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Potent small molecule antagonists for the PAC(1)-R have been discovered. Previously known antagonists for the PAC(1)-R were slightly truncated peptide ligands. The hydrazides reported here are the first small molecule antagonists ever reported for this class B GPCR.

The pituitary adenylate cyclase-activating polypeptide (PACAP) receptor is a class II G protein-coupled receptor that contributes to many different cellular functions including neurotransmission, neuronal survival, and synaptic plasticity. The solution structure of the potent antagonist PACAP (residues 6′–38′) complexed to the N-terminal extracellular (EC) domain of the human splice variant hPAC1-R-short (hPAC1-R S ) was determined by NMR. The PACAP peptide adopts a helical conformation when bound to hPAC1-R S with a bend at residue A18′ and makes extensive hydrophobic and electrostatic interactions along the exposed β-sheet and interconnecting loops of the N-terminal EC domain. Mutagenesis data on both the peptide and the receptor delineate the critical interactions between the C terminus of the peptide and the C terminus of the EC domain that define the high affinity and specificity of hormone binding to hPAC1-R S . These results present a structural basis for hPAC1-R S selectivity for PACAP versus the vasoactive intestinal peptide and also differentiate PACAP residues involved in binding to the N-terminal extracellular domain versus other parts of the full-length hPAC1-R S receptor. The structural, mutational, and binding data are consistent with a model for peptide binding in which the C terminus of the peptide hormone interacts almost exclusively with the N-terminal EC domain, whereas the central region makes contacts to both the N-terminal and other extracellular parts of the receptor, ultimately positioning the N terminus of the peptide to contact the transmembrane region and result in receptor activation.

The goal of this study was to identify a structurally distinct D(4)-selective agonist with superior oral bioavailability to our first-generation clinical candidate 1a (ABT-724) for the potential treatment of erectile dysfunction. Arylpiperazines such as (heteroarylmethyl)piperazine 1a, benzamide 2, and acetamides such as 3a,b exhibit poor oral bioavailability. Structure-activity relationship (SAR) studies with the arylpiperidine template provided potent partial agonists such as 4d and 5k that demonstrated no improvement in oral bioavailability. Further optimization with the (N-oxy-2-pyridinyl)piperidine template led to the discovery of compound 6b (ABT-670), which exhibited excellent oral bioavailability in rat, dog, and monkey (68%, 85%, and 91%, respectively) with comparable efficacy, safety, and tolerability to 1a. The N-oxy-2-pyridinyl moiety not only provided the structural motif required for agonist function but also reduced metabolism rates. The SAR study leading to the discovery of 6b is described herein.

We have discovered a novel, potent, and selective triazafluorenone series of metabotropic glutamate receptor 1 (mGluR1) antagonists with efficacy in various rat pain models. Pharmacokinetic and pharmacodynamic profiles of these triazafluorenone analogs revealed that brain/plasma ratios of these mGluR1 antagonists were important to achieve efficacy in neuropathic pain models. This correlation could be used to guide our in vivo SAR (structure–activity relationship) modification. For example, compound 4a has a brain/plasma ratio of 0.34, demonstrating only moderate efficacy in neuropathic pain models. On the other hand, antagonist 4b with a brain/plasma ratio of 2.70 was fully efficacious in neuropathic pain models.

SAR (structure-activity relationship) studies of triazafluorenone derivatives as potent mGluR1 antagonists are described. The triazafluorenone derivatives are non-amino acid derivatives and noncompetitive mGluR1 antagonists that bind at a putative allosteric recognition site located within the seven-transmembrane domain of the receptor. These triazafluorenone derivatives are potent, selective, and systemically active mGluR1 antagonists. Compound 1n, for example, was a very potent mGluR1 antagonist (IC50 = 3 nM) and demonstrated full efficacy in various in vivo animal pain models.

Agents that activate the dopamine D2-like family of receptors elicit emesis in humans and other species with a vomiting/emetic reflex; however, the lack of dopamine receptor subtype selective agonists has hampered an understanding of which dopamine D2-like receptor subtype(s) contributes to the emetic response. In this study, stable cell lines expressing the ferret dopamine D2-long (D2L) and D4 receptors were used to characterize known dopamine agonists via radioligand binding and calcium ion flux assays, while emetic activity of these dopamine receptor agonists was determined in male ferrets. Latencies to first emetic event, average number of emetic episodes, and stereotypical behaviors which may be indicative of nausea were also determined. Agonists at dopamine D1-like and D4 receptors had no emetic effect in ferrets. Conversely, stimulation of dopamine D2 and/or D3 receptors resulted in a robust emetic response characterized by a relatively short latency (

The first selective dopamine D4 agonist radioligand is described. The synthesis of these piperazine radioligands relied on the transformation of brominated precursors 4a and 4b with tritium gas in the presence of a sensitive cyano functional group. The specific activity of these two radioligands was measured and [3H]6b found to be suitable for use in D4 saturation and competition binding studies. The synthesis, biological, and radioactivity of this new agonist radioligand as well as preliminary SAR will be discussed.

The goal of this study was to develop a new approach to study the pharmacology of the dopamine D 4 receptor that could be used in comparative studies with dopamine D 2 and D 3 receptors. Stable HEK-293 cell lines co-expressing recombinant human D 2L , D 3 or D 4 receptors along with Gα qo5 cDNA were prepared. Dopamine induced a robust, transient calcium signal in these cell lines with EC 50 s for D 2L , D 3 and D 4 of 18.0, 11.9 and 2.2 nM, respectively. Reported D 4 -selective agonists CP226269 and PD168077 were potent, partial D 4 agonists exhibiting 31–1700-fold selectivity for D 4 over D 3 or D 2 . Non-selective D 2 -like agonists apomorphine and quinpirole showed full efficacy but did not discriminate across the three receptors. D 3 -selective agonists 7-hydroxy-DPAT and PD128907 were potent but non-selective D 2 -like agonists. The reported D 3 partial agonist BP-897 exhibited minimal agonist activity at D 3 but was a potent D 3 antagonist and a partial D 4 agonist. Other D 2 -like antagonists, haloperidol, clozapine, and domperidone showed concentration-dependent inhibition of dopamine responses at all three receptors with K i ranging from 0.05 to 48.3 nM. The D 3 selective antagonist S33084 and D 4 -selective antagonist L-745870 were highly selective for D 3 and D 4 receptors with K b of 0.7 and 0.1 nM, respectively. Stable co-expression of D 2 -like receptors with chimeric Gα qo5 proteins in HEK-293 cells is an efficient method to study receptor activation in a common cellular background and an efficient method for direct comparison of ligand affinity and efficacy across human D 2L , D 3 and D 4 receptors.

Tritiation of the dopamine D(4) receptor selective agonist A-369508 ([2-[4-(2-cyanophenyl)-1-piperazinyl]-N-(3-methylphenyl) acetamide) has provided a radioligand for the characterization of dopamine D(4) receptors. [(3)H] A-369508 binds with high affinity to the major human dopamine D(4) receptor variants D(4.2), D(4.4) and D(4.7) (K(d)=1.7, 4, and 1.2 nM, respectively). It also binds to the rat dopamine D(4) receptor, (K(d)=4.4 nM), implying similar binding affinity across human and rat receptors. A-369508 shows400-fold selectivity over D(2L),350-fold selectivity over 5-HT(1A) and700-1,000-fold selectivity over all other receptors tested. Agonist activity determined by inhibition of forskolin-induced cAMP in Chinese hamster ovary cells transfected with the human dopamine D(4.4) receptor (EC(50)=7.5 nM, intrinsic activity=0.71) indicates that A-369508 is a potent agonist at the human dopamine D(4) receptor. Similar data was observed in other functional assays. [(3)H] A-369508 binds to a single, high affinity site on membranes containing the human dopamine D(4.4) receptor. When compared to the D(2)-like antagonist [(3)H] spiperone, competition binding for agonists like dopamine and apomorphine were 2-10-fold more potent with [(3)H] A-369508, while the antagonists clozapine, haloperidol and L-745870 bind with similar affinity to both ligands. Binding to rat brain regions demonstrated that the most abundant area was cerebral cortex (51.2 fmol/mg protein) followed by hypothalamus, hippocampus, striatum and cerebellum. [(3)H] A-369508 is a useful tool to define the localization and physiological role of dopamine D(4) receptors in central nervous system and can facilitate measuring accurate affinities (K(i)) for structure/activity relationship studies designed to identify dopamine D(4) receptor selective agonists.

Rapid desensitization of ligand-gated ion channel receptors can alter the apparent activity of receptor modulators, as well as make detection of fast-channel activation difficult. Investigation of the antagonist pharmacology of ATP-sensitive homomeric P2X3 receptors is limited by agonist-evoked fast-desensitization kinetics. In the present studies, chimeric receptors were created using the coding sequence for the N-terminus and the first transmembrane domain of either the nondesensitizing human P2X2a or fast-desensitizing P2X3 receptor joined to the sequence encoding the extracellular loop, second transmembrane domain, and C-terminus of the other receptor (designated P2X2–3 and P2X3–2, respectively). These clones were stably transfected into 1321N1 astrocytoma cells for biophysical and pharmacological experiments using both electrophysiological and calcium-imaging methods. Chimeric P2X2–3 and P2X3–2 receptors were inwardly rectifying and agonist responses showed desensitization properties similar to the wild-type human P2X2a and P2X3 receptors, respectively. The P2X2–3 chimera displayed an agonist pharmacological profile similar to the P2X3 wild-type receptor being activated by low concentrations of both ATP and α,β-meATP. In contrast, the P2X3–2 chimera had markedly reduced sensitivity to both agonists. The P2X3 receptor antagonists TNP-ATP and A-317491 were shown to be potent, competitive antagonists of the P2X2–3 chimera (Ki=2.2 and 52.1 nM, respectively), supporting the hypothesis that rapid receptor desensitization can mask the competitive antagonism of wild-type homomeric P2X3 receptors. British Journal of Pharmacology (2003) 140, 202–210. doi:10.1038/sj.bjp.0705411

Thrombopoietin (TPO) is a primary regulator of megakaryocytopoiesis, a process through which megakaryocytes proliferate and mature into platelets. Recombinant human TPO (rhTPO) was expressed in Chinese hamster ovary (CHO) cells and purified from the culture medium. The cDNA encoding full-length TPO, including the native signal peptide sequence, was amplified by PCR from a human fetal liver cDNA library. The product was cloned into a mammalian expression vector under the control of the SV40 early promoter and enhancer. Secreted rhTPO was purified in three conventional chromatography steps. It migrates on SDS-PAGE as a broad band, characteristic of a heavily glycosylated protein, with an average molecular mass of 85 kDa. rhTPO expressed in CHO cells is biologically active in vitro as demonstrated by its ability to stimulate the proliferation of a megakaryocytic cell line and to trigger the JAK/STAT signal transduction pathway. rhTPO also shows activity in vivo as judged by the elevation of platelet count in treated mice.

A new series of dopamine D4 receptor agonists, 1-aryl-3-(4-pyridinepiperazin-1-yl)propanone oximes, was designed through the modification of known dopamine D4 receptor agonist PD 168077. Replacement of the amide group with a methylene-oxime moiety produced compounds with improved stability and efficacy. Structure-activity relationsips (SAR) of the aromatic ring linked to the N-4-piperazine ring confirmed the superiority of 2-pyridine as a core for D4 agonist activity. A two-methylene linker between the oxime group and the N-1-piperazine ring displayed the best profile. New dopamine D4 receptor agonists, exemplified by (E)-1-(4-chlorophenyl)-3-(4-pyridin-2-ylpiperazin-1-yl)propan-1-one O-methyloxime (59a) and (E)-1-(3-chloro-4-fluorophenyl)-3-(4-pyridin-2-ylpiperazin-1-yl)propan-1-one O-methyloxime (64a), exhibited favorable pharmacokinetic profiles and showed oral bioavailability in rat and dog. Subsequent evaluation of 59a in the rat penile erection model revealed in vivo activity, comparable in efficacy to apomorphine. Our results suggest that the oximes provide a novel structural linker for 4-arylpiperazine-based D4 agonists, possessing leadlike quality and with potential to develop a new class of potent and selective dopamine D4 receptor agonists.

A series of 3-aryl piperidine analogs with 2-piperidinoalkylamino or 2-piperidinoalkyloxy fused bicyclic rings were prepared and found to be potent and efficacious human dopamine D 4 agonists. The synthesis and structure–activity relationship (SAR) studies that led to the identification of these compounds are discussed.

Agents that activate the dopamine D2-like family of receptors elicit emesis in humans and other species with a vomiting/emetic reflex; however, the lack of dopamine receptor subtype selective agonists has hampered an understanding of which dopamine D2-like receptor subtype(s) contributes to the emetic response. In this study, stable cell lines expressing the ferret dopamine D2-long (D2L) and D4 receptors were used to characterize known dopamine agonists via radioligand binding and calcium ion flux assays, while emetic activity of these dopamine receptor agonists was determined in male ferrets. Latencies to first emetic event, average number of emetic episodes, and stereotypical behaviors which may be indicative of nausea were also determined. Agonists at dopamine D1-like and D4 receptors had no emetic effect in ferrets. Conversely, stimulation of dopamine D2 and/or D3 receptors resulted in a robust emetic response characterized by a relatively short latency (15 min) and multiple emetic events. Competitive antagonists of dopamine D2-like receptors (domperidone, haloperidol) dose-dependently blocked the emetic response to PNU95666E, a dopamine D2 receptor selective agonist. Thus, dopamine D2 and/or D3 receptor agonists elicit emesis, while dopamine D1/D5 or D4 receptor-selective agonists are devoid of emetic properties.

2-[4-(3,4-Dimethylphenlyl)piperazin-1-ylmethyl]-1H benzoimidazole (A-381393) was identified as a potent dopamine D4 receptor antagonist with excellent receptor selectivity. [3H]-spiperone competition binding assays showed that A-381393 potently bound to membrane from cells expressing recombinant human dopamine D4.4 receptor (Ki=1.5 nM), which was 20-fold higher than that of clozapine (Ki=30.4 nM). A-381393 exhibited highly selective binding for the dopamine D4.4 receptor (>2700-fold) when compared to D1, D2, D3 and D5 dopamine receptors. Furthermore, in comparison to clozapine and L-745870, A-381393 exhibits better receptor selectivity, showing no affinity up to 10 microM for a panel of more than 70 receptors and channels, with the exception of moderate affinity for 5-HT2A (Ki=370 nM). A-381393 potently inhibited the functional activity of agonist-induced GTP-gamma-S binding assay and 1 microM dopamine induced-Ca2+ flux in human dopamine D4.4 receptor expressing cells, but not in human dopamine D2L or D3 receptor cells. In contrast to L-745870, A-381393 did not exhibit any significant intrinsic activity in a D4.4 receptor. In vivo, A-381393 has good brain penetration after subcutaneous administration. A-381393 inhibited penile erection induced by the selective D4 agonist PD168077 in conscious rats. Thus, A-381393 is a novel selective D4 antagonist that will enhance the ability to study dopamine D4 receptors both in vitro and in vivo.

A new class of agents with potential utility for the treatment of erectile dysfunction has been discovered, guided by the hypothesis that selective D4 agonists are erectogenic but devoid of the side effects typically associated with dopaminergic agents. The lead agent 2-(4-pyridin-2-ylpiperazin-1-ylmethyl)-1H-benzimidazole (1, ABT-724) was discovered by optimization of a series of benzimidazole arylpiperazines. This highly selective D4 agonist was found to be very potent and efficacious in vivo, eliciting penile erections in rats at a dose of 0.03 micromol/kg, with a positive response rate of 77% erectile incidence. Even at high doses, it was devoid of side effects in animal models of central nervous system behaviors, emesis, or nausea. The structure-activity relationship of the parent benzimidazole series leading to 1 is described, with the detailed in vitro and in vivo profiles described. Distinctive structural features were discovered that are associated with D4 selective agonism in this series of analogues.

Diaryl piperazine acetamides were identified as potent and selective dopamine D(4) receptor agonists. Our strategy is based on an amide bond reversal of an acid sensitive, dopamine D(4) receptor partial agonist, PD 168077. This reversal provided compounds with excellent potency and improved stability. Systematic evaluation of the substitution on the aryl piperazine portion revealed a significant effect on functional activity. The synthesis and biological activity of these new dopamine D(4) agonists is discussed.

(1) Rapid desensitization of ligand-gated ion channel receptors can alter the apparent activity of receptor modulators, as well as make detection of fast-channel activation difficult. Investigation of the antagonist pharmacology of ATP-sensitive homomeric P2X3 receptors is limited by agonist-evoked fast-desensitization kinetics. (2) In the present studies, chimeric receptors were created using the coding sequence for the N-terminus and the first transmembrane domain of either the nondesensitizing human P2X2a or fast-desensitizing P2X3 receptor joined to the sequence encoding the extracellular loop, second transmembrane domain, and C-terminus of the other receptor (designated P2X2-3 and P2X3-2, respectively). These clones were stably transfected into 1321N1 astrocytoma cells for biophysical and pharmacological experiments using both electrophysiological and calcium-imaging methods. (3) Chimeric P2X2-3 and P2X3-2 receptors were inwardly rectifying and agonist responses showed desensitization properties similar to the wild-type human P2X2a and P2X3 receptors, respectively. (4) The P2X2-3 chimera displayed an agonist pharmacological profile similar to the P2X3 wild-type receptor being activated by low concentrations of both ATP and alpha,beta-meATP. In contrast, the P2X3-2 chimera had markedly reduced sensitivity to both agonists. (5) The P2X3 receptor antagonists TNP-ATP and A-317491 were shown to be potent, competitive antagonists of the P2X2-3 chimera (Ki=2.2 and 52.1 nm, respectively), supporting the hypothesis that rapid receptor desensitization can mask the competitive antagonism of wild-type homomeric P2X3 receptors.

Chemical modification of cell proliferation and fluid secretion in renal cysts. We used an in vitro model, MDCK cyst, to determine the extent to which pharmacologic compounds known to inhibit plasma membrane solute transport mechanisms could alter the enlargement of renal epithelial cysts. Solitary MDCK cells cultured within collagen gel undergo clonal growth to form true epithelial cysts in which a single layer of polarized cells (apex toward lumen) encloses a fluid-filled cavity. Repeated observations by light microscopy were used to quan-titate the rate of cyst growth in diameter, and demonstrated that cyst enlargement involved an increase in cell number (proliferation) and a net increase in intracystic volume (fluid secretion). Intracyst pressure was greater than the interstitium (6.7mm H2O ± 3.1SD), indicating that fluid entry was secondary to net solute accumulation. Amiloride and seven amiloride analogs that inhibited to different degrees conductive Na+ transport, Na+-dependent H+ transport and Na+-dependent Ca++ transport reversibly decreased the rate of cyst enlargement. The effectiveness of these agents to retard cyst enlargement correlated with their relative potencies to inhibit Na+-dependent Ca++ transport. Morphologic examination indicated that amiloride and amiloride analogs decreased cell proliferation and fluid secretion to the same degree. Ouabain and vanadate (Na+K,ATPase inhibitors), and L-645,695 (Na+-dependent Cl−/HCO3− inhibitor) potently slowed cyst expansion. In contrast to amiloride and amiloride analogs, these agents caused an unusual degree of cellular stratification within the cyst walls, a finding consistent with the notion that fluid secretion was inhibited to a greater extent then cellular proliferation. We conclude that chemical inhibitors of primary and secondary active solute transport can diminish or halt the enlargement of epithelial cysts in vitro by decreasing the rate of cellular proliferation and/or net fluid secretion.

Transepithelial fluid secretion has been postulated to account for the accumulation of fluid within hereditary and acquired renal cysts, but no such mechanism has been demonstrated in human kidney epithelium. It is shown here that transepithelial fluid secretion was stimulated by prostaglandin E1 (PGE1), forskolin, 8-Br-cyclic AMP, and 1-methyl-3-isobutylxanthine in polarized monolayers of established renal cell lines (MDCK and rat glomerular epithelial cells) and in monolayer cultures derived from the cyst walls of human autosomal dominant polycystic kidney disease and from epithelial cells of normal human renal cortex. Treatment with cyclic AMP agonists caused the same cells, when dispersed within a gel matrix of type I collagen (Vitrogen), to proliferate and form spherical fluid-filled monolayered cysts. Our findings suggest that increased intracellular cyclic AMP levels may have a critical role in the formation and expansion of hereditary and acquired renal cysts.

The enzymatic polymerization by polynucleotide phosphorylase of 6-chloro-9-(beta-D-ribofuranosyl)purine 5'-diphosphate to poly(6-chloropurinylic acid) and its conversion to poly(6-thioninosinic acid) is described. The sulfur isostere of poly(I) was found not to form a complex with poly(C), but to form a self-association complex with a Tm around 295 degrees K. The sedimentation velocities, pKa and Tm values of the polymer have been examined under various conditions. A two (or more) stranded helical array is suggested as the most probable structure. Thermal loss of the thione chromophore was noted for poly- (S6I), S6IMP and S6I; the degradation product from S6I was shown to be inosine.

Cysts, a common abnormality of kidneys, are collections of urine-like fluid enclosed by a continuous layer of epithelial cells. Renal cysts derive from nephrons and collecting ducts and progressively enlarge as a consequence of epithelial proliferation and transepithelial fluid secretion. The initiation of cyst formation and the factors that control cyst enlargement are unknown. We used an in vitro model of renal cysts to explore the role of the cAMP signal transduction system in the formation and expansion of cysts. MDCK cells, cultured in hydrated-collagen gel, produced polarized monolayered epithelial cysts when intracellular cAMP was increased by prostaglandin E1, arginine vasopressin, cholera toxin, forskolin, or 8-bromoadenosine 3',5'-cyclic monophosphate. All agonists were potentiated by 3-isobutyl-1-methylxanthine, a nucleotide phosphodiesterase inhibitor. The cell proliferation component of cyst enlargement was accelerated by cAMP agonists, as shown by the increased growth of MDCK cells in subconfluent monolayers. The fluid secretion component, reflected by the transepithelial movement of fluid across polarized monolayers of MDCK cells grown on permeable supports, was stimulated by cAMP agonists in the basolateral medium. Chloride levels were higher in the cyst fluid and the secreted fluid than in the bathing medium. We conclude that the development of MDCK cysts is dependent on cAMP. This signal transduction system may be an important modulator of epithelial cell proliferation and transepithelial fluid secretion in the kidney.

The enzymatic synthesis of a high molecular weight (s 20,w ∼10) copolymer of inosinic and 6-thioinosinic acids [poly(I:s 6 I)] has been achieved. Poly (I:s 6 I) forms a double stranded complex with poly C which has a Tm significantly lower than a poly I · ply C complex of equivalent molecular weight.