Ethynylbicycloorthobenzoate Binding Research Articles

Hops compounds modulatory effects and 6-prenylnaringenin dual mode of action on GABAA receptors

Hops (Humulus lupulus L.), a major component of beer, contain potentially neuroactive compounds that made it useful in traditional medicine as a sleeping aid. The present study aims to investigate the individual components in hops acting as allosteric modulators in GABAA receptors and bring further insight into the mode of action behind the sedative properties of hops. GABA-potentiating effects were measured using [3H]ethynylbicycloorthobenzoate (EBOB) radioligand binding assay in native GABAA receptors. Flumazenil sensitivity of GABA-potentiating effects, [3H]Ro 15–4513, and [3H]flunitrazepam binding assays were used to examine the binding to the classical benzodiazepines site. Humulone (alpha acid) and 6-prenylnaringenin (prenylflavonoid) were the most potent compounds displaying a modulatory activity at low micromolar concentrations. Humulone and 6-prenylnaringenin potentiated GABA-induced displacement of [3H]EBOB binding in a concentration-dependent manner where the IC50 values for this potentiation in native GABAA receptors were 3.2 μM and 3.7 μM, respectively. Flumazenil had no significant effects on humulone- or 6-prenylnaringenin-induced displacement of [3H]EBOB binding. [3H]Ro 15–4513 and [3H]flunitrazepam displacements were only minor with humulone but surprisingly prominent with 6-prenylnaringenin despite its flumazenil-insensitive modulatory activity. Thus, we applied molecular docking methods to investigate putative binding sites and poses of 6-prenylnaringenin at the GABAA receptor α1β2γ2 isoform. Radioligand binding and docking results suggest a dual mode of action by 6-prenylnaringenin on GABAA receptors where it may act as a positive allosteric modulator at α+β- binding interface as well as a null modulator at the flumazenil-sensitive α+γ2- binding interface.

Positive allosteric modulation of native and recombinant GABAA receptors by hops prenylflavonoids

Hops are a major component of beer that is added during brewing. In addition to its wide range of bioactivity, it exhibits neuroactive properties as a sedative and sleeping aid. The compounds responsible for this activity are yet to be revealed and understood in terms of their pharmacological properties. Here we evaluated the potential of several hops flavonoids in modulating the GABAergic activity and assessed their selectivity to GABAA receptors subtypes. GABA-potentiating effects were measured using [3H]ethynylbicycloorthobenzoate (EBOB) radioligand binding assay in native and recombinant α1β3γ2, α2β3γ2 and α6β3δ receptors expressed in HEK293 cells. Flumazenil sensitivity of GABA-potentiating effects and [3H]Ro 15-4513 binding assay were used to examine the flavonoids binding to benzodiazepine site. The prenylflavonoids xanthohumol (XN), isoxanthohumol (IXN) and 8-prenylnaringenin (8PN) potentiated GABA-induced displacement of [3H]EBOB binding in a concentration-dependent manner. The IC50 for this potentiation in native GABAA receptors were 29.7 µM, 11.6 µM, 7.3 µM, respectively. In recombinant receptors, the sensitivity to prenylflavonoid potentiation of GABA-induced displacement of [3H]EBOB binding followed the order α6β3δ > α2β3γ2 > α1β3γ2 with the strongest inhibition observed by 8PN in α6β3δ (IC50 = 3.6 μM). Flumazenil had no significant effect on the prenylflavonoid-induced displacement of [3H]EBOB binding and [3H]Ro 15-4513 displacement from native GABAA receptors was only detected at high micromolar concentrations (100 µM). We identified potent prenylflavonoids in hops that positively modulate GABA-induced responses in native and αβγ/δ recombinant GABAA receptors at low micromolar concentrations. These GABAergic modulatory effects were not mediated via the high-affinity benzodiazepine binding site.

Differential effects of two major neurosteroids on cerebellar and cortical GABA A receptor binding and function

Cerebellar and cerebrocortical A-type γ-aminobutyric acid (GABA A) receptors were examined in mice and rats. In wild-type mouse cerebellum, the agonists GABA and gaboxadol exerted heterogeneous displacement of [ 3H]ethynylbicycloorthobenzoate (EBOB) binding with nanomolar and submicromolar affinities. In mouse cerebella lacking α6 subunits (α6KO), nanomolar displacement by GABA agonists was absent, while micromolar displacement was potentiated to 12-fold by 0.3 μM 5α-tetrahydrodeoxycorticosterone (5α-THDOC). In α6KO cerebellum, 60% of [ 3H]EBOB binding was neurosteroid-insensitive, while 5α-THDOC elicited enhancement with EC 50 = 150 nM instead of nanomolar displacement. In conclusion, nanomolar displacement of cerebellar [ 3H]EBOB binding by GABA agonists and neurosteroids can be attributed to GABA A receptors containing α6 and δ subunits. In contrast, [ 3H]EBOB binding to rat cerebral cortex was affected by allopregnanolone and 5α-THDOC in bidirectional manner with nanomolar enhancement (EC 50 ~ 80 nM) and micromolar displacement. Nonequilibrium binding conditions with decreased incubation time tripled the maximal enhancement of [ 3H]EBOB binding by 5α-THDOC. 5ß-THDOC enhanced the cortical [ 3H]EBOB binding with EC 50 ~ 0.5 μM and it attenuated bidirectional modulation by 5α-THDOC. Allopregnanolone and 5α-THDOC produced biphasic enhancements of chloride currents elicited by 1 μM GABA in cerebellar granule cells, for 5α-THDOC with EC 50,1 ~ 16 nM and EC 50,2 ~ 1.3 μM. Differences in peak current enhancements in the absence minus presence of 0.1 mM furosemide corresponding to α6ßδ GABA A receptors were augmented only by micromolar 5α-THDOC while the difference curve for allopregnanolone was polyphasic as without furosemide. Consequently, these neurosteroids differentially affected the binding and function of various GABA A receptor populations.

A 17β‐derivative of allopregnanolone is a neurosteroid antagonist at a cerebellar subpopulation of GABAA receptors with nanomolar affinity

High-affinity, subtype-selective antagonists of the neurosteroid binding sites of GABA(A) receptors are not available. We have characterized an allopregnanolone derivative as an antagonist of cerebellar GABA(A) receptors with nanomolar affinity. Receptor binding and electrophysiological methods were used for the allosteric modulation of cerebellar GABA(A) receptors by an allopregnanolone derivative, (20R)-17beta-(1-hydroxy-2,3-butadienyl)-5alpha-androstane-3alpha-ol (HBAO). GABA(A) receptors of rat cerebellar membranes were labelled with the chloride channel blocker [(3)H]ethynylbicycloorthobenzoate (EBOB). The ionophore function of GABA(A) receptors was studied by whole-cell patch clamp electrophysiology in cultured rat cerebellar granule and cortical cells. Partial displacement of cerebellar [(3)H]EBOB binding by nanomolar HBAO was attenuated by 0.1 mM furosemide, an antagonist of alpha(6) and beta(2-3) subunit-containing GABA(A) receptors. Displacement curves of HBAO were reshaped by 30 nM GABA and shifted to the right. However, the micromolar potency of full displacement by allopregnanolone was not affected by 0.1 mM furosemide or 30 nM GABA. The nanomolar, but not the micromolar phase of displacement of [(3)H]EBOB binding by GABA was attenuated by 100 nM HBAO. Submicromolar HBAO did not affect [(3)H]EBOB binding to cortical and hippocampal GABA(A) receptors. HBAO up to 1 microM did not affect chloride currents elicited by 0.3-10 microM GABA, while it abolished potentiation by 1 microM allopregnanolone with nanomolar potency in cerebellar but not in cortical cells. Furosemide attenuated cerebellar inhibition by 100 nM HBAO. HBAO is a selective antagonist of allopregnanolone, a major endogenous positive modulator via neurosteroid sites of cerebellar (probably alpha(6)beta(2-3)delta) GABA(A) receptors.

Allosteric modulation of [ 3H]EBOB binding to GABA A receptors by diflunisal analogues

Allosteric modulatory effects of 12 biphenyl derivatives of diflunisal and two fenamates were studied on A-type receptors of GABA (GABA AR) via [ 3H]4′-ethynylbicycloorthobenzoate (EBOB) binding to synaptic membrane preparations of rat forebrain. A simplified ternary allosteric model was used to determine binding affinities of the compounds and the extents of cooperativity with GABA. Structure activity analysis revealed that 4-hydroxy substituents of the biphenyls contribute to their micromolar binding affinities more than 3-carboxyl groups. Electron-withdrawing fluorinated substituents, especially in ortho position, were also advantageous. These factors also strongly enhanced the cooperativity with GABA binding. The correlation between displacing potency of the allosteric agents and cooperativity with GABA suggests that these processes are associated with common mechanisms. The pharmacological relevance of these interactions is discussed. These data help to differentiate the structural requirements of these agents to act on GABAergic neurotransmission versus nonsteroidal anti-inflammatory effects.

High affinity, heterogeneous displacement of [ 3H]EBOB binding to cerebellar GABA A receptors by neurosteroids and GABA agonists

Heterogeneous binding interactions of cerebellar GABA A receptors were investigated with GABA agonists and neurosteroids. GABA A receptors of rat cerebellum were labelled with [ 3H]ethynylbicycloorthobenzoate (EBOB), a convulsant radioligand. Saturation analysis revealed a homogenous, nanomolar population of [ 3H]EBOB binding. Both GABA and 5α-tetrahydrodeoxycorticosterone (5α-THDOC) displaced [ 3H]EBOB binding heterogeneously, with nanomolar and micromolar potencies. The nanomolar phase of displacement by GABA was selectively abolished by 100 μM furosemide. Physiological concentrations of allopregnanolone (8 nM) and 5α-THDOC (20 nM) increased the displacing effects of nanomolar GABA. GABA (0.3 μM) and 5α-THDOC (0.3 μM) potentiated the micromolar population of displacement by the other. Taurine inhibited [ 3H]EBOB binding also heterogeneously, with micromolar and millimolar potencies, and 0.3 μM 5α-THDOC potentiated this inhibition. 5β-THDOC did not affect [ 3H]EBOB binding significantly but in 1 μM it antagonised selectively the nanomolar displacement by 5α-THDOC. [ 3H]EBOB binding to hippocampal GABA A receptors was inhibited by GABA and allopregnanolone with low (micromolar) potencies and with slope values higher than unity referring to allosteric interaction. High affinity displacement of cerebellar [ 3H]EBOB binding by GABA agonists and neurosteroids can be associated with constitutively open α 6βδ GABA A receptors, tonic GABAergic inhibitory neurotransmission and its modulation by physiological concentrations of neurosteroids.

Nanomolar allopregnanolone potentiates rat cerebellar GABA A receptors

The ionophore function of γ-aminobutyric acid A (GABA A) receptors was studied by whole-cell patch clamp electrophysiology in primary cultures of rat cerebellar cortex. Chloride currents elicited by 1 μM GABA were potentiated by allopregnanolone with a plateau of high affinity (EC 50 = 14 nM) and a peak of potentiation around 1 μM allopregnanolone. Furosemide (0.1 mM) eliminated the high affinity phase and increased the EC 50 to 685 nM. GABA A receptors of rat cerebellar synaptosomal membranes were labelled with [ 3H]ethynylbicycloorthobenzoate (EBOB). Allopregnanolone displaced [ 3H]EBOB binding with IC 50 = 320 nM. The displacing potency of allopregnanolone was strongly enhanced (IC 50 = 39 nM) in the presence of 400 nM GABA and 60 nM SR 95531. Nanomolar potentiation by allopregnanolone can be associated with cerebellar GABA A receptors containing α 6, β 2–3 and δ subunits. This might be suitable for physiological modulation of tonic inhibitory neurotransmission via extrasynaptic GABA A receptors in cerebellar granule cells by neurosteroids.

[formula omitted]]Ethynylbicycloorthobenzoate ([ [formula omitted]]EBOB) Binding in Recombinant GABA A Receptors

Ethynylbicycloorthobenzoate (EBOB) is a recently developed ligand that binds to the convulsant site of the GABA A receptor. While a few studies have examined the binding of [ 3H ]EBOB in vertebrate brain tissue and insect preparations, none have examined [ 3H ]EBOB binding in preparations that express known configurations of the GABA A receptor. We have thus examined [ 3H ]EBOB binding in HEK293 cells stably expressing human α1β2γ2 and α2β2γ2 GABA A receptors, and the effects of CNS convulsants on its binding. The ability of the CNS convulsants to displace the prototypical convulsant site ligand, [ 35S ]TBPS, was also assessed. Saturation analysis revealed [ 3H ]EBOB binding at a single site, with a K d of approximately 9 nM in α1β2γ2 and α2β2γ2 receptors. Binding of both [ 3H ]EBOB and [ 35S ]TBPS was inhibited by dieldrin, lindane, tert-butylbicycloorthobenzoate (TBOB), PTX, TBPS, and pentylenetetrazol (PTZ) at one site in a concentration-dependent fashion. Affinities were in the high nM to low μM range for all compounds except PTZ (low mM range), and the rank order of potency for these convulsants to displace [ 3H ]EBOB and [ 35S ]TBPS was the same. Low [GABA] stimulated [ 3H ]EBOB binding, while higher [GABA] (greater than 10 μM) inhibited [ 3H ]EBOB binding. Overall, our data demonstrate that [ 3H ]EBOB binds to a single, high affinity site in α1β2γ2 and α2β2γ2 GABA A receptors, and modulation of its binding is similar to that seen with [ 35S ]TBPS. [ 3H ]EBOB has a number of desirable traits that may make it preferable to [ 35S ]TBPS for analysis of the convulsant site of the GABA A receptor.

Regional Modification of [3H]Ethynylbicycloorthobenzoate Binding in Mouse Brain GABAA Receptor by Endosulfan, Fipronil, and Avermectin B1a

[3H]Ethynylbicycloorthobenzoate ([3H]EBOB), a high affinity radioligand for the noncompetitive blocker site of the GABAA receptor, is used here for quantitative autoradiography to determine regional binding in mouse brain and the effects on this binding of administering toxic doses of endosulfan, fipronil, and avermectin B1a (AVM). Animals were euthanized 4–8 min after 1 LD50 or 2 LD50 doses of the two channel blockers and 32 min after 1 LD50 or 4 LD50 doses of the channel activator AVM. Specific binding of [3H]EBOB was determined for 20-μm brain sections as the difference in labeling on incubation with 2 nM [3H]EBOB either alone (total binding) or with 5 μM α-endosulfan (nonspecific binding). The highest specific labeling was observed for layers I and IV of the cerebral cortex, the globus pallidus, and the medial septal nucleus/nucleus of the vertical limb of the diagonal band. Dose-dependent inhibition by endosulfan was highest in the nucleus accumbens and least in the cerebellum and periaqueductal gray matter. Fipronil had much less effect on binding even at severely toxic doses. AVM increased [3H]EBOB binding in most regions and was the only one of the three agents inhibiting in vitro [3H]strychnine binding to the glycine receptor. In summary, the noncompetitive blocker site was strongly inhibited with dose dependence and regional selectively by α-endosulfan but was generally poorly inhibited or activated by fipronil and was activated by avermectin.

House fly head GABA-gated chloride channel: Toxicologically relevant binding site for avermectins coupled to site for ethynylbicycloorthobenzoate

[5- 3H]Avermectin B 1a ([ 3H]AVM) binds in house fly head membranes to a single saturable, high affinity site ( K d 1.9 n M, B max 9.5 pmol/mg protein, 65% specific binding). This radioligand is not displaced by GABA and several noncompetitive blockers of the GABA-gated chloride channel [picrotoxinin, lindane, and 4- n-propyl-4′-ethynylbicycloorthobenzoate (EBOB)]. AVM and 11 analogs thereof including MK-243 and moxidectin vary in IC 50s for displacement of [ 3H]AVM from 26 to 1025 n M and in LD 50s to adult female house flies [topical, 24 hr, pretreated with piperonyl butoxide to minimize oxidative detoxification] from 0.01 to 45 μg/g. The IC 50 is a good predictor of the LD 50 ( r = 0.83, n = 11) establishing the toxicological relevance of this [ 3H]AVM binding assay. These AVM analogs are also noncompetitive displacers of [ 3H]EBOB binding with EC 50 values of 1.7 to 336 n M for the sensitive portion (45–80%) of [ 3H]EBOB binding. The relative potencies of a series of AVM analogs as inhibitors of [ 3H]AVM binding correlate well with those for [ 3H]EBOB binding ( r = 0.95, n = 12). It therefore appears that AVM and its analogs (channel openers) act in the GABA-gated chloride channel of house fly head at a site closely coupled to that for EBOB (a channel blocker).