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Abstract
Dermorphin (Tyr-d-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) is a heptapeptide isolated from amphibian skin. With a very high affinity and selectivity for μ-opioid receptors, dermorphin shows an extremely potent antinociceptive effect. The structure–activity relationship studies of dermorphin analogs clearly suggest that the N-terminal tetrapeptide is the minimal sequence for agonistic activity at μ-opioid receptors, and that the replacement of the d-Ala2 residue with d-Arg2 makes the tetrapeptides resistant to enzymatic metabolism. At present, only a handful of dermorphin N-terminal tetrapeptide analogs containing d-Arg2 have been developed. The analogs show potent antinociceptive activity that is greater than that of morphine with various injection routes, and retain high affinity and selectivity for μ-opioid receptors. Interestingly, some analogs show pharmacological profiles that are distinct from the traditional μ-opioid receptor agonists morphine and [d-Ala2,NMePhe4,Gly-ol5]enkephalin (DAMGO). These analogs stimulate the release of dynorphins through the activation of μ-opioid receptors. The activation of κ-opioid receptors by dynorphins is suggested to reduce the side effects of μ-opioid receptor agonists, e.g., dependence or antinociceptive tolerance. The dermorphin N-terminal tetrapeptide analogs containing d-Arg2 may provide a new target molecule for developing novel analgesics that have fewer side effects.
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2 Dermorphin
Dermorphin (Tyr-d-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) is a heptapeptide isolated from amphibian skin [26]. As shown in Table 1, the amino acid sequence of dermorphin is quite different from the other endogenous opioid peptides. Dermorphin does not contain the common N-terminal sequence for the traditional endogenous opioid peptides (Tyr-Gly-Gly-Phe), and its sequence is completely different from that of the endomorphins, which were identified as endogenous μ-opioid peptides that also do not contain the common sequence. Interestingly, dermorphin contains d-Ala (d-isomer amino acid) in its sequence. In the opioid receptor binding assay using the mouse brain, dermorphin shows a very high affinity for μ-opioid receptors with a low affinity for δ-opioid receptors (Table 2) [12]. Without any significant affinity for κ-opioid receptors, dermorphin shows a higher selectivity for μ-opioid receptors than morphine. The intrinsic activity of dermorphin for G-protein activation is similar to that of DAMGO and higher than that of morphine (Fig. 1), suggesting that dermorphin, as well as DAMGO, is a full agonist for μ-opioid receptors, whereas morphine is a partial agonist for that receptor. With a high affinity and intrinsic activity for μ-opioid receptors, dermorphin shows a strong and prolonged antinociceptive effect with various injection routes. After i.c.v. injection, dermorphin is approximately 200 times more potent than morphine for antinociception [11]. Unlike other peptidic analgesics, dermorphin shows potent antinociceptive effects with peripheral injection routes, such as s.c. and i.v. injections [40]. Although the blood–brain barrier (BBB) permeability of dermorphin is markedly lower than for morphine, dermorphin still has a four-fold more potent antinociceptive effect than morphine after s.c. injection [31]. The peripherally-administered dermorphin is mainly degraded to the deamidated N-terminal tetrapeptide (dermorphin[1-4]-OH) by cleaving enzymes in the blood, lever, brain, or spinal cord [30]. However, its rate of degradation is more than 30 times slower than for other endogenous opioid peptides [50]. Moreover, dermorphin[1-4]-OH, the main metabolite of dermorphin, does not have remarkable agonistic activity for μ-opioid receptors [17]. The potency of dermorphin[1-4]-OH for inhibiting the guinea pig ileum contraction is only 1/525 of dermorphin. Therefore, the potent and prolonged antinociceptive effect of dermorphin may be due to its slow degradation, but not to its active metabolites.
Table 1. Endogenous opioid peptides.
Endogenous μ-opioid peptides Endomorphin-1 Tyr1-Pro-Trp-Phe4-NH2 Endomorphin-2 Tyr1-Pro-Phe-Phe4-NH2 β-Endorphin Tyr1-Gly-Gly-Phe-Met5-Thr-Ser-Glu-Lys-Ser10-Gln-Thr-Pro-Leu-Val15-Thr-Leu-Phe-Lys-Asn20-Ala-Ile-Ile-Lys-Asn25-Ala-Tyr-Lys-Lys-Gly30-Gln31-OH Dermorphin Tyr1-d-Ala-Phe-Gly-Tyr5-Pro-Ser7-NH2 Endogenous δ-opioid peptides [Met5]Enkephalin Tyr1-Gly-Gly-Phe-Met5-OH [Leu5]Enkephalin Tyr1-Gly-Gly-Phe-Leu5-OH Endogenous κ-opioid peptides Dynorphin A Tyr1-Gly-Gly-Phe-Leu5-Arg-Arg-Ile-Arg-Pro10-Lys-Leu-Lys-Trp-Asp15-Asn-Gln17-OH Dynorphin B Tyr1-Gly-Gly-Phe-Leu5-Arg-Arg-Gln-Phe-Lys10-Val-Val-Thr13-OH α-Neo-endorphin Tyr1-Gly-Gly-Phe-Leu5-Arg-Lys-Tyr-Pro-Lys10-OH β-Neo-endorphin Tyr1-Gly-Gly-Phe-Leu5-Arg-Lys-Tyr-Pro9-OH
Table 2. Affinities of ligands for μ-, δ- and κ-opioid receptors in mouse brain.
Ligands IC50 values with their 95% confidence intervals (nM) Empty Cell [3H]DAMGO (μ) [3H]DPDPE (δ) [3H]U-69,593 (κ) Morphine 2.84 (2.57–3.14) 133 (100–176) 141 (114–174) Dermorphin 0.76 (0.69–0.83) 73.5 (59.1–91.4) >1000 Endomorphin-1 2.86 (2.49–3.28) n.d. n.d. [d-Ala2]Deltorphin II n.d. 2.29 (1.98–2.66) n.d. U-50,488H n.d. n.d. 0.76 (0.60–0.95)
Membranes obtained from ICR mouse whole brain without the cerebellum were incubated at 25 °C for 2 h with 1 nM of [3H]DAMGO, [3H]DPDPE or [3H]U-69,593 in the absence/presence of various concentrations (0.03–1000 nM) of the agonists. Non-specific binding was measured in the presence of 10 μM naloxone, DPDPE or U-69,593 for the [3H]DAMGO, [3H]DPDPE or [3H]U-69,593 binding assays, respectively. The IC50 values with their 95% confidence intervals for the agonists in each binding assay were calculated with GraphPad Prism, a computer-assisted curve-fitting program. n.d., not determined.
Fig. 1. The increase in [35S]GTPγS binding by DAMGO, dermorphin or morphine in spinal cord membranes obtained from ICR mice. Membranes were incubated with 50 pM [35S]GTPγS and 30 μM GDP in the absence/presence of various concentrations (1 nM–10 μM) of DAMGO, dermorphin or morphine for 2 h at 25 °C. Non-specific binding was measured in the presence of 10 μM unlabeled GTPγS. The data are expressed as the percentage of basal [35S]GTPγS binding measured in the presence of GDP and in the absence of agonist, and represent the mean ± S.E.M. for at least three independent experiments. The concentration-effect curves were calculated with GraphPad Prism, a computer-assisted curve-fitting program. The maximum stimulation with DAMGO, dermorphin or morphine is 93.77%, 94.90% or 49.55%, respectively.
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4.4 PWT2-dermorphin
Dermorphin (Table 1) is a potent opioid peptide isolated from amphibian skin by the Espamer group in the early 80′ [65]. Dermorphin displayed potent inhibitory effects in the guinea pig bioassay and long lasting, and naloxone sensitive, antinociceptive activity in rodents [66]. The dermorphin peptide sequence has been the subject to a large number of structure activity studies that increased our knowledge about the chemical requirement for mu opioid receptor binding and activation [67,68]. We synthesised and pharmacologically characterised in vitro PWT2-dermorphin, the tetrabranched PWT derivative of the mu opioid peptide dermorphin.
PWT2-dermorphin was assayed in calcium mobilization studies performed on cells coexpressing the human opioid receptors and chimeric G proteins. This assay has been previously validated [69,70] and then used for investigating novel compounds. On mu receptor expressing cells, dermorphin produced stimulatory effects in line with literature data. PWT2-dermorphin mimicked the stimulatory effects of dermorphin, showing slightly lower efficacy and being 3 fold less potent (Table 2). In cells expressing kappa, delta, or NOP receptors the standard agonists dynorphin A, DPDPE, N/OFQ stimulated the release of intracellular calcium with maximal effects and potency values in line with previous findings [69,70]. In these cells, dermorphin and its PWT derivative were either inactive (kappa and NOP) or produced a modest stimulation at micromolar concentrations (delta) (Table 2). Therefore, the high selectivity displayed by the natural peptide dermorphin on the mu receptor is maintained by its PWT derivative. The ability of dermorphin and PWT2-dermorphin to promote mu/G protein and mu/β-arrestin 2 interaction has been assessed using a BRET assay [51,71]. Dermorphin and PWT2-dermorphin promoted receptor interaction with G protein and β-arrestin 2 with similar efficacy and potency values (Fig. 4A and B). Thus PWT2-dermorphin behaves as an unbiased mu agonist. This result is somewhat different from those previously obtained with PWT2-N/OFQ [48] and PWT2-[Dmt1]N/OFQ(1-13) [36] where the PWT chemical modification confers to the molecules G protein biased agonism.
Table 2. Effects of dermorphin and PWT2-dermorphin in CHO cells expressing the human recombinant receptors and chimeric G proteins in the calcium mobilization assay.
Empty Cell mu kappa delta NOP Empty Cell pEC50 (CL95%) Emax ± sem pEC50 (CL95%) Emax ± sem pEC50 (CL95%) Emax ± sem pEC50 (CL95%) Emax ± sem dermorphin 8.19 (7.80–8.58) 345 ± 14% inactive 5.98 (5.76–6.19) 180 ± 31% inactive PWT2-dermorphin 7.66 (7.26–8.06) 293 ± 12% inactive inactive inactive
Fig. 4. Pharmacological profile of PWT2-dermorphin. Concentration response curves to dermorphin (panel A) and PWT2-dermorphin (panel B) in the BRET assay measuring mu/G protein and mu/β-arrestin 2 interaction. Concentration response curve to dermorphin (panel C) and PWT2-dermorphin (panel D) obtained in the absence (control) and presence of naloxone 100 nM in the electrically stimulated guinea pig ileum.
In order to investigate the pharmacological features of PWT2-dermorphin at native animal receptors experiments were performed in the electrically stimulated guinea pig ileum bioassay. In this preparation dermorphin inhibited the electrically induced contractions (Fig. 4C) with efficacy and potency in line with literature data [50]. PWT2-dermorphin mimicked the inhibitory effect of the natural peptide showing similar efficacy but approximately 10 fold lower potency (Fig. 4D). Interestingly, similar to previous reports on PWT2-N/OFQ [33], the kinetic of action of PWT2-dermorphin was slower than that of dermorphin and its inhibitory effects were only partially reversible by washing. The opioid receptor antagonist naloxone produced a rightward shift of the concentration response curve to dermorphin and PWT2-dermorphin without modifying the maximal effects induced by the agonists; pA2 values of 9.07 and 8.82 were obtained for naloxone against dermorphin and PWT2-dermorphin, respectively. The high pA2 value of naloxone suggests that the biological effects of both dermorphin and PWT2-dermorphin in this preparation are solely due to their ability to activate the mu opioid receptor.
In summary, PWT2-dermorphin displays in vitro a profile, in terms of pharmacological activity, unbiased agonism, potency and selectivity of action, similar if not superimposable to that of the parent peptide. This result further corroborates the proposal that the application of the PWT strategy does not affect the in vitro pharmacological features of biologically active peptides. Further studies are under way to investigate the in vivo actions of PWT2-dermorphin.
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3 Endogenous dermorphins
At present, seven endogenous analogs of dermorphin have been identified (Table 3). Unlike dermorphin, three of the endogenous analogs do not have a C-terminus that is protected by amidation. Without the C-terminal amidation, the affinity of these peptides for μ-opioid receptors is dramatically reduced by 5–63 fold [29]. Dermorphin is degraded by enzymes that cleave its C-terminal amino acid [30]. Since C-terminal amidation prevents this degradation by cleaving enzymes, endogenous dermorphin analogs that contain C-terminal amidation may have more potent agonistic activity for μ-opioid receptors than the analogs that lack C-terminal amidation.
Table 3. Dermorphin and its endogenous analogs.
Dermorphin Tyr1-d-Ala-Phe-Gly-Tyr5-Pro-Ser7-NH2 [Hyp6]Dermorphin Tyr1-d-Ala-Phe-Gly-Tyr5-Hyp6-Ser7-NH2 [Lys7]Dermorphin Tyr1-d-Ala-Phe-Gly-Tyr5-Pro-Lys7-NH2 [Lys7]Dermorphin-OH Tyr1-d-Ala-Phe-Gly-Tyr5-Pro-Lys7-OH [Trp4,Ans7]Dermorphin Tyr1-d-Ala-Phe-Trp4-Tyr5-Pro-Ans7-NH2 [Trp4,Ans7]Dermorphin-OH Tyr1-d-Ala-Phe-Trp4-Tyr5-Pro-Ans7-OH [Trp4,Ans5]Dermorphin(1-5)-OH Tyr1-d-Ala-Phe-Trp4-Ans5-OH
Among the endogenous dermorphin analog identified, [Lys7]dermorphin has been suggested to have high BBB permeability [31]. The antinociceptive effect of s.c.-administered [Lys7]dermorphin is 6.6 times stronger than dermorphin, whereas the effect of i.c.v.-administered [Lys7]dermorphin is almost identical to dermorphin, suggesting that BBB permeability of [Lys7]dermorphin is higher than dermorphin. Moreover, the duration of the antinociceptive effect of [Lys7]dermorphin is 2 times longer than dermorphin. Since [Lys7]dermorphin is more resistant than dermorphin to the cleaving enzymes in the brain [29], the slow degradation of [Lys7]dermorphin may be responsible for its prolonged antinociceptive effect [31]. It is note worthy that [Lys7]dermorphin has two binding sites on μ-opioid receptors: one is a [Lys7]dermorphin high-affinity site and the other is a [Lys7]dermorphin low-affinity site [29]. Interestingly, [Trp4,Asn7]dermorphin, one of the endogenous dermorphin analogs, has a very high affinity for the [Lys7]dermorphin low-affinity μ-opioid receptors. [Trp4,Asn7]dermorphin administered i.c.v. is 7 times more potent than [Lys7]dermorphin as an inducer of catalepsy, whereas the antinociceptive effect of i.c.v.-administered [Trp4,Asn7]dermorphin is only 1/80 of [Lys7]dermorphin. Moreover, [Trp4,Asn7]dermorphin is 2 times more potent than [Lys7]dermorphin as a μ-opioid receptor agonist in the mouse vas deference or guinea pig ileum. This evidence clearly suggests that the pharmacological profile of the [Lys7]dermorphin high-affinity μ-opioid receptors is quite different with that of the [Lys7]dermorphin low-affinity ([Trp4,Asn7]dermorphin high-affinity) μ-opioid receptors. In fact, the antinociceptive effect of i.c.v.-administered [Lys7]dermorphin is potently suppressed by pretreatment with the μ1-opioid receptor antagonist naloxonazine, whereas the cataleptic response of i.c.v.-administered [Lys7]dermorphin is not affected by naloxonazine [31]. The [Lys7]dermorphin high-affinity μ-opioid receptors may be μ1-opioid receptors that are mainly involved in central antinociception, whereas the [Lys7]dermorphin low-affinity ([Trp4,Asn7]dermorphin high-affinity) μ-opioid receptors may be μ2-opioid receptors that are involved in the cataleptic response and smooth muscle contraction.
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Chapter
Properties
Mr 803. Soluble in water (up to 2 mg/ml) and acetonitrile. Dermorphin should be stored at −20°C. It is also hygroscopic and must be protected from light.
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The second residue side-chain protons in deltorphins containing Tyr–d-Xaa–Phe at the N-terminus were shifted to unusually high field in 1H-NMR (Balboni et al., 1990; Tancredi et al., 1991; Amodeo et al., 1992). This was interpreted as an indication that this side chain lies between the aromatic rings of Tyr and Phe as a result of a type II′β-turn (Tancredi et al., 1991); d-isomers and Pro residues have a tendency to form turns or kinks when flanked by l-isomers. A similar conclusion was obtained for the structure of dermorphin (Pattabiraman et al., 1986), which contains the same N-terminal tripeptide sequence as deltorphins B and C. In fact, Tancredi et al. (1991) further suggested that the topography of deltorphins in solution was characterized not only by the type II′ β-turn in N-terminal residues 1–4, but also that the sequences C-terminal to this region consisted of a series of either β-turns or γ-turns supporting the earlier assessment of deltorphin A that indicated a solution conformation of d-Met2–Phe3 as a β-turn (Temussi et al., 1989). Moreover, the negative charge on Asp4 appeared to stabilize the β-turn of the N-terminal region of deltorphin C through hydrogen bonding (Balboni et al., 1990). The C-terminal tetrapeptide of deltorphin B and its inactive l-Ala2 analogue were also characterized by 1H-NMR as a 310 helix due to the interaction of Phe3 and Val5 (Ji et al., 1995). Further, Crescenzi et al. (1996) reported that a strong conformational preference exists at Val5 NH (amide) due to its solvent inaccessibility.
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Considering the requirement for a protonated nitrogen of the Tyr1 amine and the hydrophobic centers of Tyr1 and Phe3, a model for a rigid `T-shaped' backbone molecule was initially proposed for a dermorphin tetrapeptide (μ agonist) in comparison to the rigid alkaloid fentanyl based on 1H-NMR studies (Pastore et al., 1985; Castiglione-Morelli et al., 1987). This model was applied in the design of dermorphin analogues (Salvadori et al., 1992b) and later adopted for a topographical model of the deltorphins (Balboni et al., 1990; Tancredi et al., 1991). The important features of the model are a hard base (the amine adjacent to Tyr side chain) and a soft base (adjacent to the Phe ring) and with a proper spatial orientation of the two aromatic pharmacophore residues (Castiglione-Morelli et al., 1987).
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Amphibian opioids, particularly the deltorphins, are of considerable interest on several grounds, notwithstanding their unique position in the world of peptides (Erspamer, 1994). The Matses of the upper Amazon basin, for example, apply the dried skin secretions of Phyllomedusa bicolor, called `sapo',2 to cuts in their skin for shamanistic hunting rituals. The presence of low concentrations of deltorphin in these secretions suggests that although they may not directly cause analgesia (Erspamer et al., 1993), it might act synergistically with caerulein, phyllokinin, phyllomedusin, sauvagine, [Lys–OH7]dermorphin (Erspamer et al., 1993) and adenoregulin (Daly et al., 1992) in addition to a large array of unknown substances (Erspamer et al., 1993) to produce their marked physiological and psychological effects.
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Cloning the dermorphin cDNA derived from P. sauvagei skin led to the discovery of a potential translatable heptapeptide region in one isolate (Richter et al., 1987) (Section 1.2). Further studies revealed a cDNA for deltorphins B and C from the skin of P. bicolor that, like the dermorphin cDNA (Richter et al., 1987) and the precursor cDNAs for the mammalian opioids (enkephalins and dynorphins), contained information for multiple copies of opioid peptides. The nucleotide sequence contained 781 base pairs with an open reading frame which could code for a precursor polypeptide of 227 amino acids (Richter et al., 1990). However, unlike the proenkephalin or prodynorphin precursors [reviewed by Lazarus et al. (1994)], the deltorphin precursor polypeptide contained four copies of 36-amino acid repeats, three of which were deltorphin C and one was deltorphin B; the preprohormone also contained two shorter variants encoding new dermorphin analogues. The amino acid sequences were distributed over the entire length of the prohormone (Richter et al., 1990) rather than being clustered in the C-terminal half of the precursor as with the enkephalin and dynorphin preprohormones. In each case, a normal codon for l-Ala encoded the d-Ala isomer isolated in the translated peptide (Richter et al. 1987, 1990) suggesting the existence of an unknown post-translational conversion mechanism of the l- to the d-amino acid (Kreil 1994, 1997). Similar to mammalian prohormones, all opioid peptides in amphibian skin precursors are preceded by paired dibasic amino acids; in amphibian skin polypeptides this sequence is Lys–Arg. Immediately following the deltorphin sequence, the precursor sequence reads Gly–Glu–Arg–Ala–Lys–Lys in which Gly is converted post-translationally to a C-terminal amide (Mollay et al. 1986a, b; Kreil, 1990) by a multistep enzymatic process (Iwasaki et al., 1991; Prigge et al., 1997).
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Specific dermorphin[53]and deltorphin[57]analogs revealed receptor-subsite binding preferences on brain membranes. In particular, binding-site models for these dual-affinity peptides based on stringent statistical (iterative) calculations (which are not merely the slopes of the competitive binding curves[53, 57]) revealed significant comparisons and deviations from the model for deltorphin C (Fig. 2). The length[57], chirality of the side-chain[49, 57]and backbone conformation affected binding preferences for the δ-receptor[57]. For example, analogs with Glu, d-Asp and Pro at position 4 had Hill coefficients (nH) of 0.802, 0.670 and 0.770, respectively[57]. These results, coupled with a narrow log of the 95% confidence interval (≤0.1) and P < 0.0001, indicate two-site binding models[53, 57](supporting the existence of δ1- and δ2-receptor subtypes). However, interaction of these peptides with μ-receptors occurred as a simple, bimolecular mechanism fitting a one-site binding model[57].
Fig. 2. Competition binding curves of deltorphin opioids and their analyses for one-site and two-site binding models. (A) Deltorphin C. (B) [Abu4]-deltorphin C. (C) Deltorphin B. (D) [Aib4]deltorphin C. The solid lines indicate one-site binding models and the broken lines represent two-site models.
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