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.     

Benzodiazepine receptors on human blood platelets

Binding studies conducted on membrane preparation from human platelets using (3H) Ro5-4864 and (3H) diazepam showed specific and saturable binding. Scatchard analysis revealed a single class of binding sites with KD = 10.8 +/- 0.9 nM and Bmax = 775 +/- 105 fmol/mg protein for (3H) Ro5-4864 and KD = 10.5 +/- 1.1 nM and Bmax = 133 +/- 19 fmol/mg for (3H) diazepam. We were unable to detect any GABA binding site on crude membrane preparation, nor did GABA enhance the binding of (3H) Ro5-4864 or (3H) diazepam. This suggests that benzodiazepine receptors are uncoupled to GABA system on human platelets. Ro15-1788, a specific antagonist for "central type" benzodiazepine (BDZ) binding sites was inactive in displacing (3H) Ro5-4864 from membrane receptors, while PK 11195 (a specific ligand for the "peripheral type" receptor) was the most potent of the drugs tested in inhibiting (3H) Ro5-4864 binding. These results indicate that human blood platelets bear "peripheral-type" BDZ receptor. Moreover, we could not detect any (3H) propyl beta carboline specific binding on platelet membranes. Results on benzodiazepine receptors on human circulating lymphocytes are also reported and similarity in pharmacological properties with platelet benzodiazepine receptors is suggested.     

Solubilization of the Benzodiazepine/γ-Aminobutyric Acid Receptor Complex: Comparison of the Detergents Octylglucopyranoside and 3-[(3-Cholamidopropyl)-Dimethylammonio] 1-Propanesulfonate (CHAPS)

Abstract Octylglucopyranoside (OCTG) was three times more efficient than 3-[(3-cholamidopropyl)-dimethylammonio] 1-propanesulfonate (CHAPS) in solubilizing the benzodiazepine (BDZ)/γ-aminobutyric acid (GABA) receptor complex from rat cerebellar synaptic membranes. OCTG-solubilized receptor preparations had ligand binding characteristics that were significantly different from those of the CHAPS-solubilized receptors. The inclusion of phospholipids in the solubilization media improved the binding characteristics of both soluble receptor preparations and appeared absolutely necessary for the maintenance of chloride facilitation of flunitrazepam (FNZ) binding to OCTG-solubilized receptors. FNZ and ethyl-β-carboline-3-carboxylate bound to OCTG-solubilized preparations with equilibrium dissociation constants of 2.2 nM and 1.6 nM, respectively, and chloride (150 mM) and GABA (100 μM) + chloride facilitated the binding of FNZ by 15% and 55%, respectively; these ligand binding characteristics are similar to those of membrane-located BDZ receptors. Cartazolate, a pyrazolopyridine that facilitated the binding of FNZ to membrane-located and CHAPS-solubilized receptors, did not facilitate FNZ binding to OCTG-solubilized receptors. These results are discussed in terms of an interaction between the membrane lipid phosphatidylserine (PS) and cartazolate; PS appears to have the capacity to inhibit the effects of cartazolate on FNZ binding. Storage of the soluble receptor preparations for 24 h at 4° resulted in the loss of several characteristic BDZ receptor binding properties. Incorporation of the OCTG-solubilized receptor complex into liposomes prevented these losses but this procedure did not protect the CHAPS-solubilized receptors. We conclude that OCTG may have some advantages over CHAPS as the detergent of choice for the solubilization and reconstitution in liposomes of a functional BDZ/GABA receptor-chloride ionophore complex.     

Identification of amino acid residues responsible for the alpha5 subunit binding selectivity of L-655,708, a benzodiazepine binding site ligand at the GABA(A) receptor

L-655,708 is a ligand for the benzodiazepine site of the gamma-aminobutyric acid type A (GABA(A)) receptor that exhibits a 100-fold higher affinity for alpha5-containing receptors compared with alpha1-containing receptors. Molecular biology approaches have been used to determine which residues in the alpha5 subunit are responsible for this selectivity. Two amino acids have been identified, alpha5Thr208 and alpha5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at alpha5-containing receptors. L-655,708 is a partial inverse agonist at the GABA(A) receptor which exhibited no functional selectivity between alpha1- and alpha5-containing receptors and showed no change in efficacy at receptors containing alpha1 subunits where amino acids at both of the sites had been altered to their alpha5 counterparts (alpha1Ser205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA(A) receptor, and further delineate a region just N-terminal to the first transmembrane domain of the receptor alpha subunit that contributes to this binding site.     

Benzodiazepine-induced hyperphagia: development and assessment of a 3D pharmacophore by computational methods

Benzodiazepine receptor (BDZR) ligands are structurally diverse compounds that bind to specific binding sites on GABA(A) receptors and allosterically modulate the effect of GABA on chloride ion flux. The binding of BDZR ligands to this receptor system results in activity at multiple behavioral endpoints, including anxiolytic, sedative, anticonvulsant, and hyperphagic effects. In the work presented here, a computational procedure developed in our laboratory has been used to obtain a 3D pharmacophore for ligand recognition of the GABA(A)/BDZRs initiating the hyperphagic response. To accomplish this goal, 17 structurally diverse compounds, previously assessed in our laboratory for activity at the hyperphagic endpoint, were used. The result is a four-component 3D pharmacophore. It consists of two proton acceptor atoms, the centroid of an aromatic ring and the centroid of a hydrophobic moiety in a common geometric arrangement in all compounds with activity at this endpoint. This 3D pharmacophore was then assessed and successfully validated using three different tests. First, two BDZR ligands, which were included as negative controls in the set of seventeen compounds used for the pharmacophore development, did not fit the pharmacophore. Second, some benzodiazepine ligands known to have activity at the hyperphagia endpoint, but not included in the pharmacophore development, were used as positive controls and were found to fit the pharmacophore. Finally, using the 3D pharmacophore developed in the present work to search 3D databases, over 50 classical benzodiazepines were found. Among them, were benzodiazepine ligands known to have an effect at the hyperphagic endpoint. In addition, the novel compounds also found in this search are promising therapeutic agents that could beneficially affect feeding behavior.     

Biochemical Characterization of an Isolated and Functionally Reconstituted γ-Aminobutyric Acid/Benzodiazepine Receptor

We have solubilized, affinity-purified, and functionally reconstituted the gamma-aminobutyric acid/benzodiazepine (GABA/BDZ) receptor from rat brain into natural brain lipid liposomes. The detergent, 3-[(3-cholamidopropyl)-dimethylammonio] 1-propanesulphonate, was employed for the isolation of the receptor in the presence of a whole rat brain lipid extract supplemented with cholesteryl hemisuccinate. The soluble and reconstituted protein showed a homogeneous [3H]flunitrazepam binding population and the allosteric modulation of this binding site by GABA, by the pyrazolopyridine, cartazolate, and by the depressant barbiturate, pentobarbital. The purified GABA/BDZ receptor when incorporated into liposomes has been visualized by electron microscopy and reveals rosette structures, 8-9 nm in diameter, which appear to have a central pore. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the reconstituted GABA/BDZ receptor reveals three major protein bands of 41, 52-56, and 59-62 kDa, the latter two of which appears as doublets. Functional receptor reconstitution is demonstrated by the measurement of GABA-stimulated 36Cl- flux into the purified GABA/BDZ receptor incorporated liposomes and its modulation by the BDZs, barbiturates, and pyrazolopyridines.     

On the benzodiazepine binding pocket in GABAA receptors

Benzodiazepines are used for their sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsive effects. They exert their actions through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the gamma-aminobutyric acid, type A (GABA(A)) receptor channel, where they act as positive allosteric modulators. To start to elucidate the relative positioning of benzodiazepine binding site ligands in their binding pocket, GABA(A) receptor residues thought to reside in the site were individually mutated to cysteine and combined with benzodiazepine analogs carrying substituents reactive to cysteine. Direct apposition of such reactive partners is expected to lead to an irreversible site-directed reaction. We describe here the covalent interaction of alpha(1)H101C with a reactive group attached to the C-7 position of diazepam. This interaction was studied at the level of radioactive ligand binding and at the functional level using electrophysiological methods. Covalent reaction occurs concomitantly with occupancy of the binding pocket. It stabilizes the receptor in its allosterically stimulated conformation. Covalent modification is not observed in wild type receptors or when using mutated alpha(1)H101C-containing receptors in combination with the reactive ligand pre-reacted with a sulfhydryl group, and the modification rate is reduced by the binding site ligand Ro15-1788. We present in addition evidence that gamma(2)Ala-79 is probably located in the access pathway of the ligand to its binding pocket.     

Synthetic and computer assisted analysis of the pharmacophore for agonists at benzodiazepine receptors.

In order to employ rational drug design in the discovery of selective benzodiazepine receptor agonists and inverse agonists, pharmacophore/receptor models for both these activities must first be established. Recently, a pharmacophore for the inverse agonist site has been formulated employing the most recent receptor mapping techniques (22). The continuation of this approach to the pharmacophore for agonist ligands has permitted a definition of this site independently of the inverse agonist model. The agonist pharmacophore/receptor contains two hydrogen bond donating sites of interaction (H1 and H2) located about 6.5 A from each other, as well as three areas of lipophilic interaction (L1-L3). The areas L1 and L2 are critical for agonist activity; moreover, some ligands also require an interaction in a third lipophilic area termed L3. This is in agreement with previous work (12-23). In addition, an area of negative steric interaction (S1) between the ligand and receptor-binding protein is defined. In regard to the pharmacophore, it was established that the alignment rule for agonist beta-carbolines is different from that which elicits inverse agonist activity. Consideration of the pharmacophore has resulted in the synthesis of a new beta-carboline 16 which elicits agonist activity. This ligand 16 not only satisfied the requirements of the pharmacophore, but more importantly it elicited both anticonvulsant and anxiolytic activity, but was devoid of the myorelaxant/ataxic properties associated with the benzodiazepines.     

Development and assessment of a 3D pharmacophore for ligand recognition of BDZR/GABAA receptors initiating the anxiolytic response

Benzodiazepine receptor (BDZR) ligands are structurally diverse compounds that bind to specific binding sites on GABAA receptors and allosterically modulate the effect of GABA on chloride flux. The binding of BDZR ligands to this receptor system results in activity at multiple behavioral end points including anxiolytic, sedative, hyperphagic, anticonvulsant and hyperthermic effects. In the work presented here, 17 structurally diverse BDZR ligands of the receptors initiating the anxiolytic response have been studied using a systematic computational procedure developed in our laboratory. Using this procedure, a five component 3D recognition pharmacophore was obtained consisting of two proton acceptors, a hydrophobic group, an aromatic electron accepting ring and a ring containing polar moieties, all found in a common geometric arrangement in the 15 compounds with an effect at the anxiolytic end point and absent in two control compounds. The 3D pharmacophore developed was validated by searching 3D databases and finding known BDZR ligands active at the anxiolytic end point, including 1,4-BDZ derivatives, imidazo BDZ and beta-carboline ligands.     

Complementarity of delta opioid ligand pharmacophore and receptor models

The elaboration of a pharmacophore model for the delta opioid receptor selective ligand JOM-13 (Tyr-c[D-Cys-Phe-D-Pen]OH) and the parallel, independent development of a structural model of the delta receptor are summarized. Although the backbone conformation of JOM-13's tripeptide cycle is well defined, considerable conformational lability is evident in the Tyr(1) residue and in the Phe(3) side chain, key pharmacophore elements of the ligand. Replacement of these flexible features of the ligand by more conformationally restricted analogues and subsequent correlation of receptor binding and conformational properties allowed the number of possible binding conformations of JOM-13 to be reduced to two. Of these, one was chosen as more likely, based on its better superposition with other conformationally constrained delta receptor ligands. Our model of the delta opioid receptor, constructed using a general approach that we have developed for all rhodopsin-like G protein-coupled receptors, contains a large cavity within the transmembrane domain that displays excellent complementarity in both shape and polarity to JOM-13 and other delta ligands. This binding pocket, however, cannot accommodate the conformer of JOM-13 preferred from analysis of ligands, alone. Rather, only the "alternate" allowed conformer, identified from analysis of the ligands but "disfavored" because it does not permit simultaneous superposition of all pharmacophore elements of JOM-13 with other delta ligands, fits the binding site. These results argue against a simple view of a single, common fit to a receptor binding site and suggest, instead, that at least some binding site interactions of different ligands may differ.     

3-Alkyl- and 3-amido-isothiazoloquinolin-4-ones as ligands for the benzodiazepine site of GABA(A) receptors

Based on a pharmacophore model of the benzodiazepine binding site of the GABA(A) receptors, developed with synthetic flavones and potent 3-carbonylquinolin-4-ones, 3-alkyl- and 3-amido-6-methylisothiazoloquinolin-4-ones were designed, prepared and assayed. The suggestion that the interaction between the hydrogen bond donor site H1 with the 3-carbonyl oxygen in 3-carbonylquinolin-4-ones can be replaced by an interaction between H1 and N-2 in the isothiazoloquinolin-4-ones, was confirmed. As with the 3-carbonylquinolin-4-ones, the length of the chain in position 3 is critical for an efficient interaction with the lipophilic pockets of the pharmacophore model. The most potent 3-alkyl derivative, 3-pentyl-6-methylisothiazoloquinolin-4-one, has an affinity (K(i) value) for the benzodiazepine binding site of the GABA(A) receptors of 13 nM. However, by replacing the 3-pentyl with a 3-butyramido group an even more potent compound was obtained, with a K(i) value of 2.8 nM, indicating that the amide function facilitates additional interactions with the binding site.     

Tricyclic pyridones as functionally selective human GABAA alpha 2/3 receptor-ion channel ligands

A series of tricyclic pyridones has been evaluated as benzodiazepine site ligands with functional selectivity for the alpha(3) over the alpha(1) containing subtype of the human GABA(A) receptor ion channel. This investigation led to the identification of a high affinity, functionally selective, orally bioavailable benzodiazepine site ligand that demonstrated activity in rodent anxiolysis models and reduced sedation relative to diazepam.     

Azaflavones compared to flavones as ligands to the benzodiazepine binding site of brain GABA(A) receptors

A series of azaflavone derivatives and analogues were prepared and evaluated for their affinity to the benzodiazepine binding site of the GABA(A) receptor, and compared to their flavone counterparts. Three of the compounds, the azaflavones 9 and 12 as well as the new flavone 13, were also assayed on GABA(A) receptor subtypes (alpha(1)beta(3)gamma(2s), alpha(2)beta(3)gamma(2s), alpha(4)beta(3)gamma(2s) and alpha(5)beta(3)gamma(2s)), displaying nanomolar affinities as well as selectivity for alpha1- versus alpha2- and alpha3-containing receptors by a factor of between 14 and 26.     

Ivermectin Interactions with Benzodiazepine Receptors in Rat Cortex and Cerebellum In Vitro

Abstract: The anthelmintic macrolide, ivermectin, enhances the binding of benzodiazepine agonist ([³H]-diazepam) and antagonist ([³H]β-carboline ethyl ester) ligands to rat cortical and cerebellar membrane preparations. Enhancement of benzodiazepine agonist binding is partially additive with that of γ-aminobutyric acid (GABA) and is inhibited by etazolate, bicuculline, and the steroid GABA antagonist R5135. Ivermectin-stimulated benzodiazepine antagonist binding is enhanced by bicuculline and inhibited by GABA and etazolate. The modulatory effects of bicuculline are chloride-dependent. The stimulatory effects of ivermectin, while quantitatively different in cortex and cerebellum, are qualitatively similar in both brain regions and are reduced in the presence of chloride. Ivermectin effects on benzodiazepine ligand binding to the benzodiazepine receptor complex and the differences in the effects of GABA, bicuculline, and R5135 on ivermectin-stimulated agonist and antagonist binding may provide evidence for distinct differences in the recognition sites for the two classes of benzodiazepine receptor ligand and their interactions with other components of the receptor complex.     

Proximity-accelerated chemical coupling reaction in the benzodiazepine-binding site of gamma-aminobutyric acid type A receptors: superposition of different allosteric modulators

Benzodiazepines are widely used drugs. They exert sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects and act through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the gamma-aminobutyric acid type A (GABA(A)) receptor. Ligands of the benzodiazepine-binding site are classified into three groups depending on their mode of action: positive and negative allosteric modulators and antagonists. To rationally design ligands of the benzodiazepine site in different isoforms of the GABA(A) receptor, we need to understand the relative positioning and overlap of modulators of different allosteric properties. To solve these questions, we used a proximity-accelerated irreversible chemical coupling reaction. GABA(A) receptor residues thought to reside in the benzodiazepine-binding site were individually mutated to cysteine and combined with a cysteine-reactive benzodiazepine site ligand. Direct apposition of reaction partners is expected to lead to a covalent reaction. We describe here such a reaction of predominantly alpha(1)H101C and also three other mutants (alpha(1)G157C, alpha(1)V202C, and alpha(1)V211C) with an Imid-NCS derivative in which a reactive isothiocyanate group (-NCS) replaces the azide group (-N(3)) in the partial negative allosteric modulator Ro15-4513. Our results show four contact points of imidazobenzodiazepines with the receptor, alpha(1)H101C being shared by classical benzodiazepines. Taken together with previous data, a similar orientation of these ligands within the benzodiazepine-binding pocket may be proposed.     

Affinity of 3-acyl substituted 4-quinolones at the benzodiazepine site of GABA(A) receptors

The finding that alkyl 1,4-dihydro-4-oxoquinoline-3-carboxylate and N-alkyl-1,4-dihydro-4-oxoquinoline-3-carboxamide derivatives may be high-affinity ligands at the benzodiazepine binding site of the GABA(A) receptor, prompted a study of 3-acyl-1,4-dihydro-4-oxoquinoline (3-acyl-4-quinolones). In general, the affinity of the 3-acyl derivatives was found to be comparable with the 3-carboxylate and the 3-carboxamide derivatives, and certain substituents (e.g., benzyl) in position 6 were again shown to be important. As it is believed that the benzodiazepine binding site is situated between an alpha- and a gamma-subunit in the GABA(A) receptor, selected compounds were tested on the alpha(1)beta(2)gamma(2s), alpha(2)beta(2)gamma(2s) and alpha(3)beta(2)gamma(2s) GABA(A) receptor subtypes. The 3-acyl-4-quinolones display various degrees of selectivity for alpha(1)- versus alpha(2)- and alpha(3)-containing receptors, and high-affinity ligands essentially selective for alpha(1) over alpha(3) were developed.     

Two neighboring residues of loop A of the alpha1 subunit point towards the benzodiazepine binding site of GABAA receptors

Benzodiazepines are widely used drugs exerting sedative, anxiolytic, muscle relaxant, and anticonvulsant effects by acting through specific high affinity binding sites on some GABA(A) receptors. It is important to understand how these ligands are positioned in this binding site. We are especially interested here in the conformation of loop A of the alpha(1)beta(2)gamma(2) GABA(A) receptor containing a key residue for the interaction of benzodiazepines: alpha(1)H101. We describe a direct interaction of alpha(1)N102 with a diazepam- and an imidazobenzodiazepine-derivative. Our observations help to better understand the conformation of this region of the benzodiazepine pocket in GABA(A) receptor.     

Benzodiazepine-Induced Hyperphagia: Development and Assessment of a 3D Pharmacophore By Computational Methods

Abstract

Benzodiazepine receptor (BDZR) ligands are structurally diverse compounds that bind to specific binding sites on GABAA receptors and allosterically modulate the effect of GABA on chloride ion flux. The binding of BDZR ligands to this receptor system results in activity at multiple behavioral endpoints, including anxiolytic, sedative, anticonvulsant, and hyperphagic effects. In the work presented here, a computational procedure developed in our laboratory has been used to obtain a 3D pharmacophore for ligand recognition of the GABAA/BDZRS initiating the hyperphagic response. To accomplish this goal, 17 structurally diverse compounds, previously assessed in our laboratory for activity at the hyperphagic endpoint, were used. The result is a four-component 3D pharmacophore. It consists of two proton acceptor atoms, the centroid of an aromatic ring and the centroid of a hydrophobic moiety in a common geometric arrangement in all compounds with activity at this endpoint. This 3D pharmacophore was then assessed and successfully validated using three different tests. First, two BDZR ligands, which were included as negative controls in the set of seventeen compounds used for the pharmacophore development, did not fit the pharmacophore. Second, some benzodiazepine ligands known to have activity at the hyperphagia endpoint, but not included in the pharmacophore development, were used as positive controls and were found to fit the pharmacophore. Finally, using the 3D pharmacophore developed in the present work to search 3D databases, over 50 classical benzodiazepines were found. Among them, were benzodiazepine ligands known to have an effect at the hyperphagic endpoint. In addition, the novel compounds also found in this search are promising therapeutic agents that could beneficially affect feeding behavior.     

Imidazo[1,2-a]pyrimidines as functionally selective GABA(A) ligands

Imidazo[1,2-a]pyrimidines are GABA(A) receptor benzodiazepine binding site ligands which can exhibit functional selectivity for the alpha(3) subtype over the alpha(1) subtype. SAR studies to optimize this functional selectivity are described.     

Benzodiazepine ligands can act as allosteric modulators of the Type 1 cholecystokinin receptor

The cholecystokinin (CCK₁) receptor is a G protein-coupled receptor important for nutrient homeostasis. The molecular basis of CCK-receptor binding has been debated, with one prominent model suggesting occupation of the same region of the intramembranous helical bundle as benzodiazepines. Here, we used a specific assay of allosteric ligand interaction to probe the mode of binding of devazepide, a prototypic benzodiazepine ligand. Devazepide elicited marked slowing of dissociation of pre-bound CCK, only possible through binding to a topographically distinct allosteric site. This effect was disrupted by chemical modification of a cysteine in the benzodiazepine-binding pocket. Application of an allosteric model to the equilibrium interaction between a series of benzodiazepine ligands and CCK yielded quantitative estimates of each modulator’s affinity for the allosteric site, as well as the degree of negative cooperativity for the interaction between occupied orthosteric and allosteric sites. The allosteric nature of benzodiazepine binding to the CCK₁ receptor provides new opportunities for small molecule drug development.