Discriminative and aversive properties of beta-carboline-3-carboxylic acid ethyl ester, a benzodiazepine receptor inverse agonist, in rhesus monkeys

Rhesus monkeys were trained to discriminate injections of saline from those of beta-carboline-3-carboxylic acid ethyl ester (beta-CCE), a compound that binds to the benzodiazepine receptor, but often has actions opposite to those of the benzodiazepines. A benzodiazepine agonist midazolam and low doses of a specific benzodiazepine antagonist, Ro 15-1788, reversed the discriminative effects of beta-CCE. Higher doses of Ro 15-1788 produced stimulus effects similar to beta-CCE. In a separate experiment, monkeys responded to terminate intravenous infusions of beta-CCE, but not midazolam. This aversive effect of beta-CCE was reversed by Ro 15-1788. The behavioral effects of beta-CCE in these non-human primates are consistent with other data that have shown it to act on benzodiazepine receptors, and support the hypothesis that beta-CCE can be considered an inverse agonist at this receptor.     

The bovine peripheral-type benzodiazepine receptor: a receptor with low affinity for benzodiazepines.

The density of bovine peripheral-type benzodiazepine receptors (PBR) in four tissues was highest in adrenal cortex. The adrenal cortex PBR cofractionated with a mitochondrial membrane marker enzyme and could be solubilized with intact ligand binding properties using digitonin. The membrane bound and soluble mitochondrial receptors were pharmacologically characterized and showed the rank order of potency to inhibit [3H]PK 11195 binding was PK 11195 greater than protoporphyrin IX greater than benzodiazepines (clonazepam, diazepam, or Ro5-4864). [3H]PK 11195 binding to bovine adrenal mitochondria was unaffected by diethylpyrocarbonate, a histidine residue modifying reagent that decreased binding to rat liver mitochondria by 70%. [3H]PK 14105 photolabeled the bovine PBR and the Mr was estimated under nondenaturing (200 kDa) and denaturing (17 kDa) conditions. These results demonstrate the bovine peripheral-type benzodiazepine receptor is pharmacologically and biochemically distinct from the rat receptor, but the receptor component photolabeled by an isoquinoline ligand has a similar molecular weight.     

3H]DU41165: a high affinity ligand and novel photoaffinity labeling reagent for the progesterone receptor

17 alpha-Acetoxy-6-fluoro-16-methylene-(9 beta, 10 alpha)pregna-4,6-dien- 3,20-dione (DU41165), a retroprogestin (9 beta, 10 alpha) embodying a fluorine-substituted dienone system, has been prepared in high specific activity tritium-labeled form (4 Ci/mmol) and shown to be a high affinity ligand for the progesterone receptor (PgR) and a highly selective photoaffinity labeling reagent for PgR. The radiosynthesis involved conversion of DU41231 (the 17 alpha-hydroxy analog of DU41165) to DU41165 by treatment with tritium-labeled acetic anhydride. The binding affinity of DU41165 for PgR was determined by both a competitive binding assay and a direct binding assay (Scatchard analysis) to be 1.6-2.2-times higher than that of the high affinity synthetic progestin promegestone (R5020). In unlabeled form, DU41165 demonstrates photoinactivation of PgR to the extent of 60% at 60 min. In radiolabeled form [3H]DU41165 demonstrates specific covalent attachment with an efficiency of 5-7%. SDS-polyacrylamide gel electrophoresis of photoattached [3H]DU41165 confirms that there is covalent labeling of both the B subunit (Mr = 118,000), and the A subunit (Mr = 88,000) of PgR in a molar ratio of approximately 1:3.     

Photochemical derivatization of agarose: synthesis of an affinity agarose using a photoaffinity ligand specific for the peripheral-type benzodiazepine receptor

PK 14105, a photoaffinity ligand specific for the peripheral-type benzodiazepine receptor (PBZR), was photochemically coupled to omega-aminobutyl agarose (ABAg) to yield PK 14105 agarose (PKAg). 19F and 1H NMR spectroscopy were consistent with the proposed site of coupling at the 2'-fluorine of PK 14105 by the primary amine moiety of ABAg. Quantitation of the affinity gel using two different colorimetric assays for primary amines suggests approximately 50% of the available primary amine groups of ABAg were bound by PK 14105. The estimated concentration of PK 14105 bound to ABAg was 2.3 mumols/ml of settled gel (2.3 mM effective ligand concentration). PKAg specifically binds the bovine PBZR solubilized by digitonin. The affinity of PKAg for the soluble PBZR was estimated by varying the concentration of PKAg. PBZR binding to PKAg was saturable and the apparent affinity of PKAg for the bovine receptor was estimated from the saturation data. A PKAg affinity column bound 85% of the solubilized PBZR from rat adrenals partially purified by anion exchange chromatography. These results indicate PKAg is a receptor-specific affinity media which may be useful in the purification of the native PBZR from various species.     

Discriminative stimulus properties of methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), an inverse agonist at benzodiazepine receptors

Rats (N = 8) were trained to discriminate the stimulus properties of the potent benzodiazepine (BZ) receptor inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) from saline in a two-lever operant task. The initial training dose of DMCM was 0.4 mg/kg at which the discrimination developed slowly; increasing the dose to 0.8 mg/kg resulted in rapid acquisition. However, since convulsions eventually developed during further training (sensitization), the training dose was finally individualized below the convulsive threshold (0.4-0.7 mg/kg). The DMCM cue was mimicked by FG 7142 (10 mg/kg), a non-convulsant anxiogenic beta-carboline, by pentylenetrazol (20-30 mg/kg), and by the GABA antagonist bicuculline (2 mg/kg). The DMCM cue was not, or marginally, blocked by diazepam (2.5 mg/kg) or pentobarbital (10-15 mg/kg). Furthermore, the BZ receptor antagonists CGS 8216 (2.5 mg/kg), ZK 93426 (20 mg/kg), and Ro 15-1788 (20-80 mg/kg) also did not, or only marginally, block the DMCM cue. However, the receptor antagonists (alone) substituted for DMCM although Ro 15-1788 was less effective. The partial BZ receptor agonist ZK 91296 (25 mg/kg), which is structurally similar to DMCM, blocked completely the DMCM stimulus effect. THIP (4 mg/kg) did not block the DMCM cue. To explain these results, we suggest that the repeated DMCM treatment, necessary for maintaining the discrimination, shifts the balancing point ("set-point") for positive (i.e., BZ-like) agonist efficacy versus inverse agonist efficacy, towards inverse action. This hypothesis was supported by the finding of an enhanced ability of GABA to reduce 3H-DMCM binding to cortical neuronal membranes of animals treated chronically with DMCM in a regimen similar to that used to maintain the DMCM discrimination. Furthermore, this treatment did not affect baseline 3H-DMCM binding, baseline or GABA stimulated 3H-diazepam binding, or 35S-TBPS binding (to chloride channels).     

3H]Para-amino-clonidine: a novel ligand which binds with high affinity to alpha-adrenergic receptors.

Para-amino-clonidine (PAC) is an α-adrenergic agonist with extraordinarily high potency in some peripheral tissues. We have demonstrated the labeling of α-adrenergic binding sites in central and peripheral tissues with [³H]PAC and compared properties of this binding to those of [³H]clonidine. [³H]PAC binds saturably with a dissociation constant (K_D) of about 0.9 nM to rat cerebral cortex membranes. It has about 2–3 times the affinity of [³H]clonidine for α-receptor binding sites. The greater affinity is attributable mainly to a slower dissociation of [³H]PAC than [³H]clonidine from binding sites. The relative and absolute potencies of various adrenergic agonists and antagonists in competing for [³H]PAC and [³H]clonidine binding are essentially the same. [³H]PAC can also be utilized to label α-adrenergic binding sites in the kidney and spleen where the relative potencies of PAC and clonidine are the same as in the brain.     

The insulin receptor. Structural basis for high affinity ligand binding

Treatment of the soluble insulin receptor from human placenta with 1.25 mM dithiothreitol and 75 mM Tris at pH 8.5 results in complete reduction of interhalf disulfide bonds (class 1 disulfides) and dissociation of the tetrameric receptor into the dimeric alpha beta form. The alpha beta receptor halves exhibit a reduced affinity for insulin binding (B?ni-Schnetzler, M., Rubin, J. B., and Pilch, P. F. (1986) J. Biol. Chem. 261, 15281-15287). Kinetic experiments reveal that reduction of class 1 disulfides is a faster process than the loss of affinity for ligand, indicating that events subsequent to reduction of interhalf disulfides are responsible for the affinity change. We show that a third class of alpha subunit intrachain disulfides is more susceptible to reduction at pH 7.6 than at pH 8.5 and appears to form part of the ligand binding domain. Reduction of the intrachain disulfide bonds in this part of the alpha subunit leads to a loss of insulin binding. Modification of this putative binding domain by dithiothreitol can be minimized if reduction is carried out at pH 8.5. When the insulin receptor in placental membranes is reduced at pH 8.5, the receptor's affinity for insulin is not changed when binding is measured in the membrane. However, the Kd for insulin binding is reduced 10-fold when alpha beta receptor halves are subsequently solubilized. Scatchard analysis of insulin binding to reduced or intact receptors in the membrane and in soluble form together with sucrose density gradient analysis of soluble receptors suggests that alpha beta receptor halves remain associated in the membrane after reduction, but they are dissociated upon solubilization. We interpret these results to mean that the association of two ligand binding domains, 2 alpha beta receptor halves, is required for the formation of an insulin receptor with high affinity for ligand.     

11 beta-chloromethyl-[3H]estradiol-17 beta: a very high affinity, reversible ligand for the estrogen receptor

It has been suggested that binding of 11 beta-chloromethyl estradiol (11 beta-CME2) to the estrogen receptor is irreversible, since its complex with receptor fails to undergo exchange with estradiol (E2). To investigate this behavior directly, 11 beta-CME2 was prepared in high specific activity, tritium-labeled form: The binding of [3H]11 beta-CME2 to the estrogen receptor from lamb and rat uterus and MCF-7 human breast cancer cells was shown to be fully reversible; the 11 beta-CME2 complex with receptor, as well as that of a structural analog 11 beta-ethyl estradiol, however, do not dissociate or exchange with [3H]E2 over a 22 h period at 25 degrees C. By competitive or direct binding assays, the affinity of 11 beta-CME2 for the estrogen receptor can be estimated to be as much as 10- to 30-fold higher than that of E2. The complexes of estrogen receptor from MCF-7 cells with [3H]11 beta-CME2 and [3H]E2 show identical velocity sedimentation profiles on sucrose gradients, under conditions when the receptor is either a monomer of a dimer. Because of its very high affinity and unusual dissociation kinetics, [3H]11 beta-CME2 should be a very useful ligand for studies of estrogen receptor dynamics and in the assay of estrogen receptor concentrations in tumors and tissues.     

Characterization of a rat kidney thromboxane A2 receptor: High affinity for the agonist ligand I-BOP

We have cloned a rat kidney thromboxane A2 receptor (TP) cDNA. This receptor was shown to be functional in that the thromboxane A₂ mimetics, U46619 and 1-BOP, elicited calcium transients in Xenopus oocytes and HEK293 cells expressing the TP receptor, respectively. Comparison of the affinities of the rat and human TP sites for the agonist radioligand [¹²⁵I]BOP showed that the rat TP site has about a ten-fold higher affinity for this drug (K_D = 0.5 vs. 4.4 nM) while the affinities of the two sites for other compound (U46619, I-PTH-OH) were the same. Our results are significant in that they identify a cloned TP as having a picomolar affinity for [¹²⁵I]BOP.     

Irreversible inactivation of the beta-adrenoreceptor by a partial agonist. Evidence for selective loss of the agonist high affinity binding sites

The catecholamine derivatives aminomenthylnorepinephrine (compound 1) and bromoacetylaminomenthylnorepinephrine (compound 2) were synthesized and their interaction with the rat lung beta-adrenoreceptor was characterized. Compared to (-)-isoproterenol, compounds 1 and 2 were 10 and 280 times less potent, respectively, at inhibiting (-)-[3H]dihydroalprenolol binding. At pH 7.4, all 3 compounds induced a loss of receptors (40-60%) which could be recovered by treatment with guanyl-5'-yl imidodiphosphate (Gpp(NH)p). However, at pH 8.1 Gpp(NH)p treatment did not recover those receptors lost by compound 2 only. The compound 2-induced receptor loss at pH 8.1 was time-dependent, prevented by propranolol but unaffected by Gpp(NH)p or after membrane heating at 50 degrees C which prevented the formation of the agonist high affinity binding state. Although, the maximal receptor loss as measured by [3H]dihydroalprenolol was 40-60%, more than 80% of the receptors were lost when measured by direct agonist binding, and the receptors left showed little agonist high affinity binding state formation. In rat reticulocyte membranes, compounds 1 and 2 stimulated adenylate cyclase activity with intrinsic activities of 0.55 and 0.31, respectively. However, at pH 8.1, compound 2 initially stimulated the enzyme followed by a blockade. These data indicated that both compounds 1 and 2 were partial beta-adrenoreceptor agonists and, at pH 8.1, compound 2 appeared to bind irreversibly only to those lung receptors able to form the agonist high affinity binding state. Furthermore, after irreversible binding, compound 2 appeared to act as an antagonist.     

125I][D-Ala2]deltorphin-I: a high affinity, delta-selective opioid receptor ligand.

The selective delta opioid agonist [D-Ala2]deltorphin-I was radioiodinated and the product purified using reverse phase HPLC. The binding characteristics and distribution profile of [125I][D-Ala2]deltorphin-I were assessed in mouse brain using homogenate binding techniques and quantitative autoradiography. [125I][D-Ala2]deltorphin-I bound with high affinity to a single class of sites (KD = 0.5 nM) in brain membrane preparations and striatal sections. Competition studies indicated that [125I][D-Ala2]deltorphin-I was selectively labeling delta opioid receptors as shown by the ratio of apparent affinities for mu and delta receptors (KI mu/KI delta = 1388). The autoradiographical distribution profile of [125I][D-Ala2]deltorphin-I binding sites was also consistent with that of other delta-selective radioligands. The data indicate that [125I][D-Ala2]deltorphin-I binds to delta opioid receptors with high affinity and selectivity. Because of its very high specific activity, it can be detected rapidly with high sensitivity by autoradiographic emulsion.     

Cyclic, disulfide- and dithioether-containing opioid tetrapeptides: development of a ligand with high delta opioid receptor selectivity and affinity

Tetrapeptides of primary sequence Tyr-X-Phe-YNH2, where X is D-Cys or D-Pen (penicillamine) and where Y is D-Pen or L-Pen, were prepared and were cyclized via the side chain sulfurs of residues 2 and 4 to disulfide or dithioether-containing analogs. These peptides are related to previously reported penicillamine-containing pentapeptide enkephalin analogs but lack the central glycine residue of the latter and were designed to assess the effect of decreased ring size on opioid activity. Binding affinities of the tetrapeptides were determined to both mu and delta opioid receptors. Binding affinity and selectivity in the tetrapeptide series were observed to be highly dependent on primary sequence. For example, L-Pen4 analogs displayed low affinity and were nonselective, while the corresponding D-Pen4 diastereomers were of variable affinity and higher selectivity. Among the latter compounds were examples of potent analogs in which selectivity shifted from delta selective to mu selective as the ring size was increased. The relatively high binding affinity and delta receptor selectivity observed with one of the carboxamide terminal disulfide analogs led to the synthesis of the corresponding carboxylic acid terminal, Tyr-D-Cys-Phe-D-PenOH. This analog displayed delta receptor binding selectivity similar to that of the standard delta ligand, [D-Pen2,D-Pen5]enkephalin (DPDPE), and was found to have a 3.5-fold higher binding affinity than DPDPE. All the tetrapeptides were further evaluated in the isolated mouse vas deferens (mvd) assay and all displayed opioid agonist activity. In general, tetrapeptide potencies in the mouse vas deferens correlated well with binding affinities but were somewhat lower. Receptor selectivity in the mvd, assessed by examining the effect of opioid antagonists on the tetrapeptide concentration-effect curves, was similar to that determined in the binding studies.     

A series of Fas receptor agonist antibodies that demonstrate an inverse correlation between affinity and potency

Receptor agonism remains poorly understood at the molecular and mechanistic level. In this study, we identified a fully human anti-Fas antibody that could efficiently trigger apoptosis and therefore function as a potent agonist. Protein engineering and crystallography were used to mechanistically understand the agonistic activity of the antibody. The crystal structure of the complex was determined at 1.9 Å resolution and provided insights into epitope recognition and comparisons with the natural ligand FasL (Fas ligand). When we affinity-matured the agonist antibody, we observed that, surprisingly, the higher-affinity antibodies demonstrated a significant reduction, rather than an increase, in agonist activity at the Fas receptor. We propose and experimentally demonstrate a model to explain this non-intuitive impact of affinity on agonist antibody signalling and explore the implications for the discovery of therapeutic agonists in general.     

Meclizine is an agonist ligand for mouse constitutive androstane receptor (CAR) and an inverse agonist for human CAR

The constitutive androstane receptor (CAR, NR1I3) is a key regulator of xenobiotic and endobiotic metabolism. The ligand-binding domains of murine (m) and human (h) CAR are divergent relative to other nuclear hormone receptors, resulting in species-specific differences in xenobiotic responses. Here we identify the widely used antiemetic meclizine (Antivert; Bonine) as both an agonist ligand for mCAR and an inverse agonist for hCAR. Meclizine increases mCAR transactivation in a dose-dependent manner. Like the mCAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, meclizine stimulates binding of steroid receptor coactivator 1 to the murine receptor in vitro. Meclizine administration to mice increases expression of CAR target genes in a CAR-dependent manner. In contrast, meclizine suppresses hCAR transactivation and inhibits the phenobarbital-induced expression of the CAR target genes, cytochrome p450 monooxygenase (CYP)2B10, CYP3A11, and CYP1A2, in primary hepatocytes derived from mice expressing hCAR, but not mCAR. The inhibitory effect of meclizine also suppresses acetaminophen-induced liver toxicity in humanized CAR mice. These results demonstrate that a single compound can induce opposite xenobiotic responses via orthologous receptors in rodents and humans.     

Interactions between the benzodiazepine receptor antagonist Ro 15-1788 (flumazepil) and the inverse agonist beta-CCE: behavioral studies with squirrel monkeys

The effects of Ro 15-1788 and ethyl-beta-carboline-3-carboxylate (beta-CCE) were studied alone and in combination on the behavioral performances of squirrel monkeys. Under one procedure, performances maintained by food were suppressed by electric shock presentation (punishment or "conflict" procedure). Under a second procedure, responding was maintained either by food or electric shock delivery under a 5-min fixed-interval schedule. Doses of beta-CCE between 0.1 and 3.0 mg/kg, i.m., produced graded decreases in punished responding which were reversed by pretreatment with Ro 15-1788 (1.0 - 10.0 mg/kg, i.m.). Low doses of beta-CCE (0.03 - 0.3 mg/kg, i.m.) increased responding of monkeys maintained by shock presentation, but did not affect food-maintained responding; higher doses of beta-CCE decreased responding under both schedules. These effects of beta-CCE are opposite those produced by the benzodiazepines under this procedure. Ro 15-1788 (1.0 mg/kg i.m.) antagonized the effects of beta-CCE, producing a shift to the right in the dose-response curves. These findings provide further support for the view that beta-CCE and Ro 15-1788 produce effects mediated by the same benzodiazepine receptor recognition site.     

Characterization of benzodiazepine receptors with a fluorescence-quenching ligand

A conjugate of the high affinity benzodiazepine receptor ligand Ro 15-1788 and the fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) moiety was synthesized. This novel compound (BD 623) exhibited excitation and emission maxima at 486 and 542 nm, respectively, and possessed fluorescent properties that are dependent upon the polarity of its environment. BD 623 bound reversibly to benzodiazepine receptors in the central nervous system with an apparent affinity (K(i) 5.7 nM) comparable to the parent imidazobenzodiazepine (K(d) 2.8 nM). Addition of BD 623 to a suspension of brain membranes resulted in a time-dependent quenching of its fluorescence. Fluorescence quenching of this compound was readily reversed by specific benzodiazepine receptor ligands but not by a variety of other substances. Moreover, inactivation of benzodiazepine receptors by photoaffinity labeling with Ro 15-4513 resulted in a reduction in the fluorescence quenching of BD 623 consistent with the reduction in density of benzodiazepine receptors measured using a radioreceptor assay. Monitoring of fluorescence/dequenching of BD 623 in real time permitted a quantitative characterization of the ligand-receptor interaction, with both the K(d) of BD 623 (13.9 nM) and K(i) of Ro 15-1788 (5.7 nM) comparable with the estimates obtained using radioreceptor techniques. These results indicate that application of fluorescence quenching techniques with BD 623 could prove a useful adjunct for the study of benzodiazepine receptors. BD 623 may serve as a prototype for the development of other fluorescent ligands to study ligand-receptor interactions.     

6-Methyl-3'-bromoflavone, a high-affinity ligand for the benzodiazepine binding site of the GABA(A) receptor with some antagonistic properties.

6-Methyl-3'-bromoflavone inhibited [(3)H]flunitrazepam binding to the benzodiazepine binding site of the GABA(A) receptor (BDZ-bs) with Ki values between 10 and 50 nM in different brain regions.The GABA ratio of 1.03 for [(3)H]flunitrazepam binding to cerebral cortex, 0.76 for cerebellum, 0.7 for hippocampus, 0.7 for striatum, and 0.8 for spinal cord indicated an antagonistic or weak inverse agonistic profile of 6-methyl-3'-bromoflavone on BDZ-bs. Unlike classical benzodiazepines, it had no anticonvulsant, anxiolytic, myorelaxant, sedative, amnestic or motor incoordination effects. However, it antagonized the muscle relaxant, the sedative effect, and the changes in locomotor activity induced by diazepam. Taken together, these findings suggest that 6-methyl-3'-bromoflavone has an antagonistic profile on the BDZ-bs.