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Solution Conformation of Nociceptin
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
233, 640–643 (1997)
RC976285
Solution Conformation of Nociceptin S. Salvadori,* D. Picone,† T. Tancredi,‡,1 R. Guerrini,* R. Spadaccini,† L. H. Lazarus,§ D. Regoli,Ø and P. A. Temussi†,2 *Dipartimento di Scienze Farmaceutiche and ØIstituto di Farmacologia, Universita` di Ferrara, 44100 Ferrara, Italy; †Dipartimento di Chimica, Universita` di Napoli Federico II, via Mezzocannone 4, 80134 Naples, Italy; ‡Istituto di Chimica MIB del CNR, Arco Felice, Naples, Italy; and §Peptide Neurochemistry, LMNI, Research Triangle Park, North Carolina 27709
Received January 17, 1997
Nociceptin, a novel heptadecapeptide, interacts with ORL1 a G protein-coupled receptor whose sequence is closely related to that of the k opioid receptor but has no opioid activity. We have investigated the conformational preferences of Nociceptin also in comparison to Dynorphin A. The N-terminal part of Nociceptin has the same conformational preferences of the message of endogenous opioids but the C-terminal part of the sequence is more flexible than the corresponding address of Dynorphin A. [Tyr1]-Nociceptin, while retaining nociceptive activity, has also an opioid activity comparable to that of enkephalins. q 1997 Academic Press
Nociceptin (henceforth called NC), also known as orfanin FQ, is a heptadecapeptide whose sequence (PheGly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-LysLeu-Ala-Asn-Gln) is remarkably similar to that of Dynorphin A (henceforth called DA: Tyr-Gly-Gly-PheLeu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-AsnGln). It was discovered recently (1, 2) in a direct search of the possible agonist of ORL1 (Opioid receptor-like 1) a G-protein-coupled receptor whose aminoacid sequence is closely related to those of the opioid receptors. The nociceptin receptor contains an acidic extracellular loop similar to that required for high affinity binding of DA in the k-opioid receptor (3). Recently, NC has been associated with the N27K, a protein that is a source of NC in the mature brain and regulates neuronal differentiation (4). Systematic structure-activity studies on NC (5, 6) suggested that, like in the case of DA, the message domain is probably coincident with the sequence of the four N-terminal residues, thus leaving a highly basic C-terminal address domain (7) that 1
Affiliation is associated to the National Institute for the Chemistry of Biological Systems. 2 To whom correspondence should be addressed.
differs from that of DA mainly in the detailed distribution of the basic residues. In spite of these similarities, NC displays no opioid activity. This surprising behavior may be ascribed to constitutional and/or conformational differences. In either case a clear identification of the origin of this discrepancy can be revealing also for a better understanding of k opioid selectivity. It is also not inconceivable to hypothesize that the two biological activities (nociception and opioid) may be closely related. Here we present a detailed conformational analysis of NC and of [Tyr1]-NC, both based on NMR spectroscopy in solvents identical or similar to those employed for DA (8-10). MATERIALS AND METHODS Synthesis of NC was performed according to published methods using standard solid-phase synthesis techniques (11, 12) with a Milligen 9050 synthesizer. Amino acids were purchased from Novabiochem AG (Germany). Peptide synthesis was accomplished on a Rink resin (4-(2*,4*-dimethoxy-phenyl)-Fmoc-aminomethyl-phenoxy resin, 0.47 mmol/g; 0.1 g) obtained from Bachem (Torrance, CA, USA). The resin was mixed with glass beads (1:15 w/w) obtained from Sigma. The peptide is assembled using Fmoc-protected amino acids (4-fold excess) and 1,3-diisopropylcarbodiimide (DIPCDI, 4-fold excess) and 1-hydroxybenzotriazole (HOBt, 4-fold excess) as coupling agents, 1 h for each coupling. The peptides were cleaved from the resin by treatment with TFA/H2O/Et3SiH (88: 5: 7; v/v) at room temperature for 1 h and crude deprotected peptides were purified by preparative HPLC. NMR measurements. NMR samples were prepared by dissolving appropriate amounts of peptide in 0.5 ml of solvent to make 2 mM solutions. NMR spectra were run at 400 MHz on a Bruker AM-400 instrument equipped with an Aspect 3000 computer and at 500 MHz on a Bruker AMX-500 instrument equipped with a X-32 computer. TOCSY (13) and NOESY (14) experiments were run in the phasesensitive mode using quadrature detection in v1 by time-proportional phase incrementation of the initial pulse (15). Model building. Energy calculations performed to build molecular models of FGGFTGA-NH2 , the N terminal heptapeptide of NC, were based on the all atoms parametrization of the AMBER force field (16, 17) (as implemented in the SYBYL package).
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FIG. 1. Comparison of the bar diagram of dNN(i, i/1) effects of DA in dodecylphosphocholine micelles with the corresponding bar diagrams of NC in an aqueous solution of SDS micelles, in DMSO and in the 90/10 (v:v) DMSOd6/H2O cryoprotective mixture at 278 K.
Biological tests. The nociceptin peptides were tested on the stimulated guinea pig ileum (GPI) as a sensitive preparation on which NC exerts an inhibitory effect as recently reported by some of us (18, 19). The inhibitory effects of NC and its analogs on the binding of (3H)bremazocine (k) in guinea pig cerebellum membranes and of (3H)DAGO (m) and (3H)DPDPE (d) in rat synaptosomes were determined as previously described (20). Equilibrium inhibition constants (Ki) were calculated from the Cheng and Prusoff’s equation (21)
RESULTS AND DISCUSSION As mentioned above, one of the motivations in the H NMR study of NC was the possible comparison with DA. Accordingly, we used a mixture of methanol and water (9), an aqueous solution of SDS micelles, comparable to that of PTC micelles used in ref. (10) but also acetonitrile, DMSO and a DMSO/water cryomixture (22) since a conformational study currently in progress in our laboratory has shown that this solvent furnishes the best structuring conditions (23). Sequential assignment of NC resonances is hampered by many superpositions but was nonetheless accomplished by standard methods (24) for all residues. The NOESY spectra of NC in all solvent systems show a limited spread of the NH resonances, particularly for the C terminal moiety whose NH chemical shifts are close to typical random coil values. It is still possible however, to compare the conformational preferences of NC to those of DA, at least qualitatively. We chose the dNN(i, i/1) effects as most relevant for such a comparison. Figure 1 shows the comparison of the bar diagrams corresponding to the best structuring conditions for DA to be found in the literature i.e., in dodecylphoshocholine micelles (10), with the corresponding bar diagrams of NC in an aqueous solution of SDS micelles, in DMSO and in the 90/10 (v:v) DMSOd6/H2O cryoprotective mixture at 278 K. It can be seen that in spite of an increasingly ordered structure in going from DMSO to SDS micelles, in all experimental conditions the conformational state of NC is less ordered than that of DA. The 1
number of inter-residue cross peaks for the resonances of the C terminal moiety is small, insufficient for a detailed structural description. The NOEs of the N terminal part in the DMSO/water cryoprotective mixture and in SDS, on the other hand, are similar to those previously observed for Leu-enkephalin in the same solvent systems (25). Thus, it is possible to assume that the relevant conformers consistent with the dNN(i, i/1) effects observed for the first four residues are similar to the single-bend and double-bend structures that characterize several enkephalin analogs in the solid state (26) and to the helical N terminal part of DA (10). Figure 2 shows three molecular models of FGGFTGANH2 , the N terminal heptapeptide of NC, characterized by these typical conformations as obtained from MM calculations based on the X-ray structures of ref. (26). Each of them alone cannot account for all observed nmr parameters but a mixture of them is fully consistent with the NMR data. Accordingly, it is possible to postulate that the lack of opioid activity of NC may be linked, in part, to the different constitution of the message domain, i.e., the substitution of Tyr1 with Phe1 (18) and/or to the increased flexibility of the whole peptide with respect to DA. It is not clear however, whether the conformational preferences of the message (the N terminal tetrapeptide) or of the address (the C terminal sequence) have the same importance nor what’s the relative weight of specific constitutional differences with respect to DA. Trying to restore opioid activity into NC we substituted
FIG. 2. Molecular models of FGGFTGA-NH2 corresponding to the single-bend, double-bend and helical structures of the N terminal part of NC conformations as obtained from MM calculations.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Comparison of the Biological Activity of Nociceptin and Its Analogs and Dynorphin A GPI pIC50(CL95%)a
Peptide NC NC-NH2 [Tyr1]-NC-NH2 [Tyr1]-NC-NH2/Nald DA (1-17)e
8.10 8.08 8.08 8.13
(0.10) (0.22) (0.15) (0.19)
Opioid receptor binding EMAXb 048 058 095 061
{ { { {
8% 8% 5% 10%
Kid, nM
Kim, nM
K ik, nM
NDc 706 { 53 168 { 59
ND 251 { 116 3.6 { 1.2
ND 52,700 5300
6.3 { 1.2
1.55 { 0.06
0.23 { 0.02
a pIC50: negative logarithm of the molar concentration of an agonist that produces 50% of the maximal inhibitory effect. (CL95%): 95% confidence limits. b EMAX: maximal effect induced by an agonist, expressed as percent inhibition of electrically induced twitches. c ND: not detectable. d Data from ref. 18, Nal (Naloxone): used at 1 mM concentration. e Data from ref. 27.
Tyr1 in lieu of Phe1 so that the N terminal tetrapeptide sequence (the putative message of NC) corresponds to the message domain of enkephalins, dynorphins and endorphins. As reported in Table 1, [Tyr1]-NC regains an opioid activity comparable to that of enkephalin, and shows m selectivity consistent with the model of Schwyzer based on its positive charges (7), but contrary to DA (27) it lacks k selectivity: it has no opioid activity on the rabbit vas deferens (data not shown). [Tyr1]-NC on the GPI behaves as a mixed NC/opioid receptors agonist, its maximal effect (see EMAX column in Table 1) is significantly higher than those obtained with NC or NC-NH2 , reaching, as for classical opioid agonists,
the complete inhibition of electrically induced contractions (18). This maximal effect of [Tyr1]-NC was reduced with the classical opioid antagonist Naloxone at 1mM concentration. In other words, in either DA or [Tyr1]-NC, opioid activity is apparently conferred by the Tyr-Gly-Gly-Phe- message but selectivity with respect to opioid receptor subtypes seems completely due to the C terminal moiety. The flexibility of the address domain of [Tyr1]-NC was also investigated by NMR. Figure 3 shows a comparison of the NOESY spectra of NC and [Tyr1]-NC in a 90/10 (v:v) DMSOd6/H2O cryoprotective mixture at 278 K. It is clear that [Tyr1]-NC has a flexibility very similar to that of NC, so that it is not possible to ascribe its regained opioid activity to specific conformational preferences. The main conformational differences between NC and DA seem confined to the address moieties. According to the quoted conformational study on DA, currently in progress in our laboratory, the conformation of the address domain of DA is dominated by the presence of Pro10 that separates two groups of basic residues (23) and limits its flexibility, whereas the corresponding domain of NC does not contain relevant constitutional constraints. It is very interesting that [Tyr1]-NC retains a ‘‘nociceptive’’ activity nearly identical with that of NC. This finding hints that the ‘‘nociceptive’’ address does not have very stringent conformational requirements or that its conformation is completely induced by the interaction with the receptor. ACKNOWLEDGMENT The support of the CIMCF of the University of Naples for NMR measuring time is gratefully acknowledged.
REFERENCES FIG. 3. Comparison of the NOESY spectra of NC and [Tyr1]-NC in a 90/10 (v:v) DMSOd6/H2O cryoprotective mixture at 278 K.
1. Meunier, J.-C., Mollereau, C., Toll, L., Suaudeau, C., Moisand, C., Alvinerie, P., Butour, J.-L., Guillemot, J.-C., Ferrara, P.,
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Monsarrat, B., Mazargull, H., Vassart, G., Parmentier, M., and Costentin, J. (1995) Nature 377, 532–535. 2. Reinsheid, R. K., Nothacker, H., Bourson, P. A., Ardati, A., Henningsen, R. A., Bunzow, J. R., Grandy, D. K., Langen, H., Monsma, F. J. J., and Civelli, O. (1995) Science 270, 792–794.
17. 18.
3. Xue, Ji-C., Chen., C., Zhu, J., Kunapuli, S., DeRiel, J. K., Yu, L., and Liu-Chen, Lee-Y. (1994) J. Biol. Chem. 269, 30195–30199. 4. Saito, Y., Maruyama, K., Kawano, H., Hagino-Yamagishi, K., Saido, T. C., and Kawashima, S. (1996) J. Biol. Chem. 271, 15615–15622. 5. Dooley, C. T., and Houghten, R. A. (1996) Life Sciences 59, 23– 29. 6. Reinscheid, R. K., Ardati, A., Monsma, F. J., and Civelli, O. (1996) J. Biol. Chem. 271, 14163–14168.
19.
20.
7. Schwyzer, R. (1986) Biochemistry 25, 6335–6342. 8. Vaughn, J. B., and Taylor, J. W. (1989) Biochim. Biophys. Acta 999, 135–146. 9. Lancaster, C. R. D., Mishra, P. K., Hughes, D. W., St. Pierre, S. A., Bothner-By, A. A., and Epand, R. M. (1991) Biochemistry 30, 4715–4726.
21. 22. 23.
10. Kallick, D. A. (1993) J. Am. Chem. Soc. 115, 9317–9318. 11. Stewart, J. M., and Young, J. D. (1984) in Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, IL. 12. Atherton, E., and Sheppard, R. C. (1989) in Solid Phase Peptide Synthesis, pp. 25–53, IRL Oxford Univ. Press, Oxford.
24.
13. Bax, A., and Davis, D. G. (1985) J. Magn. Reson. 63, 207–213.
25.
14. Macura, S., and Ernst, R. R. (1979) Mol. Phys. 41, 95–101. 15. Marion, D., and Wu¨thrich, K. (1983) Biochem. Biophys. Res. Comm. 113, 967–971.
26. 27.
16. Weiner, S. J., Kollman, P. A., Case, D. A., Singh, U. C., Ghio, C.,
Alagona, G., Profeta, S., and Weiner, P. (1984) J. Am. Chem. Soc. 106, 765–784. Weiner, S. J., Kollman, P. A., Nguyen, D. T., and Case, D. A. (1986) J. Comp. Chem. 7, 230–252. (a) Calo`, G., Rizzi, M., Bogoni, G., Neugebauer, W., Salvadori, S., Guerrini, R., Bianchi, C., and Regoli, D. (1996) in Proceedings of ‘‘Peptide Receptors,’’ p. 50, Montreal, July 28–August 1. (b) Calo`, G., Rizzi, M., Neugebauer, W., Salvadori, S., Guerrini, R., Bianchi, C., and Regoli, D. (1996) Canadian J. Pharmacol., in press. Calo`, G., Rizzi, M., Bogoni, G., Neugebauer, W., Salvadori, S., Guerrini, R., Bianchi, C., and Regoli, D. (1996) Eur. J. Pharmacol. 311, R3–R5. Salvadori, S., Attila, M., Balboni, G., Bianchi, C., Bryant, S. D., Crescenzi, O., Guerrini, R., Picone, D., Tancredi, T., Temussi, P. A., and Lazarus, L. H. (1995) Mol. Med. 1, 678–689. Cheng, Y. C., and Prusoff, W. H. (1973) Biochem. Pharmacol. 22, 3099–3108. Amodeo, P., Motta, A., Picone, D., Saviano, G., Tancredi, T., and Temussi, P. A. (1991) J. Magn. Reson. 95, 201–207. Picone, D., Spadaccini, R., Crescenzi, O., Amodeo, P., Tancredi, T., and Temussi, P. A. (1996) Proceedings of the Fifth Naples Workshop on Bioactive Peptides Conformation–Activity in Peptides: Relationships and Interactions, p. 82, Capri, Italy, May 19–22. Wu¨thrich, K. (1986) in NMR of Proteins and Nucleic Acids, pp. 162–175, Wiley, New York. Picone, D., D’Ursi, A., Motta, A., Tancredi, T., and Temussi, P. A. (1990) Eur. J. Biochem. 192, 433–439. Deschamps, J. R., George, C., and Flippen-Anderson, J. L. (1996) Biopolymers (Peptide Science) 40, 121–139. Gairing, J. E., Mazarguil, H., Alvineire, O., and Saint-Pierre, S. (1986) J. Med.Chem. 29, 1913–1917.
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233, 640–643 (1997)
RC976285
Solution Conformation of Nociceptin S. Salvadori,* D. Picone,† T. Tancredi,‡,1 R. Guerrini,* R. Spadaccini,† L. H. Lazarus,§ D. Regoli,Ø and P. A. Temussi†,2 *Dipartimento di Scienze Farmaceutiche and ØIstituto di Farmacologia, Universita` di Ferrara, 44100 Ferrara, Italy; †Dipartimento di Chimica, Universita` di Napoli Federico II, via Mezzocannone 4, 80134 Naples, Italy; ‡Istituto di Chimica MIB del CNR, Arco Felice, Naples, Italy; and §Peptide Neurochemistry, LMNI, Research Triangle Park, North Carolina 27709
Received January 17, 1997
Nociceptin, a novel heptadecapeptide, interacts with ORL1 a G protein-coupled receptor whose sequence is closely related to that of the k opioid receptor but has no opioid activity. We have investigated the conformational preferences of Nociceptin also in comparison to Dynorphin A. The N-terminal part of Nociceptin has the same conformational preferences of the message of endogenous opioids but the C-terminal part of the sequence is more flexible than the corresponding address of Dynorphin A. [Tyr1]-Nociceptin, while retaining nociceptive activity, has also an opioid activity comparable to that of enkephalins. q 1997 Academic Press
Nociceptin (henceforth called NC), also known as orfanin FQ, is a heptadecapeptide whose sequence (PheGly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-LysLeu-Ala-Asn-Gln) is remarkably similar to that of Dynorphin A (henceforth called DA: Tyr-Gly-Gly-PheLeu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-AsnGln). It was discovered recently (1, 2) in a direct search of the possible agonist of ORL1 (Opioid receptor-like 1) a G-protein-coupled receptor whose aminoacid sequence is closely related to those of the opioid receptors. The nociceptin receptor contains an acidic extracellular loop similar to that required for high affinity binding of DA in the k-opioid receptor (3). Recently, NC has been associated with the N27K, a protein that is a source of NC in the mature brain and regulates neuronal differentiation (4). Systematic structure-activity studies on NC (5, 6) suggested that, like in the case of DA, the message domain is probably coincident with the sequence of the four N-terminal residues, thus leaving a highly basic C-terminal address domain (7) that 1
Affiliation is associated to the National Institute for the Chemistry of Biological Systems. 2 To whom correspondence should be addressed.
differs from that of DA mainly in the detailed distribution of the basic residues. In spite of these similarities, NC displays no opioid activity. This surprising behavior may be ascribed to constitutional and/or conformational differences. In either case a clear identification of the origin of this discrepancy can be revealing also for a better understanding of k opioid selectivity. It is also not inconceivable to hypothesize that the two biological activities (nociception and opioid) may be closely related. Here we present a detailed conformational analysis of NC and of [Tyr1]-NC, both based on NMR spectroscopy in solvents identical or similar to those employed for DA (8-10). MATERIALS AND METHODS Synthesis of NC was performed according to published methods using standard solid-phase synthesis techniques (11, 12) with a Milligen 9050 synthesizer. Amino acids were purchased from Novabiochem AG (Germany). Peptide synthesis was accomplished on a Rink resin (4-(2*,4*-dimethoxy-phenyl)-Fmoc-aminomethyl-phenoxy resin, 0.47 mmol/g; 0.1 g) obtained from Bachem (Torrance, CA, USA). The resin was mixed with glass beads (1:15 w/w) obtained from Sigma. The peptide is assembled using Fmoc-protected amino acids (4-fold excess) and 1,3-diisopropylcarbodiimide (DIPCDI, 4-fold excess) and 1-hydroxybenzotriazole (HOBt, 4-fold excess) as coupling agents, 1 h for each coupling. The peptides were cleaved from the resin by treatment with TFA/H2O/Et3SiH (88: 5: 7; v/v) at room temperature for 1 h and crude deprotected peptides were purified by preparative HPLC. NMR measurements. NMR samples were prepared by dissolving appropriate amounts of peptide in 0.5 ml of solvent to make 2 mM solutions. NMR spectra were run at 400 MHz on a Bruker AM-400 instrument equipped with an Aspect 3000 computer and at 500 MHz on a Bruker AMX-500 instrument equipped with a X-32 computer. TOCSY (13) and NOESY (14) experiments were run in the phasesensitive mode using quadrature detection in v1 by time-proportional phase incrementation of the initial pulse (15). Model building. Energy calculations performed to build molecular models of FGGFTGA-NH2 , the N terminal heptapeptide of NC, were based on the all atoms parametrization of the AMBER force field (16, 17) (as implemented in the SYBYL package).
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FIG. 1. Comparison of the bar diagram of dNN(i, i/1) effects of DA in dodecylphosphocholine micelles with the corresponding bar diagrams of NC in an aqueous solution of SDS micelles, in DMSO and in the 90/10 (v:v) DMSOd6/H2O cryoprotective mixture at 278 K.
Biological tests. The nociceptin peptides were tested on the stimulated guinea pig ileum (GPI) as a sensitive preparation on which NC exerts an inhibitory effect as recently reported by some of us (18, 19). The inhibitory effects of NC and its analogs on the binding of (3H)bremazocine (k) in guinea pig cerebellum membranes and of (3H)DAGO (m) and (3H)DPDPE (d) in rat synaptosomes were determined as previously described (20). Equilibrium inhibition constants (Ki) were calculated from the Cheng and Prusoff’s equation (21)
RESULTS AND DISCUSSION As mentioned above, one of the motivations in the H NMR study of NC was the possible comparison with DA. Accordingly, we used a mixture of methanol and water (9), an aqueous solution of SDS micelles, comparable to that of PTC micelles used in ref. (10) but also acetonitrile, DMSO and a DMSO/water cryomixture (22) since a conformational study currently in progress in our laboratory has shown that this solvent furnishes the best structuring conditions (23). Sequential assignment of NC resonances is hampered by many superpositions but was nonetheless accomplished by standard methods (24) for all residues. The NOESY spectra of NC in all solvent systems show a limited spread of the NH resonances, particularly for the C terminal moiety whose NH chemical shifts are close to typical random coil values. It is still possible however, to compare the conformational preferences of NC to those of DA, at least qualitatively. We chose the dNN(i, i/1) effects as most relevant for such a comparison. Figure 1 shows the comparison of the bar diagrams corresponding to the best structuring conditions for DA to be found in the literature i.e., in dodecylphoshocholine micelles (10), with the corresponding bar diagrams of NC in an aqueous solution of SDS micelles, in DMSO and in the 90/10 (v:v) DMSOd6/H2O cryoprotective mixture at 278 K. It can be seen that in spite of an increasingly ordered structure in going from DMSO to SDS micelles, in all experimental conditions the conformational state of NC is less ordered than that of DA. The 1
number of inter-residue cross peaks for the resonances of the C terminal moiety is small, insufficient for a detailed structural description. The NOEs of the N terminal part in the DMSO/water cryoprotective mixture and in SDS, on the other hand, are similar to those previously observed for Leu-enkephalin in the same solvent systems (25). Thus, it is possible to assume that the relevant conformers consistent with the dNN(i, i/1) effects observed for the first four residues are similar to the single-bend and double-bend structures that characterize several enkephalin analogs in the solid state (26) and to the helical N terminal part of DA (10). Figure 2 shows three molecular models of FGGFTGANH2 , the N terminal heptapeptide of NC, characterized by these typical conformations as obtained from MM calculations based on the X-ray structures of ref. (26). Each of them alone cannot account for all observed nmr parameters but a mixture of them is fully consistent with the NMR data. Accordingly, it is possible to postulate that the lack of opioid activity of NC may be linked, in part, to the different constitution of the message domain, i.e., the substitution of Tyr1 with Phe1 (18) and/or to the increased flexibility of the whole peptide with respect to DA. It is not clear however, whether the conformational preferences of the message (the N terminal tetrapeptide) or of the address (the C terminal sequence) have the same importance nor what’s the relative weight of specific constitutional differences with respect to DA. Trying to restore opioid activity into NC we substituted
FIG. 2. Molecular models of FGGFTGA-NH2 corresponding to the single-bend, double-bend and helical structures of the N terminal part of NC conformations as obtained from MM calculations.
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Comparison of the Biological Activity of Nociceptin and Its Analogs and Dynorphin A GPI pIC50(CL95%)a
Peptide NC NC-NH2 [Tyr1]-NC-NH2 [Tyr1]-NC-NH2/Nald DA (1-17)e
8.10 8.08 8.08 8.13
(0.10) (0.22) (0.15) (0.19)
Opioid receptor binding EMAXb 048 058 095 061
{ { { {
8% 8% 5% 10%
Kid, nM
Kim, nM
K ik, nM
NDc 706 { 53 168 { 59
ND 251 { 116 3.6 { 1.2
ND 52,700 5300
6.3 { 1.2
1.55 { 0.06
0.23 { 0.02
a pIC50: negative logarithm of the molar concentration of an agonist that produces 50% of the maximal inhibitory effect. (CL95%): 95% confidence limits. b EMAX: maximal effect induced by an agonist, expressed as percent inhibition of electrically induced twitches. c ND: not detectable. d Data from ref. 18, Nal (Naloxone): used at 1 mM concentration. e Data from ref. 27.
Tyr1 in lieu of Phe1 so that the N terminal tetrapeptide sequence (the putative message of NC) corresponds to the message domain of enkephalins, dynorphins and endorphins. As reported in Table 1, [Tyr1]-NC regains an opioid activity comparable to that of enkephalin, and shows m selectivity consistent with the model of Schwyzer based on its positive charges (7), but contrary to DA (27) it lacks k selectivity: it has no opioid activity on the rabbit vas deferens (data not shown). [Tyr1]-NC on the GPI behaves as a mixed NC/opioid receptors agonist, its maximal effect (see EMAX column in Table 1) is significantly higher than those obtained with NC or NC-NH2 , reaching, as for classical opioid agonists,
the complete inhibition of electrically induced contractions (18). This maximal effect of [Tyr1]-NC was reduced with the classical opioid antagonist Naloxone at 1mM concentration. In other words, in either DA or [Tyr1]-NC, opioid activity is apparently conferred by the Tyr-Gly-Gly-Phe- message but selectivity with respect to opioid receptor subtypes seems completely due to the C terminal moiety. The flexibility of the address domain of [Tyr1]-NC was also investigated by NMR. Figure 3 shows a comparison of the NOESY spectra of NC and [Tyr1]-NC in a 90/10 (v:v) DMSOd6/H2O cryoprotective mixture at 278 K. It is clear that [Tyr1]-NC has a flexibility very similar to that of NC, so that it is not possible to ascribe its regained opioid activity to specific conformational preferences. The main conformational differences between NC and DA seem confined to the address moieties. According to the quoted conformational study on DA, currently in progress in our laboratory, the conformation of the address domain of DA is dominated by the presence of Pro10 that separates two groups of basic residues (23) and limits its flexibility, whereas the corresponding domain of NC does not contain relevant constitutional constraints. It is very interesting that [Tyr1]-NC retains a ‘‘nociceptive’’ activity nearly identical with that of NC. This finding hints that the ‘‘nociceptive’’ address does not have very stringent conformational requirements or that its conformation is completely induced by the interaction with the receptor. ACKNOWLEDGMENT The support of the CIMCF of the University of Naples for NMR measuring time is gratefully acknowledged.
REFERENCES FIG. 3. Comparison of the NOESY spectra of NC and [Tyr1]-NC in a 90/10 (v:v) DMSOd6/H2O cryoprotective mixture at 278 K.
1. Meunier, J.-C., Mollereau, C., Toll, L., Suaudeau, C., Moisand, C., Alvinerie, P., Butour, J.-L., Guillemot, J.-C., Ferrara, P.,
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