Structure-activity relationships of arodyn, a novel acetylated kappa opioid receptor antagonist.

We previously reported that the novel dynorphin A (Dyn A, Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln) analog arodyn (Ac[Phe(1,2,3),Arg(4),d-Ala(8)]Dyn A-(1-11)NH(2), Bennett, M.A., Murray, T.F. & Aldrich, J.V. (2002) J. Med. Chem. vol. 45, pp. 5617-5619) is a kappa opioid receptor-selective peptide [K(i)(kappa) = 10 nm, K(i) ratio (kappa/mu/delta) = 1/174/583] which exhibits antagonist activity at kappa opioid receptors. In this study, a series of arodyn analogs was prepared and evaluated to explore the structure-activity relationships (SAR) of this peptide; this included an alanine scan of the entire arodyn sequence, sequential isomeric d-amino acid substitution in the N-terminal 'message' sequence, NMePhe substitution individually in positions 1-3, and modifications in position 1. The results for the Ala-substituted derivatives indicated that Arg(6) and Arg(7) are the most important residues for arodyn's nanomolar binding affinity for kappa opioid receptors. Ala substitution of the other basic residues (Arg(4), Arg(9) and Lys(11)) resulted in lower decreases in affinity for kappa opioid receptors (three- to fivefold compared with arodyn). Of particular interest, while [Ala(10)]arodyn exhibits similar kappa opioid receptor binding as arodyn, it displays higher kappa vs. mu opioid receptor selectivity [K(i) ratio (kappa/mu) = 1/350] than arodyn because of a twofold loss in affinity at mu opioid receptors. Surprisingly, the Tyr(1) analog exhibits a sevenfold decrease in kappa opioid receptor affinity, indicating that arodyn displays significantly different SAR than Dyn A; [Tyr(1)]arodyn also unexpectedly exhibits inverse agonist activity in the adenylyl cyclase assay using Chinese hamster ovary cells stably expressing kappa opioid receptors. Substitution of NMePhe in position 1 gave [NMePhe(1)]arodyn which exhibits high affinity [K(i)(kappa) = 4.56 nm] and exceptional selectivity for kappa opioid receptors [K(i) ratio (kappa/mu/delta) = 1/1100/>2170]. This peptide exhibits antagonistic activity in the adenylyl cyclase assay, reversing the agonism of 10 nm Dyn A-(1-13)NH(2). Thus [NMePhe(1)]arodyn is a highly kappa opioid receptor-selective antagonist that could be a useful pharmacological tool to study kappa opioid receptor-mediated activities.     

Dynorphin A analogs containing a conformationally constrained phenylalanine derivative in position 4: reversal of preferred stereochemistry for opioid receptor affinity and discrimination of kappa vs. delta receptors

Analogs of the opioid peptide [D-Ala8]dynorphin A-(1-11)NH2 containing optically pure (R)- and (S)-2-aminotetralin-2-carboxylic acid (Atc) in position 4 were synthesized and evaluated for opioid receptor affinity. These peptides are the first reported dynorphin A analogs containing a conformationally constrained amino acid in place of the important aromatic residue Phe4. By incorporating resolved Atc isomers, the opioid receptor affinity and the stereochemistry of the constrained residue could be unambiguously correlated. Both Dyn A analogs containing Atc in position 4 retained nanomolar affinity for kappa and mu opioid receptors. Unexpectedly the peptide containing (R)-Atc, corresponding to a conformationally constrained D-Phe analog, displaying higher affinity for both kappa and mu receptors than the peptide containing (S)-Atc. In contrast [D-Phe4,D-Ala8]Dyn A-(1-11)NH2 exhibited significantly lower affinity for kappa and mu receptors than the parent peptide, as expected. Conformational restriction of the Phe4 sidechain or incorporation of D-Phe in position 4 had the largest effect on delta receptor affinity, yielding compounds with negligible affinity for these receptors. Thus, there appear to be distinctly different structural requirements for this residue for kappa vs. delta receptors, and it is possible to completely distinguish between these two receptors by changing a single residue in Dyn A.     

Development of highly potent and selective dynorphin A analogues as new medicines.

Dynorphin A (Dyn A), a 17 amino acid peptide H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln-OH, is a potent opioid peptide which interacts preferentially with kappa-opioid receptors. Research in the development of selective and potent opioid peptide ligands for the kappa-receptor is important in mediating analgesia. Several cyclic disulphide bridge-containing peptide analogues of Dyn A, which were conformationally constrained in the putative message or address segment of the opioid ligand, were designed, synthesized and assayed. To further investigate the conformational and topographical requirements for the residues in positions 5 and 11 of these analogues, a systematic series of Dyn A(1-11)-NH2 cyclic analogues incorporating the sulphydryl-containing amino acids L- and D-Cys and L- and D-Pen in positions 5 and 11 were synthesized and assayed. Cyclic lactam peptide analogues were also synthesized and assayed. Several of these cyclic analogues, retained the same affinity and selectivity (vs. the mu- and delta-receptors) as the parent Dyn A(1-11)-NH2 peptide in the guinea-pig brain (GPB), but exhibited a much lower activity in the guinea-pig ileum (GPI), thus leading to centrally vs. peripherally selective peptides. Studies of the structure-activity relationship of Dyn A peptide provide new insights into the importance of each amino acid residue (and their configurations) in Dyn A analogues for high potency and good selectivity at kappa-opioid receptors. We report herein the progress towards the development of Dyn A peptide ligands, which can act as agonists or antagonists at cell surface receptors that modulate cell function and animal behaviour using various approaches to rational peptide ligand-based drug design.     

Structure-activity relationships of dynorphin analogs substituted in positions 2 and 3

Following up on the observation that the dynorphin analog [Pro(3)]Dyn A(1-11)-NH(2) 2 possesses high affinity and selectivity for the kappa opioid receptor, a number of related peptides were prepared and characterized by radioligand binding and [(35)S]GTPgammaS assays. While incorporation of 2-azetidine carboxylic acid in position 3 led to the equally potent analog 3, the corresponding analog containing piperidine-2-carboxylic acid showed a nearly 90-fold reduction in kappa affinity. Differential preferred bond angles phi in the three building blocks might account for these observations. Compounds 2 and 3 were kappa antagonists with IC(50) values of 380 and 350 nM, respectively. The Sar(3) analog 7 and the Sar(2) analog 8 were kappa agonists, with greater selectivity than Dyn A(1-11)-NH(2) 1. In view of their high kappa affinities (8: K(i) = 1.5 nM; 2: K(i) = 2.4 nM), the new analogs were surprisingly weak kappa agonists or antagonists, e.g., the EC(50) value for the agonist 8 was 280 nM. Different kappa receptor subtypes in binding vs functional assays can not account for these results, since both assays were performed using the same membrane preparation.     

Opioid receptor selectivity of peptide models of beta-endorphin

Two peptides, designed to contain structural models of the proposed hydrophilic linker domain (residues 6-12) and amphiphilic alpha-helical domain (residues 13-29) in beta-endorphin, have been tested for their abilities to mimic the opioid receptor selectivity profile of the natural hormone. In competitive binding assays employing guinea-pig brain membranes, both peptides displayed a much higher affinity for mu- and delta-opioid receptors than for kappa opioid receptors. Relative to beta-endorphin, the peptide models were 2-3 times more potent in the mu and kappa receptor binding assays, and about equipotent in the delta receptor binding assay. In guinea-pig ileum assays, one peptide was equipotent to beta-endorphin and the other was twice as potent. Like beta-endorphin, their actions on this tissue were highly sensitive to naloxone antagonism, indicating that they were mediated by mu receptors and not kappa receptors. In view of the design of the two peptide models, and their minimal homology to the natural hormone, these results provide additional evidence in support to our proposal for the functional conformation of beta-endorphin.     

Conversion of delta-, kappa- and mu-receptor selective opioid peptide agonists into delta-, kappa- and mu-selective antagonists

2',6'-Dimethyl substitution of the Tyr(1) residue of opioid agonist peptides and deletion of the positively charged N-terminal amino group or its replacement with a methyl group has recently been shown to represent a general structural modification to convert opioid peptide agonists into antagonists. This conversion requires the syntheses of opioid peptide analogues containing either 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in place of Tyr(1). Using this approach, delta-, kappa- and mu-selective opioid peptide agonist peptides were successfully converted into corresponding delta-, kappa- and mu-selective antagonists, whereby receptor selectivity was often maintained or even improved. Thus, two (2S)-Mdp(1)-analogues of the delta-selective cyclic enkephalin analogue H-Tyr-c[D-Pen-Gly-Phe(pF)-Pen]-Phe-OH turned out to be potent and selective delta antagonists. Most successful was the development of kappa antagonists derived from dynorphin A (Dyn A), including the highly potent and selective kappa-antagonist [(2S)-Mdp(1)]Dyn A(1-11)-NH(2) (dynantin) and the enzymatically stable octapeptide analogue [(2S)-Mdp(1),MeArg(7),D-Leu(8)]Dyn A(1-8)-NH(2). The (2S)-Mdp(1)-analogues of dynorphin B and alpha-neoendorphin also were kappa antagonists and may be useful as pharmacological tools in studies of kappa receptor subtypes. Finally, the Dhp(1)-analogues of the mu-selective cyclic enkephalin analogue H-Tyr-c[N(epsilon ),N(beta)-carbonyl-D-Lys(2),Dap(5)]enkephalinamide and of endomorphin-2 were moderately potent mu opioid antagonists.     

Opioid peptides. Synthesis and binding properties of dermorphin related heptapeptides

C-Terminal amino acid residues of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) were replaced by N alpha-methyl- or D-amino acids in order to examine the effect on opioid activity. In binding studies based on displacement of mu, delta, and kappa opioid receptor selective radiolabels from guinea pig brain membranes, the 13 new analogues showed, like dermorphin, a negligible affinity for the kappa binding site. The introduction of N alpha-methyl- or D-amino acid residues at position 5, 6, or 7 of dermorphin, when matched with C-terminal amide function modifications, generally produced analogues with reversed mu/delta specificity.     

ORL1 and opioid receptor preferences of nociceptin and dynorphin A analogues with Dmp substituted for N-terminal aromatic residues

Nociceptin (NOC) and dynorphin A (DYN) analogues containing 2',6'-dimethylphenylalanine (Dmp) in place of Phe or Tyr in position 1 and/or 4 were synthesized and their metabolic stability and receptor-binding properties were investigated. [Dmp1]NOC(1-13)-NH2 (1) possessed high ORL1 receptor affinity comparable to that of the parent peptide with substantially improved affinities for kappa-, mu-, and delta-opioid receptors. However, Dmp4 substitution of NOC peptide (2) reduced ORL1 receptor affinity. [Dmp1]DYN(1-13)-NH2 (4) and its Dmp4 analogue (5) possessed a 3-fold greater kappa-opioid receptor affinity and improved kappa-receptor selectivity compared to the parent peptide. Analogue 4 however exhibited an unexpectedly low in vitro bioactivity (GPI assay), suggesting, the phenolic hydroxyl group at the N-terminal residue in DYN peptide is extremely important for activation of the kappa-opioid receptor. Analogue 5 possessed an improved kappa-opioid receptor selectivity with an IC50 ratio of 1(kappa)/509(mu)/211598(delta); thus, this peptide may serve as a highly selective kappa-receptor agonist for pharmacological study. Dmp1 substitution in both the NOC and DYN peptides improved metabolic stability toward these peptides, while Dmp4 substitution provided no additional metabolic stability.     

Amino acid conjugates as kappa opioid receptor agonists

A novel series of kappa (kappa) opioid receptor agonists were synthesized by incorporating the key structural features of known kappa opioid agonists while replacing the aryl acetamide portion with substituted amino acid conjugates. Compounds 3j (Ki = 6.7 nM), 3k (Ki = 3.6 nM), 3l (Ki = 4.6 nM), 3m (Ki = 0.83 nM) and 3o (Ki = 2 nM) possessed potent affinities for the kappa opioid receptor in vitro with reasonable selectivity over other opioid receptors.     

Big dynorphin as a putative endogenous ligand for the kappa-opioid receptor

The diversity of peptide ligands for a particular receptor may provide a greater dynamic range of functional responses, while maintaining selectivity in receptor activation. Dynorphin A (Dyn A), and dynorphin B (Dyn B) are endogenous opioid peptides that activate the kappa-opioid receptor (KOR). Here, we characterized interactions of big dynorphin (Big Dyn), a 32-amino acid prodynorphin-derived peptide consisting of Dyn A and Dyn B, with human KOR, mu- (hMOR) and delta- (hDOR) opioid receptors and opioid receptor-like receptor 1 (hORL1) expressed in cells transfected with respective cDNA. Big Dyn and Dyn A demonstrated roughly similar affinity for binding to hKOR that was higher than that of Dyn B. Dyn A was more selective for hKOR over hMOR, hDOR and hORL1 than Big Dyn, while Dyn B demonstrated low selectivity. In contrast, Big Dyn activated G proteins through KOR with much greater potency, efficacy and selectivity than other dynorphins. There was no correlation between the rank order of the potency for the KOR-mediated activation of G proteins and the binding affinity of dynorphins for KOR. The rank of the selectivity for the activation of G proteins through hKOR and of the binding to this receptor also differed. Immunoreactive Big Dyn was detected using the combination of radioimmunoassay (RIA) and HPLC in the human nucleus accumbens, caudate nucleus, hippocampus and cerebrospinal fluid (CSF) with the ratio of Big Dyn and Dyn B being approximately 1:3. The presence in the brain implies that Big Dyn, along with other dynorphins, is processed from prodynorphin and secreted from neurons. Collectively, the high potency and efficacy and the relative abundance suggest that Big Dyn may play a role in the KOR-mediated activation of G proteins.     

Synthesis and evaluation of derivatives of leucine enkephalin as potential affinity labels for delta opioid receptors

As part of an effort to develop peptide-based affinity labels for opioid receptors, [Leu(5)]enkephalin (LeuEnk) and DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr), potent agonists for delta receptors, were selected as the parent peptides for further modification. The affinity label derivatives were prepared using standard Fmoc solid-phase peptide synthesis in conjunction with Fmoc-Phe(p-NHAlloc) (Fmoc: 9-flourenylmethoxycarbonyl;) and selective modification of the p-amino group on this residue. The electrophilic isothiocyanate and bromoacetamide groups were introduced into the para position of Phe(4); the corresponding free amine-containing peptides were also prepared for comparison. The pure peptides were evaluated in radioligand binding assays using Chinese hamster ovary (CHO) cells expressing delta and micro opioid receptors. Modification of Phe(4) in LeuEnk and DTLET significantly decreased delta-receptor binding affinity (40 to >2,000-fold). Among the synthesized analogues, [Phe(p-NH(2))(4)]DTLET showed the highest delta-receptor binding affinity (IC(50) = 39 nM) and enhanced selectivity for delta receptors compared to DTLET while other derivatives exhibited much lower delta receptor affinity. The differences in affinities between the two series of analogues and between the derivatives of LeuEnk and N,N-dibenzyl[Leu(5)]Enk reported previously suggest subtle differences in interactions of Phe(4) with delta receptors depending on other modifications in the sequences.     

Modifications of the cyclic mu receptor selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]NH2 (Et): effects on opioid receptor binding and activation.

The previously described cyclic mu opioid receptor-selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]NH2 (Et) (JOM-6) was modified at residues 1 and 3 by substitution with various natural and synthetic amino acids, and/or by alteration of the cyclic system. Effects on mu and delta opioid receptor binding affinities, and on potencies and efficacies as measured by the [35S]-GTPgammaS assay, were evaluated. Affinities at mu and delta receptors were not influenced dramatically by substitution of Tyr1 with conformationally restricted phenolic amino acids. In the [35S]-GTPgammaS assay, all of the peptides tested exhibited a maximal response comparable with that of fentanyl at the mu opioid receptor, and all showed high potency, in the range 0.4-9nM. However, potency changes did not always correlate with affinity, suggesting that the conformation required for binding and the conformation required for activation of the opioid receptors are different. At the delta opioid receptor, none of the peptides were able to produce a response equivalent to that of the full delta agonist BW 373,U86 and only one had an EC50 value of less than 100nM. Lastly, we have identified a peptide, D-Hat-c[D-Cys-Phe-D-Pen]NH2 (Et), with high potency and > 1,000-fold functional selectivity for the mu over delta opioid receptor as measured by the [35S]-GTPgammaS assay.     

Ligands for kappa-opioid and ORL1 receptors identified from a conformationally constrained peptide combinatorial library.

We have screened a synthetic peptide combinatorial library composed of 2 x 10(7) beta-turn-constrained peptides in binding assays on four structurally related receptors, the human opioid receptors mu, delta, and kappa and the opioid receptor-like ORL1. Sixty-six individual peptides were synthesized from the primary screening and tested in the four receptor binding assays. Three peptides composed essentially of unnatural amino acids were found to show high affinity for human kappa-opioid receptor. Investigation of their activity in agonist-promoted stimulation of [(35)S]guanosine 5'-3-O-(thio)triphosphate binding assay revealed that we have identified the first inverse agonist as well as peptidic antagonists for kappa-receptors. To fine-tune the potency and selectivity of these kappa-peptides we replaced their turn-forming template by other turn mimetic molecules. This "turn-scan" process allowed the discovery of compounds with modified selectivity and activity profiles. One peptide displayed comparable affinity and partial agonist activity toward all four receptors. Interestingly, another peptide showed selectivity for the ORL1 receptor and displayed antagonist activity at ORL1 and agonist activity at opioid receptors. In conclusion, we have identified peptides that represent an entirely new class of ligands for opioid and ORL1 receptors and exhibit novel pharmacological activity. This study demonstrates that conformationally constrained peptide combinatorial libraries are a rich source of ligands that are more suitable for the design of nonpeptidal drugs.     

Chemical space screening around Phe3 in opioid peptides: Modulating µ versus δ agonism by Suzuki-Miyaura cross-couplings

In this study, affinities and activities of derivatized analogues of Dmt-dermorphin[1–4] (i.e. Dmt-d-Ala-Phe-GlyNH₂, Dmt?=?2′,6′-dimethyl-(S)-tyrosine) for the µ opioid receptor (MOP) and δ opioid receptor (DOP) were evaluated using radioligand binding studies, functional cell-based assays and isolated organ bath experiments. By means of solid-phase or solution-phase Suzuki-Miyaura cross-couplings, various substituted regioisomers of the phenylalanine moiety in position 3 of the sequence were prepared. An 18-membered library of opioid tetrapeptides was generated via screening of the chemical space around the Phe³ side chain. These substitutions modulated bioactivity, receptor subtype selectivity and highly effective ligands with subnanomolar binding affinities, contributed to higher functional activities and potent analgesic actions. In search of selective peptidic ligands, we show here that the Suzuki-Miyaura reaction is a versatile and robust tool which could also be deployed elsewhere.     

Beta-endorphin. Biological activity of analogs containing dermorphin and dynorphin sequences: ileum and vas deferens assays

Biological activity of synthetic beta-endorphin (beta-EP) analogs containing dermorphin or dynorphin-A-(1-13) structure has been investigated using the guinea pig ileum and the vas deferens of the mouse, rat and rabbit. Replacement of NH2-terminal 1-7 segment of camel beta-EP [beta c-EP-(1-7)] with dermorphin caused a great increase in opiate potency of the analog. [Dermorphin (1-7)]-beta c-EP was 120 times more potent than beta c-EP in the guinea pig ileum assay, 49 times more potent in the mouse vas deferens assay; and only 4 times more potent in the rat vas deferens assay. Replacement of NH2-terminal 1-13 segment of human beta-EP [beta h-EP-(1-13)] with dynorphin-A-(1-13) caused an increase in opiate potency in both the guinea pig ileum and rabbit vas deferens assays, a complete loss of potency in the rat vas deferens assay, and no change in the mouse vas deferens assay. In comparison with dynorphin-A-(1-13), the hybrid peptide was less potent in the guinea pig ileum assay as well as in mouse and rabbit vas deferens assay. It is suggested that beta c-EP-(8-31) facilitates the dermorphin moiety to act on opiate mu and delta receptors but not on the epsilon receptor, while beta h-(14-31) reduces the action of dynorphin on mu, delta and kappa receptors.     

Synthesis of novel basic head-to-side-chain cyclic dynorphin A analogs: strategies and side reactions

Novel N-terminus-to-side-chain cyclic analogs of the opioid peptide dynorphin (Dyn) A-(1-11)NH(2) were prepared that retain the basicity of the N-terminal amine and restrict the backbone conformation around the important Tyr(1) residue. Cyclic peptides were synthesized in which the N-terminal amine and the N(epsilon)-amine of a Lys at position 3 or 5 were attached to the alpha-carbon and carbonyl of an acetyl group, respectively. Several synthetic strategies were explored with detailed analysis of the side reactions in order to obtain the desired cyclic peptides. One of the side reactions observed involved premature loss of the N-terminal 9-fluorenylmethoxycarbonyl (Fmoc) group during the neutralization step following deprotection of the Mtt (4-methyltrityl) protecting group from the side chain of Lys. The successful strategy involved the synthesis of the linear peptide up through Gly(2) and functionalization through the N(epsilon)-amine of Lys. A linear N-terminal alkylated analog was prepared by alkylation of the peptide on the resin with an equimolar amount of bromoacetamide, followed by treatment of the peptide with Fmoc-OSu prior to cleavage from the resin to facilitate separation by reversed phase high performance liquid chromatography of unreacted peptide from the desired alkylated product. The novel N-terminal cyclic Dyn A analogs and the linear analog were evaluated for their opioid receptor affinities. These peptides exhibited large losses in affinity for opioid receptors; the low affinity of the linear N-terminal alkylated peptide suggested that the alpha-acetamide group on the N-terminal amine resulted in unfavorable interactions with opioid receptors.     

Synthesis and biological activities of YkFA analogues: effects of position 4 substitutions and altered ring size on in vitro opioid activity.

Substitution in position 4 of the potent opioid peptide YkFA with aliphatic hydrophobic residues resulted in compounds that retained low nanomolar activities at both mu and delta opioid receptors, while ring contraction by incorporation of diaminobutyric acid in position 2 resulted in a more pronounced decrease in potency at both receptors for the psi[CH(2)NH] pseudopeptide as compared to the all amide parent.     

Dermorphin tetrapeptide analogues with 2',6'-dimethylphenylalanine (Dmp) substituted for aromatic amino acids have high mu opioid receptor binding and biological activities

To investigate the value of the 2',6'-dimethylphenylalanine (Dmp) residue as an aromatic amino acid substitution, we prepared analogues of the mu opioid receptor-selective dermorphin tetrapeptide Tyr-D-Arg-Phe-betaAla-NH(2) (YRFB) in which Dmp or its D-isomer replaced Tyr(1) or Phe(3). Replacing Phe(3) with Dmp essentially tripled mu receptor affinity and the receptor's in vitro biological activities as determined with the guinea pig ileum (GPI) assay but did not change delta receptor affinity. Despite an inversion of the D configuration at this position, mu receptor affinity and selectivity remained comparable with those of the L-isomer. Replacing the N-terminal Tyr residue with Dmp produced a slightly improved mu receptor affinity and a potent GPI activity, even though the substituted compound lacks the side chain phenolic hydroxyl group at the N-terminal residue. Dual substitution of Dmp for Tyr(1) and Phe(3) produced significantly improved mu receptor affinity and selectivity compared with the singly substituted analogues. Subcutaneous injection of the two analogues, [Dmp(3)]YRFB and [Dmp(1)]YRFB, in mice produced potent analgesic activities that were greater than morphine in the formalin test. These lines of evidence suggest that the Dmp residue would be an effective aromatic amino acid surrogate for both Tyr and Phe in the design and development of novel opioid mimetics.     

In vivo rat brain opioid receptor binding of LY255582 assessed with a novel method using LC/MS/MS and the administration of three tracers simultaneously

LY255582 is a pan opioid selective receptor antagonist that has been shown to have high affinity for mu, delta, and kappa receptors in vitro. In order to better understand the in vivo opioid receptor selectivity of LY255582, we developed in vivo receptor occupancy assays in the rat for the opioid mu, kappa and delta receptors using the occupancy tracers naltrexone, GR103545 and naltriben respectively. Individual assays for each target were established and then a "triple tracer" assay was created where all three tracers were injected simultaneously, taking advantage of LC/MS/MS technology to selectively monitor brain tracer levels. This is the first report of a technique to concurrently measure receptor specific occupancy at three opioid receptors in the same animal. The opioid subtype selective antagonists cyprodime, JDTic and naltrindole were used to validate selectivity of the assay. Examination of LY255582 in dose-occupancy experiments demonstrated a relative order of potency of mu>kappa>delta, reproducing the previously reported order determined with in vitro binding.     

A novel bioassay for the NK-2 neurokinin receptor: the guinea pig gallbladder

Although three neurokinin receptors (NK-1, NK-2, NK-3) have been identified by radioligand binding assays, only the NK-1 and NK-3 types have been found in smooth muscle bioassays. In this study, evidence is presented demonstrating functional NK-2 type receptors in the guinea pig gallbladder (GPGB). The potencies of the following neurokinins were determined in the GPGB and the guinea pig ileum (GPI): substance P (SP), physalaemin (PH), eledoisin (EL), substance K (SK) and kassinin (KA). ED50 values were determined by linear regression analysis of the dose-related increases in the force generated by each peptide. In the GPI, the rank order of potency was SP = PH = EL greater than SK = KA, indicating NK-1 selectivity. In the GPGB, the relative potencies were SK greater than KA greater than EL much greater than PH greater than SP, which is similar to that reported for the NK-2 receptor in radioligand binding assays. These findings demonstrate the NK-2 receptor tissue selectivity of the GPGB.