Reactivity of renal and mesenteric resistance vessels to angiotensin II is mediated by NOXA1/NOX1 and superoxide signaling.

Activation of NADPH oxidase (NOX) enzymes and the generation of reactive oxygen species and oxidative stress regulate vascular and renal function and contribute to the pathogenesis of hypertension. The present study examined the role of NOXA1/NOX1 function in vascular reactivity of renal and mesenteric resistance arteries/arterioles of wild-type and Noxa1-/- mice. A major finding was that renal blood flow is less sensitive to acute stimulation by angiotensin II (ANG II) in Noxa1-/- mice compared with wild-type mice, with a direct action on resistance arterioles independent of nitric oxide (NO) bioavailability. These functional results were reinforced by immunofluorescence evidence of NOXA1/NOX1 protein presence in renal arteries, afferent arterioles, and glomeruli as well as their upregulation by ANG II. In contrast, the renal vascular response to the thromboxane mimetic U46619 was effectively blunted by NO and was similar in both mouse genotypes and thus independent of NOXA1/NOX1 signaling. However, phenylephrine- and ANG II-induced contraction of isolated mesenteric arteries was less pronounced and buffering of vasoconstriction after acetylcholine and nitroprusside stimulation was reduced in Noxa1-/- mice, suggesting endothelial NO-dependent mechanisms. An involvement of NOXA1/NOX1/O2•- signaling in response to ANG II was demonstrated with the specific NOXA1/NOX1 assembly inhibitor C25 and the nonspecific NOX inhibitor diphenyleneiodonium chloride in cultured vascular smooth muscle cells and isolated mesenteric resistance arteries. Collectively, our data indicate that the NOX1/NOXA1/O2•- pathway contributes to acute vasoconstriction induced by ANG II in renal and mesenteric vascular beds and may contribute to ANG II-induced hypertension.NEW & NOTEWORTHY Renal reactivity to angiotensin II (ANG II) is mediated by superoxide signaling produced by NADPH oxidase (NOX)A1/NOX1. Acute vasoconstriction of renal arteries by ANG was blunted in Noxa1-/- compared with wild-type mice. NOXA1/NOX1/O2•- signaling was also observed in ANG II stimulation of vascular smooth muscle cells and isolated mesenteric resistance arteries, indicating that it contributes to ANG II-induced hypertension. A NOXA1/NOX1 assembly inhibitor (C25) has been characterized that inhibits superoxide production and ameliorates the effects of ANG II.

Design, Synthesis, and Biological Evaluation of Structurally Rigid Analogues of 4-(3-Hydroxyphenyl)piperidine Opioid Receptor Antagonists.

In order to gain additional information concerning the active conformation of the N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine (1) class of opioid receptor antagonists, procedures were developed for the synthesis of structurally rigid N-substituted-6-(3-hydroxyphenyl)3-azabicyclo[3.1.0]hexane and 3-methyl-4-(3-hydroxyphenyl)-4-azabicyclo[4.1.0]heptanes. Evaluation of the conformationally constrained series in a [35S]GTPγS assay showed that structural rigid compounds having the 3-hydroxyphenyl group locked in the piperidine equatorial orientation had potencies equal to or better than similar compounds having more flexible structures similar to 1. The studies of the rigid compounds also suggested that the 3-methyl group present in compound 1 type antagonists may not be necessary for their pure opioid antagonist properties.

Design, synthesis, and pharmacological evaluation of JDTic analogs to examine the significance of replacement of the 3-hydroxyphenyl group with pyridine or thiophene bioisosteres.

The potent and selective KOR antagonist JDTic was derived from the N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine class of pure opioid antagonists. In previous studies we reported that compounds that did not have a hydroxyl on the 3-hydroxyphenyl group and did not have methyl groups at the 3- and 4-position of the piperidine ring were still potent and selective KOR antagonists. In this study we report JDTic analogs 2, 3a-b, 4a-b, and 5, where the 3-hydroxyphenyl ring has been replaced by a 2-, 3-, or 4-pyridyl or 3-thienyl group and do not have the 3-methyl or 3,4-dimethyl groups, remain potent and selective KOR antagonists. Of these, (3R)-7-hydroxy-N-(1S)-2-methyl-[4-methyl-4-pyridine-3-yl-carboxamide (3b) had the best overall binding potency and selectivity in a [(35)S]GTPγS functional assay, with a Ke=0.18nM at the KOR and 273- and 16,700-fold selectivity for the KOR relative to the MOR and DOR, respectively. Calculated physiochemical properties for 3b suggest that it will cross the blood-brain barrier.

Quantitative Signaling and Structure-Activity Analyses Demonstrate Functional Selectivity at the Nociceptin/Orphanin FQ Opioid Receptor.

Comprehensive studies that consolidate selective ligands, quantitative comparisons of G protein versus arrestin-2/3 coupling, together with structure-activity relationship models for G protein-coupled receptor (GPCR) systems are less commonly employed. Here we examine biased signaling at the nociceptin/orphanin FQ opioid receptor (NOPR), the most recently identified member of the opioid receptor family. Using real-time, live-cell assays, we identified the signaling profiles of several NOPR-selective ligands in upstream GPCR signaling (G protein and arrestin pathways) to determine their relative transduction coefficients and signaling bias. Complementing this analysis, we designed novel ligands on the basis of NOPR antagonist J-113,397 [(±)-1-[(3R*,4R*)-1-(cyclooctylmethyl)-3-(hydroxymethyl)-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one] to explore structure-activity relationships. Our study shows that NOPR is capable of biased signaling, and further, the NOPR selective ligands MCOPPB [1-[1-(1-methylcyclooctyl)-4-piperidinyl]-2-(3R)-3-piperidinyl-1H-benzimidazole trihydrochloride] and NNC 63-0532 [8-(1-naphthalenylmethyl)-4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]decane-3-acetic acid, methyl ester] are G protein-biased agonists. Additionally, minor structural modification of J-113,397 can dramatically shift signaling from antagonist to partial agonist activity. We explore these findings with in silico modeling of binding poses. This work is the first to demonstrate functional selectivity and identification of biased ligands at the nociceptin opioid receptor.

A selective delta opioid receptor antagonist based on a stilbene core.

Studies of directed ortho metalation reactions on an aromatic substrate with multiple potential directing groups have identified conditions that favor either of two regioisomers. One of these regioisomers has been converted to an analogue of the stilbene pawhuskin A, and been shown to have high selectivity as an antagonist of the delta opioid receptor. Docking studies have suggested that this compound can adopt a conformation similar to naltrindole, a known delta antagonist.

Design, synthesis, and pharmacological evaluation of JDTic analogs to examine the significance of the 3- and 4-methyl substituents.

The design and discovery of JDTic as a potent and selective kappa opioid receptor antagonist used the N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine pharmacophore as the lead structure. In order to determine if the 3-methyl or 4-methyl groups were necessary in JDTic and JDTic analogs for antagonistic activity, compounds 4a-c, and 4d-f which have either the 3-methyl or both the 3- and 4-methyl groups removed, respectively, from JDTic and analogs were synthesized and evaluated for their in vitro opioid receptor antagonist activities using a [(35)S]GTPγS binding assay. Other ADME properties were also assessed for selected compounds. These studies demonstrated that neither the 3-methyl or 3,4-dimethyl groups present in JDTic and analogs are required to produce potent and selective κ opioid receptor antagonists.

Synthesis, nicotinic acetylcholine receptor binding, in vitro and in vivo pharmacology properties of 3'-(substituted pyridinyl)-deschloroepibatidine analogs.

Over the last several years we have synthesized and studied the in vitro and in vivo nAChR pharmacological properties of epibatidine (4) analogs. In this study we report the synthesis, nAChR in vitro and in vivo pharmacological properties of 3'-(substituted pyridinyl)-deschloroepibatidine analogs (5a-e and 6a-e). All of the analogs had high binding affinity for α4ß2(∗)-nAChRs. Several of the analogs were potent antagonists of α4ß2-nAChRs in in vitro efficacy tests and were potent antagonists of nicotine-induced antinociception in the mouse tail-flick test. Compound 6b had a Ki = 0.13 nM in the binding assay, 25- and 46-fold selectivity for the α4ß2(∗)-nAChR relative to the α3ß4- and α7-nAChR, respectively, in the in vitro efficacy test and an AD50 = 0.13 µg/kg in the tail-flick test. Combined with favorable calculated physiochemical properties compared to varenicline, our findings suggest that 6b should be considered for development as a potential pharmacotherapy for treating nicotine addiction and other CNS disorders.

Evaluation of structural effects on 5-HT(2A) receptor antagonism by aporphines: identification of a new aporphine with 5-HT(2A) antagonist activity.

A set of aporphine analogs related to nantenine was evaluated for antagonist activity at 5-HT2A and α1A adrenergic receptors. With regards to 5-HT2A receptor antagonism, a C2 allyl group is detrimental to activity. The chiral center of nantenine is not important for 5-HT2A antagonist activity, however the N6 nitrogen atom is a critical feature for 5-HT2A antagonism. Compound 12b was the most potent 5-HT2A aporphine antagonist identified in this study and has similar potency to previously identified aporphine antagonists 2 and 3. The ring A and N6 modifications examined were detrimental to α1A antagonism. A slight eutomeric preference for the R enantiomer of nantenine was observed in relation to α1A antagonism.

Stilbenes as κ-selective, non-nitrogenous opioid receptor antagonists.

The natural stilbene pawhuskin A has been shown to function as an opioid receptor antagonist, with preferential binding to the κ receptor. This finding encouraged assembly of a set of analogues to probe the importance of key structural features. Assays on these compounds determined that one (compound 29) shows potent opioid receptor binding activity and significantly improved selectivity for the κ receptor. These studies begin to illuminate the structural features of these non-nitrogenous opioid receptor antagonists that are required for activity.

Synthesis and evaluation of 1,2,4-methyltriazines as mGluR5 antagonists.

In previous studies we showed that 3-(substituted phenylethynyl)-5-methyl[1,2,4]triazine analogues of MPEP were potent antagonists of glutamate-mediated mobilization of internal calcium in an mGluR5 in vitro efficacy assay. In the present study we report the synthesis and evaluation of six 3-(substituted biphenylethynyl)-5-methyl[1,2,4]triazines (5a-f), and five 3-(substituted phenoxyphenylethynyl)-5-methyltriazines (6a-e). Compound 2-(4-fluorophenyl-5-[2-(5-methyl[1,2,4]triazine-3-yl)ethynyl]benzonitrile (5f) with an IC(50) of 28.2 nM was the most potent analogue.

New aporphinoid 5-HT2A and α1A antagonists via structural manipulations of nantenine.

A series of C1, C2, C3 and N6 analogs of nantenine (2) was synthesized and evaluated in 5-HT(2A) and α(1A) receptor functional assays. Alkyl substitution of the C1 and N6 methyl groups of nantenine provided selective 5-HT(2A) and α(1A) antagonists, respectively. The C2 alkyloxy analogs studied were generally selective for α(1A) versus 5-HT(2A). The C3 bromo analog 15 is one of the most potent aporphinoid 5-HT(2A) antagonists known presently.

Effects of hydroxymetabolites of bupropion on nicotine dependence behavior in mice.

Bupropion is an atypical antidepressant that also has utility as a smoking cessation aid. Hydroxybupropions are major metabolites of bupropion and are believed to contribute to antidepressant and perhaps smoking cessation activities. Because bupropion metabolism is more similar in humans and mice than in humans and rats, the present study investigated effects of hydroxybupropion enantiomers in mouse behavioral models measuring various aspects of nicotine dependence. Bupropion and (2S,3S)-hydroxybupropion, but not (2R,3R)-hydroxybupropion, significantly decreased the development of nicotine reward as measured in the conditioned place preference and withdrawal paradigm in mice. Bupropion and both of its metabolites reversed affective and somatic withdrawal signs in nicotine-dependent mice, but the (2S,3S)-hydroxymetabolite had higher potency. Bupropion and (2S,3S)-, but not (2R,3R)-hydroxybupropion, produced partial substitution for nicotine in drug discrimination tests. Our findings support the hypothesis that the effects of bupropion on measures of nicotine dependence reflect actions of bupropion itself, its hydroxymetabolites, or a combination and suggest that the (2S,3S)-hydroxy isomer is the most active principle, making it a potentially better drug candidate for smoking cessation than bupropion.

Affinity of aporphines for the human 5-HT2A receptor: insights from homology modeling and molecular docking studies.

Analogs of nantenine were docked into a modeled structure of the human 5-HT(2A) receptor using ICM Pro, GLIDE, and GOLD docking methods. The resultant docking scores were used to correlate with observed in vitro apparent affinity (K(e)) data. The GOLD docking algorithm when used with a homology model of 5-HT(2A), based on a bovine rhodopsin template and built by the program MODELLER, gives results which are most in agreement with the in vitro results. Further analysis of the docking poses among members of a C1 alkyl series of nantenine analogs, indicate that they bind to the receptor in a similar orientation, but differently than nantenine. Besides an important interaction between the protonated nitrogen of the C1 alkyl analogs and residue Asp155, we identified Ser242, Phe234, and Gly238 as key residues responsible for the affinity of these compounds for the 5-HT(2A) receptor. Specifically, the ability of some of these analogs to establish a H-bond with Ser242 and hydrophobic interactions with Phe234 and Gly238 appears to explain their enhanced affinity as compared to nantenine.

Synthesis, nicotinic acetylcholine receptor binding, and antinociceptive properties of 3'-(substituted phenyl)epibatidine analogues. Nicotinic partial agonists.

In 1992, John Daly et al. reported the isolation and structure determination of epibatidine. Epibatidine's unique structure and its potent nicotinic agonist activity have had a tremendous impact on nicotine receptor research. This research has led to a better understanding of the nicotinic acetylcholine receptor (nAChR) pharmacophore and to epibatidine analogues with potential as pharmacotherapies for treating various CNS disorders. In this study, we report the synthesis, receptor binding ([(3)H]epibatidine and [(125)I]iodoMLA), and in vivo pharmacological properties (mouse tail flick, hot plate, hypothermia, and spontaneous activity) of a series of 3'-(substituted phenyl)epibatidine analogues (5a-m). Results from these studies have added to the understanding of the nAChR pharmacophore and led to nicotinic partial agonists that may have potential for smoking cessation. All the analogues had affinities for the alpha4beta2 nAChR similar to epibatidine (1). 3'-(3-Dimethylaminophenyl)epibatidine (5m) has a nicotinic partial agonist pharmacological profile similar to the smoking cessation drug varenicline. Other analogues are partial agonists with varying degrees of nicotinic functional agonist and antagonist activity. 3'-(3-Aminophenyl)epibatidine (5j) is a more potent functional agonist and antagonist in all tests than varenicline. 3'-(3-Fluorophenyl)epibatidine and 3'-(3-chlorophenyl)epibatidine (5c and 5e) are more potent than varenicline when tested as agonists in four pharmacological tests and antagonists when evaluated against nicotine in the analgesia hot-plate test.

Amiodarone and its putative metabolites fail to activate wild type hTAAR1.

The ability of amiodarone and its putative metabolites to activate unmodified human trace amine associated receptor 1 (hTAAR1) stably expressed in CHO cell lines was evaluated. Receptor activation was monitored by measuring the accumulation of cAMP, the putative hTAAR1 native second messenger, or calcium mobilization in cells where the receptor was coupled to the promiscuous Gq, Galpha16. Despite literature reports of activation of rodent TAAR1 by these agents, no response was seen in either cell line although robust activation was obtained with the endogenous ligand beta-phenethylamine. These results indicate that TAAR1 activation by amiodarone and its analogs is species specific.

(+/-)-Nantenine analogs as antagonists at human 5-HT(2A) receptors: C1 and flexible congeners.

C1 and flexible analogs of (+/-)-nantenine were synthesized and evaluated for antagonist activity at human 5-HT(2A) receptors in a calcium mobilization assay. This work has resulted in the identification of the most potent 5-HT(2A) antagonist known based on an aporphine. Our results also suggest that the C1 position may be a key site for increasing 5-HT(2A) antagonist activity in this compound series. In addition, the structural rigidity of the aporphine core appears to be required for nantenine to function as a 5-HT(2A) antagonist.

Synthesis and structure-activity relationship of 3beta-(4-alkylthio, -methylsulfinyl, and -methylsulfonylphenyl)tropane and 3beta-(4-alkylthiophenyl)nortropane derivatives for monoamine transporters.

Early studies led to the identification of 3beta-(4-methoxyphenyl)tropane-2beta-carboxylic acid methyl ester (5) with high affinity at the DAT (IC(50)=6.5nM) and 5-HTT (K(i)=4.3nM), while having much less affinity at the NET (K(i)=1110nM). In the present study, we replaced the 4'-methoxy group of the 3beta-phenyl ring with a bioisosteric 4'-methylthio group to give 7a. We also synthesized a number of 3beta-(4-alkylthiophenyl)tropanes 7b-e, 3beta-(4-methylsulfinylphenyl) and 3beta-(4-methylsulfonylphenyl)tropane analogues 7f-h as well as the 3beta-(4-alkylthiophenyl)nortropane derivatives 8-11 to further characterize the structure-activity relationship of this type of compound for binding at monoamine transporters. With exception of the 4'-methylsulfonyl analogue 7h, all the tested compounds possessed high binding affinities at the 5-HTT. The K(i) values ranged from 0.19nM to 49nM. The 3beta-(4-methylthiophenyl)tropane 7a and its N-(3-fluoropropyl) analogue 9a and N-allyl analogue 10a are the most selective compounds for the 5-HTT over the NET (NET/5-HTT=314-364) in the series. However, none of the compounds showed selectivity similar to 5 for both the DAT and 5-HTT relative to the NET. This study provided useful SAR information for rational design of potent and selective monoamine transporter inhibitors.

Synthesis and in vitro opioid receptor functional antagonism of analogues of the selective kappa opioid receptor antagonist (3R)-7-hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (JDTic).

In previous structure-activity relationship (SAR) studies, we identified (3 R)-7-hydroxy- N-((1 S)-1-{[(3 R,4 R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (JDTic, 1) as the first potent and selective kappa opioid receptor antagonist from the trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine class of opioid antagonist. In the present study, we report the synthesis and in vitro opioid receptor functional antagonism of a number of analogues of 1 using a [ (35) S]GTPgammaS binding assay. The results from the studies better define the pharmacophore for this class of kappa opioid receptor antagonist and has identified new potent and selective kappa antagonist. (3 R)-7-Hydroxy- N-[(1 S,2 S)-1-{[(3 R,4 R)-4-(3-hydroxyphenyl)-3,4-dimethylpiperidin-1-yl]methyl}-2-methylbutyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxamide ( 3) with a K e value of 0.03 nM at the kappa receptor and 100- and 793-fold selectivity relative to the mu and delta receptors was the most potent and selective kappa opioid receptor antagonist identified.

mGluR5 antagonists that block calcium mobilization in vitro also reverse (S)-3,5-DHPG-induced hyperalgesia and morphine antinociceptive tolerance in vivo.

The present study comparatively evaluated the potency of a series of new phenylethyl[1,2,4]methyltriazines which are analogues of the classical metabotropic glutamate (mGlu) receptor subtype 5 (mGluR5) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) in blocking hyperalgesia induced by the group I mGlu receptor agonist (S)-3,5-DHPG as well as in reversing morphine antinociceptive tolerance in mice. Hyperalgesia was assessed in mice using the tail immersion test. Intrathecal (i.t.) pre-treatment with the test compounds 5-methyl-3-phenylethynyl-[1,2,4]triazine (RTI-4229-707), 5-methyl-3-(4-phenoxy-phenylethynyl-[1,2,4]triazine (RTI-4229-766), and 3-(3-methylphenylethynyl)-5-methyl-[1,2,4]triazine (RTI-4229-787) resulted in a dose-dependent blockade of (S)-3,5-DHPG-induced hyperalgesia. The inhibitory dose-50 (ID(50)) values were 0.49, 0.72 and 0.44 nmol/mouse, for RTI-4229-707, RTI-4229-766 and RTI-4229-787, respectively, compared to 18.63 nmol/mouse for MPEP. The other two compounds tested 3-(2,5-dimethylphenylethynyl)-5-methyl[1,2,4]triazine (RTI-4229-785) and 3-(2-methylphenylethynyl)-5-methyl[1,2,4]triazine (RTI-4229-828) were totally inactive. Morphine tolerance was induced in mice by implanting a 75 mg morphine pellet and assessing morphine-induced antinociception 72-h later. The morphine-pelleted mice showed a 5.5-fold tolerance to the antinociceptive effect of acute morphine compared to placebo-pelleted mice in the tail immersion test. Intracerebroventricular (i.c.v.) administration of the three active mGluR5 antagonists dose-dependently reversed morphine antinociceptive tolerance. The ID(50) values were 57.7, 25.8 and 64.3 nmol/mouse, for RTI-4229-707, RTI-4229-766 and RTI-4229-787, respectively, compared to 1050 nmol/mouse for MPEP. Similar to the hyperalgesia study, test compounds RTI-4229-785 and RTI-4229-828 were totally inactive in reversing morphine tolerance. These results are in agreement with our previous study in which we demonstrated that the same active mGluR5 antagonists blocked glutamate-mediated mobilization of internal calcium in a selective mGluR5 in vitro efficacy assay.