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We previously showed that an antagonist-based peptide ligand, H-Cys(Npys)-Arg-Tyr-Tyr-Arg- Ile-Lys-NH2 , captures the free thiol groups in the ligand-binding site of the nociceptin receptor ORL1. However, the exact receptor sites of this thiol-disulfide exchange reaction have not been uncovered, although such identification would help to clarify the ligand recognition site. Since the Cys→Ala substitution prevents the reaction, we performed the so-called Ala scanning for all the Cys residues in the transmembrane (TM) domains of the ORL1 receptor. Seven different mutant receptors were soundly expressed in the COS-7 cells and examined for their specific affinity labeling by a competitive binding assay using nociceptin and [(3) H]nociceptin. The results of in vitro Ala scanning analyses revealed that the labeled residues were Cys59 in TM1, Cys215 and Cys231 in TM5, and Cys310 in TM7. The present study has provided a novel method of Cys(Npys)-affinity labeling for identification of the ligand-binding sites in the ORL1 receptor. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 460-469, 2016., (© 2016 Wiley Periodicals, Inc.)

IsoVa-RYYRIK-NH2 is a highly specific antagonist ligand of the opioid receptor-like 1 (ORL1) receptor, an endogenous ligand of which is 17-mer peptide nociceptin. ORL1 antagonists have potential for clinical use as analgesic and antineuropathic drugs, and thus information on the receptor-binding characteristics of antagonists is very important for rational drug design. In the present study, we prepared tritium-labelled isova-RYYRIK-NH2 from its precursor with the 3-methylcrotonyl (CH3)2CCHCO group by a catalytic reduction using tritium gas. The resulting [(3)H]isoVa-RYYRIK-NH2 was evaluated in a saturation binding assay using the COS-7 cell membrane preparations of transiently expressed ORL1. It exhibited more than 90% specific binding with a dissociation constant of 1.21±0.03nM. From the mutual heterologous binding assays using [(3)H]isoVa-RYYRIK-NH2 and [(3)H]nociceptin, isoVa-RYYRIK-NH2 and nociceptin were found to share the receptor-binding site, but each also had a separate specific binding site of its own. They differentiated the two different binding states or conformations of ORL1, which might represent the agonist-active and antagonist-inactive conformations of ORL1. [(3)H]isoVa-RYYRIK-NH2 is thus a key tracer to uncover the amino acid residues important for receptor inactivation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Antagonists of the neuropeptide nociceptin are expected to be potential analgesic and antineuropathic drugs acting on ORL1 GPCR receptors. The peptide library-based antagonist Ac-RYYRIK-NH2 inhibits the nociceptin activity mediated through ORL1, but preserves a considerably high level of agonist activity. We previously reported that the N-terminal acyl group is important for interaction with specific receptors, and developed isovarelyl-RYYRIK-NH2, which exhibits strong antagonist activity with negligible agonist activity. In the present study, in order to obtain a more potent antagonist, we further modified the isovarelyl group by replacing its Cβ atom with an oxygen, nitrogen, or sulfur atom to give the methyl group improved interaction ability. The methyl group bound to such heteroatoms was expected to enhance the hydrophobic interaction between the peptide and the ORL1 receptor. The RYYRIK-NH2 peptide with a methylthioacetyl group, CH3SCH2CO, revealed a higher receptor-binding affinity with strong antagonist activity, and the results suggested the presence of a receptor aromatic group as a complementary residue of this CH3S group.

All of the δ, μ, and κ opioid receptors have a free thiol group of the Cys residue in the ligand-binding site, although its functional role is not yet known. In order to examine whether or not a similar Cys is also present in the ORL1 nociceptin receptor, we attempted to identify it by affinity labeling using a specific antagonist peptide. We first treated ORL1-expressing COS-7 cell membrane preparations with the thiol-alkylation reagent N-ethylmaleimide (NEM) to perform a binding assay using [(3)H]nociceptin as a tracer and nociceptin, an ORL1 agonist, or Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2), a nociceptin/ORL1 antagonist, as a competitor. It was suggested that ORL1 has a free Cys in its ligand-binding site, since the NEM treatment reduced the population of ligand-binding sites. This was further confirmed by affinity labeling using Cys(Npys)-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) with the SNpys group that can react with a free thiol group, resulting in the formation of a disulfide bond. This affinity labeling was approximately 23 times more specific than NEM alkylation. The results revealed that the ORL1 nociceptin receptor does contain a free Cys residue in the ligand-binding site., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

[Arg(14),Lys(15)]Nociceptin is a very potent for ORL1 receptor, showing a few times stronger binding activity and much more enhanced biological activity than endogenous nociceptin. This synergistic outcome has been suggested to be due to the interaction with the receptor aromatic and/or acidic amino acid residues crucial to receptor activation. In order to identify such receptor residues in the second ORL1 extracellular loop, we prepared a series of recombinant mutant receptors. The mutant receptor Gln205Ala was found to be as active as wild-type ORL1 for both nociceptin and [Arg(14),Lys(15)]nociceptin. In contrast, Asp206Ala and Tyr207Ala exhibited considerably reduced activity for [Arg(14),Lys(15)]nociceptin, exhibiting no synergistic activity enhancement. These results suggest that Asp206 and Tyr207 are directly involved in the interaction with nociceptin-[Arg(14),Lys(15)]. Trp208Ala was found to bind strongly both nociceptin and [Arg(14),Lys(15)]nociceptin, although it elicited no biological activity. All these results indicate that the consecutive amino acid residues Asp206, Tyr207, and Trp208 are critical to the activation of the ORL1 receptor, but not to nociceptin-binding.

ORL1 is an endogenous G protein-coupled receptor for neuropeptide nociceptin. [(R/K)(14), (R/K)(15)]nociceptin is a superagonist that strongly activates the ORL1 receptor. We have previously found that substituting with Trp can reproduce the potentiation induced by Arg or Lys at position 14. In the present study, in order to ensure the effect of Trp-substitution on the activities of [(R/K)(14), (R/K)(15)]nociceptin, we synthesized [W(14), (R/K)(15)]nociceptin and [(R/K)(14), W(15)]nociceptin. [W(14), (R/K)(15)]nociceptin was found to exhibit threefold higher binding activity and 10-fold greater potency in a functional [(35)S]GTPgammaS functional assay as compared to wild-type nociceptin. However, when only Trp was placed in position 15, the resulting analogues, [(R/K)(14), W(15)]nociceptin, showed only a moderate enhancement of binding and biological activity (2-3 fold in both). These results indicate that the placement of Trp at position 14, unlike at position 15, enhances in a synergistic fashion the interaction of nociceptin with the ORL1 receptor. The results indicate that specific interactions feasible for Arg/Lys and Trp in common must be there for aromatic residues in ORL1, thus forming a cation/pi interaction or pi/pi hydrophobic interaction. The necessity for a favorable electrostatic interaction appears strict in position 15.

Nociceptin is an endogenous ligand that activates a G protein-coupled receptor ORL1 and contains two indispensable Arg-Lys (RK) dipeptide units at positions 8-9 and 12-13. By replacing an additional RK unit at positions 6-7, 10-11, 14-15, or 16-17, of the peptide we have identified the analog, [RK(14-15)]nociceptin as a superagonist. In fact, this peptide exhibits 3-fold higher binding affinity and 17-fold greater potency in a functional GTPgammaS-binding assay compared to wild-type nociceptin. Here, we have further investigated the role of basic residues in position 14-15. The replacement of three other possible basic dipeptides, KR, RR, and KK, into nociceptin at positions 14-15 resulted in similar enhancements of binding affinity (3-5-fold) and biological potency (10-12-fold in the GTPgammaS assay). However, when only a single basic residue (Arg or Lys) was replaced in either position 14 or 15, all the resulting analogs showed moderate enhancements of binding and biological activity (2-4-fold in both). These results indicate that the addition of basic charges in positions 14 and 15 enhance in a synergistic fashion the interaction of nociceptin with the receptor and only the simultaneous presence of two adjacent basic residues yields an optimal effect. This suggests that specific electrostatic interactions between both amino acids present in 14-15 and corresponding residues in the receptor are responsible for the enhancement of nociceptin activity.

Nociceptin is an endogenous agonist ligand of the ORL1 (opioid receptor-like 1) receptor, and its antagonist is a potential target of therapeutics for analgesic and antineuropathy drugs. Ac-RYYRIK-NH(2) is a hexapeptide isolated from the peptide library as an antagonist that inhibits the nociceptin activities mediated through ORL1. However, the structural elements required for this antagonist activity are still indeterminate. In the present study, we evaluated the importance of the acetyl-methyl group in receptor binding and activation, examining the peptides acyl-RYYRIK-NH(2), where acyl (R-CO) possesses a series of alkyl groups, R=C(n)H(2n+1) (n=0-5). The isovaleryl derivative with the C(4)H(9) (=(CH(3))(2)CHCH(2)-) group was found to reveal a high receptor-binding affinity and a strong antagonist nature. This peptide achieved a primary goal of eliminating the agonist activity of Ac-RYYRIK-NH(2) and producing pure antagonist activity.

Ac-RYYRIK-NH2 is a peptide isolated from the peptide library as an antagonist that inhibits the biological activities of nociceptin, a hyperalgesic neuropeptide. In order to clarify the structural requirements of this peptide for binding to the nociceptin receptor ORL1, systematic structure-activity studies were carried out. The result of Ala-scanning indicated that the N-terminal tripeptide RYY(= Arg-Tyr-Tyr) is crucially important for binding to the ORL1 receptor. Residual truncations from the N- or C-terminus revealed the special importance of the N-terminal Arg residue. The removal of protecting groups indicated that the N-terminal acetyl group is essential, but the C-terminal amide group is insignificant. These results indicated the conspicuous importance of acetyl-Arg at position 1 of Ac-RYYRIK-NH2 as a key structure allowing binding to the receptor. To investigate the binding site of this peptide in the ORL1 receptor, we synthesized and assayed a series of analogues of the nociceptin dibasic repeat region, residues 8-13 of RKSARK. None of the derivatives were active. Ac-RYYRIK-NH2 was inactive for the mu opioid receptor to which nociceptin binds with considerable strength. All the results suggested that the mode of binding between Ac-RYYRIK-NH2 and the ORL1 receptor is different to that between the ORL1 receptor and nociceptin, and that it may consist of interaction with the receptor site to which nociceptin(1-7) or -(14-17) binds.

The affinity-labeling technique is an extremely important method in receptor biochemistry. The 3-nitro-2-pyridinesulfenyl (Npys) group, attached to a mercapto group, can react only with a free thiol group (the beta-mercapto group of cysteine residue) of the target receptor molecules, forming a disulfide bond. This disulfide bonding is mediated through the thiol-disulfide exchange reaction. Unlike other labeling methods, the approach utilizing such chemically activated thiol-containing ligands is able to reproduce an unlabeled protein by treatment with dithiothreitol, a reducing reagent. This provides several unique aspects for the studies elucidating the structure-function relationships between the peptide and the receptor. Based on the SNpys affinity technique, we have achieved the discriminative disulfide-bonding affinity labeling of the three different subtypes of opioid receptors: mu, delta and kappa. This article reviews our novel affinity techniques in the in vitro receptor biochemistry.

A ligand containing an SNpys group, i.e. 3-nitro-2-pyridinesulfenyl linked to a mercapto (or thiol) group, can bind covalently to a free mercapto group to form a disulfide bond via the thiol-disulfide exchange reaction. This SNpys chemistry has been successfully applied to the discriminative affinity labeling of mu and delta opioid receptors with SNpys-containing enkephalins [Yasunaga, T. et al. (1996) J. Biochem. 120, 459-465]. In order to explore the mercapto groups conserved at or near the ligand binding sites of three opioid receptor subtypes, we synthesized two Cys(Npys)-containing analogs of dynorphin A, namely, [D-Ala2, Cys(Npys)8]dynorphin A-(1-9) amide (1) and [D-Ala2, Cys(Npys)12]dynorphin A-(1-13) amide (2). When rat (mu and delta) or guinea pig (kappa) brain membranes were incubated with these Cys(Npys)-containing dynorphin A analogs and then assayed for inhibition of the binding of DAGO (mu), deltorphin II (delta), and U-69593 (kappa), the number of receptors decreased sharply, depending upon the concentrations of these Cys(Npys)-containing dynorphin A analogs. It was found that dynorphin A analogs 1 and 2 effectively label mu receptors (EC50 = 27-33 nM), but also label delta receptors fairly well (160-180 nM). However, for kappa receptors they showed drastically different potencies as to affinity labeling; i.e., EC50 = 210 nM for analog 1, but 10,000 nM for analog 2. Analog 2 labeled kappa receptors about 50 times more weakly than analog 1. These results suggested that dynorphin A analog 1 labels the Cys residues conserved in mu, delta, and kappa receptors, whereas analog 2 only labels the Cys residues conserved in mu and delta receptors.

Nociceptin or orphanin FQ is a novel neuropeptide that activates an opioid-like G protein-coupled receptor ORL1. This heptadecapeptide FGGFTGARKSARKLANQ resembles kappa-opioid peptide dynorphin A but exhibits an opposite effect to make animals hyperreactive to nociceptive stimulations (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; 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). In the present study, it was found that guinea pig brain contains receptors to which nociceptin binds much more strongly than to ORL1 receptors expressed in human 293 cells. Although the Tyr1 --> Phe substitution for dynorphin A eliminates almost completely an ability to bind to opioid receptors, the Phe1 --> Tyr substitution in nociceptin was found to retain almost fully both receptor binding affinity and in vivo hyperalgesic activity in tail-flick assay. Nociceptin was extremely weak to bind to opioid receptors, while Tyr1-nociceptin exhibited 10-40 times increased affinity, especially for mu receptors, due to its N-terminal sequential identity to opioid peptides. Shortened analogs of dynorphin A are known to retain receptor binding ability and analgesic activity, whereas the removal of C-terminal hexa- or decapeptide from nociceptin totally abolished the affinity for the ORL1 receptor. These results indicated that the mode of interaction between nociceptin and ORL1 receptor is quite different from that between dynorphin and opioid receptor and that the C-terminal portion of nociceptin is crucial for receptor recognition.

The thiol groups of leucinthiol, cysteamine and cysteine incorporated into opioid peptides enkephalin and morphiceptin were activated by the 3-nitro-2-pyridinesulphenyl (Npys) group to form mixed disulphides highly reactive to a free thiol. Enkephalin analogues containing Npys-leucinthiol or -cysteine at positions 4, 5 and 6 exhibited high affinities for both mu and delta receptors, while morphiceptin analogues containing Npys-cysteine at positions 4 and 5 showed relatively weak affinity only for mu receptors. When these S-activated opioid peptides were incubated with rat brain membrane preparations, it was found, by binding assay using radiolabelled and non-labelled [D-Ala2,MePhe4,Gly-ol5]enkephalin, that they label mu opioid receptors in a dose-dependent manner. The concentrations required to label half of the receptors were 0.2-2 microM for enkephalins and 10-30 microM for morphiceptins. These results suggested that the thiol group labelled by S-activated enkephalins and morphiceptins is present in the ligand binding site of receptor protein, but not in GTPase-binding protein.

Enkephalin analogs with various C-terminal modifications have been synthesized to evaluate the corresponding structural elements in the opioid receptors. The carboxyl group of the C-terminal leucine5 or glycine5 was converted into the mercaptomethyl (-CH2SH) and hydroxymethyl (-CH2OH) groups, starting from leucinthiol or leucinol for Leu5-derivatives and from cysteamine or ethanolamine for Gly5-derivatives. Interactions of synthetic peptides with the opioid receptors were examined by the radioligand receptor binding assays using rat brain and tritiated enkephalin analogs. The data suggest that the C-terminal carboxyl group in enkephalins is important, but not electrostatically, for interaction with delta-opioid receptors. With leucinthiol-enkephalin in biological assays which examine its inhibitory activity for electrically stimulated contractions of isolated smooth muscle, it was found that the reactive thiol group exists in the mu receptors present in the guinea pig ileum. Leucinthiol-enkephalin became bound covalently to this receptor-thiol group via disulfide formation after prolonged incubation.

The Tyr-Pro-Phe peptide sequence, an N-terminal tripeptide fragment of opioid peptide morphiceptin, was searched on the database SEQDB (Peptide Institute, Osaka). More than 30 proteins were drawn in a list with 15 amino acid varieties at the position adjacent to Phe. Seven morphiceptin-like peptides with the H-Tyr-Pro-Phe-Xxx-NH2 sequence, where Xxx denotes the selected amino acids (Ala, Asp, Gly, Gln, Lys, Thr and Tyr), have been synthesized. Together with the side-chain protected analogs [Asp(OBzl), Lys(Z), and Thr(Bzl)], they have been evaluated for opioid activities. For the mu opioid receptors to which morphiceptin binds specifically and selectively, analogs with Xxx = Asp, Lys, Ala, Thr, Gln, Tyr and Gly showed a considerably weaker binding affinity than morphiceptin. In contrast, the Thr(Bzl) derivative demonstrated an affinity ten times greater than morphiceptin. Because of an extremely weak affinity for the delta receptors, its mu-selectivity became very high (260-fold). The present results and the increased activity of [Val4]morphiceptin (Sakaguchi et al., 36) indicate that the mu receptors tolerate the hydrophobic or aromatic residue at the site corresponding to position 4 of morphiceptin. When the circular dichroism (CD) spectra are compared between active and inactive analogs, no significant difference is found in both water and methanol. This suggests that the activity of morphiceptin analogs with the Tyr-Pro-Phe sequence is mainly determined by the structural characters of the amino acid residue in position 4 rather than by the molecular conformation. The results suggested a possible formation of morphiceptin-like peptides in various protein digests.

Certain enkephalin analogues, including those which contain the conformationally restricted amino acid E-(2R,3S)-cyclopropylphenylalanine [2R,3S)-delta E Phe), have been shown to have high affinity for brain delta opioid receptors but are much less active in mouse vas deferens bioassays. To investigate whether there are differences between delta opioid receptors in brain and mouse was deferens, the ability of a selective delta opioid compound, [D-Pen2,pCl-Phe4,D-Pen5]enkephalin (pCl-DPDPE), and [D-Ala2,(2R,3S)-delta E Phe4,Leu5]enkephalin methyl ester (CP-OMe), to inhibit [3H]pCl-DPDPE binding in both rat brain and mouse vas deferens were measured. pCl-DPDPE recognized brain and mouse vas deferens binding sites with equal affinity, however, CP-OMe showed 33 fold lower affinity in mouse vas deferens compared to brain. This suggests that mouse vas deferens delta opioid receptors may be distinct from brain delta opioid receptors.

The intrathecal administration of several mu (morphine), delta (d-ala2-d-leu5-enkephalin, dimeric leu-enkephalin) and mixed mu/delta (beta-endorphin) agonists produced a dose-dependent inhibition of all cutaneous thermal (Tail Flick/Hot Plate) nociceptive responses in the rat. The kappa agonist U50488H had no analgesic potency in thermal nociceptive tests. On a visceral chemical test (writhing) and agonists exerted a powerful suppression of the response. In contrast at doses 10 to 50 times the ED50 on cutaneous thermal tests, delta agonists had no effect on the writhing response. At higher intrathecal doses, delta ligands produced flaccidity. These observations suggest the existence of three discriminable populations of opioid receptors in the spinal cord whose activation has different effects on the animals response to noxious stimuli.

Enkephalin analogs containing a thiol activated by a thiomethyl (SCH3)*** or 3-nitro-2-pyridinesulfenyl (Npys) group were synthesized. Incubation of such S-activated enkephalin analogs as [D-Ala2, Leu(CH2S)SCH(3)5]enkephalin or [D-Ala2,Leu(CH2S)Npys5]enkephalin with guinea pig ileum (GPI) resulted in the continuous stimulation of the mu opiate receptors. This sustained GPI-activity was completely reversed with the antagonist naloxone, while subsequent washings elicited again the full enkephalin activity. When GPI showing full enkephalin activity was incubated with 1 mM dithiothreitol, about 70% of the activity was eliminated. Examination of enkephalin analogs containing Cys(Npys) at position 1, 5, or 6 suggested that no other thiols occur near the enkephalin binding site of the mu receptor. From these results, it is considered that only one thiol group exists near the binding site of the mu receptor in GPI. Similar results were also obtained for the mu receptors in mouse vas deferens.

A dimeric pentapeptide enkephalin (DPE2) consisting of two molecules of [D-Ala 2, Leu 5] enkephalin linked at C-terminal leucine with ethylenediamine, (H-Tyr-D-Ala-Gly-Phe-Leu-NH-Ch2)2 is a bivalent ligand for the delta enkephalin receptors of rat brain and neuroblastoma-glioma hybrid (NG108-15) cells. This new enkephalin analog shows dramatically increased affinity in radioligand assays using whole brain membranes when delta but not mu specific radioligands are employed. When membranes from NG108-15 cells are used, the dimer shows greatly increased activity irrespective of the mu or delta specificity of the tracer. The dimer DPE2 shows a four-fold, "sodium shift" in its IC50 for competition with [3H]naloxone, suggestive of agonist behavior. Agonist activity was confirmed by demonstrating that DPE2 inhibits cyclic AMP production in prostaglandin E1 stimulated NG108-15 cells, and by demonstrating very high potency in the mouse vas deferens bioassay. DPE2 binds to the same delta sites as the delta-selective monomer [D-Ala2, D-Leu5] enkephalin, since the two ligands show complete crossdisplacement. Radiolabeled 3H-DPE2 shows a five-fold higher affinity constant, a 2.5-fold higher association rate constant, and a two-fold lower dissociation rate than the monomer. These results are consistent with the hypothesis that the dimeric pentapeptide enkephalin can bridge two delta receptors. This enkephalin dimer provides a valuable new probe of opiate receptors and their organization in cell membranes.

Opiate alkaloids and opioid peptides have been shown to suppress plasma LH and FSH levels via a naloxone sensitive mechanism in several species including man. Three subtypes of opiate receptors have been characterized: mu, delta and kappa. The present study was designed to investigate their role in gonadotropin release. Three highly selective opioid ligands, DAGO, MRZ and DTE12 (a dimeric tetrapeptide enkephalin), were injected intraventricularly into chronically ovariectomized rats. Injection of the mu-agonist at doses of 1 and 10 nmol produced a significant suppression of LH secretion, while the delta- and kappa-agonists had no significant effect. Thus, the mu-receptor seems to be the primary opiate receptor involved in the regulation of LH secretion. None of the opiate agonists employed had an effect on FSH secretion.

PO +Dehydrophenylalanine (delta Phe) having the E-configuration (delta EPhe ; phenyl and C = O cis) was incorporated into [Leu5]-enkephalin in order to restrict its conformation. Compared with the Z-isomer, in the radio-ligand receptor binding assays, [D-Ala2, delta EPhe4 , Leu5] enkephalin showed drastically decreased potency for the delta and mu opiate receptors, i.e., 260- and 150-fold loss of affinity, respectively. The results strongly indicate that the opiate receptors require the Z-configuration (phenyl and C = O, trans) of the delta Phe4 residue and may require a specific interrelationship between the aromatic rings of the Tyr1 and Phe4 residues in the molecule for binding. The conformation of [Leu5]-enkephalin specific for the delta receptors was analyzed and a comparison made with its crystal structure recently elucidated.

The binding of alkylendiamide dimers of the three N-terminal residues of [D-Ala2,D-Leu5]enkephalin (DADL) to rat brain and Ng108-15 neuroblastoma-glioma cell membranes was compared with that of DADL, Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAGO) and morphiceptin. Tritiated DADL and DAGO were used as labeled ligands for delta- and mu-receptors, respectively. Dimerization of the tripeptides resulted in dramatic increases in both mu and delta binding. The binding to mu-receptors showed two peaks at an alkyl chain length of n = 2 and approximately n = 16. In contrast, delta binding (NG108-15 cells) increased steadily with increasing chain length. The dimers with n less than 18 were mu-preferential, and the one with n = 2 showed the most dramatic increase in mu selectivity with a 400 fold higher affinity to mu- than to delta-receptors. For long-chain alkyl spacers the compounds became delta selective.

A series of analogs of a dimeric peptide of the inactive fragment of enkephalin (H-Tyr-D-Ala-Gly-NH-CH2-)2 (DTRE2) was synthesized by modifying one or both of tyrosine residues. The change of configuration of both tyrosines, from L to D, or the removal of both p-hydroxy groups brought about a decrease in activity, but some of the activity (about 20% of the parent enkephalin dimer's activity) was found to be retained when assayed by using guinea pig ileum and mouse vas deferens. In contrast, the analog lacking amino groups in both tyrosines was completely devoid of activity, indicating the essential importance of the tyrosine amino group in opiate receptor recognition. The activity of analogs with only one amino group was found to be higher than that of the parent enkephalin dimer having both amino groups. A possible interaction to explain this finding was discussed.

The novel fluorescent amino acid, L-1-pyrenylalanine (L-Pya), was prepared by the asymmetric hydrogenation of cyclic dehydrodipeptide. Fluorescent enkephalins containing one or two Pya residues at position 1,4 or 5 of [D-Ala2, Leu5]enkephalin were synthesized by the solution method. Mono-Pya-enkephalins showed strong fluorescence intensities and potent binding affinities with specificity and selectivity for opiate receptors. However, di-Pya-enkephalins showed markedly decreased receptor binding affinities. These results indicate that the incorporation of two Pya residues into enkephalin makes the peptide unable to interact with the opiate receptors, although introduction of one Pya residue is effective to elicit a specific receptor interaction. Di-Pya-enkephalins showed intramolecular excimer spectra, indicating that the peptides are able to take possible folded conformations.

The dehydrophenylalanine4-enkephalin having the E-configuration (delta EPhe; phenyl and C = 0, cis) was prepared by photoisomerization of the Z-isomer with 3100 A light, followed by reversed-phase HPLC separation of the resulting mixture of the Z- and E-isomers. In the radioligand receptor binding assays, the E-isomer of [D-Ala2, delta Phe4, Leu5]enkephalin exhibited an extremely diminished affinity as compared with the Z-isomer, namely 150-260-fold loss of affinity for the delta and mu opiate receptors. The results indicate that the interrelationship of the Tyr1 and Phe4 residues in the enkephalin molecule seems to be of great importance in receptor recognition.

D-Ala2-D-Leu5-enkephalin (DADL) along with dimers formed from tetra(DTE: des-Leu5-enkephalin)- or pentapeptide (DPE: Leu5-enkephalin) coupled by methylene bridges of various lengths (n) have been shown in in vitro systems to possess varying degrees of mu/delta-receptor selectivity. In the present studies we have systematically compared the intrathecal effect of these agents and morphine on the cutaneous stimuli (hot plate (HP) and tail flick (TF) and visceral-chemical (writhing) tests in the rat. The following observations were made. (1) Dimers with high delta-receptor selectivity were active in the TF(DPE2 greater than or equal to DADL greater than or equal to DTE2 greater than or equal to morphine greater than DPE12 much greater than DTE12 = 0) and HP(DPE2 greater than or equal to DTE2 greater than or equal to DADL greater than or equal to morphine greater than DPE12 greater than or equal to DTE12 greater than 0. To examine cross-tolerance, the intrathecal ED50 for morphine, DPE and DADL were determined in rats rendered tolerant by subcutaneous morphine pellets. The TF ED50 tolerant/TF ED50 naive was 18.4, 5.4 and 1.3, respectively. The ratio of activity on the HP was 14.0, 4.7 and 2.2 (2) On the visceral-chemical test, only morphine was active. The dimers or DADL in doses which totally blocked the TF or HP are at higher doses which were just below those producing motor dysfunction had no effect on the writhing response. (3) At high intrathecal doses (40 X TF ED50), morphine produced a motor rigidity which blocked the placing and stepping reflex.(ABSTRACT TRUNCATED AT 250 WORDS)

For the elucidation of structural elements in the opiate receptors, a thiol-containing enkephalin analog [D-Ala2, cysteamine 5]enkephalin, and its dimeric analog were synthesized and evaluated in the radio-ligand receptor binding assays using rat brain membranes. The dimeric analog was very potent in both delta and mu assays. Comparison of receptor affinities of the thiol-containing enkephalin with those of standard mu or delta receptor specific ligands suggested that the mu receptor contains an essential thiol group which may interact with the thiol group at the C-terminus of the enkephalin analog. It also appears that no metal-ion site, postulated for the delta receptors, is present in the delta binding site.

The Gly3 residue in Gly2- and D-Ala2-enkephalins has been replaced by delta Ala3 and Ser3 in order to examine the effect on binding to the delta and mu opiate receptors. [D-Ala2, delta Ala3, Leu5]-enkephalin maintains most of its receptor binding affinities for both sites. It is suggested that the proper conjunctions of positions 2 and 3 of the enkephalin sequence are important to receptor preference and that position 3 may have a very specific interaction with the delta receptor. The in vivo analgesic and CNS activities of the peptides are also discussed.

Two series of dimeric enkephalin analogues were assayed for opioid activity in two isolated smooth muscle preparations: the guinea pig ileum (GPI) and the mouse vas deferens (MVD). Dimers have the general structure: X-(CH2)n-X, where X is H-Tyr-D-Ala-Gly-Phe-Leu-NH-(n = 0, 2, 4, 6, 8, 10, 12), for the first series of dimeric pentapeptide enkephalins (DPEn), and H-Tyr-D-Ala-Gly-Phe-NH-(n = 2, 4, 6, 8, 12), for the series of dimeric tetrapeptide enkephalins (DTEn). Comparison of biological activities with binding affinities revealed that: (1) the DPE series with n = 2-8 showed increased potency in the MVD assay relative to monomeric [D-Ala2, Leu5]enkephalinamide (DALEA); (2) there was an associated increase affinity for the delta receptor of rat brain or neuroblastoma-glioma hybrid cells. (however, the relative potencies were higher in the MVD assay then predicted on the basis of binding affinities); (3) the DTE series also showed an increase in delta receptor affinities and MVD potencies relative to DALEA, for n = 2-12; (4) for the DTE series, the increase in MVD activities was less than that expected on the basis of delta binding affinity; (5) for both the DPE and DTE series, activities in the GPI assay and mu-receptor affinities were highly correlated: as the length of the methylene bridge increased from 2 to 12, there was a progressive loss of activity in both assays, with a similar pattern for DPE and DTE. Two selected dimers and their corresponding monomers were also assayed for antinociceptive activity in vivo: results were consistent with GPI and mu-binding but not with MVD and delta-binding. Two alkylamide analogs of penta- and tetrapeptide monomers, representing the monomer with the attached spacer of the most active dimers, were also assayed in biological and binding assays. Comparison of these compounds with the corresponding dimers suggest that the changes in activities and selectivities induced by dimerization are not a spurious effect of the presence of an akylamide derivative of the carboxy terminal of enkephalin but rather may represent a specific effect due to the bivalent nature of the ligands.

The fluorescent enkephalins in which an essential Tyr1 residue is replaced by L-1-pyrenylalanine (Pya) were synthesized and examined in the receptor binding assays. [Pya1, Leu5]Enkephalin and its methyl ester showed binding characteristics specific for the opiate receptors, exhibiting a potent inhibition of Tyr1-containing enkephalins. Surprisingly, the methyl ester displayed almost the same potencies to those of DAGO-enkephalin. This analog bound 24-fold more strongly to mu than to delta-receptors. C-terminal free analog Pya1-Enk-OH was delta-preferential with a fairly good affinity. These results indicate that Tyr1 in enkephalin is not necessary to recognition of the opiate receptors.

The fluorescent amino acid, L-1-pyrenylalanine (Pya) was incorporated into [D-Ala2,Leu5]enkephalin and its methyl ester at position 4 or 5. Pya-enkephalins showed strong fluorescent intensity and displayed high binding affinity for opiate receptors. Pya4-enkephalins showed high specificity for the mu receptors, while Pya5-enkephalins showed high specificity and selectivity for the delta receptors. Particularly, [D-Ala2,Pya5]enkephalin was as potent as the most utilized delta-specific ligand of [D-Ala2,D-Leu5]enkephalin (DADLE), and yet its delta-selectivity was about 5-times greater than that of DADLE. Thus, Pya-enkephalins per se can be utilized as a fluorescent probe or tracer for the opiate receptor-binding assays.

Conformationally restricted enkephalin analogs containing E-cyclopropylphenylalanine (delta EPhe), [D-Ala2, (2R,3S)-delta EPhe4,Leu5]enkephalin and its (2S,3R) isomer, were evaluated in receptor-binding assays using rat brain and in assays using muscle preparations. The (2S,3R) isomer was almost completely inactive in all assays. In contrast, the (2R,3S) isomer showed a very high affinity for the delta and a very weak affinity for the mu receptors in rat brain. The extent of delta affinity and the selectivity of this isomer were almost equal to those of [D-Pen2,D-Pen5]enkephalin. However, the (2R,3S) isomer was inactive in both the mouse vas deferens and guinea pig ileum assays, and showed no antagonistic activity in these tissues. These results indicate that the (2R,3S) isomer interacts with the delta receptors in rat brain, but not with those in the mouse vas deferens, and they suggest that the delta receptors in the central and peripheral nervous systems are different from each other.

[D-Ala(2)(2R,3S)-delta(E)Phe(4)Leu(5)]enkephalin (CP-OH) [delta denoting cyclopropyl; superscript E indicating the E-configuration about the cyclopropane ring], a highly selective opioid ligand for delta receptors in rat brain, but not for those in the mouse vas deferens, was examined for in vivo biological activities by intracerebroventricular administration. CP-OH (5-20 micrograms) showed no analgesic activity in the hot plate (51 degrees C) test using rats. However, it suppressed completely the analgesic effects of intraperitoneally administered morphine (3 mg/kg rat) in a dose-dependent manner. CP-OH showed no binding affinity for brain kappa receptors to which dynorphin, an opioid peptide that inhibits morphine analgesia, binds predominantly. These results suggest that, besides the conventional delta receptors which mediate analgesia, the rat brain contains another delta-like receptor which has a modulatory role to attenuate morphine-induced analgesia mediated through the mu receptors, and that this modulatory receptor does not exist in the mouse vas deferens.

The N-terminal tripeptide enkephalin analogue, Tyr-D-Ala-Gly, was dimerized at the C-terminus systematically with a series of alpha,omega-diaminoalkanes, NH2-(CH2)n-NH2 (n = 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22). The binding affinities of dimers for delta opiate receptors in rat brain were evaluated and compared with those for delta receptors in NG108-15 cells. Although the monomeric tripeptide amide was almost inactive, dimers showed a dramatic increase in binding affinity (8-900 times). The enhancement of affinity was apparently related to the number of methylene chains in the crosslinking spacer moiety, and it was maximal at n = 14-18 in the rat brain. In NG cells the activity increased progressively from n = 2 to n = 22 without reaching any apparent peak. These results suggest that delta receptors in rat brain and NG cells may have slight structural differences.

Dimeric analogues of the inactive enkephalin fragment Tyr-D-Ala-Gly were synthesized by cross-linking with alkanediamine at the C-terminus. Biological evaluation of these dimers (H-Tyr-D-Ala-Gly-NH)2.(-CH2-)n (DTREn), where n = 0-6, revealed that the fragment inactive for mu receptors was activated by its dimerization, with the maximum activation found with DTRE2, and that the dimer was highly mu-selective. So-called "handicapped" dimers, which lack one of the essential groupings required for enkephalin activity, were found to be far less active, indicating that the dimer interacts bivalently with mu receptors. It seems, therefore, that mu opiate receptors contain at least two equivalent binding sites which are extremely close to each other.

DAGO-enkephalin ([ D-Ala2, MePhe4, Gly-ol5]enkephalin), a highly selective ligand for mu opiate receptors, was dimerized with a series of alpha,omega-alkanedioic acids (n = 2-12) at the OH-terminus. In the radioligand receptor binding assays with rat brain, most of the DAGO-enkephalin dimers with cross-linking methylene chain n (DEDn) were more potent than DAGO monomer. For delta receptors, affinity of DEDn was maximized with n = 8, which might be related to an optimal distance between two binding sites. For mu receptors, an increase in chain length resulted in a progressive loss of potency. Although all of DEDn are considerably mu-selective, with a mu/delta ratio of 15-50, DEDn exhibited fairly flat binding curves with 15-50% smaller sloped than that of DAGO, suggesting that the dimers interact more strongly with one of the possible two mu binding sites.