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Highly Potent Nociceptin Analog Containing the Arg-Lys Triple Repeat
Biochemical and Biophysical Research Communications 278, 493– 498 (2000) doi:10.1006/bbrc.2000.3822, available online at http://www.idealibrary.com on
Highly Potent Nociceptin Analog Containing the Arg-Lys Triple Repeat Kazushi Okada,* Tetsujo Sujaku,† Yoshiro Chuman,* Rie Nakashima,* Takeru Nose,* Tommaso Costa,‡ Yoshinari Yamada,† Masayuki Yokoyama,† Atsushi Nagahisa,† and Yasuyuki Shimohigashi* ,1 *Laboratory of Structure–Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences, Kyushu University, Fukuoka 812-8581, Japan; †Central Research, Pfizer Pharmaceuticals Inc., 5-2, Taketoyo, Aichi 470-2393, Japan; and ‡Laboratorio di Farmacologia, Istituto Superiore di Sanita`, Viale Regina Elena 299, Rome, Italy
Received October 4, 2000
One of the structural characteristics of a neuropeptide nociceptin is the existence of Arg-Lys (RK) residues at positions 8 –9 and 12–13; both RKs have been suggested to bind to the acidic amino acid cluster in the second extracellular loop of the seven transmembrane domain receptor ORL1. With a design strategy of attempting to obtain an analog that binds more strongly to the receptor’s acidic cluster, we synthesized a series of nociceptin analogs in which the RK dipeptide unit was placed at positions 6 –7, 10 –11, or 14 –15 adjacent to the parent RKs. Among these nociceptin analogs containing the RK triple repeat, [ArgLys 6 –7]- and [Arg-Lys 10 –11]nociceptins exhibited weak activities (6 –9 and 60 –90% of nociceptin, respectively) both in the receptor binding assay and in the [ 35S]GTP␥S binding functional assay. In contrast, [ArgLys 14 –15]nociceptin was found to be very potent in both assays (3-fold in binding and 17-fold in GTP␥S functional assay). [Arg-Lys 14 –15]nociceptin was the first peptide analog found to be stronger than the parent nociceptin, and structure–activity studies have suggested that the incorporated Arg-Lys 14 –15 interacts with either the receptor acidic amino acid cluster or the receptor aromatic amino acid residues. © 2000 Academic Press Key Words: nociceptin; nociceptive peptides; opioid peptides; opioid receptors; structure–activity studies; receptor binding.
Nociceptin (1) or orphanin FQ (2) is an endogenous ligand of the G-protein-coupled seven transmembrane receptor ORL1 (opioid receptor-like 1), the structure of Abbreviations used: GTP␥S, guanosine 5⬘-(␥-thio)triphosphate; ORL1, opioid receptor-like 1; RK, Arg-Lys; RP-HPLC, reversedphase high-performance liquid chromatography; SPA, scintillation proximity assay. 1 To whom correspondence should be addressed. Fax: ⫹81-92-6422584. E-mail: [email protected].
which is very much similar to that of opioid receptors (3– 6). Human nociceptin is a heptadecapeptide, the amino acid sequence of which, FGGFTGARKSARKLANQ, has been found to be the same as those of species such as the rat, porcine, bovine, and mouse (1, 2, 7–9). Although nociceptin was originally reported to produce hyperalgesia (1), it was recently found to have various physiological effects relating, for example, to locomotion and learning (10, 11). The study using mice lacking ORL1 suggested that the nociceptin system plays suppressive roles both in learning and memory (11). Dynorphin A, YGGFLRRIRPKLKWDNQ, is an opioid peptide specific to -receptors (12, 13), and the sequences of nociceptin and dynorphin A resemble each other. Both nociceptin and dynorphin A contain several basic amino acids at their C-terminal portions, and for dynorphin A, the importance of these basic residues (Arg-6, Arg-7, Arg-9, Lys-11, and Lys-13), especially of Arg-7, has been demonstrated for the recognition and activation of receptors (14 –17). Nociceptin contains a couple of basic amino acid pairs, namely, two Arg-Lys sequences at positions 8 –9 and 12–13, and these have been acknowledged to be essential for both receptor recognition and activation (18, 19). The basic regions of dynorphin and nociceptin have been postulated to interact with the receptor segment consisting of a cluster of acidic amino acids in the second extracellular loop of the seven transmembrane structure (20 –24). A number of nociceptin analogs have been designed and synthesized to clarify the structural essentials for receptor interaction (18, 19, 25–29). However, no analogs more potent than native nociceptin have yet been announced. Since the activities of nociceptin are either hyperalgesic or to suppress learning and memory, major efforts have recently focused on the design of nociceptin antagonists to overcome these rather negative
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FIG. 1.
Amino acid sequences of nociceptin, and Arg-Lys-multiplied analogs.
cellular responses (30). On the other hand, a highly potent agonist often facilitates the receptor responses such as desensitization and internalization (31, 32). These cellular responses are a kind of suppressive or antagonistic replies of the receptor. The idea of a socalled superagonist prompted us to design and synthesize a highly potent agonist analog of nociceptin. In the present study, in order to obtain potent nociceptin analogs we chose a strategy to strengthen the electrostatic interaction between nociceptin and the second extracellular loop of the nociceptin receptor ORL1. To this end, we have synthesized a series of analogs in which Arg-Lys (RK) displaces the amino acid pairs at places adjacent to the original RK pairs (Fig. 1). These basic forms of nociceptin were evaluated for their receptor binding affinity to ORL1 and opioid receptors and for their biological activity in a functional assay using [ 35S]GTP␥S. As a result, [Arg-Lys14 –15]nociceptin emerged as the first time as an analog much more potent than native nociceptin, especially in the functional biological assay. MATERIALS AND METHODS Peptide synthesis. All peptides, namely, nociceptin and its RKmultiplied analogs, were synthesized (0.1 mmol scale) by the automated peptide synthesizer ABI 430A (Applied Biosystems Inc., Foster City, CA) with the Fmoc synthetic strategy. Peptides were liberated from the resin by Reagent K (33) at room temperature for 3 h. After evaporation, the residue was solidified with diethyl ether. The purification was carried out first by gel filtration on a column (2.0 ⫻ 100 cm) of Sephadex G-15 (Pharmacia, Uppsala, Sweden) eluted with 10% acetic acid. For further purification, reversed-phase high performance liquid chromatography (RP-HPLC) was performed on a preparative column (25 ⫻ 250 mm, Cica-Merck LiChrospher RP-18 (e), 5 m) with a linear gradient of 0.1% trifluoroacetic acid and 80% acetonitrile and the fractions containing pure peptides were lyophilized to obtain the final peptide sample. The purity was verified by analytical RP-HPLC (4 ⫻ 250 mm, Cica-Merck LiChrospher 100 RP-18, 5 m). Mass spectra of peptides were measured on a mass spectrometer Voyager DE-PRO (PerSeptive Biosystems Inc., Framingham, MA) with the method of matrixassisted laser desorption ionization time-of-flight (MALDI-TOF). Amino acid analyses of peptides were carried out on an amino acid analyzer Hitachi L-8800 system after hydrolysis in a constantboiling hydrochloric acid at 110°C for 24 h. The amino acid sequences of nociceptin and its analogs were determined by the Procise Model 491 Protein Sequencing System (PE Biosystems, Foster City, CA). Receptor binding assays. Membranes were prepared from human embryonic kidney 293 cells expressing human ORL1, , ␦, and
opioid receptors as described previously (25). Peptides were evaluated using [ 3H]nociceptin (185 Ci/mmol, Amersham, Buckinghamshire, UK) for ORL1 receptor assays. Binding assays were also carried out for opioid receptor subtypes using [ 3H]DAGO (55 Ci/ mmol, DuPont/NEN Research Products, Wilmington, DE) for human receptors, [ 3H]DPDPE (46 Ci/mmol, DuPont/NEN) for human ␦ receptors, and [ 3H]Cl-977 (42 Ci/mmol, Amersham) for human receptors. Briefly, incubations were carried out at room temperature for 60 min in Tris–HCl buffer (pH 7.55) containing 0.1% bovine serum albumin. Bacitracin (100 g/ml) was added as an enzyme inhibitor. After incubation, each incubation mixture was filtered through glass fiber filters (Whatman GF/B) and rinsed twice with 10 mM Tris–HCl buffer pH 7.55 (4 ml). Dose–response curves were analyzed by the computer program ALLFIT (34). [ 35S]GTP␥S binding to cell membranes. The [ 35S]GTP␥S binding assay was performed according to the method of SPAG-protein coupled receptor assay provided by Amersham with slight modification. The membranes were suspended in ice-cold 20 mM Hepes buffer (pH 7.4) containing 100 mM NaCl, 10 mM MgCl 2, and 1 mM EDTA. After the addition of dithiothreitol (threo-1,4-dimercapto-2,3-butanediol; 0.17 mg/ml) to the solution, membranes were incubated at 25°C for 30 min with the appropriate concentration of drugs in the presence of 5 M GDP, 0.4 nM of [ 35S]GTP␥S (200 –370 mCi/mmol, Amersham), and wheat-germ agglutinin-coated scintillation proximity assay (SPA) beads (1.5 mg) in a 0.2 ml total volume. Basal binding was assessed in the absence of agonist, and nonspecific binding was determined with 10 M GTP␥S. The activity was estimated as EC 50, the value of which exhibits the concentration inducing 50% of its own maximal stimulation.
RESULTS AND DISCUSSION Peptide design and synthesis. Nociceptin is encoded in a precursor protein, in which the sequence of the natural peptide is flanked by Lys-Arg (KR) proteolytic excision motifs at both the N and C-termini. Seventeen-peptide nociceptin consists of the inverse sequence of this KR motif at the two separated positions 8 –9 and 12–13. The RK motif is also occasionally functions as a proteolytic excision site. We noted that the KR and RK motifs in nociceptin precursors are clearly distinguished by a specific processing enzyme not to cleave the RK motif. This suggests that the RK repeats in nociceptin peptide is crucially important for receptor recognition and/or activation. Thus, we chose a RK dipeptide unit to replace the amino acid residues in nociceptin in expectation of the electrostatic interaction reinforced between nociceptin and ORL1 receptor. The resulting [Arg-Lys 6 –7]-, [Arg-Lys 10 –11]-, and [Arg-Lys 14 –15]nociceptins consist of three RK pairs in
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Analytical Data of Synthetic Peptides from Mass Spectrometry, Reversed-Phase High-Performance Liquid Chromatography, and Amino Acid Analysis MALDI-TOFMASS a
RP-HPLC b
Amino acid analysis c
Peptides
Found
Calcd
RT (min)
Asn
Thr
Ser
Gln
Gly
Ala
Leu
Phe
Lys
Arg
Nociceptin (⫽Noc)
1808.8
1810.1
33.17
1965.2
1966.3
31.89
[Arg-Lys 10–11]Noc
1935.6
1936.3
32.32
[Arg-Lys 14–15]Noc
1909.3
1910.2
31.21
1.05 (1) 1.05 (1) 1.03 (1) 1.02 (1)
1.08 (1) 1.04 (1) 1.12 (1) 1.02 (1)
1.12 (1) 1.21 (1) — — 1.02 (1)
1.06 (1) 1.04 (1) 1.13 (1) 1.08 (1)
2.95 (3) 2.03 (2) 2.81 (3) 2.94 (3)
3.00 (3) 2.00 (2) 2.00 (2) 2.00 (2)
1.07 (1) 1.10 (1) 1.08 (1) — —
2.20 (2) 2.08 (2) 2.24 (2) 2.33 (2)
1.85 (2) 2.75 (3) 2.70 (3) 2.83 (3)
1.84 (2) 2.78 (3) 2.77 (3) 2.76 (3)
[Arg-Lys
6–7
]Noc
The values express the mass number (m/z) of (M ⫹ H) ⫹. Retention time (RT) was measured on an analytical column (Cica-Merck LiChrospher 100 RP-18(e), 4 ⫻ 250 mm, 5 m) with a linear gradient of 0.1% trifluoroacetic acid and 80% acetonitrile. c The values are normalized for alanine as an internal standard. a b
each peptide molecule, and we designated these analogs as RK-multiplied nociceptin analogs. Nociceptin and its analogs were prepared by the solid-phase methodology using Fmoc-amino acids. They contains two or three Arg residues, the side chains of which were protected by the 2,2,5,7,8pentamethylchroman-6-sulfonyl (Pmc) group (35). The Pmc groups were removed completely by TFAcontaining Reagent K without any troubles. Peptides were obtained in an average yield of about 35%. Table 1 shows the analytical data of all four heptadecapeptides synthesized. The purity of peptides was verified by analytical HPLC, in which all the peptides emerged as a single peak. The mass numbers measured were coincident with the values calculated. Amino acid analyses revealed a good coincidence of the number of amino acid constituents, and this was also confirmed by the amino acid sequencing of the peptides (data not shown). Thus, synthetic nociceptin and its analogs have been identified physicochemically to reveal the desired compounds.
Receptor binding affinity of nociceptin peptides. Nociceptin was very potent to bind to ORL1 receptors expressed in human 293 cells. The half-maximal concentration (IC 50) for inhibition of the binding of radiolabeled ligand [ 3H]nociceptin was calculated to be 0.93 nM (Table 2). When Arg-Lys (RK) was placed at position 6 –7 to replace Gly-Ala, the resulting analog [ArgLys 6 –7]nociceptin exhibited about 90% decreased affinity for ORL1 receptor in the cells compared to nociceptin. [Arg-Lys 10 –11]nociceptin also showed a reduced receptor affinity (about 60% of nociceptin). In contrast to these RK-multiplied nociceptin analogs, [Arg-Lys 14 –15]nociceptin was found to be very potent, exhibiting an ability to displace [ 3H]nociceptin in ORL1 receptor about three times stronger than parent nociceptin. The activity profile of all these RKmultiplied nociceptin analogs are shown in Fig. 2 by potencies relative to that of native nociceptin. It should be noted that [Arg-Lys 14 –15]nociceptin is the first nociceptin analog showing a receptor binding affinity higher than nociceptin.
TABLE 2
Results of Receptor Binding Affinities and Biological Activity of Nociceptin and Its RK-Multiplied Analogs [ 35S]GTP␥S binding activity
Receptor binding affinity (IC 50, nM) Peptides
ORL1
␦
EC 50 (nM)
Relative potency (%)
Nociceptin (⫽Noc) [Arg-Lys 6–7]Noc [Arg-Lys 10–11]Noc [Arg-Lys 14–15]Noc
0.93 ⫾ 0.50 11 ⫾ 7.9 1.5 ⫾ 0.78 0.32 ⫾ 0.13
290 ⫾ 50 220 ⫾ 85 190 ⫾ 41 280 ⫾ 87
⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000
3500 ⫾ 140 240 ⫾ 78 940 ⫾ 600 1,500 ⫾ 1,300
17 ⫾ 2.3 260 ⫾ 120 19 ⫾ 4.8 1.0 ⫾ 0.30
100 6.6 89 1,700
Note. Binding assays were carried out for human ORL1, , ␦, and opioid receptor expressed in 293 cells and the biological assay was performed with [ 35S]GTP␥S against human ORL1 receptor expressed in 293 cells. Each value represents the mean ⫾ SD from three independent experiments. 495
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(residues 6 –13) of dynorphin A was suggested to interact with the cluster of acidic amino acids in the second extracellular loop of the receptors (20, 21). Since RK-multiplied nociceptin analogs have three RK pairs in a molecule, they must be more preferable to interact with the receptors than nociceptin itself. In particular, [Arg-Lys 6 –7]nociceptin, which is the most potent analog for the receptors, possessed a sequence very much similar to that of dynorphin A at positions 6 –13: i.e., RKRKSARK for [Arg-Lys 6 –7]nociceptin and RRIRPKLK for dynorphin A. Despite such increased binding affinities, however, RK-multiplied nociceptins are still much more potent for ORL1 than for receptors (Table 2).
FIG. 2. Relative potencies of Arg-Lys-multiplied nociceptin analogs for ORL1 receptors in the human embryonic kidney 293 cell membrane preparations: Receptor binding potencies (A) and GTP␥S binding potencies (B). The potencies were calculated by dividing the IC 50 or EC 50 values with that of nociceptin (⫽100) in each assay.
Nociceptin and RK-multiplied analogs were also assayed to for the ordinary ␦, , and opioid receptors to assess their binding ability. For ␦ receptors, they were almost completely inactive. For the opioid receptors, nociceptin exhibited a distinct but weak binding with the IC 50 value of about 300 nM as reported previously (25). It was found that all RK-multiplied nociceptin analogs bind to receptors as well as nociceptin. Their binding affinities (190 –280 nM) to the receptors are, however, 20- to 870-fold weaker than those to ORL1 receptor, indicating that they are unable to construct a bioactive structure specific for the receptors. For receptors, nociceptin was extremely weak with the IC 50 values of three orders of magnitude larger than that for ORL1 receptor (Table 2). In contrast, RK-multiplied nociceptin analogs exhibited increased binding affinity (2- to 15-fold) for the receptors. This is apparently the positive effect of incorporation of Arg-Lys into nociceptin. The basic amino acid region
GTP␥S binding affinity. In the present study, [ 35S]GTP␥S binding assay was carried out to evaluate the functional activation of the ORL1 receptor by nociceptin and RK-multiplied nociceptin analogs. The opioid receptors including ORL1 are coupled with G-proteins, the stimulation of which results in its conformation change that allows cytoplasmic GTP to displace GDP in the ␣-subunit and further facilitates a dissociation of GTP-bound ␣-subunit from ␥-subunits. Thus, the GDP/GTP exchange represents a receptormediated G-protein activation of a receptor ligand. Guanocine 5⬘-(␥-thio)triphosphate, GTP␥S, is a GTP analog resistant to the GTPase enzyme activity of G-protein, and dissociates very slowly. The amount of [ 35S]GTP␥S bound to G-protein thus represents the receptor activity of a ligand as demonstrated in many studies of signaling properties in G-protein coupled receptors (36 –39). Nociceptin dose-dependently stimulated the nucleotide binding in membranes with an apparent EC 50 of 17 nM (Table 2), which compared well with the values (9 –20 nM) as found in other studies (40, 41). Under the same experimental conditions, the affinity (0.93 nM) of nociceptin for ORL1 was about 18-fold higher than its potency in stimulating the GTP binding. This is a rather general phenomenon as observed in both the brain and cell membrane preparations (22, 40, 41). [Arg-Lys 6 –7]nociceptin was very weak (EC 50 ⫽ 260 nM) to stimulate the GTP␥S binding. Compared with nociceptin, this analog reached about 40 – 60% activity in stimulating [ 35S]GTP␥S binding at its 10 M concentration. [Arg-Lys 10 –11]nociceptin was considerably potent (19 nM), whereas [Arg-Lys 14 –15]nociceptin was extremely potent with a small half-maximal concentration for GTP␥S binding (1.0 nM). These two analogs exhibited a full activity in GTP binding (data not shown). When the specificity of the coupling between receptor binding and GTP␥S binding was assessed, the order of magnitude of the receptor affinities of nociceptin, [ArgLys 6 –7]nociceptin, and [Arg-Lys 10 –11]nociceptin (IC 50 1:0.085:0.62 as seen in Fig. 2A) was nearly the same as
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that of their potencies in stimulating the [ 35S]GTP␥S binding (EC 50 1:0.066:0.89, Fig. 2B). By contrast, [ArgLys 14 –15]nociceptin was found to be considerably more active in stimulating the [ 35S]GTP␥S binding than expected from its binding affinity. When compared with the activities of nociceptin, [Arg-Lys 14 –15]nociceptin was 17 times more potent than nociceptin in the GTP␥S functional assay. Since [Arg-Lys 14 –15]nociceptin was about 3 times more potent than nociceptin in the receptor binding assay, this RK-multiplied analog is about 6-fold stronger in the GTP␥S binding assay than in receptor binding assay. This suggests that [ArgLys 14 –15]nociceptin is involved in a molecular mechanism to facilitate very effectively a basal level of receptor activation with its lower concentrations. It should be noted that the affinity (0.32 nM) of [Arg-Lys 14 –15]nociceptin for ORL1 was only 3-fold higher than its potency in stimulating the GTP binding under the same conditions. Structure–activity relationships of [Arg-Lys 14 –15 ]nociceptin. Extremely high activity of [Arg-Lys 14 –15]nociceptin, 17-fold stronger than native nociceptin, should be explained by an appropriate molecular interaction mode. The structure–activity studies of [ArgLys 14 –15]nociceptin suggest two possible interaction modes for Arg-Lys 14 –15: i.e., the electrostatic interaction with the receptor acidic amino acid cluster and the cation/ interaction with the receptor aromatic groups. Recent studies have suggested that Arg-Lys 8 –9 and Arg-Lys 12–13, which are native constituents of the nociceptin peptide, interact with the cluster of acidic amino acids in the second extracellular loop of nociceptin receptor ORL1 (22–24). It is thus likely that newly incorporated Arg-Lys 14 –15 arrests the negative charges of the receptor molecule by the electrostatic interaction. The original amino acids at positions 14 and 15 are Leu and Ala, respectively. Arg-Lys 14 –15 would fill the space of the original Leu-Ala 14 –15 and further cap the cationic groups such as guanidino and amino groups, eliciting an additional interaction with ORL1. This extra electrostatic interaction may stabilize the total ligand/ receptor interaction to induce a reinforced activation. Unlike Lys/Ala 15 replacement, Asp or Trp/Ala 15 replacement has been found to result in a sharp drop in receptor binding and biological activities (27, 42). In contrast, position 14 permits the placement of various kind of amino acids such as Leu (native nociceptin), Tyr (18, 25), Trp (42), and now even Arg. Apparently, Leu, Tyr, and Trp do not engage in the electrostatic interaction. Given that all these amino acids interact with the same site of ORL1, the receptor site must possess a structural component that interacts specifically with isobutyl (Leu), phenol (Tyr), indolyl (Trp), and guanidino (Arg) groups. This is only possible with aromatic amino acid residues such as Phe, Tyr, Trp, and His, which consist of systems. In the second
extracellular loop of the ORL1 receptor, three aromatic amino acids exist: i.e., Tyr-210, Trp-211, and Phe-215 (the numbers based on the amino acid sequence of the human ORL1 receptor). Arg-Lys 14 –15 might interact with these receptor aromatic groups, producing the so-called cation/ interaction. This cation/ interaction is also a kind of electrostatic interaction. Identification of the receptor-binding site for Arg-Lys 14 –15 might afford important structural information regarding the design of receptor agonists and antagonists. REFERENCES 1. Meunier, J.-C., Mollereau, C., Toll, L., Suaudeau, C., Moisand, C., Alvinerie, P., Butour, J.-L., Guillemot, J.-C., Ferrara, P., Monsarrat, B., Mazarguil, H., Vassart, G., Parmentier, M., and Costentin, J. (1995) Nature 377, 532–535. 2. Reinscheid, R. K., Nothacker, H.-P., Bourson, A., Ardati, A., Henningsen, R. A., Bunzow, J. R., Grandy, D. K., Langen, H., Monsma, F. J., Jr., and Civelli, O. (1995) Science 270, 792–794. 3. Mollereau, C., Parmentier, M., Mailleux, P., Butour, J.-L., Moisand, C., Chalon, P., Caput, D., Vassart, G., and Meunier, J.-C. (1994) FEBS Lett. 341, 33–38. 4. Chen, Y., Fan, Y., Liu, J., Mestek, A., Tian, M., Kozak, C. A., and Yu, L. (1994) FEBS Lett. 347, 279 –283. 5. Wang, J. B., Johnson, P. S., Imai, Y., Persico, A. M., Ozenberger, B. A., Eppler, C. M., and Uhl, G. R. (1994) FEBS Lett. 348, 75–79. 6. Nishi, M., Takeshima, H., Mori, M., Nakagawara, K., and Takeuchi, T. (1994) Biochem. Biophys. Res. Commun. 205, 1353–1357. 7. Mollereau, C., Simons, M.-J., Soularue, P., Liners, F., Vassart, G., Meunier, J.-C., and Parmentier, M. (1996) Proc. Natl. Acad. Sci. USA 93, 8666 – 8670. 8. Okuda-Ashitaka, E., Tachibana, S., Houtani, T., Minami, T., Masu, Y., Nishi, M., Takeshima, H., Sugimoto, T., and Ito, S. (1996) Mol. Brain Res. 43, 96 –104. 9. Pan Y.-X., Xu, J., and Pasternak, G. W. (1996) Biochem. J. 315, 11–13. ¨ gren, S. O., and Tere10. Sandin, J., Georgieva, J., Scho¨tt, P. A., O nius, L. (1997) Eur. J. Neurosci. 9, 194 –197. 11. Manabe, T., Noda, Y., Mamiya, T., Katagiri, H., Houtani, T., Nishi, M., Noda, T., Takahashi, T., Sugimoto, T., Nabeshima, T., and Takeshima, H. (1998) Nature 394, 577–581. 12. Goldstein, A., Fischli, W., Lowney, L. I., Hunkapiller, M., and Hood, L. (1981) Proc. Natl. Acad. Sci. USA 78, 7219 –7223. 13. Tachibana, S., Araki, K., Ohya, S., and Yoshida, S. (1982) Nature 295, 339 –340. 14. Chavkin, C., and Goldstein, A. (1981) Proc. Natl. Acad. Sci. USA 78, 6543– 6547. 15. Chavkin, C., James, I. F., and Goldstein, A. (1982) Science 215, 413– 415. 16. Weber. E., Evans. C. J., and Barchas, J. D. (1982) Nature 299, 77–79. 17. Corbett, A. D., Paterson, S. J., McKnight, A. T., Magnan, J., and Kosterlitz, H. W. (1982) Nature 299, 79 – 81. 18. Reinscheid, R. K., Ardati, A., Monsma, F. J., Jr., and Civelli, O. (1996) J. Biol. Chem. 271, 14163–14168. 19. Butour, J.-L., Moisand, C., Mazarguil, H., Mollereau, C., and Meunier, J.-C. (1997) Eur. J. Pharmacol. 321, 97–103. 20. Wang, J. B., Johnson, P. S., Wu, J. M., Wang, W. F., and Uhl, G. R. (1994) J. Biol. Chem. 269, 25966 –25969. 21. Naqvi, T., Haq, W., and Mathur, K. B. (1998) Peptides 19, 1277– 1292.
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22. Dooley, C. T., Spaeth, C. G., Berzetei-Gurske, I. P., Craymer, K., Adapa, I. D., Brandt, S. R., Houghten, R. A., and Toll, L. (1997) J. Pharmacol. Exp. Ther. 283, 735–741. 23. Topham, C. M., Moule´dous, L., Poda, G., Maigret, B., and Meunier, J.-C. (1998) Protein Eng. 11, 1163–1179. 24. Mollereau, C., Mouledous, L., Lapalu, S., Cambois, G., Moisand, C., Butour, J.-L., and Meunier, J.-C. (1999) Mol. Pharmacol. 55, 324 –331. 25. Shimohigashi, Y., Hatano, R., Fujita, T., Nakashima, R., Nose, T., Sujaku, T., Saigo, A., Shinjo, K., and Nagahisa, A. (1996) J. Biol. Chem. 271, 23642–23645. 26. Dooley, C. T., and Houghten, R. A. (1996) Life Sci. 59, PL23– PL29. 27. Lapalu, S., Moisand, C., Mazarguil, H., Cambois, G., Mollereau, C., and Meunier, J.-C. (1997) FEBS Lett. 417, 333–336. 28. Reinscheid, R. K., Higelin, J., Henningsen, R. A., Monsma, F. J., Jr., and Civelli, O. (1998) J. Biol. Chem. 273, 1490 –1495. 29. Calo’, G., Guerrini, R., Bigoni, R., Rizzi, A., Bianchi, C., Regoli, D., and Salvadori, S. (1998) J. Med. Chem. 41, 3360 –3366. 30. Barlocco, D., Cignarella, G., Giardina, G. A. M., and Toma, L. (2000) Eur. J. Med. Chem. 35, 275–282. 31. Sternini, C., Spann, M., Anton, B., Keith, D. E., Jr., Bunnett, N. W., von Zastrow, M., Evans, C., and Brecha, N. C. (1996) Proc. Natl. Acad. Sci. USA 93, 9241–9246.
32. Xiao, Z., Yao, Y., Long, Y., and Devreotes, P. (1999) J. Biol. Chem. 274, 1440 –1448. 33. King, D. S., Fields, C. G., and Fields, G. B. (1990) Int. J. Peptide Protein Res. 36, 255–266. 34. De Lean, A., Munson, P. J., and Rodbard. D. (1978) Am. J. Physiol. 235, E97–E102. 35. Ramage, R., and Green, J. (1987) Tetrahedron Lett. 28, 2287– 2290. 36. Lazareno, S., and Birdsall, N. J. M. (1993) Br. J. Pharmacol. 109 1120 –1127. 37. Lorenzen, A., Fuss, M., Vogt, H., and Schwabe, U. (1993) Mol. Pharmacol. 44, 115–123. 38. Gardner, B., Hall, D. A., and Strange, P. G. (1996) Br. J. Pharmacol. 118, 1544 –1550. 39. Befort, K., Tabbara, L., and Kieffer, B. L. (1996) Neurochem. Res. 21, 1301–1307. 40. Sim, L. J., Xiao, R. Y., and Childers, S. R. (1996) NeuroReport 7, 729 –733. 41. Albrecht, E., Samovilova, N. N., Oswald, S., Baeger, I., and Berger, H. (1998) J. Pharmacol. Exp. Ther. 286, 896 –902. 42. Okada, K., Sujaku, T., Nakashima, R., Nose, T., Yamada, Y., Yokoyama, M., Nagahisa, A., and Shimohigashi, Y (1999) Bull. Chem. Soc. Jpn. 72, 1899 –1904.
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Highly Potent Nociceptin Analog Containing the Arg-Lys Triple Repeat Kazushi Okada,* Tetsujo Sujaku,† Yoshiro Chuman,* Rie Nakashima,* Takeru Nose,* Tommaso Costa,‡ Yoshinari Yamada,† Masayuki Yokoyama,† Atsushi Nagahisa,† and Yasuyuki Shimohigashi* ,1 *Laboratory of Structure–Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences, Kyushu University, Fukuoka 812-8581, Japan; †Central Research, Pfizer Pharmaceuticals Inc., 5-2, Taketoyo, Aichi 470-2393, Japan; and ‡Laboratorio di Farmacologia, Istituto Superiore di Sanita`, Viale Regina Elena 299, Rome, Italy
Received October 4, 2000
One of the structural characteristics of a neuropeptide nociceptin is the existence of Arg-Lys (RK) residues at positions 8 –9 and 12–13; both RKs have been suggested to bind to the acidic amino acid cluster in the second extracellular loop of the seven transmembrane domain receptor ORL1. With a design strategy of attempting to obtain an analog that binds more strongly to the receptor’s acidic cluster, we synthesized a series of nociceptin analogs in which the RK dipeptide unit was placed at positions 6 –7, 10 –11, or 14 –15 adjacent to the parent RKs. Among these nociceptin analogs containing the RK triple repeat, [ArgLys 6 –7]- and [Arg-Lys 10 –11]nociceptins exhibited weak activities (6 –9 and 60 –90% of nociceptin, respectively) both in the receptor binding assay and in the [ 35S]GTP␥S binding functional assay. In contrast, [ArgLys 14 –15]nociceptin was found to be very potent in both assays (3-fold in binding and 17-fold in GTP␥S functional assay). [Arg-Lys 14 –15]nociceptin was the first peptide analog found to be stronger than the parent nociceptin, and structure–activity studies have suggested that the incorporated Arg-Lys 14 –15 interacts with either the receptor acidic amino acid cluster or the receptor aromatic amino acid residues. © 2000 Academic Press Key Words: nociceptin; nociceptive peptides; opioid peptides; opioid receptors; structure–activity studies; receptor binding.
Nociceptin (1) or orphanin FQ (2) is an endogenous ligand of the G-protein-coupled seven transmembrane receptor ORL1 (opioid receptor-like 1), the structure of Abbreviations used: GTP␥S, guanosine 5⬘-(␥-thio)triphosphate; ORL1, opioid receptor-like 1; RK, Arg-Lys; RP-HPLC, reversedphase high-performance liquid chromatography; SPA, scintillation proximity assay. 1 To whom correspondence should be addressed. Fax: ⫹81-92-6422584. E-mail: [email protected].
which is very much similar to that of opioid receptors (3– 6). Human nociceptin is a heptadecapeptide, the amino acid sequence of which, FGGFTGARKSARKLANQ, has been found to be the same as those of species such as the rat, porcine, bovine, and mouse (1, 2, 7–9). Although nociceptin was originally reported to produce hyperalgesia (1), it was recently found to have various physiological effects relating, for example, to locomotion and learning (10, 11). The study using mice lacking ORL1 suggested that the nociceptin system plays suppressive roles both in learning and memory (11). Dynorphin A, YGGFLRRIRPKLKWDNQ, is an opioid peptide specific to -receptors (12, 13), and the sequences of nociceptin and dynorphin A resemble each other. Both nociceptin and dynorphin A contain several basic amino acids at their C-terminal portions, and for dynorphin A, the importance of these basic residues (Arg-6, Arg-7, Arg-9, Lys-11, and Lys-13), especially of Arg-7, has been demonstrated for the recognition and activation of receptors (14 –17). Nociceptin contains a couple of basic amino acid pairs, namely, two Arg-Lys sequences at positions 8 –9 and 12–13, and these have been acknowledged to be essential for both receptor recognition and activation (18, 19). The basic regions of dynorphin and nociceptin have been postulated to interact with the receptor segment consisting of a cluster of acidic amino acids in the second extracellular loop of the seven transmembrane structure (20 –24). A number of nociceptin analogs have been designed and synthesized to clarify the structural essentials for receptor interaction (18, 19, 25–29). However, no analogs more potent than native nociceptin have yet been announced. Since the activities of nociceptin are either hyperalgesic or to suppress learning and memory, major efforts have recently focused on the design of nociceptin antagonists to overcome these rather negative
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FIG. 1.
Amino acid sequences of nociceptin, and Arg-Lys-multiplied analogs.
cellular responses (30). On the other hand, a highly potent agonist often facilitates the receptor responses such as desensitization and internalization (31, 32). These cellular responses are a kind of suppressive or antagonistic replies of the receptor. The idea of a socalled superagonist prompted us to design and synthesize a highly potent agonist analog of nociceptin. In the present study, in order to obtain potent nociceptin analogs we chose a strategy to strengthen the electrostatic interaction between nociceptin and the second extracellular loop of the nociceptin receptor ORL1. To this end, we have synthesized a series of analogs in which Arg-Lys (RK) displaces the amino acid pairs at places adjacent to the original RK pairs (Fig. 1). These basic forms of nociceptin were evaluated for their receptor binding affinity to ORL1 and opioid receptors and for their biological activity in a functional assay using [ 35S]GTP␥S. As a result, [Arg-Lys14 –15]nociceptin emerged as the first time as an analog much more potent than native nociceptin, especially in the functional biological assay. MATERIALS AND METHODS Peptide synthesis. All peptides, namely, nociceptin and its RKmultiplied analogs, were synthesized (0.1 mmol scale) by the automated peptide synthesizer ABI 430A (Applied Biosystems Inc., Foster City, CA) with the Fmoc synthetic strategy. Peptides were liberated from the resin by Reagent K (33) at room temperature for 3 h. After evaporation, the residue was solidified with diethyl ether. The purification was carried out first by gel filtration on a column (2.0 ⫻ 100 cm) of Sephadex G-15 (Pharmacia, Uppsala, Sweden) eluted with 10% acetic acid. For further purification, reversed-phase high performance liquid chromatography (RP-HPLC) was performed on a preparative column (25 ⫻ 250 mm, Cica-Merck LiChrospher RP-18 (e), 5 m) with a linear gradient of 0.1% trifluoroacetic acid and 80% acetonitrile and the fractions containing pure peptides were lyophilized to obtain the final peptide sample. The purity was verified by analytical RP-HPLC (4 ⫻ 250 mm, Cica-Merck LiChrospher 100 RP-18, 5 m). Mass spectra of peptides were measured on a mass spectrometer Voyager DE-PRO (PerSeptive Biosystems Inc., Framingham, MA) with the method of matrixassisted laser desorption ionization time-of-flight (MALDI-TOF). Amino acid analyses of peptides were carried out on an amino acid analyzer Hitachi L-8800 system after hydrolysis in a constantboiling hydrochloric acid at 110°C for 24 h. The amino acid sequences of nociceptin and its analogs were determined by the Procise Model 491 Protein Sequencing System (PE Biosystems, Foster City, CA). Receptor binding assays. Membranes were prepared from human embryonic kidney 293 cells expressing human ORL1, , ␦, and
opioid receptors as described previously (25). Peptides were evaluated using [ 3H]nociceptin (185 Ci/mmol, Amersham, Buckinghamshire, UK) for ORL1 receptor assays. Binding assays were also carried out for opioid receptor subtypes using [ 3H]DAGO (55 Ci/ mmol, DuPont/NEN Research Products, Wilmington, DE) for human receptors, [ 3H]DPDPE (46 Ci/mmol, DuPont/NEN) for human ␦ receptors, and [ 3H]Cl-977 (42 Ci/mmol, Amersham) for human receptors. Briefly, incubations were carried out at room temperature for 60 min in Tris–HCl buffer (pH 7.55) containing 0.1% bovine serum albumin. Bacitracin (100 g/ml) was added as an enzyme inhibitor. After incubation, each incubation mixture was filtered through glass fiber filters (Whatman GF/B) and rinsed twice with 10 mM Tris–HCl buffer pH 7.55 (4 ml). Dose–response curves were analyzed by the computer program ALLFIT (34). [ 35S]GTP␥S binding to cell membranes. The [ 35S]GTP␥S binding assay was performed according to the method of SPAG-protein coupled receptor assay provided by Amersham with slight modification. The membranes were suspended in ice-cold 20 mM Hepes buffer (pH 7.4) containing 100 mM NaCl, 10 mM MgCl 2, and 1 mM EDTA. After the addition of dithiothreitol (threo-1,4-dimercapto-2,3-butanediol; 0.17 mg/ml) to the solution, membranes were incubated at 25°C for 30 min with the appropriate concentration of drugs in the presence of 5 M GDP, 0.4 nM of [ 35S]GTP␥S (200 –370 mCi/mmol, Amersham), and wheat-germ agglutinin-coated scintillation proximity assay (SPA) beads (1.5 mg) in a 0.2 ml total volume. Basal binding was assessed in the absence of agonist, and nonspecific binding was determined with 10 M GTP␥S. The activity was estimated as EC 50, the value of which exhibits the concentration inducing 50% of its own maximal stimulation.
RESULTS AND DISCUSSION Peptide design and synthesis. Nociceptin is encoded in a precursor protein, in which the sequence of the natural peptide is flanked by Lys-Arg (KR) proteolytic excision motifs at both the N and C-termini. Seventeen-peptide nociceptin consists of the inverse sequence of this KR motif at the two separated positions 8 –9 and 12–13. The RK motif is also occasionally functions as a proteolytic excision site. We noted that the KR and RK motifs in nociceptin precursors are clearly distinguished by a specific processing enzyme not to cleave the RK motif. This suggests that the RK repeats in nociceptin peptide is crucially important for receptor recognition and/or activation. Thus, we chose a RK dipeptide unit to replace the amino acid residues in nociceptin in expectation of the electrostatic interaction reinforced between nociceptin and ORL1 receptor. The resulting [Arg-Lys 6 –7]-, [Arg-Lys 10 –11]-, and [Arg-Lys 14 –15]nociceptins consist of three RK pairs in
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Analytical Data of Synthetic Peptides from Mass Spectrometry, Reversed-Phase High-Performance Liquid Chromatography, and Amino Acid Analysis MALDI-TOFMASS a
RP-HPLC b
Amino acid analysis c
Peptides
Found
Calcd
RT (min)
Asn
Thr
Ser
Gln
Gly
Ala
Leu
Phe
Lys
Arg
Nociceptin (⫽Noc)
1808.8
1810.1
33.17
1965.2
1966.3
31.89
[Arg-Lys 10–11]Noc
1935.6
1936.3
32.32
[Arg-Lys 14–15]Noc
1909.3
1910.2
31.21
1.05 (1) 1.05 (1) 1.03 (1) 1.02 (1)
1.08 (1) 1.04 (1) 1.12 (1) 1.02 (1)
1.12 (1) 1.21 (1) — — 1.02 (1)
1.06 (1) 1.04 (1) 1.13 (1) 1.08 (1)
2.95 (3) 2.03 (2) 2.81 (3) 2.94 (3)
3.00 (3) 2.00 (2) 2.00 (2) 2.00 (2)
1.07 (1) 1.10 (1) 1.08 (1) — —
2.20 (2) 2.08 (2) 2.24 (2) 2.33 (2)
1.85 (2) 2.75 (3) 2.70 (3) 2.83 (3)
1.84 (2) 2.78 (3) 2.77 (3) 2.76 (3)
[Arg-Lys
6–7
]Noc
The values express the mass number (m/z) of (M ⫹ H) ⫹. Retention time (RT) was measured on an analytical column (Cica-Merck LiChrospher 100 RP-18(e), 4 ⫻ 250 mm, 5 m) with a linear gradient of 0.1% trifluoroacetic acid and 80% acetonitrile. c The values are normalized for alanine as an internal standard. a b
each peptide molecule, and we designated these analogs as RK-multiplied nociceptin analogs. Nociceptin and its analogs were prepared by the solid-phase methodology using Fmoc-amino acids. They contains two or three Arg residues, the side chains of which were protected by the 2,2,5,7,8pentamethylchroman-6-sulfonyl (Pmc) group (35). The Pmc groups were removed completely by TFAcontaining Reagent K without any troubles. Peptides were obtained in an average yield of about 35%. Table 1 shows the analytical data of all four heptadecapeptides synthesized. The purity of peptides was verified by analytical HPLC, in which all the peptides emerged as a single peak. The mass numbers measured were coincident with the values calculated. Amino acid analyses revealed a good coincidence of the number of amino acid constituents, and this was also confirmed by the amino acid sequencing of the peptides (data not shown). Thus, synthetic nociceptin and its analogs have been identified physicochemically to reveal the desired compounds.
Receptor binding affinity of nociceptin peptides. Nociceptin was very potent to bind to ORL1 receptors expressed in human 293 cells. The half-maximal concentration (IC 50) for inhibition of the binding of radiolabeled ligand [ 3H]nociceptin was calculated to be 0.93 nM (Table 2). When Arg-Lys (RK) was placed at position 6 –7 to replace Gly-Ala, the resulting analog [ArgLys 6 –7]nociceptin exhibited about 90% decreased affinity for ORL1 receptor in the cells compared to nociceptin. [Arg-Lys 10 –11]nociceptin also showed a reduced receptor affinity (about 60% of nociceptin). In contrast to these RK-multiplied nociceptin analogs, [Arg-Lys 14 –15]nociceptin was found to be very potent, exhibiting an ability to displace [ 3H]nociceptin in ORL1 receptor about three times stronger than parent nociceptin. The activity profile of all these RKmultiplied nociceptin analogs are shown in Fig. 2 by potencies relative to that of native nociceptin. It should be noted that [Arg-Lys 14 –15]nociceptin is the first nociceptin analog showing a receptor binding affinity higher than nociceptin.
TABLE 2
Results of Receptor Binding Affinities and Biological Activity of Nociceptin and Its RK-Multiplied Analogs [ 35S]GTP␥S binding activity
Receptor binding affinity (IC 50, nM) Peptides
ORL1
␦
EC 50 (nM)
Relative potency (%)
Nociceptin (⫽Noc) [Arg-Lys 6–7]Noc [Arg-Lys 10–11]Noc [Arg-Lys 14–15]Noc
0.93 ⫾ 0.50 11 ⫾ 7.9 1.5 ⫾ 0.78 0.32 ⫾ 0.13
290 ⫾ 50 220 ⫾ 85 190 ⫾ 41 280 ⫾ 87
⬎10,000 ⬎10,000 ⬎10,000 ⬎10,000
3500 ⫾ 140 240 ⫾ 78 940 ⫾ 600 1,500 ⫾ 1,300
17 ⫾ 2.3 260 ⫾ 120 19 ⫾ 4.8 1.0 ⫾ 0.30
100 6.6 89 1,700
Note. Binding assays were carried out for human ORL1, , ␦, and opioid receptor expressed in 293 cells and the biological assay was performed with [ 35S]GTP␥S against human ORL1 receptor expressed in 293 cells. Each value represents the mean ⫾ SD from three independent experiments. 495
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(residues 6 –13) of dynorphin A was suggested to interact with the cluster of acidic amino acids in the second extracellular loop of the receptors (20, 21). Since RK-multiplied nociceptin analogs have three RK pairs in a molecule, they must be more preferable to interact with the receptors than nociceptin itself. In particular, [Arg-Lys 6 –7]nociceptin, which is the most potent analog for the receptors, possessed a sequence very much similar to that of dynorphin A at positions 6 –13: i.e., RKRKSARK for [Arg-Lys 6 –7]nociceptin and RRIRPKLK for dynorphin A. Despite such increased binding affinities, however, RK-multiplied nociceptins are still much more potent for ORL1 than for receptors (Table 2).
FIG. 2. Relative potencies of Arg-Lys-multiplied nociceptin analogs for ORL1 receptors in the human embryonic kidney 293 cell membrane preparations: Receptor binding potencies (A) and GTP␥S binding potencies (B). The potencies were calculated by dividing the IC 50 or EC 50 values with that of nociceptin (⫽100) in each assay.
Nociceptin and RK-multiplied analogs were also assayed to for the ordinary ␦, , and opioid receptors to assess their binding ability. For ␦ receptors, they were almost completely inactive. For the opioid receptors, nociceptin exhibited a distinct but weak binding with the IC 50 value of about 300 nM as reported previously (25). It was found that all RK-multiplied nociceptin analogs bind to receptors as well as nociceptin. Their binding affinities (190 –280 nM) to the receptors are, however, 20- to 870-fold weaker than those to ORL1 receptor, indicating that they are unable to construct a bioactive structure specific for the receptors. For receptors, nociceptin was extremely weak with the IC 50 values of three orders of magnitude larger than that for ORL1 receptor (Table 2). In contrast, RK-multiplied nociceptin analogs exhibited increased binding affinity (2- to 15-fold) for the receptors. This is apparently the positive effect of incorporation of Arg-Lys into nociceptin. The basic amino acid region
GTP␥S binding affinity. In the present study, [ 35S]GTP␥S binding assay was carried out to evaluate the functional activation of the ORL1 receptor by nociceptin and RK-multiplied nociceptin analogs. The opioid receptors including ORL1 are coupled with G-proteins, the stimulation of which results in its conformation change that allows cytoplasmic GTP to displace GDP in the ␣-subunit and further facilitates a dissociation of GTP-bound ␣-subunit from ␥-subunits. Thus, the GDP/GTP exchange represents a receptormediated G-protein activation of a receptor ligand. Guanocine 5⬘-(␥-thio)triphosphate, GTP␥S, is a GTP analog resistant to the GTPase enzyme activity of G-protein, and dissociates very slowly. The amount of [ 35S]GTP␥S bound to G-protein thus represents the receptor activity of a ligand as demonstrated in many studies of signaling properties in G-protein coupled receptors (36 –39). Nociceptin dose-dependently stimulated the nucleotide binding in membranes with an apparent EC 50 of 17 nM (Table 2), which compared well with the values (9 –20 nM) as found in other studies (40, 41). Under the same experimental conditions, the affinity (0.93 nM) of nociceptin for ORL1 was about 18-fold higher than its potency in stimulating the GTP binding. This is a rather general phenomenon as observed in both the brain and cell membrane preparations (22, 40, 41). [Arg-Lys 6 –7]nociceptin was very weak (EC 50 ⫽ 260 nM) to stimulate the GTP␥S binding. Compared with nociceptin, this analog reached about 40 – 60% activity in stimulating [ 35S]GTP␥S binding at its 10 M concentration. [Arg-Lys 10 –11]nociceptin was considerably potent (19 nM), whereas [Arg-Lys 14 –15]nociceptin was extremely potent with a small half-maximal concentration for GTP␥S binding (1.0 nM). These two analogs exhibited a full activity in GTP binding (data not shown). When the specificity of the coupling between receptor binding and GTP␥S binding was assessed, the order of magnitude of the receptor affinities of nociceptin, [ArgLys 6 –7]nociceptin, and [Arg-Lys 10 –11]nociceptin (IC 50 1:0.085:0.62 as seen in Fig. 2A) was nearly the same as
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that of their potencies in stimulating the [ 35S]GTP␥S binding (EC 50 1:0.066:0.89, Fig. 2B). By contrast, [ArgLys 14 –15]nociceptin was found to be considerably more active in stimulating the [ 35S]GTP␥S binding than expected from its binding affinity. When compared with the activities of nociceptin, [Arg-Lys 14 –15]nociceptin was 17 times more potent than nociceptin in the GTP␥S functional assay. Since [Arg-Lys 14 –15]nociceptin was about 3 times more potent than nociceptin in the receptor binding assay, this RK-multiplied analog is about 6-fold stronger in the GTP␥S binding assay than in receptor binding assay. This suggests that [ArgLys 14 –15]nociceptin is involved in a molecular mechanism to facilitate very effectively a basal level of receptor activation with its lower concentrations. It should be noted that the affinity (0.32 nM) of [Arg-Lys 14 –15]nociceptin for ORL1 was only 3-fold higher than its potency in stimulating the GTP binding under the same conditions. Structure–activity relationships of [Arg-Lys 14 –15 ]nociceptin. Extremely high activity of [Arg-Lys 14 –15]nociceptin, 17-fold stronger than native nociceptin, should be explained by an appropriate molecular interaction mode. The structure–activity studies of [ArgLys 14 –15]nociceptin suggest two possible interaction modes for Arg-Lys 14 –15: i.e., the electrostatic interaction with the receptor acidic amino acid cluster and the cation/ interaction with the receptor aromatic groups. Recent studies have suggested that Arg-Lys 8 –9 and Arg-Lys 12–13, which are native constituents of the nociceptin peptide, interact with the cluster of acidic amino acids in the second extracellular loop of nociceptin receptor ORL1 (22–24). It is thus likely that newly incorporated Arg-Lys 14 –15 arrests the negative charges of the receptor molecule by the electrostatic interaction. The original amino acids at positions 14 and 15 are Leu and Ala, respectively. Arg-Lys 14 –15 would fill the space of the original Leu-Ala 14 –15 and further cap the cationic groups such as guanidino and amino groups, eliciting an additional interaction with ORL1. This extra electrostatic interaction may stabilize the total ligand/ receptor interaction to induce a reinforced activation. Unlike Lys/Ala 15 replacement, Asp or Trp/Ala 15 replacement has been found to result in a sharp drop in receptor binding and biological activities (27, 42). In contrast, position 14 permits the placement of various kind of amino acids such as Leu (native nociceptin), Tyr (18, 25), Trp (42), and now even Arg. Apparently, Leu, Tyr, and Trp do not engage in the electrostatic interaction. Given that all these amino acids interact with the same site of ORL1, the receptor site must possess a structural component that interacts specifically with isobutyl (Leu), phenol (Tyr), indolyl (Trp), and guanidino (Arg) groups. This is only possible with aromatic amino acid residues such as Phe, Tyr, Trp, and His, which consist of systems. In the second
extracellular loop of the ORL1 receptor, three aromatic amino acids exist: i.e., Tyr-210, Trp-211, and Phe-215 (the numbers based on the amino acid sequence of the human ORL1 receptor). Arg-Lys 14 –15 might interact with these receptor aromatic groups, producing the so-called cation/ interaction. This cation/ interaction is also a kind of electrostatic interaction. Identification of the receptor-binding site for Arg-Lys 14 –15 might afford important structural information regarding the design of receptor agonists and antagonists. REFERENCES 1. Meunier, J.-C., Mollereau, C., Toll, L., Suaudeau, C., Moisand, C., Alvinerie, P., Butour, J.-L., Guillemot, J.-C., Ferrara, P., Monsarrat, B., Mazarguil, H., Vassart, G., Parmentier, M., and Costentin, J. (1995) Nature 377, 532–535. 2. Reinscheid, R. K., Nothacker, H.-P., Bourson, A., Ardati, A., Henningsen, R. A., Bunzow, J. R., Grandy, D. K., Langen, H., Monsma, F. J., Jr., and Civelli, O. (1995) Science 270, 792–794. 3. Mollereau, C., Parmentier, M., Mailleux, P., Butour, J.-L., Moisand, C., Chalon, P., Caput, D., Vassart, G., and Meunier, J.-C. (1994) FEBS Lett. 341, 33–38. 4. Chen, Y., Fan, Y., Liu, J., Mestek, A., Tian, M., Kozak, C. A., and Yu, L. (1994) FEBS Lett. 347, 279 –283. 5. Wang, J. B., Johnson, P. S., Imai, Y., Persico, A. M., Ozenberger, B. A., Eppler, C. M., and Uhl, G. R. (1994) FEBS Lett. 348, 75–79. 6. Nishi, M., Takeshima, H., Mori, M., Nakagawara, K., and Takeuchi, T. (1994) Biochem. Biophys. Res. Commun. 205, 1353–1357. 7. Mollereau, C., Simons, M.-J., Soularue, P., Liners, F., Vassart, G., Meunier, J.-C., and Parmentier, M. (1996) Proc. Natl. Acad. Sci. USA 93, 8666 – 8670. 8. Okuda-Ashitaka, E., Tachibana, S., Houtani, T., Minami, T., Masu, Y., Nishi, M., Takeshima, H., Sugimoto, T., and Ito, S. (1996) Mol. Brain Res. 43, 96 –104. 9. Pan Y.-X., Xu, J., and Pasternak, G. W. (1996) Biochem. J. 315, 11–13. ¨ gren, S. O., and Tere10. Sandin, J., Georgieva, J., Scho¨tt, P. A., O nius, L. (1997) Eur. J. Neurosci. 9, 194 –197. 11. Manabe, T., Noda, Y., Mamiya, T., Katagiri, H., Houtani, T., Nishi, M., Noda, T., Takahashi, T., Sugimoto, T., Nabeshima, T., and Takeshima, H. (1998) Nature 394, 577–581. 12. Goldstein, A., Fischli, W., Lowney, L. I., Hunkapiller, M., and Hood, L. (1981) Proc. Natl. Acad. Sci. USA 78, 7219 –7223. 13. Tachibana, S., Araki, K., Ohya, S., and Yoshida, S. (1982) Nature 295, 339 –340. 14. Chavkin, C., and Goldstein, A. (1981) Proc. Natl. Acad. Sci. USA 78, 6543– 6547. 15. Chavkin, C., James, I. F., and Goldstein, A. (1982) Science 215, 413– 415. 16. Weber. E., Evans. C. J., and Barchas, J. D. (1982) Nature 299, 77–79. 17. Corbett, A. D., Paterson, S. J., McKnight, A. T., Magnan, J., and Kosterlitz, H. W. (1982) Nature 299, 79 – 81. 18. Reinscheid, R. K., Ardati, A., Monsma, F. J., Jr., and Civelli, O. (1996) J. Biol. Chem. 271, 14163–14168. 19. Butour, J.-L., Moisand, C., Mazarguil, H., Mollereau, C., and Meunier, J.-C. (1997) Eur. J. Pharmacol. 321, 97–103. 20. Wang, J. B., Johnson, P. S., Wu, J. M., Wang, W. F., and Uhl, G. R. (1994) J. Biol. Chem. 269, 25966 –25969. 21. Naqvi, T., Haq, W., and Mathur, K. B. (1998) Peptides 19, 1277– 1292.
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