Discovery of 1-(1,3,5-triazin-2-yl)piperidine-4-carboxamides as inhibitors of soluble epoxide hydrolase.

1-(1,3,5-Triazin-yl)piperidine-4-carboxamide inhibitors of soluble epoxide hydrolase were identified from high through-put screening using encoded library technology. The triazine heterocycle proved to be a critical functional group, essential for high potency and P450 selectivity. Phenyl group substitution was important for reducing clearance, and establishing good oral exposure. Based on this lead optimization work, 1-[4-methyl-6-(methylamino)-1,3,5-triazin-2-yl]-N-{[[4-bromo-2-(trifluoromethoxy)]-phenyl]methyl}-4-piperidinecarboxamide (27) was identified as a useful tool compound for in vivo investigation. Robust effects on a serum biomarker, 9, 10-epoxyoctadec-12(Z)-enoic acid (the epoxide derived from linoleic acid) were observed, which provided evidence of robust in vivo target engagement and the suitability of 27 as a tool compound for study in various disease models.

NADPH oxidase-dependent and -independent mechanisms of reported inhibitors of reactive oxygen generation.

NADPH oxidase isoform-2 (NOX2) generates reactive oxygen species (ROS) that contribute to neurodegenerative and cardiovascular pathologies. However, validation of NOX2 as a pharmacotherapeutic target has been hampered by a lack of mechanistically-defined inhibitors. Using cellular and biochemical assays, we explored previously reported inhibitors of ROS production (perhexiline, suramin, VAS2870 and two Shionogi patent compounds) as direct NOX2 inhibitors. All but suramin, which presumably lacks cell penetrance, inhibit cellular ROS production. However, only perhexiline and suramin inhibit biochemical NOX2 activity. Indeed, our data suggest that NOX2 inhibition by perhexiline may contribute significantly to its demonstrated cardioprotective effects. Inhibition of protein kinase CßII explains the cellular activity of the Shionogi compounds, whereas VAS2870 inhibits by an as-yet unidentified mechanism unrelated to direct NOX2 function or subunit assembly. These data delineate the mechanisms of action of these compounds and highlight their strengths and limitations for use in future target validation studies.

Chronic urotensin II receptor antagonist treatment does not alter hypertrophy or fibrosis in a rat model of pressure-overload hypertrophy.

Urotensin II (UII) is a potential mediator in the pathogenesis of cardiovascular disease, and inhibition of its actions at the urotensin receptor (UT) has been shown to improve cardiac function and structural changes of the myocardium in a model of myocardial infarction. In this study we utilized a model of pressure-overload hypertrophy induced by abdominal aortic constriction (AAC) which resulted in hypertrophy, increased fibrosis and impaired diastolic and systolic function. These changes were associated with a 4-fold increase in UII protein expression in the myocardium. Treatment of animals with a selective UT (SB-657510) antagonist for 20 weeks at a dose of 1500 ppm did not improve cardiac function as assessed by echocardiography and pressure-volume loop analysis, nor did it inhibit left ventricular hypertrophy or fibrosis. We hypothesize that other neurohumoral pathways may have a greater involvement in the pathogenesis of this model. Targeting the UII system appears to be insufficient to observe a beneficial outcome.

Direct inotropic effects of exogenous and endogenous urotensin-II: divergent actions in failing and nonfailing human myocardium.

BACKGROUND: Urotensin-II (U-II) is an endogenous peptide upregulated in failing hearts. To date, insights into the myocardial actions of U-II have been obscured by its potent vasoconstrictor effects and interspecies differences in physiological responses to U-II. METHODS AND RESULTS: We examined the direct effects of exogenous U-II on in vitro contractility in nonfailing and failing human myocardial trabeculae (n=47). Rapid cooling contractures (RCC) were used to examine sarcoplasmic reticulum Ca(2+) load. In nonfailing myocardium, exogenous U-II increased developed force (DF), rates of force generation and decline and RCC amplitude suggesting increased sarcoplasmic reticulum Ca(2+) load. In isolated myocyte suspensions from nonfailing hearts, U-II increased phospholamban phosphorylation. In failing myocardium, exogenous U-II reduced DF and rates of force generation and decline without a significant change in RCC amplitude in trabeculae or a change in phospholamban phosphorylation in myocytes. To examine the effects of endogenous U-II, we administered the peptidic U-II receptor antagonist (UT-A) GSK248451A to isolated trabeculae. UT-A induced a decrease in DF in nonfailing myocardium and an increase in DF in failing myocardium. UT-A increased RCC amplitude slightly in both nonfailing and failing myocardium. During ongoing UT-A, exogenous U-II had little effect on DF and RCC amplitude, confirming effective receptor blockade. CONCLUSIONS: U-II modulates contractility independent of vasoconstriction with opposite effects in failing and nonfailing hearts. Positive inotropic responses to UT-A alone suggests that increased endogenous U-II constrains contractility in failing hearts via an autocrine or paracrine mechanism. These findings support a potential therapeutic role for UT-A in heart failure.

Urotensin II receptor knockout mice on an ApoE knockout background fed a high-fat diet exhibit an enhanced hyperlipidemic and atherosclerotic phenotype.

RATIONALE: Expression of the vasoactive peptide Urotensin II (UII) is elevated in a number of cardiovascular diseases. OBJECTIVE: Here, we sought to determine the effect of UII receptor (UT) gene deletion in a mouse model of atherosclerosis. METHODS AND RESULTS: UT knockout (KO) mice were crossed with ApoE KO mice to generate UT/ApoE double knockout (DKO) mice. Mice were placed on a high-fat Western-type diet for 12 weeks. We evaluated the degree of atherosclerosis and hepatic steatosis by histology. In addition, serum glucose, insulin, and lipids were determined. DKO mice exhibited significantly increased atherosclerosis compared to ApoE KO mice (P<0.05). This was associated with a significant increase in serum insulin and lipids (P<0.001) but a decrease in hepatic steatosis (P<0.001). UT gene deletion led to a significant increase in systolic pressure and pulse pressure. RT-PCR and immunoblot analyses showed significant reductions in hepatic scavenger receptors, nuclear receptors, and acyl-CoA:cholesterol acyltransferase (ACAT1) expression in DKO mice. UII induced a significant increase in intracellular cholesteryl ester formation in primary mouse hepatocytes, which was blocked by the MEK inhibitor, PD98059. Hepatocytes of UTKO mice showed a significant reduction in lipoprotein uptake compared to wild-type mice. CONCLUSIONS: We propose that UT gene deletion in an ApoE-deficient background promotes downregulation of ACAT1, which in turn attenuates hepatic lipoprotein receptor-mediated uptake and lipid transporter expression. As the liver is the main organ for uptake of lipoprotein-derived lipids, DKO leads to an increase in hyperlipidemia, with a concomitant decrease in hepatic steatosis, and consequently increased atherosclerotic lesion formation. Furthermore, the hypertension associated with UT gene deletion is likely to contribute to the increased atherosclerotic burden.

The effects of benzodiazepines on urotensin II-stimulated norepinephrine release from rat cerebrocortical slices.

BACKGROUND: Urotensin II (UII) and its receptor (UT) are implicated in mood disorders, such as stress and anxiety, and this may result, at least in part, from increased norepinephrine release from the cerebral cortex. Benzodiazepines have been widely used as hypnotics and anxiolytics, producing a decrease in cerebrocortical norepinephrine release. We hypothesized that there was some interaction between benzodiazepines and the UII system in the cerebral cortex. METHODS: In the present study, we have examined the effects of benzodiazepines on UII-increased norepinephrine release from rat cerebrocortical slices and intracellular Ca(2+) concentrations ([Ca(2+)]i) in HEK293 cells expressing rat UT receptor (HEK293-rUT cells). RESULTS: Midazolam, diazepam and flunitrazepam concentration-dependently inhibited UII-evoked norepinephrine release but did not affect [Ca(2+)]i. The IC(50) of midazolam for inhibition of UII-evoked norepinephrine release (0.32 microM, P < 0.01) was significantly lower than that of diazepam (187 microM) or flunitrazepam (40 microM). The inhibitory effects of midazolam on UII-evoked norepinephrine release were significantly attenuated by flumazenil, a benzodiazepine site antagonist. CONCLUSION: The present study suggests that midazolam, at clinically relevant concentration, significantly inhibited UII-evoked norepinephrine release. This inhibitory effect may be partially mediated via central benzodiazepine receptors.

Heme-oxygenase induction inhibits arteriolar thrombosis in vivo: effect of the non-substrate inducer cobalt protoporphyrin.

Heme oxygenase-1 (HO) metabolizes heme to form the vasodilator carbon monoxide and antioxidant biliverdin. Upregulation of HO-1 by hemin, which is also a substrate attenuates thrombosis in rodent models, however, whether protection is due to HO-1 upregulation or to increased substrate availability is unknown. This study tested the hypothesis that treatment of mice with cobalt protoporphyrin (CoPP), a non-substrate HO-1 inducer, would protect the endothelium from laser injury. C57Bl/J6 mice were treated with vehicle, CoPP (20 mg/kg), CoPP plus the HO-1 inhibitor tin protoporphyrin (SnPP; 20 mg/kg) or SnPP alone for 18 h. Intravital microscopy was used to quantitate thrombus formation in cremaster arterioles in response to laser ablation of the endothelium. CoPP treatment inhibited thrombosis by 43% compared to vehicle (P<0.05). SnPP co-treatment negated the inhibitory effect of CoPP while SnPP alone potentiated thrombosis compared to vehicle. In CoPP-treated animals, cremaster HO-1 mRNA expression was increased 59+/-17-fold over vehicle (P<0.001). Co-treatment with CoPP+SnPP attenuated this effect by 36%, however the increase in HO-1 protein induced by CoPP was unaffected by SnPP. Induction of HO-1 by the non-substrate inducer CoPP protects against laser induced endothelial injury without the need for increased substrate. Small molecule, substrate-independent upregulation of HO-1 expression represents a feasible approach to ameliorate endothelial dysfunction in cardiovascular disease.

Epoxyeicosatrienoic acids function as selective, endogenous antagonists of native thromboxane receptors: identification of a novel mechanism of vasodilation.

Epoxy- and dihydroxy-eicosatrienoic acids (EETs and DHETs) are vasoactive cytochrome P450 metabolites of arachidonic acid. Interestingly, however, the mechanism(s) by which EETs/DHETs mediate smooth muscle relaxation remains unclear. In contrast to previous reports, where dilation was purportedly large-conductance Ca(2+)-activated K(+) (BK(Ca)) and/or transient receptor potential cation channel, subfamily V, member 4 (TRPV4) channel-mediated, 14,15-EET-induced vasodilation [reversal of contractile tone established with the thromboxane receptor (TP) agonist 15-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic acid (U-46619)] was unaltered in BK(Ca) and TRPV4 knockout mouse isolated aortae compared with wild-type controls, indicating a significant BK(Ca)/TRPV4-resistant mechanism. Whereas all EET and DHET regioisomers reversed U-46619 contraction in rat aortae and mouse mesenteric resistance arteries, these eicosanoids failed to alter phenylephrine-induced contraction, suggesting that they mediated dilation via a "TP-selective" mechanism. Competitive TP antagonism was also observed in nonvascular tissue, including rat fundus and tertiary bronchus, indicating that the effect is not specific to blood vessels. Such effects were TP-selective because 14,15-EET failed to inhibit "non-TP" prostanoid receptor-mediated function in multiple cell/tissue-based assays (K(b) > 10 microM). In accordance, 14,15-EET inhibited specific [(3)H]7-(3-((2-((phenylamino)carbonyl)hydrazino)-methyl)-7-oxabicyclo(2.2.1)hept-2-yl)-5-heptenoic acid (SQ-29548) binding to human recombinant TP receptor, with a K(i) value of 3.2 microM, and it showed weaker affinity for non-TP prostanoid receptors, including DP, FP, EP(1-4), and IP receptors (K(i) values of 6.1, 5.3, 42.6, 19.7, 13.2, 20.2, and >25 microM, respectively) and no appreciable affinity (K(i) values >10 microM) for a diverse array of pharmacologically distinct receptors, including the leukotriene receptors Cys-LT(1/2) and BLT(1). As such, EETs/DHETs represent a unique class of "endogenous" G protein-coupled receptor competitive antagonists, inducing vasodilation via direct TP inhibition. Thus, EETs/DHETs represent novel autoregulatory agents, directly modulating the actions of cyclooxygenase-derived eicosanoids following arachidonic acid mobilization.

Targeted overexpression of the human urotensin receptor transgene in smooth muscle cells: effect of UT antagonism in ApoE knockout mice fed with Western diet.

Urotensin II (UII) and its receptor UT are upregulated in the pathological setting of various cardiovascular diseases including atherosclerosis. However, their exact role in atherosclerosis remains to be determined. In the present study we used four strains of mice; wild-type (WT), UT(+) (a transgenic strain expressing human UT driven by the alpha-smooth muscle-specific, SM22, promoter), ApoE knockout (ko), and UT(+)/ApoE ko. All animals were fed high fat diet for 12 weeks. Western blot analysis revealed a significant increase in aortic UT expression in UT(+) relative to WT mice (P<0.05). Aortas of ApoE ko mice expressed comparable UT protein level to that of UT(+). Immunohistochemistry revealed the presence of strong expression of UT and UII proteins in the atheroma of UT(+), ApoE ko and UT(+)/ApoE ko mice, particularly in foam cells. Serum cholesterol and triglyceride levels were significantly increased in ApoE ko and in UT(+)/ApoE ko but not in UT(+) mice when compared to WT mice (P<0.0001). Analysis of aortas showed a significant increase in atherosclerotic lesion in the UT(+), ApoE ko and UT(+)/ApoE ko compared to WT mice (P<0.05). Oral administration of the UT receptor antagonist SB-657510A (30 microg/Kg/day gavage) for 10 weeks in a group of ApoE ko mice fed on high fat diet resulted in a significant reduction of lesion (P<0.001). SB-657510A also significantly reduced ACAT-1 protein expression in the atherosclerotic lesion of ApoE ko mice (P<0.05). The present findings demonstrate an important role for UT in the pathogenesis of atherosclerosis. The use of UT receptor antagonists may provide a beneficial tool in the management of this debilitating disease process.

N-alkyl-5H-pyrido[4,3-b]indol-1-amines and derivatives as novel urotensin-II receptor antagonists.

High throughput screening of our compound collection led to the discovery of a novel series of N-alkyl-5H-pyrido[4,3-b]indol-1-amines as urotensin-II receptor antagonists. Synthesis, initial structure and activity relationships, functional and animal ortholog activities of the series are described.

Aminomethylpiperazines as selective urotensin antagonists.

Aminomethylpiperazines, reported previously as being kappa-opioid receptor agonists, were identified as lead compounds in the development of selective urotensin receptor antagonists. Optimized substitution of the piperazine moiety has provided high affinity urotensin receptor antagonists with greater than 100-fold selectivity over the kappa-opioid receptor. Select compounds were found to inhibit urotensin-induced vasoconstriction in isolated rat aortic rings consistent with the hypothesis that an urotensin antagonist may be useful for the treatment of hypertension.

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.

Urotensin-II receptor antagonists: synthesis and SAR of N-cyclic azaalkyl benzamides.

SAR exploration of the central diamine, benzyl, and terminal aminoalkoxy regions of the N-cyclic azaalkyl benzamide series led to the identification of very potent human urotensin-II receptor antagonists such as 1a with a K(i) of 4 nM. The synthesis and structure-activity relationships (SAR) of N-cyclic azaalkyl benzamides are described.

Urotensin II evokes neurotransmitter release from rat cerebrocortical slices.

Urotensin II (UII) has been reported to modulate rapid eye movement (REM) sleep via activation of brainstem cholinergic neurons and REM sleep is regulated by locus coerleus (LC)-cerebrocortical noradrenergic neurons. We hypothesized that UII may activate LC-cerebrocortical noradrenergic neurons. To test this hypothesis, we have examined the effects of UII on norepinephrine release from rat cerebrocortical slices. In addition, the effect of the putative UT receptor antagonist [Pen(5), DTrp(7), Dab(8)]UII(4-11) (UFP-803) was assessed. We have compared this with other wakefulness-promoting neurotransmitters such as dopamine, glutamate, serotonin and histamine. We also studied the effects of UII and UFP-803 on intracellular Ca(2+) ([Ca(2+)]i) in HEK293 cells stably expressing rat UT receptor (HEK293-rUT cells). UII produced a time- (peaking at approximately 10 min following stimulation with 10nM) and concentration-dependent increase in norepinephrine release with pEC(50) and E(max) (% of basal) values of 8.78+/-0.17 (1.65 nM) and 138+/-2%, respectively. UII also evoked dopamine, serotonin and histamine release with similar pEC(50) values. UII increased glutamate release but only at high concentrations (<100 nM) and this failed to saturate. UII markedly increased [Ca(2+)](i) in HEK293-rUT cells in a concentration-dependent manner with pEC(50) of 8.26+/-0.24. The UT antagonist UFP-803 reversed both UII-increased norepinephrine release from the cerebrocortical slices (pK(B)=8.98) and [Ca(2+)](i) (pK(B)=8.87) in HEK293-rUT cells. Collectively these data suggest that UII evokes the release of norepinephrine via UT receptor activation and produces similar effects on other wakefulness-promoting neurotransmitters: these neurochemical actions of UII may be important for the control of the sleep-wake cycle.

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.

2-Aminomethyl piperidines as novel urotensin-II receptor antagonists.

A series of 2-aminomethyl piperidines has been discovered as novel urotensin-II receptor antagonists. The synthesis, initial structure-activity relationships, and optimization of the initial hit that resulted in the identification of potent, cross-species active, and functional urotensin-II receptor antagonists such as 1a and 11a are described.

Effects of p38 MAPK Inhibitor on angiotensin II-dependent hypertension, organ damage, and superoxide anion production.

Angiotensin II (Ang II) activates p38 mitogen-activated protein kinase (p38 MAPK) and increases reactive oxygen species (ROS), but the nature of the relationship in vivo is not fully understood. We assess the effect of SB239063AN, a highly selective, orally active, p38 MAPK inhibitor, on Ang II-dependent hypertension, target-organ damage and ROS production. Sprague-Dawley rats and MAPKAP kinase-2 knockout mice were infused with Ang II. Ang II infusion increased the levels of phosphorylated p38 MAPK in the heart and aorta. Production of superoxide anion and expression of NAD(P)H oxidase subunit gp91 in the aorta were increased 4- and 5-fold, respectively. In addition, Ang II infusion led to endothelial dysfunction, progressive and sustained hypertension, and cardiac hypertrophy. Treatment with SB239063AN (800 ppm in the diet) significantly attenuated the levels of phosphorylated p38 MAPK in the heart and aorta, reduced superoxide anion generation by 57% (P < 0.01), markedly suppressed gp91 mRNA expression, prevented endothelial dysfunction, and blunted both the hypertension and cardiac hypertrophy. Ang II-dependent hypertension was also significantly attenuated in MAPKAP kinase-2 knockout mice. The results suggest that Ang II induced hypertension, organ damage, and ROS production are possibly mediated by p38 MAPK and inhibition of p38 MAPK may offer a therapeutic approach for cardiovascular disease.

Dendroaspis natriuretic peptide binds to the natriuretic peptide clearance receptor.

Dendroaspis natriuretic peptide (DNP) is a newly-described natriuretic peptide which lowers blood pressure via vasodilation. The natriuretic peptide clearance receptor (NPR-C) removes natriuretic peptides from the circulation, but whether DNP interacts with human NPR-C directly is unknown. The purpose of this study was to test the hypothesis that DNP binds to NPR-C. ANP, BNP, CNP, and the NPR-C ligands AP-811 and cANP(4-23) displaced [(125)I]-ANP from NPR-C with pM-to-nM K(i) values. DNP displaced [(125)I]-ANP from NPR-C with nM potency, which represents the first direct demonstration of binding of DNP to human NPR-C. DNP showed high pM affinity for the GC-A receptor and no affinity for GC-B (K(i)>1000 nM). DNP was nearly 10-fold more potent than ANP at stimulating cGMP production in GC-A expressing cells. Blockade of NPR-C might represent a novel therapeutic approach in augmenting the known beneficial actions of DNP in cardiovascular diseases such as hypertension and heart failure.

Lysophosphatidylcholine induces inflammatory activation of human coronary artery smooth muscle cells.

Lysophosphatidylcholine (LPC) is the major bioactive lipid component of oxidized LDL, thought to be responsible for many of the inflammatory effects of oxidized LDL described in both inflammatory and endothelial cells. Inflammation-induced transformation of vascular smooth muscle cells from a contractile phenotype to a proliferative/secretory phenotype is a hallmark of the vascular remodeling that is characteristic of atherogenesis; however, the role of LPC in this process has not been fully described. The present study tested the hypothesis that LPC is an inflammatory stimulus in coronary artery smooth muscle cells (CASMCs). In cultured human CASMCs, LPC stimulated time- and concentration-dependent release of arachidonic acid that was sensitive to phospholipase A2 and C inhibition. LPC stimulated the release of arachidonic acid metabolites leukotriene-B4 and 6-keto-prostaglandin F1alpha, within the same time course. LPC was also found to stimulate basic fibroblast growth factor release as well as stimulating the release of the cytokines GM-CSF, IL-6, and IL-8. Optimal stimulation of these signals was obtained via palmitic acid-substituted LPC species. Stimulation of arachidonic acid, inflammatory cytokines and growth factor release, implies that LPC might play a multifactorial role in the progression of atherosclerosis, by affecting inflammatory processes.

Urotensin-II Immunoreactivity in Normolipidemic and Hyperlipidemic New Zealand White Rabbits Following Balloon Angioplasty and Stenting.

Treatment for symptomatic atherosclerosis is being carried out by balloon mediated angioplasty, with or without stent implantation, more and more frequently. Although advances with the development of drug eluting stents have improved prognosis, restenosis is still the most limiting factor for this treatment modality. Urotensin-II (UII), a small pleiotropic vasoactive peptide is increasingly being recognized as a contributory factor in cardiovascular diseases. We qualitatively evaluated UII immunoreactivity (IR) in three models of balloon angioplasty mediated restenosis. Specifically, we performed balloon angioplasty in the ilio-femoral arteries of New Zealand White Rabbits (NZWR) fed either a normal chow or high fat diet. In addition, UIIIR was also assessed in stent implanted abdominal aortae of NZWR fed a high fat diet. UII was constitutively expressed in the endothelium of all arterial segments evaluated. Abundant expression of UII was associated with lesion progression, particularly in myointimal cells, and less so in medial smooth muscle cells (SMC). The strongest UII-IR was observed in foam cells of animals fed a high fat diet. We demonstrate abundant expression of UII in regenerating endothelial cells and myointimal cells in vascular lesions following balloon mediated angioplasty and stent implantation in both animals fed a normal chow and high fat diet.