Inverse Agonist Ligand Research Articles

Modulation of acetaminophen-induced hepatotoxicity by the xenobiotic receptor CAR

Zhang J, Huang W, Chua SS, et al (Baylor College of Medicine, Houston, TX) Overdoses of acetaminophen are the leading cause of hospital admission for acute liver failure in the United States. Ingestion of only 2 to 3 times the maximum daily recommended dose of acetaminophen can cause hepatotoxicity, and higher doses result in potentially fatal centrilobular necrosis. Acetaminophen toxicity is increased in both human beings and rodents by pretreatment with various inducers of CYP gene expression, including ethanol and phenobarbital. Because the xenobiotic receptor CAR has recently been shown to mediate the effects of phenobarbital and other phenobarbital-like inducers, the effects of phenobarbital and the potent CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) on acetaminophen toxicity were examined in wild-type and CAR knockout mice. Both strains of mice were treated with inducers or the vehicle control, followed by acetaminophen (250 mg/kg of body weight). The known CAR activators and the high dose of acetaminophen induced expression of 3 acetaminophen-metabolizing enzymes in wild-type mice but not in the CAR null mice. The CAR null mice were resistant to acetaminophen toxicity. Inhibition of CAR activity by administration by the inverse agonist ligand androstanol 1 hour after acetaminophen treatment blocked hepatotoxicity in wild-type but not in CAR null mice. These findings demonstrate that CAR is a central mediator of acetaminophen toxicity in mice and potentially also in human beings. It would appear that CAR is not involved in the toxicity associated with ethanol-dependent induction of CYP2E1 and other targets because CAR activation caused a modest decrease in CYP2E1 mRNA levels. However, activation of either mouse or human CAR by appropriate inducers, including acetaminophen itself, increased the production of APAP-metabolizing enzymes and increased toxicity. Fatal outcomes have been reported to result from the combination of phenobarbital and acetaminophen in human beings. The findings of this study suggest a new strategy for treatment of acetaminophen toxicity based on the use of CAR agonists.

Plasmon-waveguide resonance spectroscopy: a new tool for investigating signal transduction by G-protein coupled receptors

Plasmon-waveguide resonance (PWR) spectroscopy provides a highly sensitive method for characterizing the kinetics, affinities and conformational changes involved in ligand binding to G-protein coupled receptors, without the need for radioactive or other labeling strategies. In the case of the cloned δ-opioid receptor from human brain incorporated into a lipid bilayer, we have shown that affinities determined in this way are consistent with those measured by standard binding procedures using membranes or whole cells containing the receptors, and that the spectral and kinetic properties of the binding processes allow facile distinction between agonist, inverse agonist, and antagonist ligands. We have also shown by direct measurements that G-protein binding affinities and the ability to undergo GTP/GDP exchange are dependent upon the type of ligand pre-bound to the receptor. PWR spectroscopy thus provides a powerful new approach to investigating signal transduction in biological membrane systems.

Inverse agonism at neurotensin receptors NTS1 and NTS2

Neurotensin (NT) is a peptide expressed in the brain and small intestines. Its central effects include analgesia, inhibition of food intake, modulation of central dopaminergic systems, and modulation of pituitary hormones secretion. In the gut, neurotensin is released upon food ingestion to regulate digestive functions. In addition, the peptide acts as a trophic factor on human colon, pancreatic, prostate, and lung cancer cell lines. Thus, neurotensin receptors represent interesting targets for the treatment of pain, obesity, schizophrenia, and cancer. Two neurotensin receptors, NTS1 and NTS2, that belong to the family of G protein-coupled receptors have been identified. A nonpeptide antagonist with selectivity for the NTS1, SR 48692, has been characterized. Recent mutagenesis and modeling studies allowed us to map agonist and antagonist binding sites in the NTS1 and to identify extracellular and transmembrane (TM) residues involved in receptor activation. In particular, we identified a transmembrane residue whose mutation confers constitutive activity to the NTS1 and reveals inverse agonist properties of SR 48692. Quite recently, we demonstrated that the human NTS2 is constitutively active and we characterized agonist, inverse agonist, and neutral antagonist ligands at this receptor. These results and their implications are the focus of the present paper.

Car Unlikely to Significantly Modulate Acetaminophen Hepatotoxicity in Most Humans

We have identified the xenobiotic receptor CAR (constitutive androstane receptor) as a key regulator of acetaminophen metabolism and hepatotoxicity. Known CAR activators as well as high doses of acetaminophen induced expression of three acetaminophen–metabolizing enzymes in wild–type but not in CAR null mice, and the CAR null mice were resistant to acetaminophen toxicity. Inhibition of CAR activity by administration of the inverse agonist ligand androstanol 1 hour after acetaminophen treatment blocked hepatotoxicity in wild–type but not CAR null mice. These results suggest an innovative therapeutic approach for treating the adverse effects of acetaminophen and potentially other hepatotoxic agents.

Complex effects of rexinoids on ligand dependent activation or inhibition of the xenobiotic receptor, CAR

BackgroundCAR/RXR heterodimers bind a variety of hormone response elements and activate transcription in the absence of added ligands. This constitutive activity of murine CAR can be inhibited by the inverse agonist ligand androstanol or increased by the agonist TCPOBOP. RXR agonists activate some RXR heterodimer complexes, which are termed permissive, while other non-permissive complexes are not responsive to such ligands.ResultsDirect protein-protein interaction studies demonstrate that the RXR agonist 9-cis-RA increases interaction of CAR/RXR heterodimers with the coactivator SRC-3, but also inhibits the ability of TCPOBOP to increase and androstanol to decrease coactivator binding. CAR transactivation of a response element with a five nucleotide spacer (DR-5) is unaffected by 9-cis-RA or the synthetic RXR agonist LG1069. In agreement with the inhibitory effect observed in vitro, these rexinoids block both the TCPOBOP mediated transactivation of this element and the androstanol dependent inhibition. In contrast, CAR transactivation of other response elements is increased by rexinoids. Stable expression of CAR in a HepG2 derived cell line increases expression of the endogenous CAR target CYP2B6. This expression is further increased by TCPOBOP but decreased by either androstanol or LG1069, and LG1069 blocks the stimulatory effect of TCPOBOP but not the inhibitory effect of androstanol.ConclusionWe conclude that CAR/RXR heterodimers are neither strictly permissive nor non-permissive for RXR signaling. Instead, rexinoids have distinct effects in different contexts. These results expand the potential regulatory mechanisms of rexinoids and suggest that such compounds may have complex and variable effects on xenobiotic responses.

Modulation of acetaminophen-induced hepatotoxicity by the xenobiotic receptor CAR.

We have identified the xenobiotic receptor CAR (constitutive androstane receptor) as a key regulator of acetaminophen metabolism and hepatotoxicity. Known CAR activators as well as high doses of acetaminophen induced expression of three acetaminophen-metabolizing enzymes in wild-type but not in CAR null mice, and the CAR null mice were resistant to acetaminophen toxicity. Inhibition of CAR activity by administration of the inverse agonist ligand androstanol 1 hour after acetaminophen treatment blocked hepatotoxicity in wild type but not in CAR null mice. These results suggest an innovative therapeutic approach for treating the adverse effects of acetaminophen and potentially other hepatotoxic agents.

Altered GABAA Receptor Subunit and Splice Variant Expression in Rats Treated With Chronic Intermittent Ethanol

Background: Intermittent chronic administration of ethanol to rats has been shown previously to produce a hyperexcitable, kindling-like state, accompanied by reduced inhibitory synaptic transmission in the hippocampus and changes in γ-aminobutyric acid type A (GABAA) receptors. Further information is needed on the detailed changes in GABAA receptors and their time course and persistence, as is comparison to changes after chronic, continuous ethanol. Methods: GABAA receptors were analyzed in the rat brain after chronic intermittent ethanol (CIE) by using radioligand binding, photoaffinity labeling of polypeptides, and estimates of messenger RNA (mRNA) levels of receptor subunits by reverse transcriptase–polymerase chain reaction (RT-PCR) and in situ hybridization. Results: CIE rats were confirmed to have increased GABAA receptor binding of the benzodiazepine partial inverse agonist and ethanol antidote ligand Ro15-4513, due to increased expression of the α6 subunit polypeptide in the cerebellum, shown by photoaffinity labeling. Estimates of mRNA levels by use of RT-PCR did not reveal any significant increase in α6 or in several other receptor subunits in several brain regions, but a decrease in the ratio of the long and short splice variants (L/S) of the γ2 subunit was detected in the hippocampus, especially the CA1 region. Conclusions: Changes in GABAA receptors were found in rats given CIE. Increased α6 subunit in the cerebellum was demonstrated by using both the binding to diazepam-insensitive sites for [3H]Ro15-4513 and increased levels of the 57-kDa α6 polypeptide after photoaffinity labeling with this ligand. This increase appeared after 30 doses of ethanol and decayed to normal 1 week after ethanol was discontinued. The transient change in cerebellar α6 subunit-containing receptors, also reportedly seen after chronic continuous ethanol, is thus unlikely to account for the persistently hyperexcitable, kindled, seizure-susceptible state seen in CIE. However, the significant decrease in γ2 subunit L/S splice variant ratio in the hippocampus implies changes in GABAA receptor function, possibly involving protein phosphorylation by protein kinase C. Altered receptor trafficking and turnover associated with synaptic plasticity may contribute to the observed reduced inhibition in the hippocampus and other signs of alcohol dependence produced by CIE.

Altered GABAA Receptor Subunit and Splice Variant Expression in Rats Treated With Chronic Intermittent Ethanol

Intermittent chronic administration of ethanol to rats has been shown previously to produce a hyperexcitable, kindling-like state, accompanied by reduced inhibitory synaptic transmission in the hippocampus and changes in gamma-aminobutyric acid type A (GABAA) receptors. Further information is needed on the detailed changes in GABAA receptors and their time course and persistence, as is comparison to changes after chronic, continuous ethanol. GABAA receptors were analyzed in the rat brain after chronic intermittent ethanol (CIE) by using radioligand binding, photoaffinity labeling of polypeptides, and estimates of messenger RNA (mRNA) levels of receptor subunits by reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. CIE rats were confirmed to have increased GABAA receptor binding of the benzodiazepine partial inverse agonist and ethanol antidote ligand Ro15-4513, due to increased expression of the alpha6 subunit polypeptide in the cerebellum, shown by photoaffinity labeling. Estimates of mRNA levels by use of RT-PCR did not reveal any significant increase in alpha6 or in several other receptor subunits in several brain regions, but a decrease in the ratio of the long and short splice variants (L/S) of the gamma2 subunit was detected in the hippocampus, especially the CA1 region. Changes in GABAA receptors were found in rats given CIE. Increased alpha6 subunit in the cerebellum was demonstrated by using both the binding to diazepam-insensitive sites for [3H]Ro15-4513 and increased levels of the 57-kDa alpha6 polypeptide after photoaffinity labeling with this ligand. This increase appeared after 30 doses of ethanol and decayed to normal 1 week after ethanol was discontinued. The transient change in cerebellar alpha6 subunit-containing receptors, also reportedly seen after chronic continuous ethanol, is thus unlikely to account for the persistently hyperexcitable, kindled, seizure-susceptible state seen in CIE. However, the significant decrease in gamma2 subunit L/S splice variant ratio in the hippocampus implies changes in GABAA receptor function, possibly involving protein phosphorylation by protein kinase C. Altered receptor trafficking and turnover associated with synaptic plasticity may contribute to the observed reduced inhibition in the hippocampus and other signs of alcohol dependence produced by CIE.

Monitoring Receptor Oligomerization Using Time-resolved Fluorescence Resonance Energy Transfer and Bioluminescence Resonance Energy Transfer: THE HUMAN δ-OPIOID RECEPTOR DISPLAYS CONSTITUTIVE OLIGOMERIZATION AT THE CELL SURFACE, WHICH IS NOT REGULATED BY RECEPTOR OCCUPANCY

Oligomerization of the human delta-opioid receptor and its regulation by ligand occupancy were explored following expression in HEK293 cells using each of co-immunoprecipitation of differentially epitope-tagged forms of the receptor, bioluminescence resonance energy transfer and time-resolved fluorescence resonance energy transfer. All of the approaches identified constitutively formed receptor oligomers, and the time-resolved fluorescence studies confirmed the presence of such homo-oligomers at the cell surface. Neither the agonist ligand [d-Ala(2),d-Leu(5)]enkephalin nor the inverse agonist ligand ICI174864 were able to modulate the oligomerization status of this receptor. Interactions between co-expressed delta-opioid receptors and beta(2)-adrenoreceptors were observed in co-immunoprecipitation studies. Such hetero-oligomers could also be detected using bioluminescence resonance energy transfer although the signal obtained was substantially smaller than for homo-oligomers of either receptor type. Signal corresponding to the delta-opioid receptor-beta(2)-adrenoreceptor hetero-oligomer was increased in the presence of agonist for either receptor. However, substantial levels of this hetero-oligomer were not detected at the cell surface using time-resolved fluorescence resonance energy transfer. These studies demonstrate that, following transient transfection of HEK293 cells, constitutively formed oligomers of the human delta-opioid receptor can be detected by a variety of approaches. However, these are not regulated by ligand occupancy. They also indicate that time-resolved fluorescence resonance energy transfer represents a means to detect such oligomers at the cell surface in populations of intact cells.

2-[N-Acylamino(C1-C3)alkyl]indoles as MT1 melatonin receptor partial agonists, antagonists, and putative inverse agonists.

The synthesis of several novel indole melatonin analogues substituted at the 2-position with acylaminomethyl (8-11), acylaminoethyl (5a-k), or acylaminopropyl (13) side chains is reported. On the basis of a novel in vitro functional assay (specific binding of [35S]GTPgammaS), which can discriminate agonist from partial agonist, antagonist, and inverse agonist ligands, 5a,g, h,j and 13 were shown to be partial agonists, 5d,e and 8-11 competitive antagonists, and 5b,c,k putative inverse agonists. Binding and functional assays were performed on cloned human MT1 receptor. Structure-activity relationship considerations indicate that N-[1-aryl-2-(4-methoxy-1H-indol-2-yl)(C1-C2)alkyl]alkanamides represent a lead structure for this type of ligands.

Efficacy of inverse agonists in cells overexpressing a constitutively active beta2-adrenoceptor and type II adenylyl cyclase.

1 Maximal stimulant output from the adenylyl cyclase cascade in neuroblastoma x glioma hybrid, NG108-15, cells is limited by the levels of expression of isoforms of adenylyl cyclase. Stable expression in these cells of a constitutively active mutant (CAM) version of the human beta2-adrenoceptor resulted in higher basal adenylyl cyclase activity than following expression of the human wild type beta2-adrenoceptor. Isoprenaline acted as a full agonist in membranes from both wild type and CAM beta2-adrenoceptor expressing clones. 2 Expression of type II adenylyl cyclase resulted in a substantially elevated capacity of isoprenaline to stimulate [3H]-forskolin binding, whereas in CAM beta2-adrenoceptor expressing cells the basal high affinity [3H]-forskolin binding represented a markedly greater % of the maximal effect which could be produced by addition of isoprenaline, and the EC50 for isoprenaline was some 10 fold lower than in cells expressing the wild type beta2-adrenoceptor. 3 Further transfection of the CAM beta2-adrenoceptor expressing cells with type II adenylyl cyclase greatly increased both absolute basal and agonist-stimulated levels of adenylyl cyclase activity. 4 Betaxolol, ICI 118,551, sotalol and timolol acted as inverse agonists with varying degrees of efficacy, whereas propranolol functioned as a neutral antagonist and alprenolol as a partial agonist. 5 Pretreatment of the CAM beta2-adrenoceptor and type II adenylyl cyclase expressing clones with the irreversible alkylating agent BAAM (1 microM) did not reduce the efficacy of isoprenaline but eliminated efficacy from all the inverse agonist ligands. This effect was dependent upon the concentration of BAAM employed, with half-maximal effects being produced between 10 nM and 100 nM of the alkylating agent, which is similar to the concentrations required to prevent subsequent ligand access to some 50% of the CAM beta2-adrenoceptor population. 6 These data demonstrate that inverse agonist efficacy can be modulated by receptor availability and also indicate why in physiological systems, inverse agonism can be difficult to detect.

Up-regulation of a constitutively active form of the β2-adrenoceptor by sustained treatment with inverse agonists but not antagonists

In neuroblastoma × glioma hybrid, NG108-15, cells transfected to stably express a constitutively active mutant (CAM) form of the human R2-adrenoceptor, the β-adrenoceptor ligands sotalol and betaxolol functioned as inverse agonists as they reduced basal adenylyl cyclase activity whereas the antagonists dihydroalprenolol and propranolol did not. Maintained presence of the CAM β 2-adrenoceptor inverse agonists but not the antagonists in the culture medium of the cells resulted in a substantial, concentration-dependent, up-regulation of the CAM β 2-adrenoceptor. Up-regulation of the CAM β 2-adrenoceptor by the inverse agonists was prevented by co-incubation of the cells with either propranolol or dihydroalprenolol. Neither maintained elevation of cAMP levels nor the inhibition of adenylyl cyclase activity altered the ability of the inverse agonist ligands to cause receptor up-regulation.

Structure – activity relationships in antagonist and inverse agonist ligands for the benzodiazepine receptor

The X-ray crystal and molecular structures of the three benzodiazepine (BZD) receptor ligands are presented and the electronic character of inverse agonist ligands is probed through molecular orbital calculations. Two of the ligands have a 6-benzylamino substituent: 6-benzylamino-β-carboline-3-carboxylic acid methyl ester, 1, which is a high affinity antagonist with IC50 = 10 nM, and 6-benzylamino-β-carboline, 2, which is a moderate affinity inverse agonist with IC50 = 106 nM. The third compound, 3-ethoxy-β-carboline hydrochloride, 3, displays partial inverse agonist activity with an IC50of 24 nM. Intermolecular interactions, including extensive hydrogen bonding involving both the pyridyl nitrogen atom and the indole N—H as well as π stacking of aromatic rings, are characteristic of β-carbolines and are found in these three structures. In addition, two of these compounds are protonated in the crystalline state, thereby providing a model for interactions in the absence of the 3-carboxylic acid ester function. Electronic calculations show that (1) the partial inverse agonist ligand has the highest charge on the N(2) atom and (2) high affinity β-carbolines possess two neighboring sites that have high electrostatic attraction for a hydrogen atom in an intermolecular interaction. These findings suggest that modifications to the 3-position side chain to enhance the charge on the pyridyl N atom and provide a hydrogen bond acceptor site will facilitate the development of partial inverse agonist ligands. Keywords: β-carbolines, benzodiazepine receptor, crystallography, structure–activity relationships.

Stable expression of cloned rat GABA A receptor subunits in a human kidney cell line

A predominant form of the GABA A/benzodiazepine receptor-Cl − channel complex is believed to consist of three different 48–55 kDa subunits (α, β, γ) with unknown stoichiometry. Plasmids containing the rat GABA A receptor cDNAs coding for α 1, β 2, and γ 2 were co-transfected, along with a plasmid encoding G418 resistance, into human embryonic kidney cells previously transformed with Adenovirus 5 (HEK-293) [J. Gen. Virol., 36 (1977) 59–72]. Four percent of the G418 resistant colonies were found to express mRNA for all three of the GABA A subunits constitutively. A single cell clone derived from one of the α 1 β 2 γ 2 expressors has demonstrated stable electrophysiological characteristics over 25 passages. The GABA-activated Cl − current in this cell line is blocked by picrotoxin and bicuculline, and is modulated by a variety of agonist and inverse agonist ligands including diazepam, Ro 154513, zolpidem, and β-CCE. The cell line has been used successfully over a 12-month period as a screen for novel drugs modulating GABA-mediated polarization of neuronal cells.

Opposite effects of agonist and inverse agonist ligands of benzodiazepine receptor on self-defensive and submissive postures in the rat.

The effects of benzodiazepine receptor ligands on different types of defensive behaviours were examined in intruder male rats confronted with offensive residents. Chronic administration, via a subcutaneous silastic pellet, of a full agonist (diazepam) for 15 days increased self-defensive postures as well as social and non-social behaviour whereas submissive postures and flight were reduced. Acute administration of a partial agonist (ZK 91296) resulted in a similar increase in self-defensive postures and a decrease of submission and non-social elements. Acute administration of a partial inverse agonist (FG 7142) reduced defensive postures and social behaviour whereas submissive postures were increased. These results show that activation of benzodiazepine receptors by full or partial agonists increased self-defensive responses to attacks by a conspecific, while decreasing submissive postures. On the contrary, "inverse activation" of these receptors by an inverse agonist increased submissive postures while decreasing self-defensive responses. These data suggest that benzodiazepine receptors are involved in the control of the animal's strategy to respond to an attack of another rat.

Flumazenil Provocation of Panic Attacks

The possibility that panic disorder might be due to abnormal activity of endogenous ligands of the benzodiazepine receptor was investigated with the use of the benzodiazepine antagonist flumazenil. Physiological and subjective psychological responses to this selective antagonist were measured in 10 patients with panic disorder and in 10 control subjects, by using a placebo-controlled crossover study design. Subjective anxiety responses after flumazenil infusion were significantly higher in the patient group with panic disorder than in the controls, and eight patients with panic disorder but no controls had panic attacks. This anxiogenic effect of flumazenil in the patients argues against the presence of endogenous anxiogenic (inverse agonist) ligands. Possible explanations include the differential production of an anxiolytic endogenous ligand or an altered benzodiazepine receptor "set-point." Such an abnormality may contribute to the pathogenesis of panic disorder.