Tetrahydro-4-quinolinamines identified as novel P2Y(1) receptor antagonists.

High-throughput screening of the GSK compound collection against the P2Y(1) receptor identified a novel series of tetrahydro-4-quinolinamine antagonists. Optimal substitution around the piperidine group was pivotal for ensuring activity. An exemplar analog from this series was shown to inhibit platelet aggregation.

Potent and selective small-molecule human urotensin-II antagonists with improved pharmacokinetic profiles.

Lead compound 1 was successfully redesigned to provide compounds with improved pharmacokinetic profiles for this series of human urotensin-II antagonists. Replacement of the 2-pyrrolidinylmethyl-3-phenyl-piperidine core of 1 with a substituted N-methyl-2-(1-pyrrolidinyl)ethanamine core as in compound 7 resulted in compounds with improved oral bioavailability in rats. The relationship between stereochemistry and selectivity for hUT over the kappa-opioid receptor was also explored.

Development of potent and selective small-molecule human Urotensin-II antagonists.

This work describes the development of potent and selective human Urotensin-II receptor antagonists starting from lead compound 1, (3,4-dichlorophenyl)methyl{2-oxo-2-[3-phenyl-2-(1-pyrrolidinylmethyl)-1-piperidinyl]ethyl}amine. Several problems relating to oral bioavailability, cytochrome P450 inhibition, and off-target activity at the kappa opioid receptor and cardiac sodium channel were addressed during lead development. hUT binding affinity relative to compound 1 was improved by more than 40-fold in some analogs, and a structural modification was identified which significantly attenuated both off-target activities.

The peptidic urotensin-II receptor ligand GSK248451 possesses less intrinsic activity than the low-efficacy partial agonists SB-710411 and urantide in native mammalian tissues and recombinant cell systems.

Several peptidic urotensin-II (UT) receptor antagonists exert 'paradoxical' agonist activity in recombinant cell- and tissue-based bioassay systems, likely the result of differential urotensin-II receptor (UT receptor) signal transduction/coupling efficiency between assays. The present study has examined this phenomenon in mammalian arteries and recombinant UT-HEK (human embryonic kidney) cells.BacMam-mediated recombinant UT receptor upregulation in HEK cells augmented agonist activity for all four peptidic UT ligands studied. The nominal rank order of relative intrinsic efficacy was U-II>urantide ([Pen(5)-DTrp(7)-Orn(8)]hU-II(4-11))>SB-710411 (Cpa-c[DCys-Pal-DTrp-Lys-Val-Cys]-Cpa-amide)>>GSK248451 (Cin-c[DCys-Pal-DTrp-Orn-Val-Cys]-His-amide) (the relative coupling efficiency of recombinant HEK cells was cat>human>>rat UT receptor). The present study further demonstrated that the use of high signal transduction/coupling efficiency isolated blood vessel assays (primate>cat arteries) is required in order to characterize UT receptor antagonism thoroughly. This cannot be attained simply by using the rat isolated aorta, an artery with low signal transduction/coupling efficiency in which low-efficacy agonists appear to function as antagonists. In contrast to the 'low-efficacy agonists' urantide and SB-710411, GSK248451 functioned as a potent UT receptor antagonist in all native isolated tissues studied (UT receptor selectivity was confirmed in the rat aorta). Further, GSK248451 exhibited an extremely low level of relative intrinsic activity in recombinant HEK cells (4-5-fold less than seen with urantide). Since GSK248451 (1 mg kg(-1), i.v.) blocked the systemic pressor actions of exogenous U-II in the anaesthetized cat, it represents a suitable peptidic tool antagonist for delineating the role of U-II in the aetiology of mammalian cardiometabolic diseases.

Identification and characterization of binding sites for human urotensin-II in Sprague-Dawley rat renal medulla using quantitative receptor autoradiography.

Urotensin-II (U-II), a ligand for the G-protein-coupled receptor UT, has been characterized as the most potent mammalian vasoconstrictor identified to date. Although circulating levels of U-II are altered in lower species (e.g., fish) upon exposure to hypo-osmotic stress, little is known about the actions of this cyclic undecapeptide within the kidney, an organ that plays a pivotal role in the control of cardiovascular homeostasis, influencing both cardiac preload (plasma volume) and after load (peripheral resistance). The present study reports the identification of specific, high affinity [125I]hU-II binding sites in Sprague-Dawley rat kidney outer medulla by autoradiography and also through membrane radioligand binding (Kd 1.9 +/- 0.9 nM and Bmax 408 +/- 47 amol mm(-2) and Kd 1.4 +/- 0.3 nM and Bmax 51.3 +/- 7.8 fmol mg(-1) protein, respectively). Differences were observed in the binding characteristics within rat strains. Compared to the Sprague-Dawley, Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rat kidney outer medulla displayed low density < 20 fmol mg(-1) protein and low affinity (> 1 microM) [125I]hU-II binding sites. Thus, the relative contribution of specific U-II binding sites to the physiological actions of U-II in the control of cardiorenal homeostasis is worthy of further investigation.

Inhibitory effects of putative peptidic urotensin-II receptor antagonists on urotensin-II-induced contraction of cat isolated respiratory smooth muscle.

Urotensin-II is purported to influence pulmonary function by modulating smooth muscle tone/growth. In the present study, Northern blot and reverse transcription polymerase chain reaction (RT-PCR) analysis indicated the presence of UT receptor mRNA in cat trachea, bronchi and lung parenchyma. Urotensin-II contracted cat isolated trachea and bronchi with similar potencies (pEC(50)s 8.61+/-0.07-8.81+/-0.10). Contractile efficacies ranged from 19+/-9% to 63+/-11% KCl in the primary and secondary bronchi. The peptidic UT receptor antagonists BIM-23127, SB-710411 and GSK248451 (7.18+/-0.12, 7.52+/-0.08 and 9.05+/-0.16 cat recombinant UT pK(i)s) inhibited urotensin-II-induced contraction of cat isolated trachea with pK(b)s 6.36+/-0.11, 6.74+/-0.07 and 9.27+/-0.12, respectively. As such, feline lung contains significant amounts of UT mRNA and this receptor appears to be functionally coupled to bronchoconstriction (the peptidic tool compound GSK248451 representing a sub-nanomolar inhibitor of such effects). These findings suggest that the cat represents a suitable species for future studies designed to assess the effects of the urotensin-II receptor on pulmonary (patho)physiology.

Nonpeptidic urotensin-II receptor antagonists I: in vitro pharmacological characterization of SB-706375.

1. SB-706375 potently inhibited [(125)I]hU-II binding to both mammalian recombinant and 'native' UT receptors (K(i) 4.7+/-1.5 to 20.7+/-3.6 nM at rodent, feline and primate recombinant UT receptors and K(i) 5.4+/-0.4 nM at the endogenous UT receptor in SJRH30 cells). 2. Prior exposure to SB-706375 (1 microM, 30 min) did not alter [(125)I]hU-II binding affinity or density in recombinant cells (K(D) 3.1+/-0.4 vs 5.8+/-0.9 nM and B(max) 3.1+/-1.0 vs 2.8+/-0.8 pmol mg(-1)) consistent with a reversible mode of action. 3. The novel, nonpeptidic radioligand [(3)H]SB-657510, a close analogue of SB-706375, bound to the monkey UT receptor (K(D) 2.6+/-0.4 nM, B(max) 0.86+/-0.12 pmol mg(-1)) in a manner that was inhibited by both U-II isopeptides and SB-706375 (K(i) 4.6+/-1.4 to 17.6+/-5.4 nM) consistent with the sulphonamides and native U-II ligands sharing a common UT receptor binding domain. 4. SB-706375 was a potent, competitive hU-II antagonist across species with pK(b) 7.29-8.00 in HEK293-UT receptor cells (inhibition of [Ca(2+)](i)-mobilization) and pK(b) 7.47 in rat isolated aorta (inhibition of contraction). SB-706375 also reversed tone established in the rat aorta by prior exposure to hU-II (K(app) approximately 20 nM). 5. SB-706375 was a selective U-II antagonist with >/=100-fold selectivity for the human UT receptor compared to 86 distinct receptors, ion channels, enzymes, transporters and nuclear hormones (K(i)/IC(50)>1 microM). Accordingly, the contractile responses induced in isolated aortae by KCl, phenylephrine, angiotensin II and endothelin-1 were unaltered by SB-706375 (1 microM). 6. In summary, SB-706375 is a high-affinity, surmountable, reversible and selective nonpeptide UT receptor antagonist with cross-species activity that will assist in delineating the pathophysiological actions of U-II in mammals.

Differential agonistic and antagonistic effects of the urotensin-II ligand SB-710411 at rodent and primate UT receptors.

SB-710411 (Cpa-c[d-Cys-Pal-d-Trp-Lys-Val-Cys]-Cpa-amide) inhibited [(125)I]urotensin-II rat and monkey UT receptor binding (pK(i)s 7.50+/-0.07 and 6.82+/-0.06). However, whereas SB-710411 antagonized urotensin-II-induced inositol phosphate formation at the rat UT receptor (pK(b) 6.54+/-0.05), this ligand functioned as an agonist at the monkey UT receptor (pEC(50) 6.56+/-0.35, E(max) 5.27+/-0.65-fold over basal). Indeed, in contrast to the rat UT receptor (and rat isolated arteries), SB-710411 exhibited intrinsic activity in monkey arteries acting as an efficacious vasoconstrictor (pEC(50)s 5.03+/-0.18 to 5.71+/-0.21, E(max)s 101+/-4 to 218+/-58% KCl). These data demonstrate that caution must be taken when extrapolating the pharmacology of a specific ligand(s) between the rodent and primate UT receptors.

Identification and pharmacological characterization of native, functional human urotensin-II receptors in rhabdomyosarcoma cell lines.

1 In an effort to identify endogenous, native mammalian urotensin-II (U-II) receptors (UT), a diverse range of human, primate and rodent cell lines (49 in total) were screened for the presence of detectable [125I]hU-II binding sites. 2 UT mRNA (Northern blot, PCR) and protein (immunocytochemistry) were evident in human skeletal muscle tissue and cells. 3 [(125)I]hU-II bound to a homogenous population of high-affinity, saturable (Kd 67.0+/-11.8 pm, Bmax 9687+/-843 sites cell(-1)) receptors in the skeletal muscle (rhabdomyosarcoma) cell line SJRH30. Radiolabel was characteristically slow to dissociate (< or =15% dissociation 90 min). A lower density of high-affinity U-II binding sites was also evident in the rhabdomyosarcoma cell line TE671 (1667+/-165 sites cell(-1), Kd 74+/-8 pm). 4 Consistent with the profile recorded in human recombinant UT-HEK293 cells, [125I]hU-II binding to SJRH30 cells was selectively displaced by both mammalian and fish U-II isopeptides (Kis 0.5+/-0.1-1.2+/-0.3 nm) and related analogues (hU-II[4-11]>[Cys(5,10)]Acm hU-II; Kis 0.4+/-0.1 and 864+/-193 nm, respectively). 5 U-II receptor activation was functionally coupled to phospholipase C-mediated [Ca2+]i mobilization (EC50 6.9+/-2.2 nm) in SJRH30 cells. 6 The present study is the first to identify the presence of 'endogenous' U-II receptors in SJRH30 and TE671 cells. SJRH30 cells, in particular, might prove to be of utility for (a) investigating the pharmacological properties of hU-II and related small molecule antagonists at native human UT and (b) delineating the role of this neuropeptide in the (patho)physiological regulation of mammalian neuromuscular function.

Production and characterization of monoclonal antibodies against the vasoconstrictive peptide human urotensin-II.

We report the production and characterization of four monoclonal antibodies (MAbs) against human urotensin-II (hU-II). The antibodies were raised against human hU-II, which contains the C-terminus cyclic ring (CFWKYC) that is conserved across species. Multiple selection assays were applied to ensure antibody potency and reactivity against the ring structure. The MAbs reacted via ELISA with hU-II bound to plastic, immunoprecipitated [(125)I-Y(9)] hU-II, bound to biotinylated hU-II in BIAcore analysis and, by Western analysis, recognized the full-length human preprourotensin-II expressed in transfected HEK293 cells. All four MAbs cross-reacted with porcine A, porcine B, rat, mouse, and goby U-II in ELISA. By competitive RIA, hU-II(5-11) (identical to the C-terminus of goby U-II) reacted equivalently to hU-II and goby U-II. The IC(50)s were 0.8 nM for one MAb and 1.6 nM for the others. All four MAbs reacted 15-fold less potently with hU-II(5-10) and 50-fold less potently with hU-II(5-10) amide. Thus, the ring structure and terminal Val/Ile comprise the binding site for this group of MAbs. This panel of antibodies could be useful tools to help delineate the biology and pharmacology of U-II. They may also be of diagnostic value in monitoring hU-II in body fluids.

Molecular and pharmacological characterization of genes encoding urotensin-II peptides and their cognate G-protein-coupled receptors from the mouse and monkey.

Urotensin-II (U-II) and its receptor (UT) represent novel therapeutic targets for management of a variety of cardiovascular diseases. To test such hypothesis, it will be necessary to develop experimental animal models for the manipulation of U-II/UT receptor system. The goal of this study was to clone mouse and primate preproU-II and UT for pharmacological profiling. Monkey and mouse preproU-II genes were identified to encode 123 and 125 amino acids. Monkey and mouse UT receptors were 389, and 386 amino acids, respectively. Genomic organization of mouse genes showed that the preproU-II has four exons, while the UT receptor has one exon. Although initially viewed by many exclusively as cardiovascular targets, the present study demonstrates expression of mouse and monkey U-II/UT receptor mRNA in extra-vascular tissue including lung, pancreas, skeletal muscle, kidney and liver. Ligand binding studies showed that [125I]h U-II bound to a single sites to the cloned receptors in a saturable/high affinity manner (Kd 654+/-154 and 214+/-65 pM and Bmax of 1011+/-125 and 497+/-68 fmol mg-1 for mouse and monkey UT receptors, respectively). Competition binding analysis demonstrated equipotent, high affinity binding of numerous mammalian, amphibian and piscine U-II isopeptides to these receptors (Ki=0.8 - 3 nM). Fluorescein isothiocyanate (FITC) labelled U-II, bound specifically to HEK-293 cells expressing mouse or monkey UT receptor, confirming cell surface expression of recombinant UT receptor. Exposure of these cells to human U-II resulted in an increase in intracellular [Ca2+] concentrations (EC50 3.2+/-0.8 and 1.1+/-0.3 nM for mouse and monkey UT receptors, respectively) and inositol phosphate (Ip) formation (EC50 7.2+/-1.8 and 0.9+/-0.2 nM for mouse and monkey UT receptors, respectively) consistent with the primary signalling pathway for UT receptor involving phospholipase C activation.