higher than those of DPDPE. In fact, the affinity of deltorphin for 8 sites was practically identical to that of DTLET, but its selectivity was about 50 times greater. In these assays, DER showed the highest affinity and selectivity for # binding sites.
4. Discussion
These results demonstrate that, besides dermorphin, a second peptide containing a D-amino acid is present in the skin of Phyllomedusa sauvagei. The new peptide, termed deltorphin, appears to be present in rather low amounts, about 1 # g / g of fresh skin, corresponding to about 1 / 5 0 t h of the content of dermorphin. The detection of a peptide with another D-amino acid in its sequence prompts certain suggestions about the biosynthesis of these residues. It seems likely that the enzyme(s) catalyzing the epimerization of certain amino acids recognizes the sequence common to both dermorphin and deltorphin, i.e. a Tyr-X-Phe
128 structure a n d possibly a free a - a m i n o group, r a t h e r t h a n the side c h a i n of the a m i n o acid to be c o n v e r t e d from the L to the D isomer. A whole family of p e p t i d e with the a m i n o - t e r m i n a l sequence T y r - D - X - P h e m a y thus exist in the skin of Phyllomedusa species a n d p o s s i b l y in o t h e r biological systems as well. The two peptides, d e r m o r p h i n a n d d e l t o r p h i n , differed significantly in their affinity for o p i o i d receptors. D e l t o r p h i n is a n a t u r a l l y o c c u r r i n g linear o p i o i d with the highest affinity a n d selectivity for 6 b i n d i n g sites yet observed. In a g r e e m e n t with the m o d e l of 8 r e c e p t o r r e c o g n i t i o n suggested b y F o u r n i 6 - Z a l u s k i et al. (1981), this m o r e flexible linear p e p t i d e showed a 20 times higher affinity for 6 b i n d i n g sites t h a n the c o n f o r m a t i o n ally restricted cyclic e n k e p h a l i n a n a l o g u e D P D P E . T h u s the suggestion that the highest level of selectivity t o w a r d s a given o p i o i d b i n d i n g site m a y o n l y be achieved b y i m p o s i n g c o n f o r m a t i o n a l restrictions on p e p t i d e chains ( M o s b e r g et al., 1983) is not s u p p o r t e d b y these observations. In o u r view, the two p e p t i d e s isolated from skin of Phyllomedusa sauvagei, d e r m o r p h i n a n d d e l t o r p h i n , m a y well represent the most a p p r o p r i a t e , p o t e n t a n d selective tools for b i n d i n g a n d p h a r m a c o l o g i cal studies on /z a n d 8 o p i o i d receptors, respectively.
Acknowledgements These studies were supported by a Grant from the Austrian Funds zur Forderung ($29T4 to G.K.), from Italian Ministero
Pubblica Istruzione and National Research Council of Italy. M.S. is a recipient of an Anna Villa Rusconi fellowship.
References Broccardo, M., V. Erspamer, G. Falconieri Erspamer, G. Improta, G. Linari, P. Melchiorri and P.C. Montecucchk 198l, Pharmacological data on dermorphins, a new class of potent opioid peptides from amphibian skin, Br. J. Pharmacol. 73, 625. Corbett, A~D., M.G.C. Gillan, H.W. Kosterlitz, A.T. McKnight, S.J. Paterson and L.E. Robson, 1984, Selectivities of opioid peptide analogues as agonists and antagonists at the &receptor, Br. J. Pharmacol. 83, 271. Delay-Goyet, P., C. Seguin, G. Gacel and B.P. Roques, 1988, [3H][D-Ser2(O-tert-butyl), LeuS]enkephalyl-Thr 6 and [DSer 2(O-tert-butyl), Leu5 ]enkephalyl-Thr 6(O-tert-butyl). Two new enkephalin analogs with both a good selectivity and a high affinity toward 6-opioid binding sites, J. Biol. Chem. 263, 4124. Fourni~-Zaluski, M.C., G. Gacel, B. Maigret, S. Premilat and B.P. Roques, 1981, Structural requirements for specific recognition of /z and 6 opiate receptors, Mol. Pharmacol. 20, 484. Mosberg, H.I., R. Hurst, V.J. Hruby, K. Gee, H.I. Yamamura, J.J. Gallican and T.F. Burks, 1983, Bis-penicillamine enkephalins possess highly improved specificity toward 6opioid receptors, Proc. Natl. Acad. Sci. U.S.A. 80, 5871. Richter, K., R. Egger and G. Kreil, 1987, D-Alanine in the frog skin peptide dermorphin is derived from L-alanine in the precursor, Science 238, 200. Rossi, A.C, R. De Castiglione and G. Perseo, 1986, Opioid receptor binding profile of selected dermorphin-like peptides, Peptides 7, 755.
123
EJP 50688
Deltorphin, a novel amphibian skin peptide with high selectivity and affinity for 8 opioid receptors G u n t h e r Kreil t, D o n a t e l l a B a r r a 2, M a u r i z i o S i m m a c o 2, V i t t o r i o E r s p a m e r 3 G i u l i a n a F a l c o n i e r i E r s p a m e r 3, L u c i a N e g r i 3, Cinzia Severini 3, R i t a Corsi 3' a n d Pietro M e l c h i o r r i 3,. t Institute of Molecular Biology, Salzburg, Austria, : Department of Biochemical Sciences and Center for Molecular Biology of the National Research Council, and 3 Institute of Medical Pharmacology, University La Sapienza, Rome, Italy Received 28 July 1988, revised MS received 18 November 1988, accepted 13 December 1988
With a cDNA library prepared from skin of Phyllomedusa sauvagei, the sequence of the precursor of dermorphin was elucidated recently. The sequence suggested the existence of another peptide, distantly related to dermorphin. Two variants of this peptide have now been synthesized containing either L- or D-methionine as the second amino acid. The peptide containing the D-methionine exhibited high-affinity and selectivity for 8 opioid receptors in the mouse vas deferens and in rat brain homogenates. Moreover, using the synthetic peptide as marker, we could isolate small quantities of the corresponding natural peptide containing D-methionine as the second amino acid from skin extracts of Phyllomedusa sauvagei. The name deltorphin is proposed for this new peptide and its sequence is Tyr-D-Met-PheHis-Leu-Met-Asp-NH> Deltorphin; 80pioid receptor agonists; (Isolation, Sequencing)
1. Introduction
The heptapeptide dermorphin, isolated from the skin of the South American frog Phyllomedusa sauvagei, displays unusually high opioid activity. It binds selectively and with high affinity to the/~ binding site of opioid receptors (Rossi et al., 1986). The biological activity of dermorphin is dependent on the presence of a D-alanine residue in the second position; homologues with an L-amino acid in this position are inactive (Broccardo et al., 1981). Using recombinant D N A techniques, Richter et al. (1987) showed that dermorphin, like numerous other peptides, is derived in multiple
* To whom all correspondence should be addressed: Institute of Medical Pharmacology III, University of Rome 'La Sapienza', P.za A. Moro 5, 00185 Rome, Italy.
copies from larger precursors. In the cloned cDNAs of these precursors, a normal alanine codon was found at the position where D-alanine is present in the end product, pointing to a posttranslational conversion of specific alanine residues from the L to the D isomer. From the sequence of one of these cloned cDNAs, the existence of another heptapeptide, only distantly related to dermorphin, could be predicted, assuming that the same processing mechanism as that which liberates dermorphin from its precursors operated (fig. 1). Two variants of this peptide have now been synthesized containing either L- or Dmethionine as the second amino acid. We now report that the peptide with the D isomer selectively bound to 8 opioid receptors with an affinity that was an order of magnitude higher than that of the synthetic cyclic bis-penicillamine enkephalin analogues (Mosberg et al., 1983) and which
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
124 a.......
Lys-Arg-Tyr-Ala-Phe-Gly-Tyr-Pro-Ser-Gly-Glu-Ala-Lys-Lys...
b.......
Lys-Arg-Tyr-Met-Phe-His-Leu-Met-Asp-Gly-Glu-Ala-Lys-Lys...
Fig. 1. Segment of the amino acid sequence of prepro-dermorphin as predicted from the nucleotide sequence of clone D - l / 2 (for details see Richter et al., 1987). Amino acids present in the end product dermorphin (a) and the predicted peptide deltorphin (b) are in bold type.
was in the same range of affinity exhibited by the recently described, highly &selective linear hexapeptides D S T B U L E T and BUBU (Delay-Goyet et al., 1988). Moreover, the peptide could be isolated from skin extracts of Phyllomedusa sauvagei. This peptide is thus the first naturally occurring peptide with a high selectivity for 6 receptors. This peptide has accordingly been named deltorphin. 2. Materials and methods
The heptapeptides Tyr-Met-Phe-His-Leu-MetAsp-NH 2 with either L- or D-methionine as the second amino acid were custom synthesized by Biotec Nordhorn (Nordhorn, FRG). Samples of these peptides were also kindly supplied by Drs. Gozzini and De Castiglione (Farmitalia Carlo Erba, Milano, Italy). Dermorphin (DER), [DAlaZ,D-Leu 5]enkephalin (DADLE), [D-Ala 2,MePhe4,Gly-olS]enkephalin (DAGO) and [D-Pen2,D PenS]enkephalin (DPDPE) were purchased from Peninsula Labs (Belmont, CA, USA), [D-Thr 2, Leu 5]enkephalyl-Thr 6 (DTLET) and AllylZ-TyrAib-Aib-Phe-Leu-PH (ICI 174,864) from CRB (Cambridge, England). The peptides were purified by HPLC (see below) to a purity of more than 95%, and their claimed sequence was confirmed by automatic Edman degradation carried out on an Applied Biosystems peptide sequencer model 470A (see below) before they were used in the biological assays and as marker substances for the isolation of deltorphin from skin extracts. Radioactive peptides were purchased from Amersham (England).
2.1. Isolation and characterization of deltorphin from skin extracts of Phyllomedusa sauvagei Frog skin was minced with scissors and extracted at room temperature for 24 h with four volumes ( v / w ) of methanol. This procedure was
repeated three times and the supernatants were combined and evaporated under vacuum. The dried methanol extract was dissolved in 50% aqueous acetonitrile and fractionated (extract from 2 g skin per run) by reverse phase high performance liquid chromatography (RP-HPLC) (Aquapore RP 300, C 18, 7 × 250 mm, 7 t~m, Brownlee Labs, USA) with a linear gradient of solvent B (0.2% trifluoroacetic acid (TFA, Pierce Chemical Company, USA) in acetonitrile-isopropyl alcohol 4:1, v / v ) on solvent A (0.2% TFA) in water) at a flow rate of 2.8 m l / m i n (fig. 2a). The biological activity of the collected fractions was tested on in vitro preparations of electrically stimulated mouse vas deferens and expressed as nmol of synthetic deltorphin (fig. 2a: black bars). The fraction that eluted between 25 and 30% of solvent B, corresponding to the elution zone of the biological activity, was further purified by RP-HPLC (Spheri 5, C18, 4.6 × 250 mm, 5 /1, Brownlee Labs) at a flow rate of 1 ml/min, with a 40 rain gradient from 30 to 70% acetonitrile on 0.2% aqueous TFA (fig. 2b). Pooled bioactive fractions were submitted to a further RP-HPLC fractionation (Supelcosil LC18DB, 4.6 × 250 mm, 5 /~, 100 A °, Supelco, USA) with a different acetonitrile gradient in aqueous TFA at a flow rate of 1.5 m l / m i n (solvent A, 0.1% TFA in water, solvent B, 0.1% TFA in acetonitrile; 10-100% B in four steps: 10-15% over 10 min, 15-25% over 30 min, 25-50% over 25 min, and 50-100% over 20 min). The elution profiles of the two synthetic deltorphins in this system are shown by the dashed upper trace in fig. 2c. The retention times were 47.7 rain for [L-Met2]deltorphin and 51.4 min for deltorphin. The peptide co-eluting with synthetic deltorphin after the third purification step was used for the sequence analysis as well as for the biological assays. Sequence analysis of this peptide was performed on an Applied Biosystems Model 470A gas-phase protein sequencer equipped with an Ap-
125
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Fig. 2. Purification of deltorphin from skin extracts of Phyllomedusasauvagei. (a) HPLC on an Aquapore RP 300 column of the crude extract from 2 g of the amphibian skin. (b) Further HPLC of bioactive fractions collected in the previous chromatogram (see a black columns) on Spheri 5, C18, column. (c) Final chromatography of active fractions from b on Supelcosil LC18DB column. The elution profiles of the two synthetic deltorphins, [L-Met2]deltorphin (e) and deltorphin (11), used as reference standards in thL, system, are shown by dashed upper trace.
126 plied Biosystems Model 120A P T H Analyzer for the on-line detection of phenylthiohydantoins. The sample was dissolved in 30% acetonitrile and loaded onto a glass fiber filter coated with polybrene and pre-washed according to the manufacturer's instructions.
Non-specific binding was defined by 1 t~M D A G O and 1 /~M D T L E T in /~ and 8 binding assays, respectively.
2.2. Assays of biological activity
The synthetic peptides Tyr-Met-Phe-His-LeuM e t - A s p - N H 2 with either L- or D-methionine as the second amino acid were used as markers to search for their natural counterparts in amphibian skin. A methanol extract from the skin of PhylIomedusa sauvagei was fractionated by R P - H P L C as described in the Methods. Fractions were tested for biological activity on the mouse vas deferens (fig. 2). The fraction that co-eluted with deltorphin, the D-MetZ-containing peptide, after the third purification step (fig. 2c) was used for sequence analysis, The automated Edman degradation of the skin peptide gave the sequence: TyrMet-Phe-His-Leu-Met-Asp. The possible presence of a D-methionine and the amidation of the COOH-terminal aspartate could not be determined from this analysis. However, the peptide isolated from skin extracts not only co-eluted with synthetic Tyr-D-Met-Phe-His-Leu-Met-Asp-NH 2, but also had the same biological activity. We tested the inhibitory action of synthetic deltorphin, [L-MetZ]deltorphin and reference peptides on electrically evoked contractions of the myenteric plexus-longitudinal muscle of the guinea pig ileum and of the mouse, rat and rabbit vas deferens. The results obtained with these peptides in the bioassays are summarized in table 1. The high 8 receptor selectivity and affinity of del-
The peptides were tested for their inhibitory action on electrically evoked contractions in isolated preparations of myenteric plexus-longitudinal muscle of guinea-pig ileum and of mouse, rat~ and rabbit vas deferens. The synthetic reference peptides were DER, D A D L E and DPDPE. The assays were performed in the presence or absence of naloxone and the 6 opioid receptor antagonist ICI 174,864. The equilibrium constant K e for the antagonists naloxone and ICI 174,864 was calculated by using the formula Ke = Q / ( D R - 1 ) , where Q is the antagonist concentration (nM) and D R is the dose ratio in antagonist-treated versus antagonist-free preparations.
2.3. Receptor-binding assays The binding of the peptides to opioid receptors in rat brain homogenates was assayed as described by Mosberg et al. (1983). The /~ binding site was selectively labeled with [3H]DAGO (1 nM), the 8 binding site with [3H]DTLET (1 nM) in the presence of 100 nM D A G O to suppress ~t binding, and the k-binding site with [3H](-)-bremazocine (0.15 nM) in the presence of 100 nM each of D A G O and D T L E T to suppress t~ and ~ binding.
3. Results
TABLE 1 Inhibitory potencies (ICso) of dermorphin, DADLE, DPDPE, synthetic dehorphin, natural deltorphin and L[Meta]dehorphin in guinea pig ileum (GPI) and mouse vas deferens (MVD) asays. Peptides Dermorphin DADLE DPDPE Synthetic deltorphin Natural deltorphin [L-Met2]dehorphin
ICs0 (riM _+S.E.) MVD
GPI
MVD/GPI
23.16_+ 4.56 0.63_+ 0.066 4.74+_ 0.393 1.09 +_ 0.078 0.85_+ 0.047 1 186 +_282
1.07 + 0.08 9.12_+ 0.85 2698 _+645 2460 _+735 2500 ± 880 > 100.000
21.54 0.07 0.0018 0.0004 0.0004
127 TABLE 2 Receptor binding affinity (Ki) and selectivity of dermorphin, DADLE, DTLET. D P D P E and deltorphin for ~t, 6 and ~ opioid receptors. Peptides
Dermorphin DADLE DTLET DPDPE Deltorphin
K i (nM-+ S.E.)
0.35_+ 0.03 11.36_+ 0.89 28.45_+ 8.24 990 -+ 98 1 630 + 114
8
g
Receptor selectivity (Ki6/Ki'u')
1730 _+28 2.31_+ 0.33 2.22_+ 0.57 18.87-+ 0.64 2.41 -+ 0.03
18200_+ 350 10350_+ 895 22380_+1042 >25000 > 25 000
4943 0.20 0.08 0.02 0.0015
torphin was evident from these tests. On the mouse vas deferens, the ICs0 for deltorphin was about 1000, 20 and 6 times lower than that for [LMet2]deltorphin, D E R and DPDPE, respectively, and only about 25% higher than the ICs0 for DADLE. In the same assay, the K e values (nM) for naloxone were: 76.9 _+ 2.1 for deltorphin, 43.2 +_ 3.4 for D P D P E and 5.3 + 0.6 for DER. This confirms that naloxone is a weak antagonist of 8 receptor selective opioids. In the mouse vas deferens, ICI 174,864 was a slightly better antagonist of deltorphin and D P D P E than naloxone was, but was completely inactive with D E R as agonist. The K e values (riM) for ICI 174,864 were: 23.2 + 4.5 for deltorphin, 19.5 + 3.8 for D P D P E and 150 + 5.2 for DER. On the guinea pig ileum, deltorphin was 2 300 and 270 times less active than D E R and DADLE, respectively. In this assay, the D-Met2-containing peptide was again at least 100 times more potent than the L isomer. The ratio of the ICs0 value in the mouse vas deferens to the ICs0 value in the guinea pig ileum is considered to give an indication of the selectivity of a compound for 8 opioid receptors (Corbett et al., 1984). The very low ratios obtained for deltorphin and D P D P E (table 1) further demonstrate their high selectivity in this respect. Conversely, the selective/~ receptor ligand DER had the highest ICs0 ratio in these two assays. Both deltorphin and [L-Met2]deltorphin were completely inactive on the rat and rabbit vas deferens (data not shown). This confirms that these peptides do not bind to/~ receptors (rat vas deferens) and also indicates that they have no significant affinity for x receptors (rabbit vas deferens).
The deltorphin isolated from skin extracts was only tested on the mouse vas deferens and guinea pig ileum because of the low amount present. In these assays, the activity of the natural compound was indistinguishable from that of the synthetic peptide (table 1). Table 2 shows the apparent affinity (Ki, nM) of these peptides at /~, 8 and x sites in rat brain homogenates. Receptor selectivity was calculated by the ratio of the K i for 8 sites to the K i for/~ sites. It is evident that both the affinity and the selectivity of deltorphin for the 8 binding sites in brain homogenates were more than one order of magnitude higher than those of DPDPE. In fact, the affinity of deltorphin for 8 sites was practically identical to that of DTLET, but its selectivity was about 50 times greater. In these assays, DER showed the highest affinity and selectivity for # binding sites.
4. Discussion
These results demonstrate that, besides dermorphin, a second peptide containing a D-amino acid is present in the skin of Phyllomedusa sauvagei. The new peptide, termed deltorphin, appears to be present in rather low amounts, about 1 # g / g of fresh skin, corresponding to about 1 / 5 0 t h of the content of dermorphin. The detection of a peptide with another D-amino acid in its sequence prompts certain suggestions about the biosynthesis of these residues. It seems likely that the enzyme(s) catalyzing the epimerization of certain amino acids recognizes the sequence common to both dermorphin and deltorphin, i.e. a Tyr-X-Phe
128 structure a n d possibly a free a - a m i n o group, r a t h e r t h a n the side c h a i n of the a m i n o acid to be c o n v e r t e d from the L to the D isomer. A whole family of p e p t i d e with the a m i n o - t e r m i n a l sequence T y r - D - X - P h e m a y thus exist in the skin of Phyllomedusa species a n d p o s s i b l y in o t h e r biological systems as well. The two peptides, d e r m o r p h i n a n d d e l t o r p h i n , differed significantly in their affinity for o p i o i d receptors. D e l t o r p h i n is a n a t u r a l l y o c c u r r i n g linear o p i o i d with the highest affinity a n d selectivity for 6 b i n d i n g sites yet observed. In a g r e e m e n t with the m o d e l of 8 r e c e p t o r r e c o g n i t i o n suggested b y F o u r n i 6 - Z a l u s k i et al. (1981), this m o r e flexible linear p e p t i d e showed a 20 times higher affinity for 6 b i n d i n g sites t h a n the c o n f o r m a t i o n ally restricted cyclic e n k e p h a l i n a n a l o g u e D P D P E . T h u s the suggestion that the highest level of selectivity t o w a r d s a given o p i o i d b i n d i n g site m a y o n l y be achieved b y i m p o s i n g c o n f o r m a t i o n a l restrictions on p e p t i d e chains ( M o s b e r g et al., 1983) is not s u p p o r t e d b y these observations. In o u r view, the two p e p t i d e s isolated from skin of Phyllomedusa sauvagei, d e r m o r p h i n a n d d e l t o r p h i n , m a y well represent the most a p p r o p r i a t e , p o t e n t a n d selective tools for b i n d i n g a n d p h a r m a c o l o g i cal studies on /z a n d 8 o p i o i d receptors, respectively.
Acknowledgements These studies were supported by a Grant from the Austrian Funds zur Forderung ($29T4 to G.K.), from Italian Ministero
Pubblica Istruzione and National Research Council of Italy. M.S. is a recipient of an Anna Villa Rusconi fellowship.
References Broccardo, M., V. Erspamer, G. Falconieri Erspamer, G. Improta, G. Linari, P. Melchiorri and P.C. Montecucchk 198l, Pharmacological data on dermorphins, a new class of potent opioid peptides from amphibian skin, Br. J. Pharmacol. 73, 625. Corbett, A~D., M.G.C. Gillan, H.W. Kosterlitz, A.T. McKnight, S.J. Paterson and L.E. Robson, 1984, Selectivities of opioid peptide analogues as agonists and antagonists at the &receptor, Br. J. Pharmacol. 83, 271. Delay-Goyet, P., C. Seguin, G. Gacel and B.P. Roques, 1988, [3H][D-Ser2(O-tert-butyl), LeuS]enkephalyl-Thr 6 and [DSer 2(O-tert-butyl), Leu5 ]enkephalyl-Thr 6(O-tert-butyl). Two new enkephalin analogs with both a good selectivity and a high affinity toward 6-opioid binding sites, J. Biol. Chem. 263, 4124. Fourni~-Zaluski, M.C., G. Gacel, B. Maigret, S. Premilat and B.P. Roques, 1981, Structural requirements for specific recognition of /z and 6 opiate receptors, Mol. Pharmacol. 20, 484. Mosberg, H.I., R. Hurst, V.J. Hruby, K. Gee, H.I. Yamamura, J.J. Gallican and T.F. Burks, 1983, Bis-penicillamine enkephalins possess highly improved specificity toward 6opioid receptors, Proc. Natl. Acad. Sci. U.S.A. 80, 5871. Richter, K., R. Egger and G. Kreil, 1987, D-Alanine in the frog skin peptide dermorphin is derived from L-alanine in the precursor, Science 238, 200. Rossi, A.C, R. De Castiglione and G. Perseo, 1986, Opioid receptor binding profile of selected dermorphin-like peptides, Peptides 7, 755.