A 1,536-well [(35)S]GTPgammaS scintillation proximity binding assay for ultra-high-throughput screening of an orphan galphai-coupled GPCR

Members of the superfamily of seven transmembrane receptors, known as G protein-coupled receptors (GPCRs), are important targets for many therapeutic areas in drug discovery. A homogeneous guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) scintillation proximity assay (SPA) binding assay targeting a Galphai-coupled GPCR recombinantly expressed in membranes of Chinese hamster ovary (CHO) cells was developed and miniaturized into 1,536-well plate format. The primary ultra-high-throughput screen of the entire compound collection was accomplished on the Kalypsys (San Diego, CA) robotic platform at a concentration of 8 muM using the 1,536-well [(35)S]GTPgammaS SPA binding functional assay. The signal-to-noise ratio of the primary screen was approximately 2.1-fold, and the plate coefficient of variation for the compound field was approximately 11%. The hit rate from the primary screen for receptor agonists at >35% activity was approximately 0.3%. Primary hits were cherry-picked, confirmed in triplicate, counterscreened against untransfected CHO cell membranes, and further analyzed in a cyclic AMP functional assay, resulting in 34 leads for optimization.     

A fully automated [35S]GTPgammaS scintillation proximity assay for the high-throughput screening of Gi-linked G protein-coupled receptors

The diversity of physiological functions mediated by the GPCR superfamily provides a rich source of molecular targets for drug discovery programs. Consequently, a variety of assays have been designed to identify lead molecules based on ligand binding or receptor function. In one of these, the binding of [(35)S]GTPgammaS, a nonhydrolyzable analogue of GTP, to receptor-activated G-protein alpha subunits represents a unique functional assay for GPCRs and is well suited for use with automated HTS. Here we compare [(35)S]GTPgammaS scintillation proximity binding assays for two different G(i)-coupled GPCRs, and describe their implementation with automated high-throughput systems.     

Mu-opioid receptor specific antagonist cyprodime: characterization by in vitro radioligand and [35S]GTPgammaS binding assays

The use of compounds with high selectivity for each opioid receptor (mu, delta and kappa) is crucial for understanding the mechanisms of opioid actions. Until recently non-peptide mu-opioid receptor selective antagonists were not available. However, N-cyclopropylmethyl-4,14-dimethoxy-morphinan-6-one (cyprodime) has shown a very high selectivity for mu-opioid receptor in in vivo bioassays. This compound also exhibited a higher affinity for mu-opioid receptor than for delta- and kappa-opioid receptors in binding assays in brain membranes, although the degree of selectivity was lower than in in vitro bioassays. Cyprodime has recently been radiolabelled with tritium resulting in high specific radioactivity (36.1 Ci/mmol). We found in in vitro binding experiments that this radioligand bound with high affinity (K(d) 3. 8+/-0.18 nM) to membranes of rat brain affording a B(max) of 87. 1+/-4.83 fmol/mg. Competition studies using mu, delta and kappa tritiated specific ligands confirmed the selective labelling of cyprodime to a mu-opioid receptor population. The mu-opioid receptor selective agonist [D-Ala(2),N-MePhe(4),Gly(5)-ol]enkephalin (DAMGO) was readily displaced by cyprodime (K(i) values in the low nanomolar range) while the competition for delta- ([D-Pen(2), D-Pen(5)]enkephalin (DPDPE)) and kappa- (5alpha,7alpha, 8beta-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4, 5)dec-8-yl]-benzene-acetamide (U69,593)) opioid receptor selective compounds was several orders of magnitude less. We also found that cyprodime inhibits morphine-stimulated [35S]GTPgammaS binding. The EC(50) value of morphine increased about 500-fold in the presence of 10 microM cyprodime. These findings clearly indicate that cyprodime is a useful selective antagonist for mu-opioid receptor characterization.     

High affinity acylating antagonists for the A1 adenosine receptor: identification of binding subunit

Two isomeric isothiocyanate derivatives of the A1 adenosine receptor antagonist xanthine amine cogener (XAC) have been synthesized and found to be potent affinity labels (irreversibly bound ligands) for A1 adenosine receptors. The interaction of m- and p-isomers of 1,3-dipropyl-8-isothiocyanatophenyl(aminothiocarbonyl (2-aminoethylaminocarbonyl(4-methyloxy(phenyl]])-xanthine (DITC-XAC) with rat brain A1 receptors is of high affinity (EC50 = 27 and 52 nM, respectively) as determined by radioligand competition curves. These compounds reduced the number of A1 receptors (greater than 90% at 500 nM m-DITC-XAC) in brain membranes, without any change in the affinity of the remaining receptors for [125I]N6-2-(4-aminophenyl)ethyladenosine. Prior reaction of the isothiocyanate moiety with ethylenediamine did not alter the affinity of the XAC derivative for the A1 receptor but eliminated its ability to covalently incorporate into the receptor. Incubation of brain membranes with radiolabeled p- and m-DITC-XAC results in the specific labeling of a Mr 38,000 peptide. This labeling can be blocked with both an A1 adenosine receptor-specific agonist and an antagonist. This specific protein has the same molecular weight as the protein labeled with A1-selective photoaffinity probes. The much higher efficiency of incorporation of these affinity probes compared with photoaffinity probes should make them extremely useful for structural studies of A1 adenosine receptors.     

WGA-coated yttrium oxide beads enable an imaging-based adenosine 2a receptor binding scintillation proximity assay suitable for high throughput screening

Adenosine receptors belong to the superfamily of G protein-coupled receptors and are involved in a variety of physiologic functions. Traditionally, binding assays to detect adenosine 2a (A2a) antagonists and agonists have used filtration methods that are cumbersome to run and not amenable to HTS. We developed scintillation proximity assays (SPA trade mark ) utilizing HEK293 RBHA2AM cell membranes, either wheat germ agglutinin (WGA)-coated yttrium silicate (YSi) or red-shifted yttrium oxide (YO) beads and the A2a-selective radioligand [(3)H]SCH 58261. Both beads gave windows (total binding/nonspecific binding) of >5 and K(d) values of 2-3 nM for the radioligand, in agreement with results obtained by filtration. In contrast, WGA-polyvinyltoluene as well as other bead types had windows of <3 and significant radioligand binding to the uncoated beads. A 384-well WGA-YO bead SPA was optimized utilizing a LEADseeker imaging system and an automated trituration process for dispensing the dense yttrium-based beads. Signals were stable after 4 h, and Z' values were 0.7-0.8. The LEADseeker imaging assay tolerated 2% dimethyl sulfoxide and generated IC(50) values of 3-5 nM for the A2a antagonist CGS 15943, comparable to that obtained by the filtration method. A number of adenosine and xanthine analogues were identified as hits in the Library of Pharmacologically Active Compounds (LOPAC). This imaging-based A2a SPA enables HTS and is a major improvement over the filtration method.     

Development of a complex scintillation proximity assay for highthroughput screening of PPARγmodulators

Aim: To develop a complex high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel peroxisome proliferator-activated receptor gamma (PPARγ) modulators. Methods: Fulllength PPARγand retinoid X receptor alpha (RXRα), biotinylated PPAR response element (PPRE), [^3H]BRL49653 and streptavidin-coated FlashPlate or microbead were used to develop an HTS assay based on SPA technology. This ‘ABCDE‘ method was validated against conventional hydroxyapatite (HA) assay and applied to large-scale screening of 16 000 synthetic compounds and natural product extracts. Results: (1) IC50 values of positive control compounds (BRL49653 and troglitazone) obtained from the ‘ABCDE‘ method and HA assay were comparable and consistent with those reported elsewhere; (2) Approximately 178 compounds, showing more than 70% competitive inhibition on BRL49653 binding to PPARγ, were identified initially by the ‘ABCDE‘ method (microbead); (3) Secondary screening using FlashPlate and cross-reactivity studies with RARα, β,γand RXRα,β,γconfirmed that 12 compounds possessed specific PPARγbinding properties including 2 with IC50 values less than 0.5μmol/L and novel chemical structures. Conclusions: The ‘ABCDE‘ method using either FlashPlate or microbead, is a highly efficient, automatable, and robust tool to screen potential PPARγmodulators in HTS setting. Its application may be expanded to other nuclear receptors that form heterodimers upon activation.     

Development of homogeneous high-affinity agonist binding assays for 5-HT2 receptor subtypes

The serotonin (5-hydroxytryptamine) 5-HT2 receptor subfamily consists of three members, 5-HT2A, 5-HT2B, and 5-HT2C. These receptors share high homology in their amino acid sequence, have similar signaling pathways, and have been indicated to play important roles in feeding, anxiety, aggression, sexual behavior, mood, and pain. Subtype-selective agonists and antagonists have been explored as drugs for hypertension, Parkinson's disease, sleep disorders, anxiety, depression, schizophrenia, and obesity. In this study, we report the development of homogeneous agonist binding assays in a scintillation proximity assay (SPA) format to determine the high-affinity binding state of agonist compounds for the human 5-HT2C, 5-HT2A, and 5-HT2B receptors. The 5-HT2 agonist 1-(4- [125I]iodo-2,5-dimethoxyphenyl)-2-aminopropane ([125I]DOI) was used to label the high-affinity sites for the 5-HT2A and 5-HT2C receptors. The high-affinity sites for the 5-HT2B receptor were labeled with [3H]lysergic acid diethylamide. Total receptor expression was determined with the 5-HT2 antagonist [3H]mesulergine for the 5-HT2B and 5-HT2C receptors, and [3H]ketanserin for the 5-HT2A receptor. The agonist high-affinity binding sites accounted for 2.3% (5-HT(2C) receptor), 4.0% (5-HT2A receptor), and 22% (5-HT2B receptor) of the total receptor population. Competition binding studies using known agonists indicated high Z' values of the agonist binding assays in SPA format (Z' > 0.70). The Ki values of 5-HT, (R)(-)DOI, and VER-3323 for the 5-HT2A, 5-HT2B, and 5-HT2C receptors by SPA format were equivalent to published data determined by filtration binding assays. These results indicate that agonist binding assays in SPA format can be easily adapted to a high throughput assay to screen for selective 5-HT2C receptor agonists, as well as for selectivity profiling of the compounds.     

Development and application of a scintillation proximity assay (SPA) for identification of selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1

Pre-receptor metabolism of glucocorticoids by the 11beta-hydroxysteroid dehydrogenase (11betaHSD) enzymes has been implicated in the etiology of the metabolic syndrome. Recent studies have shown that alterations in the activity of the type 1 isozyme can affect many aspects of the disease. This paper describes the optimization and application of a high-throughput scintillation proximity assay (SPA) developed to identify selective specific inhibitors of 11betaHSD1. Microsomes containing 11betaHSD1 were incubated in the presence of NADPH and [3H]cortisone, and the product, [3H]cortisol, was specifically detected in the mixture by a monoclonal antibody coupled to protein A-coated SPA beads with greater than 2 log higher affinity for cortisol than cortisone. Dimethyl sulfoxide and NADPH co-substrate additions were optimized for 11betaHSD1 reductase activity. Titrated test compound, when introduced into the optimized assay, reproducibly inhibited the enzyme and yielded consistent IC50 data in either 96- or 384-well format. An 11betaHSD2 counterscreen was performed by incubating 11betaHSD2 microsomes with [3H]cortisol and NAD+ and monitoring substrate disappearance.     

Pharmacology of quinpirole-stimulated [35S]GTPgammaS binding: discrepancy with receptor binding profile

Functional consequences of receptor stimulation by quinpirole, a dopamine D(2)-like receptor agonist, were assessed using agonist-stimulated [35S]GTPgammaS binding in rat striatal membranes. Dopamine receptor antagonists inhibited quinpirole-stimulated [35SCH 23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4, 5-tetrahydro-1H-3-benzazepine), consistent with a dopamine D(2)-like profile. In contrast, the monoamine oxidase inhibitors Ro 41-1049 (N-(2-aminoethyl)-5-(3-fluorophenyl)-4-thiazolecarboxemide), and (+)- and (-)-deprenyl, which inhibit [3H]quinpirole binding, had no effect on agonist-independent or quinpirole-stimulated [35S]GTPgammaS binding. Clorgyline inhibited [35S]GTPgammaS binding by a non-dopamine D(2) receptor-mediated mechanism. These findings demonstrate a notable discrepancy between the pharmacological profile of [3H]quinpirole binding and quinpirole-stimulated [35S]GTPgammaS binding.     

delta-Opioid receptor agonists produce antinociception and [35S]GTPgammaS binding in mu receptor knockout mice

We examined the effects of [D-Pen(2),D-Pen(5)]enkephalin (DPDPE), [D-Ala(2),Glu(4)]deltorphin (DELT), and (+)-4-[(alphaR)-alpha((2S, 5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N, N-diethylbenzamide (SNC80) on [35S]GTPgammaS binding in brain membranes prepared from micro-opioid receptor knockout (-/-) mice. The potency and maximal response (E(max)) of these agonists were unchanged compared to control mice. In contrast, while the potency of [D-Pen(2),pCl-Phe(4),D-Pen(5)]enkephalin (pCl-DPDPE) was not significantly different, the E(max) was reduced as compared to controls. In the tail-flick test, intracerebroventricular (i.c.v.) or intrathecal (i.th.) DELT produced antinociceptive effects in -/- mice with potency that did not differ significantly from controls. In contrast, the antinociceptive potency of i.c.v. and i.th. DPDPE was displaced to the right by 4- and 9-fold in -/- compared to control mice, respectively. Reduced DPDPE antinociceptive potency in -/- mice, taken together with reduced DPDPE- and pCl-DPDPE- stimulated G protein activity in membranes prepared from -/- mice, demonstrate that these agonists require mu-opioid receptors for full activity. However, because DELT mediated G protein activation and antinociception were both comparable between -/- and wild type mice, we conclude that the mu-opioid receptor is not a critical component of delta-opioid receptor function.     

In vitro optimization of structure activity relationships of analogues of A-331440 combining radioligand receptor binding assays and micronucleus assays of potential antiobesity histamine H3 receptor antagonists

A-331440 [4'-[3-(3(R)-(dimethylamino)-pyrrolidin-1-yl)-propoxy]-biphenyl-4-carbonitrile], a potent and selective antagonist of histamine H3 receptors, yielded positive results in an in vitro micronucleus assay, predictive of genotoxicity in vivo. Because this compound has highly favourable properties and potential as an antiobesity agent, new compounds of this general chemical class were sought that would retain or improve upon the high potency and selectivity of A-331440 for H3 receptors, but would lack the potential for genotoxicity obtained with that compound. Our working hypothesis was that the biphenyl rings in A-331440 might contribute to interactions with DNA and thereby predispose toward genotoxicity. Toward this end, several analogues were prepared, with substituents introduced onto the biaryl ring to alter the orientation, electronegativity, and polarity of this moiety, and were tested for their radioligand binding potency and selectivity and their propensity to induce genotoxicity in the in vitro micronucleus assay. Using this strategy, novel compounds were discovered that retained or improved upon the potency and selectivity of A-331440 for H3 receptors and were devoid of genotoxicity in vitro. Of these, the simple mono- and di-fluorinated analogues (A-417022 [4'-[3-[(3R)-3-(dimethylamino)-1-pyrrolidinyl]propoxy]-3'-fluoro-1,1'-biphenyl-4-carbonitrile] and A-423579 [4'-[3-[(3R)-3-(dimethylamino)-1-pyrrolidinyl]-propoxy]-3',5'-difluoro-1,1'-biphenyl-4-carbonitrile], respectively) were found to bind to H3 receptors at least as potently as A-331440, while lacking genotoxicity in the micronucleus assay. The reason of the lack of genotoxicity of the fluorinated analogues is unclear, but is especially noteworthy in light of the general principle that fluorine and hydrogen are very similar in size. Therefore, these fluorinated analogues of A-331440 represented the most potent and potentially safest compounds for further evaluation as antiobesity leads. Preliminary findings with one of these examples, A-417022, in a mouse model of obesity are presented.     

Affinity of muscarinic receptor antagonists for three putative muscarinic receptor binding sites

1. A range of muscarinic receptor antagonists were examined for affinity at the M1 muscarinic binding site, present in rat cerebrocortical membranes and the M2 muscarinic binding sites of rat cardiac and submaxillary gland membranes. 2. The results obtained were consistent with the presence of three classes of muscarinic binding site. 3. Both the M1 binding site, labelled by [3H]-pirenzepine ([3H]-Pir) in rat cerebrocortical membranes, and the M2 gland binding site, labelled by [3H]-N-methyl scopolamine ([3H]-NMS) in rat submaxillary gland membranes, displayed higher affinity for pirenzepine, dicyclomine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and cyclohexylphenyl (2-piperidinoethyl) silanol (CPPS) than did the M2 binding sites of cardiac membranes labelled by [3H]-NMS. 4. The M2 cardiac sites displayed higher affinity for methoctramine, himbacine and AF-DX 116 than did either the M1 binding site of cerebrocortical membranes or the M2 gland binding site present in rat submaxillary gland membranes. 5. The M1 and M2 gland binding sites could only be distinguished by considering the absolute affinity of compounds for these two sites. Thus, all compounds, with the exception of 4-DAMP, displayed between a 2 and 8 fold higher affinity for the M1 than for the M2 gland binding site. There were no antagonists with higher M2 gland than M1 affinity.     

Development of a high-throughput assay for two inositol-specific phospholipase Cs using a scintillation proximity format

Inositol-specific PLCs comprise a family of enzymes that utilize phosphoinositide substrates, e.g., PIP(2), to generate intracellular second messengers for the regulation of cellular responses. In the past, monitoring this reaction has been difficult due to the need for radiolabeled substrates, separation of the reaction products by organic-phase extraction, and finally radiometric measurements of the segregated products. In this report, we have studied the enzymatic characteristics of two novel PLCs that were derived from functional genomic analyses using a phospholipid-modified solid scintillating support. This method allows for the hydrophobic capture of the [(3)H]phosphoinositide substrate on a well defined scintillation surface and the homogenous measurement of the enzymatic hydrolysis of the substrate by proximity effects. Our results show that the assay format is robust and well suited for this class of lipid-metabolizing enzymes.     

Characterization of the kinase domain of the ephrin-B3 receptor tyrosine kinase using a scintillation proximity assay

We have characterized a recombinantly expressed N-terminally tagged GST fusion of the tyrosine kinase domain of human EphB3. The EphB3 kinase domain was shown to phosphorylate a group of synthetic tyrosine-containing peptides derived from a proprietary biotinylated kinase-biased peptide substrate library. In addition, the enzyme activity was stimulated by the divalent cation, manganese, and inhibited by addition of magnesium. The most active tyrosine-containing peptide, a biotinylated 49-mer, displayed saturation kinetics with an apparent K(m) of approximately 0.4 microM. The apparent K(m) for ATP was determined to be approximately 3 microM. The kinetics of the reaction was linear from concentrations of enzyme of 0.5 to 2 nM, and at or below the K(m) concentrations of the two substrates for at least 2 h at room temperature. Moreover, the tryrosine kinase inhibitor, PP2, produced an IC(50) of roughly 0.8 microM. In addition, the enzyme tolerated the solvent DMSO and was stable to multiple freeze/thaw cycles. Stability of the enzyme at 4 degrees C storage was seen out to 6 h with an approximately 50% reduction of activity by 24 h. Formatting the assay in a 384-well microtiter plate produced good uniformity of signal at 100% inhibition, 50% inhibition, and no inhibition. The coefficient of variance was at or below 10% with a signal-to-background ratio of approximately 24 and a z value of 0.72. Collectively, these results showed the ability to configure a robust HTS for a truncated recombinantly expressed family member of the Ephrin tyrosine kinases.     

Development of cell-based high-throughput assays for the identification of inhibitors of receptor activator of nuclear factor-kappa B signaling

Bone loss due to metabolic or hormonal disorders and osteolytic tumor metastasis continues to be a costly health problem, but current therapeutics offer only modest efficacy. Unraveling of the critical role for the receptor activator of nuclear factor-kappa B (RANK) and its ligand, RANK ligand (RANKL), in osteoclast biology provides an opportunity to develop more effective antiresorptive drugs. The in vivo effectiveness of RANKL inhibitors demonstrates the potency of the RANKL/RANK system as a drug target. Here, we report the development of cell-based assays for high-throughput screening to identify compounds that inhibit signaling from two RANK cytoplasmic motifs (PVQEET(559-564) and PVQEQG(604-609)), which play potent roles in osteoclast formation and function. Inhibitors of these motifs' signaling have the potential to be developed into new antiresorptive drugs that can complement current therapies. The cell-based assays consist of cell lines generated from RAW264.7 macrophages stably expressing a nuclear factor-kappa B-responsive luciferase reporter and a chimeric receptor containing the human Fas external domain linked to a murine RANK transmembrane and intracellular domain in which only one of the RANK motifs is functional. With these cells, specific RANK motif activation after chimeric receptor stimulation can be measured as an increase in luciferase activity. These assays demonstrated >300% increases in luciferase activity after RANK motif activation and Z?'-factor values over 0.55. Our assays will be used to screen compound libraries for molecules that exhibit inhibitory activity. Follow-up assays will refine hits to a smaller group of more specific inhibitors of RANK signaling.     

Insight into the binding mode for cyclopentapeptide antagonists of the CXCR4 receptor

The finding that the chemokine receptor CXCR4 is involved in T‐cell HIV entry has encouraged the development of antiretroviral drugs targeting this receptor. Additional evidence that CXCR4 plays a crucial role in both angiogenesis and metastasis provides further motivation for the development of a CXCR4 inhibitor for therapeutic applications in oncology. To facilitate the design of such ligands, we have investigated the possible binding modes for cyclopentapeptide CXCR4 antagonists by docking 11 high/medium affinity cyclopentapeptides to a developed three‐dimensional model of the CXCR4 G‐protein‐coupled receptor's transmembrane region. These ligands, expected to bind in the same mode to the receptor, were docked in the previously deduced receptor‐bound conformation [Våbenøet al., in press; doi 10.1002/bip.20508]. Ligand–receptor complexes were generated using an automated docking procedure that allowed ligand flexibility. By comparing the resulting ligand poses, only two binding modes common for all 11 compounds were identified. Inspection of these two ligand–receptor complexes identified several CXCR4 contact residues shown by mutation to be interaction sites for ligands and important for HIV gp120 binding. Thus, the results provide further insight into the mechanism by which these cyclopentapeptides block HIV entry as well as a basis for rational design of CXCR4 mutants to map potential contacts with small peptide ligands.     

Analysis of the behaviour of selected CCKB/gastrin receptor antagonists in radioligand binding assays performed in mouse and rat cerebral cortex

1. The previously described complex behaviour of the CCKB/gastrin receptor antagonist, L-365,260, in radioligand binding assays could be explained by a variable population of two binding sites. We have investigated whether other CCKB/gastrin receptor ligands (PD134,308, PD140,376, YM022 and JB93182) can distinguish between these sites. 2. In the mouse cortex assay, Hill slopes were not different from unity and the ligand pKI values did not differ when either [125I]-BH-CCK-8S or [3H]-PD140,376 was used as label as expected for a single site (G2). 3. In the rat cortex, where previous analysis of replicate (n=48) L-365,260 data indicated the presence of two CCKB/gastrin sites (G1 and G2), the competition data for PD134,308, PD140,376, YM022 and JB93182 could be explained by a homogeneous population of CCKB/gastrin sites because the Hill slope estimates were not significantly different from unity. However, the estimated affinity values for JB93182 and YM022 were significantly higher and that for PD134,308 was significantly lower than those obtained in the mouse cortex when the same radioligand was used. In view of our previous data obtained with L-365,260, the rat cortex data were also interpreted using a two-site model. In this analysis, SR27897 expressed approximately 9 fold, PD134,308 approximately 13 fold and PD140,376 approximately 11 fold selectivity for the G2 site. In contrast, JB93182 expressed approximately 23 fold and YM022 approximately 4 fold selectivity for the G1 site. If the two-site interpretation of the data is valid then, because of its reverse selectivity to L-365,260, JB93182 has been identified as a compound which if radiolabelled could provide a test of this receptor subdivision.     

Structural determinants required for the bioactivities of prokineticins and identification of prokineticin receptor antagonists

Prokineticins are cysteine-rich secreted proteins that regulate diverse biological processes, including gastrointestinal motility, angiogenesis, and circadian rhythms. Two closely related G protein-coupled receptors that mediate signal transduction of prokineticins have recently been cloned. The structural elements required for prokineticins' bioactivities are still unknown. We show here that both the N-terminal hexapeptide (AVITGA) and C-terminal cysteine-rich domains are critical for the bioactivities of prokineticins. Substitutions, deletions, and insertions to the conserved N-terminal hexapeptides result in the loss of agonist activity. Mutant prokineticins with the substitution of the first N-terminal alanine with methionine or the addition of a methionine to the N terminus inhibit the activation of prokineticin receptors and thus are considered as antagonists of prokineticin receptors. We have further shown that mutations in selected cysteine residues in the C-terminal domain result in prokineticins without biological activity. The essential role of C-terminal domain is reinforced by two observations: that peptides without the carboxyl domain and proteins with the N-terminal hexapeptide fused to the carboxyl domains of colipase or dickkopf are devoid of biological activity. We demonstrate that limited structural changes of C-terminal cysteine-rich regions of prokineticins are tolerable because chimeric prokineticins with swapped cysteine-rich domains between prokineticin 1 and prokineticin 2, as well as a splice variant of prokineticin 2 that contains extra 21 residue insertion in its C-terminal domain, are biologically active.     

Receptor binding properties and antinociceptive effects of chimeric peptides consisting of a micro-opioid receptor agonist and an ORL1 receptor antagonist

Receptor binding properties and antinociceptive activities of chimeric peptides linked by spacers were investigated. The peptides consisted of the micro-opioid receptor ligand dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) or its analog YRFB (Tyr-D-Arg-Phe-betaAla-NH(2)) linked to the ORL1 receptor ligand Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) (Ac-RYYRIK-NH(2)). All chimeric peptides were found to possess high receptor binding affinities for both micro-opioid and ORL1 receptors in mouse brain membranes although their binding affinities for both receptors in spinal membranes were significantly lower. Among them, chimeric peptide 2, which consists of dermorphin and Ac-RYYRIK-NH(2) connected by a long spacer, had the highest binding affinity towards both receptors. In the tail-flick test following intrathecal (i.t.) administration to mice, all chimeric peptides showed potent and dose-dependent antinociceptive activities with an ED(50) of 1.34-4.51 (pmol/mouse), nearly comparable to dermorphin alone (ED(50); 1.08 pmol/mouse). In contrast to their micro-opioid receptor binding profiles, intracerebroventricular (i.c.v.) administration of the chimeric peptides resulted in much less potent antinociceptive activity (ED(50) 5.55-100< pmol/mouse) than when administered i.t. (ED(50): 1.34-4.51 pmol/mouse). These results suggest the involvement of nociceptin-like agonistic effects of the Ac-RYYRIK pharmacophore in the peptides, and the regulation of mu-opioid receptor-mediated antinociception in brain. The present chimeric peptides may be useful as pharmacological tools for studies on micro-opioid receptor/ORL1 receptor heterodimers.