Synthesis and Structure-Activity Relationships of (-)-cis-N-Normetazocine-Based LP1 Derivatives.

(−)-cis-N-Normetazocine represents a rigid scaffold able to mimic the tyramine moiety of endogenous opioid peptides, and the introduction of different N-substituents influences affinity and efficacy of respective ligands at MOR (mu opioid receptor), DOR (delta opioid receptor), and KOR (kappa opioid receptor). We have previously identified LP1, a MOR/DOR multitarget opioid ligand, with an N-phenylpropanamido substituent linked to (−)-cis-N-Normetazocine scaffold. Herein, we report the synthesis, competition binding and calcium mobilization assays of new compounds 10⁻16 that differ from LP1 by the nature of the N-substituent. In radioligand binding experiments, the compounds 10⁻13, featured by an electron-withdrawing or electron-donating group in the para position of phenyl ring, displayed improved affinity for KOR (Ki = 0.85⁻4.80 μM) in comparison to LP1 (7.5 μM). On the contrary, their MOR and DOR affinities were worse (Ki = 0.18⁻0.28 μM and Ki = 0.38⁻1.10 μM, respectively) with respect to LP1 values (Ki = 0.049 and 0.033 μM). Analogous trends was recorded for the compounds 14⁻16, featured by indoline, tetrahydroquinoline, and diphenylamine functionalities in the N-substituent. In calcium mobilization assays, the compound 10 with a p-fluorophenyl in the N-substituent shared the functional profile of LP1 (pEC50MOR = 7.01), although it was less active. Moreover, the p-methyl- (11) and p-cyano- (12) substituted compounds resulted in MOR partial agonists and DOR/KOR antagonists. By contrast, the derivatives 13⁻15 resulted as MOR antagonists, and the derivative 16 as a MOR/KOR antagonist (pKBMOR = 6.12 and pKBKOR = 6.11). Collectively, these data corroborated the critical role of the N-substituent in (−)-cis-N-Normetazocine scaffold. Thus, the new synthesized compounds could represent a template to achieve a specific agonist, antagonist, or mixed agonist/antagonist functional profile.

Synthesis and Structure-Activity Relationships of Triazaspirodecanone Derivatives as Nociceptin/Orphanin FQ Receptor Ligands.

Several spiroxatrine derivatives were synthesized and evaluated as potential NOP receptor ligands. Structural modifications of the 1,4-benzodioxane moiety of spiroxatrine have been the focus of this research project. The structure-activity relationships that emerged indicate that the presence of an H-bond donor group (hydroxyl group) is more favorable for NOP activity when it is positioned α with respect to the CH2 linked to the 1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one portion. Moreover, cis diastereoisomers of the hydroxyl derivatives4 and 22 show a moderately higher degree of stereoselectivity than trans isomers. In particular, the spiropiperidine derivative cis-4 has submicromolar agonistic activity, and it will be the reference compound for the design and synthesis of new NOP agonists.

Chimeric G proteins in fluorimetric calcium assays: experience with opioid receptors.

High throughput calcium mobilization assays are extensively used for pharmacological characterization of GPCR ligands. These approaches, initially developed for G(q)-coupled receptors, can be extended to G(i) coupled GPCRs using chimeric G proteins. Here we used the Gα(qi5) protein to force the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor, as well as the classical opioid receptors to signal through the PLC-IP(3)-Ca(2+) pathway in CHO cells. Calcium levels were monitored using the fluorometric imaging plate reader FlexStation II and the Ca(2+) dye Fluo 4 AM. For investigating the pharmacology of the NOP receptor a panel of full and partial agonists and antagonists were assessed, while a small panel of agonists and antagonists was used for evaluating the pharmacological profile of opioid receptors. Some limitations of this assay and differences in the results obtained in comparison with those with G(i) based biochemical assays are described. Overall, the present results confirm that the chimeric G protein strategy is useful for studying the pharmacological activity of G(i) coupled receptor ligands and that the aberrant signaling does not produce any measurable change in the pharmacological profile of the receptor under study. Thus, this G protein strategy is extremely useful for setting up primary screening assays for NOP and classical opioid receptors and likely for other members of the GPCR family.

In vitro and in vivo pharmacological characterization of the novel NK1 receptor selective antagonist Netupitant.

The novel NK(1) receptor ligand Netupitant has been characterized in vitro and in vivo. In calcium mobilization studies CHO cells expressing the human NK receptors responded to a panel of agonists with the expected order of potency. In CHO NK(1) cells Netupitant concentration-dependently antagonized the stimulatory effects of substance P (SP) showing insurmountable antagonism (pK(B) 8.87). In cells expressing NK(2) or NK(3) receptors Netupitant was inactive. In the guinea pig ileum Netupitant concentration-dependently depressed the maximal response to SP (pK(B) 7.85) and, in functional washout experiments, displayed persistent (up to 5h) antagonist effects. In mice the intrathecal injection of SP elicited the typical scratching, biting and licking response that was dose-dependently inhibited by Netupitant given intraperitoneally in the 1-10mg/kg dose range. In gerbils, foot tapping behavior evoked by the intracerebroventricular injection of a NK(1) agonist was dose-dependently counteracted by Netupitant given intraperitoneally (ID(50) 1.5mg/kg) or orally (ID(50) 0.5mg/kg). In time course experiments in gerbils Netupitant displayed long lasting effects. In all the assays Aprepitant elicited similar effects as Netupitant. These results suggest that Netupitant behaves as a brain penetrant, orally active, potent and selective NK(1) antagonist. Thus this molecule can be useful for investigating the NK(1) receptor role in the control of central and peripheral functions. Netupitant has clinical potential in conditions such as chemotherapy induced nausea and vomiting, in which the blockade of NK(1) receptors has been demonstrated valuable for patients.

Synthesis and separation of the enantiomers of the neuropeptide S receptor antagonist (9R/S)-3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide (SHA 68).

This study reports the synthesis, chromatographic separation, and pharmacological evaluation of the two enantiomers of the neuropeptide S receptor (NPSR) antagonist (9R/S)-3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide (SHA 68). The (9R)-3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide (compound 10) and (9S)-3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide (compound 10a) were synthesized and their purity assessed by chiral chromatography. The absolute configuration of the enantiomer 10 has been assigned from the crystal structure of the corresponding (S)-phenyl ethyl amine derivative 8. Calcium mobilization studies performed on cells expressing the recombinant NPSR demonstrated that compound 10 is the active enantiomer while the contribution of 10a to the NPSR antagonist properties of the racemic mixture is negligible.

Pharmacological profile and antiparkinsonian properties of the novel nociceptin/orphanin FQ receptor antagonist 1-[1-cyclooctylmethyl-5-(1-hydroxy-1-methyl-ethyl)-1,2,3,6-tetrahydro-pyridin-4-yl]-3-ethyl-1,3-dihydro-benzoimidazol-2-one (GF-4).

In this study we provided a pharmacological characterization of the recently synthesized nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) antagonist 1-[1-Cyclooctylmethyl-5-(1-hydroxy-1-methyl-ethyl)-1,2,3,6-tetrahydro-pyridin-4-yl]-3-ethyl-1,3-dihydro-benzoimidazol-2-one (GF-4) and investigated its antiparkinsonian properties. GF-4 inhibited N/OFQ binding to CHO(hNOP) cell membranes (pK(i) 7.46), and antagonized N/OFQ effects in a calcium mobilization assay and electrically stimulated isolated tissues (pK(B) 7.27-7.82), showing a approximately 5-fold selectivity over classical opioid receptors. In vivo, GF-4 dually modulated stepping activity in wild-type mice, causing facilitation in the 0.01-10mg/kg dose range and inhibition at 30mg/kg. These effects were mediated by NOP receptors since GF-4 was ineffective in NOP receptor knock-out mice. Antiparkinsonian properties of GF-4 were investigated in 6-hydroxydopamine hemilesioned rats. GF-4 ameliorated akinesia, bradykinesia and overall gait ability in the 0.1-10mg/kg dose range, but inhibited motor activity at 30mg/kg. To investigate the circuitry underlying motor facilitating and inhibitory effects of GF-4, microdialysis coupled to behavioral testing (akinesia test) was performed. An anti-akinetic dose of GF-4 (1mg/kg) reduced glutamate (GLU) and enhanced GABA release in SNr, while the pro-akinetic dose of GF-4 (30mg/kg) evoked opposite effects. Moreover, the anti-akinetic dose of GF-4 reduced GABA and increased GLU release in ventro-medial thalamus, the pro-akinetic dose decreasing GABA without affecting GLU release in this area. We conclude that GF-4 is an effective NOP receptor antagonist able to attenuate parkinsonian-like symptoms in vivo via inhibition of the nigro-thalamic pathway.

Further studies on the pharmacological profile of the neuropeptide S receptor antagonist SHA 68.

Neuropeptide S (NPS) regulates various biological functions by selectively activating the NPS receptor (NPSR). Previous studies demonstrated that the non-peptide molecule SHA 68 acts as a selective NPSR antagonist. In the present study the pharmacological profile of SHA 68 has been further investigated in vitro and in vivo. In cells expressing the mouse NPSR SHA 68 was inactive per se up to 10microM while it antagonized NPS-stimulated calcium mobilization in a competitive manner showing a pA(2) value of 8.06. In the 10-50mg/kg range of doses, SHA 68 counteracted the stimulant effects elicited by NPS, but not those of caffeine, in mouse locomotor activity experiments. In the mouse righting reflex assay SHA 68 fully prevented the arousal-promoting action of the peptide. The anxiolytic-like effects of NPS were slightly reduced by SHA 68 in the mouse open field, fully prevented in the rat elevated plus maze and partially antagonized in the rat defensive burying paradigm. Finally, SHA 68 was found poorly active in antagonizing the NPS inhibitory effect on palatable food intake in rats. In all assays SHA 68 did not produce any effect per se. In conclusion, the present study demonstrated that SHA 68 behaves as a selective NPSR antagonist that can be used to characterize the in vivo actions of NPS. However the usefulness of this research tool is limited by its poor pharmacokinetic properties.

Comparative biochemical and pharmacological characterization of a novel, NOP receptor selective hexapeptide, Ac-RYYRIR-ol.

Nociceptin/orphanin FQ (N/OFQ) is an endogenous neuropeptide, which is widely distributed in central and peripheral nervous system. Some N/OFQ sequence unrelated hexapeptides can effectively bind to the N/OFQ peptide (NOP) receptor and they were used as template for structure-activity studies that lead to discovery of the new NOP selective ligands. In the present study, the pharmacological profile of the novel hexapeptide Ac-RYYRIR-ol was investigated using various in vitro assays including receptor binding and G-protein activation in rat brain membranes, mouse and rat vas deferens, guinea pig ileum, mouse colon and Ca(2+) mobilization assay in chinese hamster ovary (CHO) cells co-expressing the human recombinant NOP receptor and the C-terminally modified Galpha(qi5) protein. In rat brain membranes Ac-RYYRIR-ol displaced both [(3)H]nociceptin/OFQ and [(3)H]Ac-RYYRIK-ol with high affinity (pK(i) 9.35 and 8.81, respectively) and stimulated [(35)S]GTPgammaS binding showing however lower maximal effects than N/OFQ (alpha=0.28). The stimulatory effect of Ac-RYYRIR-ol was antagonized by the selective NOP receptor antagonist UFP-101. In the electrically stimulated mouse vas deferens Ac-RYYRIR-ol displayed negligible agonist activity while antagonizing in a competitive manner (pA(2) 7.99) the inhibitory effects of N/OFQ. Similar results were obtained in the rat vas deferens. In the mouse colon Ac-RYYRIR-ol produced concentration dependent contractile effects with similar potency and maximal effects as N/OFQ. Finally, in the Ca(2+) mobilization assay performed with CHO-hNOP-Galpha(qi5) cells Ac-RYYRIR-ol displayed lower potency and maximal effects (alpha=0.87) compared with N/OFQ. In conclusion, the novel NOP receptor selective hexapeptide Ac-RYYRIR-ol has been shown to have fine selectivity, high potency, furthermore agonist and antagonist effects toward the NOP receptors were measured in various assays; this is likely due to its partial agonist pharmacological activity.

Structure-activity studies on the nociceptin/orphanin FQ receptor antagonist 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4'-piperidin-1-yl]propyl} pyrrolidine-2-carboxamide.

Twelve derivatives of the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) antagonist 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4'-piperidin-1-yl]propyl} pyrrolidine-2-carboxamide (Comp 24) were synthesized and tested in binding experiments performed on CHO(hNOP) cell membranes. Among them, a novel interesting NOP receptor antagonist (compound 35) was identified by blending chemical moieties taken from different NOP receptor ligands. In vitro in various assays, Compound 35 consistently behaved as a pure, highly potent (pA(2) in the range 8.0-9.9), competitive and NOP selective antagonist. However compound 35 was found inactive when challenged against N/OFQ in vivo in the mouse tail withdrawal assay. Thus, the usefulness of the novel NOP ligand compound 35 is limited to in vitro investigations.

Pharmacological characterization of the nociceptin/orphanin FQ receptor non peptide antagonist Compound 24.

Compound 24, 1-benzyl-N-[3-[spiroisobenzofuran-1(3H),4'-piperidin-1-yl]propyl] pyrrolidine-2-carboxamide was recently identified as a nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) ligand. In this study, the in vitro and in vivo pharmacological profiles of Compound 24 were investigated. In vitro studies were performed measuring receptor and [(35)S]GTPgammaS binding and calcium mobilization in cells expressing the recombinant NOP receptor as well as using N/OFQ sensitive tissues. In vivo studies were conducted using the tail withdrawal assay in mice. Compound 24 produced a concentration-dependent displacement of [(3)H]N/OFQ binding to CHO(hNOP) cell membranes showing high affinity (pK(i) 9.62) and selectivity (1000 fold) over classical opioid receptors. Compound 24 antagonized with high potency the following in vitro effects of N/OFQ: stimulation of [(35)S]GTPgammaS binding in CHO(hNOP) cell membranes (pA(2) 9.98), calcium mobilization in CHO(hNOP) cells expressing the Galpha(qi5) chimeric protein (pK(B) 8.73), inhibition of electrically evoked twitches in the mouse (pA(2) 8.44) and rat (pK(B) 8.28) vas deferens, and in the guinea pig ileum (pK(B) 9.12). In electrically stimulated tissues, Compound 24 up to 1 microM did not modify the effects of classical opioid receptor agonists. Finally in vivo, in the mouse tail withdrawal assay, Compound 24 at 10 mg/kg antagonized the pronociceptive and antinociceptive effects of 1 nmol N/OFQ given supraspinally and spinally, respectively. Under the same experimental conditions Compound 24 did not affect the antinociceptive action of 3 nmol endomorphin-1 injected intrathecally. The present study demonstrated that Compound 24 is a pure, competitive, and highly potent non-peptide NOP receptor selective antagonist.

Structure-activity relationship study on Tyr9 of urotensin-II(4-11): identification of a partial agonist of the UT receptor.

Urotensin-II (U-II) activates the U-II receptor (UT) to modulate a range of biological responses at both central and peripheral sites. Previous studies have demonstrated that the sequence Trp(7)-Lys(8)-Tyr(9) of the cyclic portion of the peptide is crucial for biological activity. Here, we describe a focused structure-activity study of Tyr(9) which has been replaced with a series of non-coded amino acids in the U-II(4-11) template. Thirteen analogs were synthesized and pharmacologically tested for intracellular calcium mobilization in HEK293 cells stably expressing the rat UT receptor. The results of this study demonstrated the following Tyr(9) structure-activity features: (i) the position of the OH group of the side chain is not important for biological activity, (ii) the distance of the phenol moiety from the peptide backbone and its conformational freedom are crucial for UT receptor recognition, (iii) this position is important not only for receptor occupation but also for its activation since the 3,5-diiodoTyr(9) chemical modification generated a potent partial agonist. This pharmacological activity of [3,5-diiodoTyr(9)]U-II(4-11) was confirmed in bioassay experiments performed using the rat thoracic aorta as a U-II sensitive preparation.

Further studies at neuropeptide s position 5: discovery of novel neuropeptide S receptor antagonists.

Neuropeptide S (NPS) regulates various biological functions by activating the NPS receptor (NPSR). Previous studies demonstrated that the substitution of Gly(5) with d-amino acids generates NPSR antagonists. Eleven [d-Xaa(5)]NPS derivatives were synthesized and pharmacologically tested measuring [Ca(2+)](i) in HEK293(mNPSR) cells. The results confirmed that the [d-Xaa(5)] substitution promotes antagonist activity with potency inversely related to the side chain size and allowed identification of the novel potent NPSR peptide antagonist [(t)Bu-d-Gly(5)]NPS.

Pharmacological profile of NOP receptors coupled with calcium signaling via the chimeric protein G alpha qi5.

In this study, the Galpha(qi5) protein was used to force the human nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor to signal through the Ca(2+) pathway in CHO cells. [Ca(2+)](i) levels were monitored using the fluorometer FlexStation II and the Ca(2+) dye Fluo 4 AM. Concentration response curves were generated with a panel of full and partial agonists, while NOP antagonists were assessed in inhibition-response curves. The following rank order of potency of antagonists was measured: SB - 612111 > J - 113397 = Trap - 101 > or = UFP - 101 > [Nphe1]N/OF Q(1 - 13)NH2 >> naloxone, which is superimposable to literature findings. The rank order of potency of full and partial agonists is also similar to that obtained in previous studies with the exception of a panel of ligands (UFP-112, Ro 64-6198, ZP120, UFP-113) whose potency was relatively low in the Galpha(qi5)-NOP receptor calcium assay. Interestingly, these NOP ligands are characterized by slow kinetic of interaction with the NOP receptor, as demonstrated by bioassay experiments. These results demonstrated that the FlexStation II-Galpha(qi5)-NOP receptor calcium assay represents an adequate and useful screening for NOP receptor ligands, particularly for antagonists.

In vitro and in vivo pharmacological characterization of the neuropeptide s receptor antagonist [D-Cys(tBu)5]neuropeptide S.

Neuropeptide S (NPS) was identified as the endogenous ligand of an orphan receptor now referred to as the NPS receptor (NPSR). In the frame of a structure-activity study performed on NPS Gly5, the NPSR ligand [D-Cys(tBu)(5)]NPS was identified. [D-Cys(tBu)(5)]NPS up to 100 microM did not stimulate calcium mobilization in human embryonic kidney (HEK) 293 cells stably expressing the mouse NPSR; however, in a concentration-dependent manner, the peptide inhibited the stimulatory effects elicited by 10 and 100 nM NPS (pK(B), 6.62). In Schild analysis experiments [D-Cys(tBu)(5)]NPS (0.1-100 microM) produced a concentration-dependent and parallel rightward shift of the concentration-response curve to NPS, showing a pA(2) value of 6.44. Ten micromolar [D-Cys(tBu)(5)]NPS did not affect signaling at seven NPSR unrelated G-protein-coupled receptors. In the mouse righting reflex (RR) recovery test, NPS given at 0.1 nmol i.c.v. reduced the percentage of animals losing the RR in response to 15 mg/kg diazepam and their sleeping time. [d-Cys(tBu)(5)]NPS (1-10 nmol) was inactive per se but dose-dependently antagonized the arousal-promoting action of NPS. Finally, NPSR-deficient mice were similarly sensitive to the hypnotic effects of diazepam as their wild-type littermates. However, the arousal-promoting action of 1 nmol NPS could be detected in wild-type but not in mutant mice. In conclusion, [D-Cys(tBu)(5)]NPS behaves both in vitro and in vivo as a pure and selective NPSR antagonist but with moderate potency. Moreover, using this tool together with receptor knockout mice studies, we demonstrated that the arousal-promoting action of NPS is because of the selective activation of the NPSR protein.

Synthesis and biological activity of human neuropeptide S analogues modified in position 5: identification of potent and pure neuropeptide S receptor antagonists.

Neuropeptide S (NPS), the endogenous ligand of a previously orphan receptor now named NPSR, regulates various biological functions in the brain, including arousal, locomotion, anxiety, and food intake. Here we report on a focused structure-activity study of Gly5, which has been replaced with L and D amino acids. Fifteen NPS related peptides were synthesized and pharmacologically tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPSR. The results of this study demonstrated that peptide potency is inversely related to the side chain size, while peptide efficacy strongly depends on the relative L and D configuration, with the L amino acids favoring agonist while D amino acids display antagonist pharmacological activity. [D-Val5]NPS behaved as NPSR pure antagonist in HEK293(mNPSR) cells showing the highest potency (pK(B) 7.56) among this series of peptides. The antagonist action of [D-Val5]NPS was confirmed in vivo in mice, where the peptide at a dose of 10 nmol completely blocked the stimulatory effect of 0.1 nmol NPS on locomotor activity.

Structure-activity study at positions 3 and 4 of human neuropeptide S.

Neuropeptide S (NPS) has been identified as the endogenous ligand of a previously orphan receptor now named NPSR. Previous studies demonstrated that the N-terminal sequence Phe(2)-Arg(3)-Asn(4) of the peptide is crucial for biological activity. Here, we report on a focused structure-activity study of Arg(3) and Asn(4) that have been replaced with a series of coded and non-coded amino acids. Thirty-eight human NPS analogues were synthesized and pharmacologically tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPSR. The results of this study demonstrated the following NPS position 3 structure-activity features: (i) the guanidine moiety and its basic character are not crucial requirements, (ii) an aliphatic amino acid with a linear three carbon atom long side chain is sufficient to bind and fully activate NPSR, (iii) the receptor pocket allocating the position 3 side chain can accommodate slightly larger side chains at least to a certain degree [hArg, Arg(NO2) or Arg(Me)2 but not Arg(Tos)]. Position 4 seems to be more sensitive to amino acids replacement compared to position 3; in fact, all the amino acid replacements investigated produced either an important decrease of biological activity or generated inactive derivatives suggesting a pivotal role of the Asn(4) side chain for NPS bioactivity.

Synthesis and biological activity of human neuropeptide S analogues modified in position 2.

Neuropeptide S (NPS) has been identified as the endogenous ligand of a previously orphan receptor now named NPSR. Previous studies demonstrated that the N-terminal sequence Phe (2)-Arg(3)-Asn(4) of the peptide is crucial for biological activity. Here we report on a focused structure-activity study of Phe(2) which has been replaced with a series of coded and noncoded amino acids. Thirty-one human NPS analogues were synthesized and pharmacologically tested for intracellular calcium mobilization by using HEK293 cells stably expressing the mouse NPSR. The results of this study demonstrated the following NPS position 2 structure-activity features: (i) lipophilicity but not aromaticity is crucial, (ii) both the size of the chemical moiety and its distance from the peptide backbone are important for biological activity, and (iii) this position plays a role in both receptor binding and activation, since [4,4'-biphenyl-Ala(2)]hNPS behaved as a partial agonist.

Structure-activity relationship study of position 4 in the urotensin-II receptor ligand U-II(4-11).

In the present study we describe the synthesis and biological evaluation of 24 analogues of the urotensin II (U-II) fragment U-II(4-11) substituted in position 4 with coded and non-coded aromatic amino acids. All of the new analogues behaved as full U-II receptor (UT) agonists. Our results indicated that aromaticity is well tolerated, size, length and chirality of the side chain are not important, while substituents with a nitrogen atom are preferred. Thus acylation of U-II(5-11) with small groups bearing nitrogen atoms could be instrumental in future studies for the identification of novel potent UT receptor ligands.

Conformation-activity relationship of neuropeptide S and some structural mutants: helicity affects their interaction with the receptor.

Neuropeptide S (NPS) is the endogenous ligand of the previously orphan G-protein coupled receptor now named NPSR. The NPS-NPSR receptor system regulates important biological functions such as sleep/waking, locomotion, anxiety and food intake. Recently, exhaustive Ala scan and d-amino acid scan studies, together with systematic N- and C-terminal truncation, led to the identification of key residues for biological activity. Because conformational preferences might also play an important role, we undertook a detailed conformational analysis of NPS and several analogues in solution. We show that helicity induced by substitution of three flexible residues in the 5-13 regulatory region abolishes biological activity. A parallel pharmacological and conformational study of single and multiple substitutions of glycines 5, 7, and 9 showed that helicity can be tolerated in the C-terminal part of the peptide but not around Gly7. The identification of hNPSR partial agonists heralds the possibility of designing pure NPS receptor antagonists.

Cell and tissue responses of a range of Urotensin II analogs at cloned and native urotensin II receptors. Evidence for coupling promiscuity.

Urotensin II (U-II) is the peptide ligand for the G-protein-coupled U-II receptor (UT). U-II has been dubbed "the most potent vasoconstrictor identified to date". However, in vivo studies with this system are hampered by the paucity of available ligands. Here, we characterise Chinese hamster ovary (CHO) cells expressing the human UT receptor in the following assays; (1) [(125)I]U-II binding, (2) GTPgamma[(35)S] binding, (3) cAMP formation, and (4) intracellular Ca(2+). We assess activity of 9 U-II analogues using these paradigms and examine their ability to contract isolated rat aorta. CHO(hUT) cells bound [(125)I]U-II with a B (max) and K (d) of 1,110+/-70 fmol/mg protein and 742 pM, respectively. hU-II stimulated GTPgamma[(35)S] binding (pEC(50) 8.38), optimal at low (0.1 muM) GDP concentrations. The hU-II GTPgamma[(35)S] response was partially PTx sensitive and there was a potent (pEC(50) 9.23) low efficacy ( approximately 20% inhibition) coupling to adenylyl cyclase. In CHO(hUT) cells hU-II stimulates calcium release from intracellular stores (pEC(50) 8.80) and calcium influx in a PTx-insensitive manner. In our structure-activity relationship study most ligands acted as full agonists. However, urantide behaved as a partial agonist (pEC(50) 7.67/pK(B) 7.55) in GTPgamma[(35)S] binding, a full agonist (pEC(50) 8.11) for increases in intracellular Ca(2+) and a competitive antagonist in the rat aorta bioassay (pK(B) 8.59). Collectively, these data show promiscuity at high expression and indicate the need for careful multi-assay evaluation of novel U-II analogues. Further modification of urantide, in order to eliminate residual agonist activity and to identify novel ligands for in vivo cardiovascular studies are clearly warranted.