Tert-butylbicyclophosphorothionate Binding Research Articles

Pharmacological properties of AC-3933, a novel benzodiazepine receptor partial inverse agonist

We investigated in this study the pharmacological properties of AC-3933 (5-(3-methoxyphenyl)-3-(5-methyl-1,2,4-oxadiazol-3-yl)-1,6-naphthyridin-2(1H)-one), a novel benzodiazepine receptor (BzR) partial inverse agonist. AC-3933 potently inhibited [3H]-flumazenil binding to rat whole brain membrane with a Ki value of 5.15±0.39nM and a GABA ratio of 0.84±0.03. AC-3933 exhibited almost no affinity for the other receptors, transporters and ion channels used in this study. In addition, AC-3933, in the presence of GABA (1μM), gradually but significantly increased [35S] tert-butylbicyclophosphorothionate binding to rat cortical membrane to 117.1% of the control (maximum increase ratio) at 3000nM. However, this increase reached a plateau at 30nM with hardly any change at a concentration range of 100–3000nM (from 115.2% to 117.1%). AC-3933 (0.1–10μM) significantly enhanced KCl-evoked acetylcholine (ACh) release from rat hippocampal slices in a concentration-dependent manner. Moreover, in vivo brain microdialysis showed that intragastric administration of AC-3933 at the dose of 10mg/kg significantly increased extracellular ACh levels in the hippocampus of freely moving rats (area under the curve (AUC0–2h) of ACh level; 288.3% of baseline). These results indicate that AC-3933, a potent and selective BzR inverse agonist with low intrinsic activity, might be useful in the treatment of cognitive disorders associated with degeneration of the cholinergic system.

AZD-3043

Background Propofol can be associated with delayed awakening after prolonged infusion. The aim of this study was to characterize the preclinical pharmacology of AZD-3043, a positive allosteric modulator of the γ-aminobutyric acid type A (GABA(A)) receptor containing a metabolically labile ester moiety. The authors postulated that its metabolic pathway would result in a short-acting clinical profile. Methods The effects of AZD-3043, propofol, and propanidid were studied on GABA(A) receptor-mediated chloride currents in embryonic rat cortical neurons. Radioligand binding studies were also performed. The in vitro stability of AZD-3043 in whole blood and liver microsomes was evaluated. The duration of the loss of righting reflex and effects on the electroencephalograph evoked by bolus or infusion intravenous administration were assessed in rats. A mixed-effects kinetic-dynamic model using minipigs permitted exploration of the clinical pharmacology of AZD-3043. Results AZD-3043 potentiated GABA(A) receptor-mediated chloride currents and inhibited [(35)S]tert-butylbicyclophosphorothionate binding to GABA(A) receptors. AZD-3043 was rapidly hydrolyzed in liver microsomes from humans and animals. AZD-3043 produced hypnosis and electroencephalograph depression in rats. Compared with propofol, AZD-3043 was shorter acting in rats and pigs. Computer simulation using the porcine kinetic-dynamic model demonstrated that AZD-3043 has very short 50 and 80% decrement times independent of infusion duration. Conclusions AZD-3043 is a positive allosteric modulator of the GABA(A) receptor in vitro and a sedative-hypnotic agent in vivo. The esterase dependent metabolic pathway results in rapid clearance and short duration of action even for long infusions. AZD-3043 may have clinical potential as a sedative-hypnotic agent with rapid and predictable recovery.

AZD-3043

Propofol can be associated with delayed awakening after prolonged infusion. The aim of this study was to characterize the preclinical pharmacology of AZD-3043, a positive allosteric modulator of the γ-aminobutyric acid type A (GABA(A)) receptor containing a metabolically labile ester moiety. The authors postulated that its metabolic pathway would result in a short-acting clinical profile. The effects of AZD-3043, propofol, and propanidid were studied on GABA(A) receptor-mediated chloride currents in embryonic rat cortical neurons. Radioligand binding studies were also performed. The in vitro stability of AZD-3043 in whole blood and liver microsomes was evaluated. The duration of the loss of righting reflex and effects on the electroencephalograph evoked by bolus or infusion intravenous administration were assessed in rats. A mixed-effects kinetic-dynamic model using minipigs permitted exploration of the clinical pharmacology of AZD-3043. AZD-3043 potentiated GABA(A) receptor-mediated chloride currents and inhibited [(35)S]tert-butylbicyclophosphorothionate binding to GABA(A) receptors. AZD-3043 was rapidly hydrolyzed in liver microsomes from humans and animals. AZD-3043 produced hypnosis and electroencephalograph depression in rats. Compared with propofol, AZD-3043 was shorter acting in rats and pigs. Computer simulation using the porcine kinetic-dynamic model demonstrated that AZD-3043 has very short 50 and 80% decrement times independent of infusion duration. AZD-3043 is a positive allosteric modulator of the GABA(A) receptor in vitro and a sedative-hypnotic agent in vivo. The esterase dependent metabolic pathway results in rapid clearance and short duration of action even for long infusions. AZD-3043 may have clinical potential as a sedative-hypnotic agent with rapid and predictable recovery.

Status epilepticus in mice deficient for succinate semialdehyde dehydrogenase: GABAA receptor–mediated mechanisms

The epilepsy that occurs in SSADH deficiency has a seizure phenotype similar to that occurring in the SSADH(-/-) mouse. We examined the expression and function of the GABA(A) receptor (GABA(A)R) in SSADH-deficient mice. A selective decrease in binding of [(35)S]tert-butylbicyclophosphorothionate was observed in SSADH(-/-) mice at postnatal day 7 that was progressive until the third postnatal week of life when, at the nadir of the decreased [(35)S]tert-butylbicyclophosphorothionate binding, generalized convulsive seizures emerged that rapidly evolved into status epilepticus. We also observed a substantial downregulation of the beta(2) subunit of GABA(A)R, a reduction in GABA(A)-mediated inhibitory postsynaptic potentials, and augmented postsynaptic population spikes recorded from hippocampal slices. The SSADH(-/-) mouse model represents a powerful investigative tool for understanding the pathophysiology of the seizures associated with human SSADH deficiency. These data raise the possibility that progressive dysfunction of the GABA(A)R may be involved in the development of seizures in SSDAH-deficient mice. Elucidation of the precise fundamental mechanisms of the perturbation of the GABA(A)R-mediated function in SSADH(-/-) mice could lead to the development of novel treatment modalities designed to reduce the neurological morbidity in children with SSADH deficiency.

Neurosteroid Analogues. 10. The Effect of Methyl Group Substitution at the C-6 and C-7 Positions on the GABA Modulatory and Anesthetic Actions of (3α,5α)- and (3α,5β)-3-Hydroxypregnan-20-one

The planar 5alpha-reduced steroid (3alpha,5alpha)-3-hydroxypregnan-20-one and the nonplanar 5beta-reduced steroid (3alpha,5beta)-3-hydroxypregnan-20-one act at GABA(A) receptors to induce general anesthesia. The structural features of the binding sites for these anesthetic steroids on GABA(A) receptors have not been determined. To determine how structural modifications at the steroid C-6 and C-7 positions effect the actions of these anesthetic steroids, an axial or equatorial methyl group was introduced at these positions. The analogues were evaluated (1) in [(35)S]-tert-butylbicyclophosphorothionate binding experiments, (2) in electrophysiological experiments using rat alpha(1)beta(2)gamma(2L) GABA(A) receptors expressed in Xenopus laevis oocytes, and (3) as tadpole anesthetics. The effects of methyl group substitution in the 5alpha- and 5beta-reduced series of compounds were strikingly similar. In both series, a 6beta-Me group gave compounds with actions similar to or greater than those of the parent steroids. A 6alpha-, 7beta- or 7alpha-Me substituent resulted in reduced potency for inhibition of radioligand binding, GABA(A) receptor modulation and tadpole anesthesia. Because of the similar effects of methyl group substitution in the two series of compounds and previous results from other studies showing that structural modifications in the steroid D ring/side chain region produce similar effects regardless of the stereochemistry of the A,B-ring fusion, we propose that either the 3alpha-hydroxyl groups of planar and nonplanar anesthetic steroids hydrogen bond to different amino acids on GABA(A) receptors or that this critical hydrogen bonding group interacts with membrane lipids instead of the receptor.

Neurosteroid analogues. 9. Conformationally constrained pregnanes: structure-activity studies of 13,24-cyclo-18,21-dinorcholane analogues of the GABA modulatory and anesthetic steroids (3alpha,5alpha)- and (3alpha,5beta)-3-hydroxypregnan-20-one.

The hydrogen-bond-acceptor properties of the carbonyl moiety in the 17beta-acetyl group on the D-ring of the anesthetic steroids (3alpha,5alpha)- and (3alpha,5beta)-3-hydroxypregan-20-one form an important part of the anesthetic steroid pharmacophore. 13,24-Cyclo-18,21-dinorcholanes containing a ketone or conjugated ketone group at C-20, C-22, C-23, or C-24 were prepared as conformationally constrained analogues of these anesthetic steroids and were used to probe for alternate locations for the D-ring hydrogen-bond-accepting carbonyl group. The analogues were evaluated (1). in [(35)S]-tert-butylbicyclophosphorothionate binding experiments, (2). in electrophysiological experiments using rat alpha(1)beta(2)gamma(2L) GABA(A) receptors expressed in Xenopus laevis oocytes, and (3). as tadpole anesthetics. In the binding assay, the relative order of potencies for the analogues in the 5alpha- and 5beta-series is identical. For the ketones, the order is 24-one >or= 23-one > 20-one > 22-one. Likewise, for the enones, the order is delta(22)-24-one > delta(20(22))-23-one > delta(22)-20-one > delta(23)-22-one. Similar relative orders of potencies are also found in the other two bioassays. The activities of the 24-one and delta(22)-24-one compounds were expected to be very low, because the carbonyl group in these compounds is located over the steroid C-ring and oriented toward C-8. Instead, these compounds have the highest activities in their respective series, with the delta(22)-24-one compounds having activities comparable to those of the reference anesthetic steroids. The electrophysiology results obtained with the 24-oxo-cyclosteroids suggest that rat alpha(1)beta(2)gamma(2L) GABA(A) receptors contain more than one donor for the hydrogen-bond-acceptor group of anesthetic steroids. The family of cyclosteroids should be useful for future structure-activity relationship studies of steroid modulation of other GABA(A) receptor subtypes.

Allopregnanolone-stimulated GABA-mediated chloride ion flux is inhibited by 3β-hydroxy-5α-pregnan-20-one (isoallopregnanolone)

Altered gamma-aminobutyric acid (GABA)-ergic function is associated with neurological and psychiatric disorders. Certain progesterone metabolite 3α-hydroxy-5α-pregnan-20-one, or allopregnanolone (ALLO), increases the GABA-mediated chloride ion (Cl −) flux through GABA A receptors in a similar fashion as benzodiazepines and barbiturates. We have studied the effect of its 3β diastereomer, 3β-hydroxy-5α-pregnan-20-one, or isoallopregnanolone (ISO), on the Cl − flux and investigated the interaction between ISO and ALLO on GABA-mediated Cl − uptake in cortical homogenates from adult male Wistar rats. We found that ISO from 1 μM to 1 mM does not affect baseline Cl − uptake in rat cortical homogenates. Neither does ISO at dose range of 100 nM to 100 μM interact with 10 μM GABA in the Cl − uptake assay. In addition, ISO at the dose range of 1–30 μM does not affect flunitrazepam and pentobarbital-induced increase of Cl − uptake. We conclude that ISO selectively inhibits the ALLO-induced Cl − uptake with respect to ALLO concentrations. The IC 50 of ISO inhibition on 1 μM ALLO-induced Cl − uptake was calculated to be 12.25 μM. On the other hand, we have studied the effect of 30 μM ISO on ALLO (0.01 nM to 1 μM) induced displacement of tert-butylbicyclophosphorothionate (TBPS) binding. We did not note any interaction between ALLO and ISO on TBPS binding assay. These results indicate that ISO may be useful functional blocker of GABA A receptor potentiating steroid ALLO when used at concentrations that do not affect baseline GABAergic neurotransmission.

Age-related changes of GABA-activated chloride channel properties in brain cortex synaptic plasma membrane: evidence for phospholipase involvement.

Brain aging decreases binding of tert-butylbicyclophosphorothionate (TBPS), a specific ligand for Cl- channels, but has no effect on Cl- influx. Detailed studies on the kinetics of TBPS dissociation allowed the characterization of Cl- channel properties. Aging lowers, exclusively in the presence of GABA agonist, muscimol, the half-life of the fast phase of TBPS dissociation, indicating an opening time of receptor-dependent Cl- channels shorter than that in adult brain. The half-life of the slow phase of TBPS dissociation is significantly lower in aged brain in the presence and absence of muscimol. These results suggest a sustained Cl- current, including also the other channel(s) not connected with GABAA receptor activation. The analysis of biphasic TBPS dissociation demonstrates a lowered number of binding sites resulted in the reduction of the number of Cl- channels in the "open" state. This may explain an observed decrease of TBPS binding in aged brain. One of the possible factors involved in modification of GABAA receptor behavior during aging may be arachidonic acid or diacylglycerol, known to be accumulated in aged brain. The action of these compounds on the Cl- channel, observed in this study, correlates well with the effect of aging.

Modulation Of [35S]-tert-butylbicyclophosphorothionate binding by somatostatin in rat hypothalamus.

1. The present study was designed to assess the effect of the tetradecapeptide somatostatin on the GABA(A) receptor complex in the rat hypothalamus. 2. GABA(A) receptors were labelled with [35S]-tert-butylbicyclophosphorothionate (TBPS), which binds in or near the chloride channel, and binding as assessed by in vitro quantitative autoradiography using a computer-assisted image analysis system. 3. Somatostatin inhibited the binding of [35S]-TBPS to the convulsant site of the hypothalamic GABA(A) receptor complex of rat slide-mounted hypothalamic structures in a concentration-dependent manner with an affinity in the micromolar range (10(-6) to 3 x 10(-6) mol/L). Somatostatin appeared to mimic the effects of the neurosteroid 5alpha-pregnane-3alpha ol-one (5alpha3alphaP), GABA and picrotoxin on [35S]-TBPS binding in the rat hypothalamus in all structures examined. Furthermore, GABA or muscimol (a GABA(A) receptor agonist), when added to the incubation medium, enhanced the capacity of somatostatin to inhibit [35S]-TBPS binding, with an IC50 of 10(-7) mol/L. However, incubation with bicuculline (a GABA(A) receptor antagonist) led to the abolition of the inhibitory effect of somatostatin on [35S]-TBPS specific binding in rat hypothalamus. 4. The present results demonstrate the presence of a modulatory effect of somatostatin on the GABA(A) receptor complex in rat hypothalamic structures. Furthermore, the data suggest that somatostatin allosterically modifies [35S]-TBPS binding through a mechanism similar to that of GABA. Taken together, these results provide evidence for the presence of somatostatin- GABA interactions in rat hypothalamus.

Identification of amino acid residues of GABA(A) receptor subunits contributing to the formation and affinity of the tert-butylbicyclophosphorothionate binding site.

A chimeric GABA(A) receptor subunit was constructed that contained the beta3 sequence from the N-terminus to the first two amino acids of the second transmembrane (TM2) domain. The remaining part of this chimera had the sequence of the alpha1 subunit. On co-expression with alpha1 subunits, this chimera was able to form heterooligomeric channels that were open in the absence of GABA. Picrotoxin and tert-butylbicyclophosphorothionate (TBPS) were able to block these channels with low potency. These channels exhibited high-affinity [3H]muscimol but no high-affinity [35S]TBPS binding sites. Introduction of V251, A252, and L253 of the beta3 subunit into the chimera resulted in the formation of closed channels that could be opened by GABA. The introduction of A252 and L253 of the beta3 subunit into this chimera was sufficient to reconstitute the specific high-affinity [35S]TBPS binding site in receptors composed of the chimera and alpha1 subunits. Replacement of other amino acids of the TM2 region of the chimera with corresponding amino acids of the beta3 subunit modulated the affinity of this [35S]TBPS binding site. Results obtained provide important information on the structure-function relationship of GABA(A) receptors.

Effects of the chlorotriazine herbicide, cyanazine, on GABA A receptors in cortical tissue from rat brain

Chlorotriazine herbicides disrupt luteinizing hormone (LH) release in female rats following in vivo exposure. Although the mechanism of action is unknown, significant evidence suggests that inhibition of LH release by chlorotriazines may be mediated by effects in the central nervous system. GABA A receptors are important for neuronal regulation of gonadotropin releasing hormone and LH release. The ability of chlorotriazine herbicides to interact with GABA A receptors was examined by measuring their effects on [ 3H]muscimol, [ 3H]Ro15-4513 and [ 35S] tert-butylbicyclophosphorothionate (TBPS) binding to rat cortical membranes. Cyanazine (1–400 μM) inhibited [ 3H]Ro15-4513 binding with an IC 50 of approximately 105 μM ( n=4). Atrazine (1–400 μM) also inhibited [ 3H]Ro15-4513 binding, but was less potent than cyanazine (IC 50=305 μM). However, the chlorotriazine metabolites diaminochlorotriazine, 2-amino-4-chloro-6-ethylamino- s-triazine and 2-amino-4-chloro-6-isopropylamino- s-triazine were without significant effect on [ 3H]Ro15-4513 binding. Cyanazine and the other chlorotriazines were without effect on [ 3H]muscimol or [ 35S]TBPS binding. To examine whether cyanazine altered GABA A receptor function, GABA-stimulated 36Cl − flux into synaptoneurosomes was examined. Cyanazine (50–100 μM) alone did not significantly decrease GABA-stimulated 36Cl − flux. Diazepam (10 μM) and pentobarbital (100 μM) potentiated GABA-stimulated 36Cl − flux to 126 and 166% of control, respectively. At concentrations of 50 and 100 μM, cyanazine decreased potentiation by diazepam to 112 and 97% of control, respectively, and decreased potentiation by pentobarbital to 158 and 137% of control ( n=6). Interestingly, at lower concentrations (5 μM), cyanazine shifted the EC 50 for GABA-stimulated 36Cl − flux into synaptoneurosomes from 28.9 to 19.4 μM, respectively ( n=5). These results suggest that cyanazine modulates benzodiazepine, but not the muscimol (GABA receptor site) or TBPS (Cl − channel), binding sites on GABA A receptors. Furthermore, at low concentrations, cyanazine may slightly enhance function of GABA A receptors, but at higher concentrations, cyanazine antagonizes GABA A receptor function and in particular antagonizes the positive modulatory effects of diazepam and pentobarbital.

Synthesis, structure-activity relationships, and molecular modeling studies of N-(indol-3-ylglyoxylyl)benzylamine derivatives acting at the benzodiazepine receptor.

A number of N-(indol-3-ylglyoxylyl)benzylamine derivatives were synthesized and tested for [3H]flunitrazepam displacing activity in bovine brain membranes. Some of these derivatives (9, 12, 14, 15, 17, 27, 34, 35, 38, 41, and 45) exhibited high affinity for the benzodiazepine receptor (BzR) with Ki values ranging from 67 to 11 nM. The GABA ratio and [35S]-tert-butylbicyclophosphorothionate binding data, determined for the most active compounds, showed that they elicit an efficacy profile at the BzR which depends on the kind of substituent present on the phenyl ring of the benzylamine moiety. Moreover, lengthening (propylamine derivatives 1-3) and shortening (aniline derivatives 46-54) of the distance between the phenyl ring and the amide group of the side chain gave compounds with a drastically lower binding potency. The biological results are discussed in the light of a recently proposed pharmacophore model and compared, by molecular modeling studies, with those obtained from effective BzR ligands.

Effect of free fatty acids on GABA A receptor ligand binding

Phospholipase A 2 (PLA 2) treatment of synaptosomal membranes, which causes the release of fatty acids, particularly unsaturated fatty acids, inhibits the flux of chloride ions through the γ aminobutyric acid (GABA) benzodiazepine receptor ion channel in response to activation by agonists. PLA; treatment has also been shown to affect ligand binding to the receptor. In the present study, we have investigated the effect of unsaturated free fatty acids, arachidonic acid and oleic acid and saturated free fatty acids, arachidic acid and stearic acid on various characteristics of GABA A receptor ligand binding. Only the unsaturated fatty acids showed any effect: arachidonic acid and oleic acid enhanced flunitrazepam binding and muscimol binding but inhibited tert butylbicyclophosphorothionate (TBPS) binding in a dose-dependent manner. The effects on muscimol and TBPS binding were shown to be due to changes in receptor density by saturation analysis. Oleic acid and arachidonic acid also decreased the enhancement of flunitrazepam and muscimol binding by cartazolate and pentobarbital but did not affect GABA enhancement of flunitrazepam binding. These data indicate that unsaturated free fatty acids can mimic the effects of PLA 2 treatment and underline the importance of the lipid microenvironment on ligand binding to the Gabaa receptor.