Haesook K. Im

Interaction of β‐carboline inverse agonists for the benzodiazepine site with another site on GABAA receptors

1 We examined the effects of methyl 6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate (DMCM), a β‐carboline inverse agonist for the benzodiazepine site, on γ‐aminobutyric acid (GABA)‐induced Cl− currents in several cloned rat GABAA receptor subtypes expressed in human embryonic kidney cells. The Cl− currents were measured in the whole cell configuration of patch clamp techniques. 2 DMCM at low concentrations (<0.5 μm) occupying only the benzodiazepine site decreased GABA‐induced Cl currents in the α1β2γ2 and α3β2γ2 subtypes as expected from an inverse agonist, but produced no change in the α6β2γ2 subtype (perhaps a neutral antagonist). The drug at higher concentrations (> 0.5 μm) enhanced Cl− currents in all the subtypes, with a half maximal concentration of 6 to 20 μm, depending on the α isoform. In the α1β2 subtype, which is without the benzodiazepine site, DMCM monophasically increased Cl− currents with a half maximal concentration of 1.9 μm. 3 Ro 15–1788 (a classical benzodiazepine antagonist) had no effect on Cl− current enhancement by DMCM and, in fact, increased the current level through blocking current inhibition by DMCM via the benzodiazepine site. Also, Cl− current enhancement by pentobarbitone or by 3α, 21‐dihydroxy‐5α‐pregnan‐20‐one was additive to that by DMCM at saturating doses. It appears that the agonist site for DMCM is distinct from those for benzodiazepines, barbiturates and neurosteroids. 4 Among β‐carboline analogues, methyl‐β‐carboline‐3‐carboxylate and propyl‐β‐carboline‐3‐carboxylate markedly enhanced GABA‐induced Cl currents in the α1β2γ2 subtype, while N‐methyl‐β‐carboline‐3‐carboxamide and 1‐methyl‐7‐methoxy‐3,4‐dihydro‐β‐carboline did not. It appears that the 3‐carboxyl ester moiety is necessary for β‐carbolines to interact with a novel site on GABAA receptors as agonists.

Characterization of U-101017 as a GABAA receptor ligand of dual functionality

Drugs acting on the benzodiazepine site of GABA(A) receptors are much safer than barbiturates, but are still liable to abuse. Recently, we have reported that a benzodiazepine site agonist, U-97775 (a dihydroimidazoquinoxaline analog), may have minimal abuse liability because of its interaction with a second, low-affinity site on GABA(A) receptors, the occupancy of which, at high drug concentrations, leads to a reversal of its agonistic activity on the benzodiazepine site and inhibition of GABA-induced Cl- currents [Br. J. Pharmacol. 115 (1995)19-24]. Here we report that U-101017 (7-chloro-5[(cis-3,5-dimethylpiperazine)carbonyl]imidazo[1,5a]quinoline- 3-carboxylate) is another similar benzodiazepine site agonist possessing the ability to reverse its agonistic activity at higher concentrations, but its ability to inhibit GABA currents is considerably milder than that of U-97775. In the alpha 6 beta 2 gamma 2 subtype where these drugs have no agonistic activity, for instance, U-101017 at concentrations up to 80 mu M, showed no appreciable effect on GABA currents, whereas U-97775 inhibited the currents with an IC(50) value of 10 mu M as measured with the whole cell patch clamp techniques in human embryonic kidney cells expressing recombinant receptors. Similar, milder inhibition of GABA currents by U-101017 was observed in the alpha 1 beta 2 gamma 2 and alpha 3 beta 2 gamma 2 subtypes. Furthermore, U-101017 was of higher efficacy in the alpha 1 beta 2 gamma 2 than alpha 3 beta 2 gamma 2 subtypes as compared to diazepam, although its binding affinity was not appreciably different in the two subtypes. We conclude that U-101017 is a partial benzodiazepine agonist, somewhat selective to the alpha 1 beta 2 gamma 2 subtype, and with the ability to limit its own agonistic activity over a wide range of doses through its interaction with the low affinity site, but without potential convulsant activity, inherent to agents which block GABA currents.

Acceleration of GABA-dependent desensitization by mutating threonine 266 to alanine of the α6 subunit of rat GABAA receptors

Various GABAA receptor subunits share four highly homologous putative transmembrane domains (M1 to M4) and have been proposed to form an ion channel of pentameric structure with M2 lining the pore. The carboxyl terminal side of M2 contains three amino acid residues containing a hydroxyl group, which are Thr 265, 266 and serine 268 in the alpha 6 subunit. In order to study their functional role, we generated mutants of the alpha 6 subunit carrying a single point mutation of threonine 265 or 266 to alanine, or serine 268 to glycine. Co-expression of the mutants with beta 2 and gamma 2 subunits in human embryonic kidney cells produced functional receptors which are similar to the wild type in their sensitivity to a benzodiazepine agonist (U-92330), insensitivity to Zn, anion permeability, and GABA dose-response profiles as monitored with the whole cell patch clamp technique. Only in the alpha 6T266A beta 2 gamma 2 subtype, however, GABA-induced Cl- currents decayed much more rapidly than the wild type (about 10 times faster). Analysis of the GABA dependency of desensitization indicates that the T266A mutation enhanced the desensitization rate with little effect on the recovery rate from desensitization or on the half-maximal GABA concentration. We conclude that threonine 266 in the alpha 6 subunit plays a pivotal role in desensitization processes of GABAA receptors.

Alterations of the benzodiazepine site of rat α6β2γ2-GABAA receptor by replacement of several divergent amino-terminal regions with the αl counterparts

1 The benzodiazepine site of the α6β2γ2 subtype of γ‐aminobutyric acidA (GABAA) receptors is distinguishable from that of the α1β2γ2 subtype by its inability to interact with classical benzodiazepines (i.e., diazepam) and its agonistic response to Ro 15–1788, which behaves as an antagonist in the α1β2γ2 subtype. 2 The point mutation of Arg 100 of the α6 subunit to histidine (the corresponding residue in α1) has been shown to enable the α6β2γ2 subtype to interact with diazepam but failed in this study to abolish the ability of Ro 15–1788 to enhance GABA‐induced Cl− currents. 3 Here we identified the segment of P161 to L187 of α6 to contain the functional region responsible for the agonistic action of Ro 15–1788. Its replacement with the corresponding α1 sequence abolished the ability of Ro 15–1788 to enhance GABA currents without appreciable effects on its binding affinity to the benzodiazepine site or on the functionality of the other benzodiazepine site ligands such as diazepam, U‐92330 and 6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate. These data support the evidence that the functionality of a given ligand could arise from a single region of the benzodiazepine site, not shared by others. 4 In addition we have learned that several residues in the N‐terminal region of α6, such as R100, V142 and N143, have the ability to influence GABA‐dependent Cl current induction probably by allosterically modulating low affinity GABA sites.

Differential affinity of dihydroimidazoquinoxalines and diimidazoquinazolines to the α1β2γ2 and α6β2γ2 subtypes of cloned GABAA receptors

1 In this study, we compared two series of newly discovered ligands for their selectivity to benzodiazepine sites in the α1β2γ2 and the α6β2γ2 subtypes of cloned γ‐aminbutyric acidA (GABAA) receptors, the latter being unique in not interacting with classical benzodiazepines. 2 The prototype compounds, U‐85575 (12‐chloro‐5‐(5‐cyclopropyl‐1′,2′,4′‐oxadiazol‐3′‐yl)‐2,3‐dihydro‐diimidazo[1,5‐a;1,2‐c]quinazoline), and U‐92330 (5‐acetyl‐3‐(5′‐cyclopropyl‐1′,2′,4′‐oxadiazole‐3′‐yl)‐7‐chloro‐4,5‐dihydro[1,5‐a]quinoxaliné), appear to share an overlapping recognition site with classical benzodiazepines on the GABAA receptor, because their potentiation of GABA‐mediated Cl− currents in both subtypes were sensitive to Ro 15–1788, a classical benzodiazepine antagonist. 3 Minor changes in the ring substituents of the drugs reduced their affinity to the α6β2γ2 subtype more pronouncedly than to the α1β2γ2 subtype. The diimidazoquinazoline containing a 2‐methyl group which projected below the plane of the rigid ring showed a markedly lower affinity to the α6β2γ2 subtype as compared to its stereoisomer having the methyl group above the plane of the ring. Also, the dihydroimidazoquinoxalines containing the 5‐benzoyl group showed a lower affinity to the α6β2γ2 subtype than the 5‐acetyl counterpart. In particular, the 5‐benzoyl analogue containing a 6‐fluoro group showed no interaction with the α6β2γ2 subtype even at the concentration of 10 μm, probably due to stabilization of the benzoyl group in the out‐of‐plane region by the steric and electrostatic effects of the 6‐fluoro group. 4 We propose that the benzodiazepine site of the α6β2γ2 subtype shares overlapping regions with that of the α1β2γ2 subtype, but has a sterically restricted out‐of‐plane region, which may be also incompatible with the 5‐phenyl group of classical benzodiazepines.

Characterization of U‐97775 as a GABAA receptor ligand of dual functionality in cloned rat GABAA receptor subtypes

1 U‐97775 (tert‐butyl 7‐chloro‐4,5‐dihydro‐5‐[(1‐(3,4,5‐trimethyl)piperazino)carbonyl]‐imidazo[1,5‐a])quinoxaline‐3‐carboxylate) is a novel GABAA receptor ligand of dual functionality and was characterized for its interactions with cloned rat GABAA receptors expressed in human embryonic kidney cells. 2 The drug produced a bell‐shaped dose‐response profile in the α1β2γ2 receptor subtype as monitored with GABA‐induced C1− currents in the whole cell patch‐clamp technique. At low concentrations (<0.5 μM), U‐97775 enhanced the currents with a maximal increase of 120% as normalized to 5 μM GABA response (control). An agonist interaction of U‐97775 with the benzodiazepine site is suggested, because Ro 15–1788 (an antagonist at the benzodiazepine site) abolished the current increase and [3H]‐flunitrazepam binding was inhibited by U‐97775 with a Ki of 1.2 nM. 3 The enhancement of GABA currents progressively disappeared as the U‐97775 concentration was raised above 1 μM, and the current amplitude was reduced to 40% below the control at 10 μM U‐97775. The current inhibition by U‐97775 (10 μM) was not affected by Ro 15–1788. It appears that U‐97775 interacts with a second site on GABA receptors, distinct from the benzodiazepine site, to reverse its agonistic activity on the benzodiazepine site and also to inhibit GABA currents. 4 U‐97775 at low concentrations reduced and at high concentrations enhanced [35S]‐TBPS binding. Ro 15–1788 selectively blocked the effect of U‐97775 at low concentrations. Analysis of the binding data in the presence of Ro 15–1788 yielded a single low affinity site with an estimated Kd of 407 nM. 5 In other αβγ receptor subtypes, U‐97775 at low concentrations enhanced C1− currents in the α3β2γ2 but not in the α6β2γ2 subtype. On the other hand, U‐97775 at high concentrations reduced C1− currents in all the receptor subtypes we examined, including those of two subunits, α1β2, β2γ2 and α1gamma;2 subtypes. 6 Therapeutically, U‐97775 could be unique among benzodiazepine ligands because of its ability to limit its own agonistic activity such that, at high doses the appearance of agonistic activity would be delayed until occupancy of its second site wanes. This property should make the total agonistic activity of U‐97775 relatively constant over a wide range of drug doses, and may minimize its liability to abuse.

Two imidazoquinoxaline ligands for the benzodiazepine site sharing a second low affinity site on rat GABAA receptors but with the opposite functionality

Imidazoquinoxaline PNU‐97775 and imidazoquinoline PNU‐101017 are benzodiazepine site ligands with a second low affinity binding site on GABAA receptors, the occupancy of which at high drug concentrations reverses their positive allosteric activity via the benzodiazepine site, and may potentially minimize abuse liability and physical dependence. In this study we discovered, with two imidazoquinoxaline analogues, that the functionality of the second site was altered by the nitrogen substituent on the piperazine ring moiety: PNU‐100076 with a hydrogen substituent on the position produced a negative allosteric effect via the second low affinity site, like the parent compounds, while PNU‐100079 with a trifluoroethyl substituent produced a positive allosteric response. These functional characteristics were monitored with Cl− currents measurements in cloned rat αxβ2γ2 subtypes of GABAA receptors expressed in human embryonic kidney 293 cells, and further confirmed in rat cerebrocortical membranes containing complex subtypes of GABAA receptors with binding of [35S]‐TBPS, which is a high affinity ligand specific for GABAA receptors with exquisite sensitivity to allosteric modulations. This structure‐functional relationship could be exploited to further our understanding of the second allosteric site of imidazoquinoxaline analogues, and to develop more effective benzodiazepine site ligands without typical side effects associated with those currently available on the market.

Differential potentiation of GABAA receptor function by two stereoisomers of diimidazoquinazoline analogues

1 U‐84935, diimidazo[1,5‐a;1′,2′‐C]quinazoline,5‐(5‐cyclopropyl‐1,2,4‐oxidiazol‐3yl)‐2,3‐dihydro, is a ligand of high affinity for the benzodiazepine site of the GABAA receptor composed of α1β2γ2 subunits. 2 The efficacy of its analogues was measured with their ability to potentiate GABA‐mediated Cl− currents in the whole cell configuration of the patch clamp techniques in human kidney cells (A293 cells) expressing the subtype of the GABAA receptor. 3 The analogues displayed various levels of efficacy including agonists, partial agonists and antagonists without marked changes in their affinity for the receptors. 4 The major determinant of their efficacy was the spacial configuration of a methyl substituent of the C2 atom of the rigid and planar diimidazoquinazoline ring: U‐90167, containing the methyl substituent projected below the plane of the ring, markedly enhanced the GABA current with a maximal potentiation of 220 ± 25%, while its stereoisomer, U‐90168, marginally increased the GABA response with a maximal potentiation of 45 ± 10%, to which its methyl group appeared to contribute very little. 5 U‐90167 potentiated the GABA response with an EC50 of 8.1 nm and a Hill coefficient of 1.1 and did not alter the reversal potential for the Cl− current. 6 From computational modelling, the sensitive methyl group of U‐90167 could be assigned to the general region for the 5‐phenyl group of diazepam. The diimidazoquinazoline, because of its rigid and plantar ring structure, may be useful to define further the out‐of‐plane region responsible for agonistic activity and to pinpoint other areas pivotal to the functionality of benzodiazepine ligands.

Alterations of the benzodiazepine site of rat alpha 6 beta 2 gamma 2-GABAA receptor by replacement of several divergent amino-terminal regions with the alpha 1 counterparts.

1 The benzodiazepine site of the α6β2γ2 subtype of γ‐aminobutyric acidA (GABAA) receptors is distinguishable from that of the α1β2γ2 subtype by its inability to interact with classical benzodiazepines (i.e., diazepam) and its agonistic response to Ro 15–1788, which behaves as an antagonist in the α1β2γ2 subtype. 2 The point mutation of Arg 100 of the α6 subunit to histidine (the corresponding residue in α1) has been shown to enable the α6β2γ2 subtype to interact with diazepam but failed in this study to abolish the ability of Ro 15–1788 to enhance GABA‐induced Cl− currents. 3 Here we identified the segment of P161 to L187 of α6 to contain the functional region responsible for the agonistic action of Ro 15–1788. Its replacement with the corresponding α1 sequence abolished the ability of Ro 15–1788 to enhance GABA currents without appreciable effects on its binding affinity to the benzodiazepine site or on the functionality of the other benzodiazepine site ligands such as diazepam, U‐92330 and 6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate. These data support the evidence that the functionality of a given ligand could arise from a single region of the benzodiazepine site, not shared by others. 4 In addition we have learned that several residues in the N‐terminal region of α6, such as R100, V142 and N143, have the ability to influence GABA‐dependent Cl current induction probably by allosterically modulating low affinity GABA sites.

Alterations of the benzodiazepine site of rat α6β2γ2-GABAAreceptor by replacement of several divergent amino-terminal regions with the α1 counterparts

1 The benzodiazepine site of the α6β2γ2 subtype of γ‐aminobutyric acidA (GABAA) receptors is distinguishable from that of the α1β2γ2 subtype by its inability to interact with classical benzodiazepines (i.e., diazepam) and its agonistic response to Ro 15–1788, which behaves as an antagonist in the α1β2γ2 subtype. 2 The point mutation of Arg 100 of the α6 subunit to histidine (the corresponding residue in α1) has been shown to enable the α6β2γ2 subtype to interact with diazepam but failed in this study to abolish the ability of Ro 15–1788 to enhance GABA‐induced Cl− currents. 3 Here we identified the segment of P161 to L187 of α6 to contain the functional region responsible for the agonistic action of Ro 15–1788. Its replacement with the corresponding α1 sequence abolished the ability of Ro 15–1788 to enhance GABA currents without appreciable effects on its binding affinity to the benzodiazepine site or on the functionality of the other benzodiazepine site ligands such as diazepam, U‐92330 and 6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate. These data support the evidence that the functionality of a given ligand could arise from a single region of the benzodiazepine site, not shared by others. 4 In addition we have learned that several residues in the N‐terminal region of α6, such as R100, V142 and N143, have the ability to influence GABA‐dependent Cl current induction probably by allosterically modulating low affinity GABA sites.