Characterization of two new ETB selective radioligands, [125I]-BQ3020 and [125I]-[Ala1,3,11,15]ET-1 in human heart.

Two new endothelin receptor radioligands, [125I]-BQ3020 and [125I]-[Ala1,3,11,15]ET-1, were characterized in tissue sections of human right atrium and left ventricle. Both radioligands had high affinity ([125I]-BQ3020 right atrium: KD = 0.145 +/- 0.037 nM, left ventricle: KD = 0.107 +/- 0.004 nM; [125I]-[Ala1,3,11,15]ET-1 right atrium: KD = 0.239 +/- 0.036 nM, left ventricle: KD = 0.199 +/- 0.027 nM). Competition binding experiments were performed in the left ventricle. The selective ETA receptor compound BQ123 competed with low affinity against [125I]-BQ3020 (KD = 28.7 +/- 2.7 microM) and [125I]-[Ala1,3,11,15]ET-1 (KD = 28.5 +/- 4.2 microM). The selective ETB receptor compound BQ3020 competed with high affinity against [125I]-BQ3020 (KD = 40.8 +/- 6.6 pM) and [125I]-[Ala1,3,11,15]ET-1 (KD = 0.276 +/- 0.099 nM). Another selective ETB receptor compound, [Ala1,3,11,15]ET-1 also competed with high affinity against [125I]-BQ3020 (KD = 0.663 +/- 0.120 nM) and [125I]-[Ala1,3,11,15]ET-1 (KD = 0.643 +/- 0.124 nM). These results indicate that [125I]-BQ3020 and [125I]-[Ala1,3,11,15]ET-1 are selective ETB receptor radioligands. [Ala1,3,11,15]ET-1 competed with the non-selective radioligand [125I]-ET-1 in left ventricle and revealed the presence of ETA and ETB receptors in the proportions of 76:24% respectively in the human left ventricle.     

Structural characterizations of neuropeptide tyrosine (NPY) and its agonist analog [Ahx5-17]NPY by NMR and molecular modeling

The structures of human NPY and of its centrally truncated agonist analog [Ahx^5–17]NPY have been investigated in DMSO-d₆ by two-dimensional NMR and by molecular modeling. For both peptides, a complete resonance assignment was achieved and a large number (more than 200) of inter-residue NOE connectivities were observed, including long-range connectivities between the N- and C-terminal ends of the chain. Molecular models were calculated using NOE constraints by distance geometry, simulated annealing and conjugate gradient energy minimization. The results indicate that both peptides are folded in the center of their chain, NPY adopting the hairpin shape, whereas the central portion of [Ahx^5–17]NPY is characterized by relatively large loops. In contrast to previous models, practically no α-helical structure exists for these peptides under our conditions, but two β-turns are found in NPY and one in [Ahx^5–17]NPY. The proximity of the terminal ends could be the determinant factor for their activity. © Munksgaard 1995.     

Neuropeptide Y (NPY) Y4 receptor selective agonists based on NPY(32-36): development of an anorectic Y4 receptor selective agonist with picomolar affinity

We have previously shown [Cys-Trp-Arg-Nva-Arg-Tyr-NH(2)](2), 1, to be a moderately selective neuropeptide Y (NPY) Y(4) receptor agonist. Toward improving the selectivity and potency for Y(4) receptors, we studied the effects of dimerizing H-Trp-Arg-Nva-Arg-Tyr-NH(2) using various diamino-dicarboxylic acids containing either di-, tri-, or tetramethylene spacers. These parallel dimers, 2A, 2B, 3, 4A, and 4B, and the corresponding linear tandem dimer and trimer analogues, 5 and 6, had enhanced selectivity and affinity for Y(4) receptors compared to 1 (Table 1). Substitution of Trp and Nva with Tyr and Leu, respectively, as in 2,7-d/l-diaminosuberic acid derivatized dimer, 7, resulted in a superior Y(4) selective agonist with picomolar affinity. Intraperitoneal (ip) injection of 7 potently inhibited food intake in fasted mice. Moreover, 7 (ip) inhibited the food intake in wild-type mice and not in Y(4)(-/-) knock-out mice, confirming that the actions of 7 on food intake are not due to global effects, but specifically mediated Y(4) receptors.     

[3H]-F13640, a novel, selective and high-efficacy serotonin 5-HT1A receptor agonist radioligand

F13640 is a selective and high-efficacy serotonin 5-HT_1A receptor agonist that demonstrates outstanding analgesic potential in different animal models. Here, we use the radiolabelled compound to further characterise its binding properties at 5-HT_1A receptors. F13640 was tritium-labelled to 47 and 64 Ci/mmol specific activity and used as radioligand at membrane preparations of CHO cells expressing human (h) 5-HT_1A receptors. The K _d of [³H]-F13640 was 1.8 nM at h5-HT_1A receptors as determined from saturation binding experiments. In association time-course experiments, k _obs of [³H]-F13640 was 0.06 min^?1. Dissociation experiments performed in the presence of unlabelled F13640 as competing ligand yielded a k _off value of 0.05 min^?1, resulting in a calculated K _d of 1.4 nM. In comparison, [³H]-8-OH-DPAT had a k _obs of 0.50 min^?1, a k _off of 0.25 min^?1 and a calculated K _d of 0.37 nM. Surprisingly, [³H]-F13640 dissociation kinetics were distinctly slower in the presence of WAY-100635 and spiperone as competing ligands when compared with the agonist competitors, F13640 and (+)8-OH-DPAT. The competitive binding profile of [³H]-F13640 with eight chemically diverse 5-HT_1A receptor agonists and antagonists correlated highly (r?=?0.996) with that of [³H]-8-OH-DPAT. In conclusion, [³H]-F13640 is a potent agonist radioligand at 5-HT_1A receptors and may be a useful tool in pharmacological studies at native and recombinant 5-HT_1A receptors. In addition, [³H]-F13640 dissociates more slowly from h5-HT_1A receptors than [³H]-8-OH-DPAT, a kinetic property that might be related to its powerful analgesic effects as observed in vivo.     

Three-dimensional structure of the Y1 receptor agonist [Leu31, Pro34]NPY as determined by NMR and molecular modeling

The solution structure of the Y1 receptor agonist, porcine [Leu³¹, Pro³⁴]NPY, has been investigated by two-dimensional NMR and molecular modeling. A complete assignment of the NMR resonances was achieved and 201 inter-residue nuclear Overhauser enhancement spectroscopy (NOESY) connectivities could be identified, comprising several connectivities between the N- and C-terminal segments. A molecular model was calculated by distance geometry, simulated annealing and conjugate gradients energy minimization using the NOE constraints. The results indicate that, like NPY and other peptides of the family, [Leu³¹, Pro³⁴]NPY adopts a folded hairpin structure with the terminal segments in close proximity. Analysis of the secondary chemical shifts for the CHα's and of the temperature dependence of the NH chemical shifts combined with the NOE constraints indicates a tendency toward helix structure for the segment 18-30 and the presence of turn structure for the C-terminal segment (residues 31-36). Native NPY and [Leu³¹, Pro³⁴]NPY have most of their structures in common but differ slightly in their C-terminal portion. Based on the structures of NPY and of its specific agonists, [Leu³¹, Pro³⁴]NPY and NPY 13-36, conclusions can be drawn about the structural requirements for binding to the Y1 and Y2 receptor subtypes.     

[125I]-PD151242: a selective radioligand for human ETA receptors.

Our aim was to synthesize a new endothelin ETA selective radioligand, [125I]-PD151242 and characterize the compound in human vascular tissue. Binding of [125I]-PD151242 to sections of human aorta was time-dependent and reached equilibrium after 120 min at 23 degrees C with an association rate constant of 1.26 +/- 0.17 x 10(8) M-1 min-1 (n = 3 individuals +/- s.e.mean). The binding was reversible at 23 degrees C with an observed dissociation rate constant of 0.0025 +/- 0.0006 min-1 (n = 3). Saturation binding assays using [125I]-PD151242 revealed a single population of high affinity ET receptors (n = 3) in aorta (KD = 0.76 +/- 0.17 nM; Bmax = 5.98 +/- 1.56 fmol mg-1 protein), pulmonary (KD = 1.75 +/- 0.20 nM; Bmax = 12.78 +/- 1.39 fmol mg-1 protein) and coronary arteries (KD = 0.51 +/- 0.07 nM; Bmax = 44.9 +/- 1.67 fmol mg-1 protein). ETA selective ligands competed for [125I]-PD151242 binding in aorta with nanomolar affinity (BQ123, KD = 0.41 +/- 0.26 nM; FR139317, KD = 0.55 +/- 0.11 nM) whereas the ETB selective compound, BQ3020, competed with micromolar affinity (KD = 1.36 +/- 0.25 microM). In isolated coronary arteries, PD151242 was a functional antagonist and caused a significant, parallel rightward shift of the ET-1 dose-response curve with a pA2 value of 5.92 (n = 5) and a slope of unity. The high affinity and selectivity of [125I]-PD151242 for ETA receptors will facilitate the characterization of this sub-type in human tissues.     

(125)I]-GR231118: a high affinity radioligand to investigate neuropeptide Y Y(1) and Y(4) receptors

GR231118 (also known as 1229U91 and GW1229), a purported Y(1) antagonist and Y(4) agonist was radiolabelled using the chloramine T method. [(125)I]-GR231118 binding reached equilibrium within 10 min at room temperature and remained stable for at least 4 h. Saturation binding experiments showed that [(125)I]-GR231118 binds with very high affinity (K(d) of 0.09 - 0.24 nM) in transfected HEK293 cells with the rat Y(1) and Y(4) receptor cDNA and in rat brain membrane homogenates. No specific binding sites could be detected in HEK293 cells transfected with the rat Y(2) or Y(5) receptor cDNA demonstrating the absence of significant affinity of GR231118 for these two receptor classes. Competition binding experiments revealed that specific [(125)I]-GR231118 binding in rat brain homogenates is most similar to that observed in HEK293 cells transfected with the rat Y(1), but not rat Y(4), receptor cDNA. Autoradiographic studies demonstrated that [(125)I]-GR231118 binding sites were fully inhibited by the Y(1) antagonist BIBO3304 in most areas of the rat brain. Interestingly, high percentage of [(125)I]-GR231118/BIBO3304-insensitive binding sites were detected in few areas. These [(125)I]-GR231118/BIBO3304-insensitive binding sites likely represent labelling to the Y(4) receptor subtype. In summary, [(125)I]-GR231118 is a new radiolabelled probe to investigate the Y(1) and Y(4) receptors; its major advantage being its high affinity. Using highly selective Y(1) antagonists such as BIBO3304 or BIBP3226 it is possible to block the binding of [(125)I]-GR231118 to the Y(1) receptor allowing for the characterization and visualization of the purported Y(4) subtype. British Journal of Pharmacology (2000) 129, 37 - 46     

Purification and characterization of (-)[125I]hydroxyphenylisopropyladenosine, an adenosine R-site agonist radioligand and theoretical analysis of mixed stereoisomer radioligand binding

(-)-N6-(R-4-Hydroxyphenylisopropyl)adenosine (HPIA) was iodinated with NaI and trace 125I. Mono- and diiodinated reaction products and the starting material were separated by high pressure liquid chromatography and the structures of the reaction products were verified by NMR. (-)-N6-(R-Phenylisopropyl)adenosine (PIA), IHPIA, and I2HPIA decreased rat atrial contractility with ED50 values of 24, 28, and 33 nM, respectively. The contractile effects of these compounds were competitively blocked by theophylline (KI = 7.9 microM), but were not affected by adenosine deaminase. IHPIA also inhibited (-)isoproterenol-stimulated cyclic AMP accumulation in adipocytes with an ED50 (10 nM) and to an extent (83%) nearly identical to PIA. [125I]HPIA prepared using carrier-free 125I bound to adenosine receptors on membranes from rat cerebral cortex, adipocyte ghosts, and heart ventricles. Binding was inhibited stereospecifically by PIA and by other adenosine analogues and alkylxanthines. The KD of [125I]HPIA determined kinetically using brain membranes at 21 degrees was 0.94 nM (K1 = 2.55 X 10(7) M-1 min-1; K-1 = 0.024 min-1) in good agreement with the equilibrium determination of 1.94 nM. The density of adenosine receptors in brain membranes was found to be 871 fmol/mg of protein. When normalized to protein, the density of receptors in heart membranes and adipocyte ghosts, respectively, was found to be 39- and 2.3-fold less than in brain membranes. We conclude that [125I]HPIA can be rapidly synthesized and purified, binds to adenosine R-sites and is an agonist radioligand resistant to adenosine deaminase. Computer modeling of the equilibrium binding resulting from the use of mixed stereoisomers of a radioligand indicates that the combined use of (-)[125I]HPIA and (+)[125I]HPIA would result in the generation of nonlinear Scatchard plots.     

Synthesis and pharmacological characterization of [I]MRS5127, a high affinity, selective agonist radioligand for the A3 adenosine receptor

A recently reported selective agonist of the human A₃ adenosine receptor (hA₃AR), MRS5127 (1′R,2′R,3′S,4′R,5′S)-4′-[2-chloro-6-(3-iodobenzylamino)-purine]-2′,3′-O-dihydroxy-bicyclo-[3.1.0]hexane, was radioiodinated and characterized pharmacologically. It contains a rigid bicyclic ring system in place of a 5′-truncated ribose moiety, and was selected for radiolabeling due to its nanomolar binding affinity at both human and rat A₃ARs. The radioiodination of the N⁶-3-iodobenzyl substituent by iododestannylation of a 3-(trimethylstannyl)benzyl precursor was achieved in 73% yield, measured after purification by HPLC. [¹²⁵I]MRS5127 bound to the human A₃AR expressed in membranes of stably transfected HEK 293 cells. Specific binding was saturable, competitive, and followed a one-site binding model, with a K_d value of 5.74 ± 0.97 nM. At a concentration equivalent to its K_d, non-specific binding comprised 27 ± 2% of total binding. In kinetic studies, [¹²⁵I]MRS5127 rapidly associated with the hA₃AR (t_1/2 = 0.514 ± 0.014 min), and the affinity calculated from association and dissociation rate constants was 3.50 ± 1.46 nM. The pharmacological profile of ligands in competition experiments with [¹²⁵I]MRS5127 was consistent with the known structure-activity-relationship profile of the hA₃AR. [¹²⁵I]MRS5127 bound with similar high affinity (K_d, nM) to recombinant A₃ARs from mouse (4.90 ± 0.77), rabbit (2.53 ± 0.11), and dog (3.35 ± 0.54). For all of the species tested, MRS5127 exhibited A₃AR agonist activity based on negative coupling to cAMP production. Thus, [¹²⁵I]MRS5127 represents a new species-independent agonist radioligand for the A₃AR. The major advantage of [¹²⁵I]MRS5127 compared with previously used A₃AR radioligands is its high affinity, low degree of non-specific binding, and improved A₃AR selectivity.     

BIIE0246, a potent and highly selective non-peptide neuropeptide Y Y(2) receptor antagonist

1. BIIE0246, a newly synthesized non-peptide neuropeptide Y (NPY) Y(2) receptor antagonist, was able to compete with high affinity (8 to 15 nM) for specific [(125)I]PYY(3 - 36) binding sites in HEK293 cells transfected with the rat Y(2) receptor cDNA, and in rat brain and human frontal cortex membrane homogenates. 2. Interestingly, in rat brain homogenates while NPY, C2-NPY and PYY(3 - 36) inhibited all specific [(125)I]PYY(3 - 36) labelling, BIIE0246 failed to compete for all specific binding suggesting that [(125)I]PYY(3 - 36) recognized, in addition to the Y(2) subtype, another population of specific NPY binding sites, most likely the Y(5) receptor. 3. Quantitative receptor autoradiographic data confirmed the presence of [(125)I]PYY(3 - 36)/BIIE0246-sensitive (Y(2)) and-insensitive (Y(5)) binding sites in the rat brain as well as in the marmoset monkey and human hippocampal formation. 4. In the rat vas deferens and dog saphenous vein (two prototypical Y(2) bioassays), BIIE0246 induced parallel shifts to the right of NPY concentration-response curves with pA(2) values of 8.1 and 8.6, respectively. In the rat colon (a Y(2)/Y(4) bioassay), BIIE0246 (1 microM) completely blocked the contraction induced by PYY(3 - 36), but not that of [Leu(31), Pro(34)]NPY (a Y(1), Y(4) and Y(5) agonist) and hPP (a Y(4) and Y(5) agonist). Additionally, BIIE0246 failed to alter the contractile effects of NPY in prototypical Y(1) in vitro bioassays. 5. Taken together, these results demonstrate that BIIE0246 is a highly potent, high affinity antagonist selective for the Y(2) receptor subtype. It should prove most useful to establish further the functional role of the Y(2) receptor in the organism.     

(S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-[125I]iodo- 2-methoxybenzamide hydrochloride, a new selective radioligand for dopamine D-2 receptors

From salicyclic acid, the two enantiomers of N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2-methoxybenzamide (6b) were prepared in a five-step synthesis. With use of Heindel's triazene method for introduction of the radionuclide, the iodine-125-labeled substituted benzamide was obtained with a calculated specific activity of 136 Ci/mmol and 14% radiochemical yield. For the preparation of the iodine-125-labeled benzamide with higher specific activity, this method was unsuccessful and utilization of the corresponding tri-n-butyltin derivative was required. Treatment of the latter in dilute hydrochloric acid with sodium iodide-125 and chloramine-T gave [125I](S)-6b in 56% radiochemical yield and at least 97% radiochemical purity. The displacement of [125I](S)-6b and [3H](S)-sulpiride from their respective binding sites in striatal rat brain homogenates using various neuroleptic agents showed that (S)-6b has the same binding profile but more potent binding for dopamine D-2 receptors than has sulpiride. These experiments also indicate that the S enantiomer of 6b is a specific ligand (KD = 1.2 nM) for the D-2 receptor. Further, the octanol-water partition coefficient of (S)-6b as determined by reverse-phase high-performance liquid chromatography was found to be 40 times greater than that for sulpiride. Thus (S)-6b has a lipophilicity that will allow a relatively higher uptake into the brain compared to sulpiride. In vivo experiments with rats show that [125I](S)-6b penetrates readily into the brain and is preferentially localized in the striatum as compared to the cerebellum, the ratio of uptake being 7.2 to 1, 60 min after injection. These observations of good brain penetration and high affinity and selectivity for D-2 receptors indicate that the corresponding iodine-123-labeled benzamide may be a useful ligand for the noninvasive visualization study of dopamine D-2 receptor sites in vivo by single photon emission computed tomography.     

Binding characteristics of [125I]Bolton-Hunter [Sar9,Met(O2)11]substance P, a new selective radioligand for the NK1 receptor.

The selective tachykinin agonist [Sar9,Met(O2)11]substance P (Sar-SP) was radioiodinated with [125I]Bolton-Hunter reagent and the product [125I]Bolton-Hunter-[Sar9,Met(O)2)11]SP (BHSar-SP) purified using reverse phase HPLC. Autoradiographic studies showed dense specific binding of BHSar-SP over the rat submandibular gland and over several regions in rat brain, with very low nonspecific binding, identical with the pattern of binding sites seen in a parallel study with [125I]Bolton-Hunter SP (BHSP). In homogenate binding experiments, BHSar-SP bound with high affinity to a single site in membranes from rat brain (KD 261 pM) and rat submandibular gland (KD 105 pM). Comparative values for BHSP were 495 and 456 pM, i.e. of two and four fold lower affinity than BHSar-SP. Association of BHSar-SP to membranes from brain (k+1 3.7 x 10(9) M-1 min-1) was faster than to membranes from salivary gland (k+1 5.6 x 10(8) M-1 min-1). In competition studies, BHSar-SP was displaced from salivary gland membranes by substance P (SP) approximately physalaemin greater than or equal to Sar-SP approximately SP-(3-11) greater than SP-(5-11) much greater than neurokinin A (NKA) approximately eledoisin = kassinin = SP-methyl ester greater than or equal to neurokinin B (NKB) much greater than [Nle10]NKA-(4-10) greater than [MePhe7]NKB-(4-10). In brain membranes, the rank potency order was SP greater than Sar-SP greater than or equal to physalaemin greater than SP-(3-11) greater than SP-(5-11) greater than NKA greater than or equal to eledoisin much greater than NKB greater than kassinin greater than SP-methyl ester: however [MePhe7]NKB-(4-10) and [Nle10]NKA-(4-10) were ineffective competitors at concentrations up to 1 microM. Both binding patterns are consistent with BHSar-SP binding to an NK1 site. With the exception of SP, Sar-SP, SP-(3-11) and physalaemin, all competitors were 5 to 54 times less potent at BHSar-SP binding sites in brain than in salivary gland. These data reveal some differences in characteristics of NK1 binding sites in brain and submandibular gland. Although of higher affinity, BHSar-SP does not appear greatly more selective than BHSP in its ability to define NK1 binding sites.     

125I]2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), a high-affinity radioligand selective for I1 imidazoline receptors

The I1 subtype of imidazoline receptors (I1R) is a plasma membrane protein that is involved in diverse physiological functions. Available radioligands used so far to characterize the I(1)R were able to bind with similar affinities to alpha2-adrenergic receptors (alpha2-ARs) and to I1R. This feature was a major drawback for an adequate characterization of this receptor subtype. New imidazoline analogs were therefore synthesized and the present study describes one of these compounds, 2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), which was of high affinity and selectivity for the I1R. LNP 911 was radioiodinated and its binding properties characterized in different membrane preparations. Saturation experiments with [125I]LNP 911 revealed a single high affinity binding site in PC-12 cell membranes (K(D) = 1.4 nM; B(max) = 398 fmol/mg protein) with low nonspecific binding. [125I]LNP 911 specific binding was inhibited by various imidazolines and analogs but was insensitive to guanosine-5'-O-(3-thio)triphosphate. The rank order of potency of some competing ligands [LNP 911, PIC, rilmenidine, 4-chloro-2-(imidazolin-2-ylamino)-isoindoline (BDF 6143), lofexidine, and clonidine] was consistent with the definition of [125I]LNP 911 binding sites as I1R. However, other high-affinity I1R ligands (moxonidine, efaroxan, and benazoline) exhibited low affinities for these binding sites in standard binding assays. In contrast, when [125I]LNP 911 was preincubated at 4 degrees C, competition curves of moxonidine became biphasic. In this case, moxonidine exhibited similar high affinities on [125I]LNP 911 binding sites as on I1R defined with [125I]PIC. Moxonidine proved also able to accelerate the dissociation of [125I]LNP 911 from its binding sites. These results suggest the existence of an allosteric modulation at the level of the I1R, which seems to be corroborated by the dose-dependent enhancement by LNP 911 of the agonist effects on the adenylate cyclase pathway associated to I1R. Because [125I]LNP 911 was unable to bind to the I2 binding site and alpha2AR, our data indicate that [125I]LNP 911 is the first highly selective radioiodinated probe for I1R with a nanomolar affinity. This new tool should facilitate the molecular characterization of the I1 imidazoline receptor.     

Establishment of robust functional assays for the characterization of neuropeptide Y (NPY) receptors: identification of 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile as selective NPY type 5 receptor antagonist

The human Neuropeptide Y (NPY) receptors 1 (hY1), 2 (hY2), 4 (hY4), and the mouse type 5 (mY5) receptor were expressed in human embryonic kidney 293 (HEK293) cells. The receptors bound a radioiodinated NPY ligand with high affinity and various NPY analogs competed for binding in a receptor selective-manner. Similarly, cAMP-inhibition and GTPgammaS binding assays were established. The four NPY receptors were further tested in the fluorimetric imaging plate reader (FLIPR) format, a cellular high-throughput assay, in the absence and presence of chimeric G proteins, Gqo5, Gqi5 and Gqi9. The receptors stimulated transient calcium release only in the presence of chimeric G proteins. While hY1, hY2 and hY4 receptors coupled to Gqo5, Gqi5 and Gqi9, the mY5 receptor stimulated transient calcium release only when co-expressed with Gqi9. Using an in silico screening approach we identified a small molecule 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile (compound 1), which bound to the mY5 receptor with high affinity (Ki=32.1+/-1.8 nM), competitively antagonized NPY-mediated GTPgammaS binding and calcium stimulation with high potency, and had no affinity for other NPY receptors. These data show that NPY receptors can be functionally coupled to the FLIPR readout, allowing for high throughput compound testing and identification of novel molecules.     

In vitro and in vivo characterization of 3-[2-[6-(2-tert-butoxyethoxy)pyridin-3-yl]-1H-imidazol-4-yl]benzonitrile hydrochloride salt,a potent and selective NPY5 receptor antagonist

To investigate the anorectic potential of NPY5 receptor antagonists, we have profiled the in vitro and in vivo properties of 3-[2-[6-(2-tert-butoxyethoxy)pyridin-3-yl]-1H-imidazol-4-yl]benzonitrile hydrochloride salt (1). This compound was found to have excellent NPY5 receptor affinity and selectivity, potent functional antagonism, and good peripheral and central nervous system exposure in rats. This compound attenuated bovine pancreatic polypeptide induced food intake in rats but failed to demonstrate anorectic activity in rodent natural feeding models.     

Discovery of N1-(6-chloroimidazo[2,1-b][1,3]thiazole-5-sulfonyl)tryptamine as a potent, selective, and orally active 5-HT(6) receptor agonist

N1-Arylsulfonyltryptamines have been identified as 5-HT6 receptor ligands. In particular, N1-(6-chloroimidazo[2,1-b][1,3]thiazole-5-sulfonyl)tryptamine (11q) is a high affinity, potent full agonist (5-HT6 Ki = 2 nM, EC50 = 6.5 nM, Emax = 95.5%). Compound 11q is selective in a panel of over 40 receptors and ion channels, has good pharmacokinetic profile, has been shown to increase GABA levels in the rat frontal cortex, and is active in the schedule-induced polydipsia model for obsessive compulsive disorders.     

In vivo pharmacology of [beta Ala8]neurokinin A-(4-10), a selective NK-2 tachykinin receptor agonist

We studied the effect of [beta Ala8]neurokinin A-(4-10), a newly developed selective NK-2 tachykinin receptor agonist, on various parameters in anaesthetized rats (blood pressure, urinary bladder motility, plasma extravasation) and guinea-pigs (salivation, increase of pulmonary insufflation pressure) as compared to the response produced by tachykinins. [beta Ala8]Neurokinin A-(4-10) was as active as, or more active than, neurokinin A (NKA) or NKA-(4-10) in producing rat bladder contraction or bronchospasm in guinea-pigs, two effects known to involve activation of NK-2 receptors. On the other hand, the synthetic peptide was weakly active, if active at all, in producing hypotension or plasma extravasation in the rat bladder as well as salivation in guinea-pigs, effects known to involve activation of NK-1 receptors. These findings provide evidence that [beta Ala8]NKA-(4-10) acts as a selective NK-2 agonist in vivo and that it can be used to explore the distribution and function of NK-2 receptors.     

p-[125I]iodoclonidine is a partial agonist at the alpha 2-adrenergic receptor

The binding properties of p-[125I]iodoclonidine [( 125I]PIC) to human platelet membranes and the functional characteristics of PIC are reported. [125I]PIC bound rapidly and reversibly to platelet membranes, with a first-order association rate constant (kon) at room temperature of 8.0 +/- 2.7 x 10(6) M-1 sec-1 and a dissociation rate constant (koff) of 2.0 +/- 0.8 x 10(-3) sec-1. Scatchard plots of specific [125I]PIC binding (0.1-5 nM) were linear, with a Kd of 1.2 +/- 0.1 nM. [125I]PIC bound to the same number of high affinity sites as the alpha 2-adrenergic receptor (alpha 2-AR) full agonist [3H] bromoxidine (UK14,304), which represented approximately 40% of the sites bound by the antagonist [3H]yohimbine. Guanosine 5'-(beta, gamma-imido)triphosphate greatly reduced the amount of [125I]PIC bound (greater than 80%), without changing the Kd of the residual binding. In competition experiments, the alpha 2-AR-selective ligands yohimbine, bromoxidine, oxymetazoline, clonidine, p-aminoclonidine, (-)-epinephrine, and idazoxan all had Ki values in the low nanomolar range, whereas prazosin, propranolol, and serotonin yielded Ki values in the micromolar range. Epinephrine competition for [125I]PIC binding was stereoselective. Competition for [3H]bromoxidine binding by PIC gave a Ki of 1.0 nM (nH = 1.0), whereas competition for [3H]yohimbine could be resolved into high and low affinity components, with Ki values of 3.7 and 84 nM, respectively. PIC had minimal agonist activity in inhibiting adenylate cyclase in platelet membranes, but it potentiated platelet aggregation induced by ADP with an EC50 of 1.5 microM. PIC also inhibited epinephrine-induced aggregation, with an IC50 of 5.1 microM. Thus, PIC behaves as a partial agonist in a human platelet aggregation assay. [125I]PIC binds to the alpha 2B-AR in NG-10815 cell membranes with a Kd of 0.5 +/- 0.1 nM. [125I]PIC should prove useful in binding assays involving tissues with a low receptor density or in small tissue samples and in studies of cloned and expressed alpha 2-AR.