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

1. Imidazoquinoxaline PNU-97775 and imidazoquinoline PNU-101017 are benzodiazepine site ligands with a second low affinity binding site on GABA_A 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.
2. 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.
3. These functional characteristics were monitored with Cl⁻ currents measurements in cloned rat αxβ2γ2 subtypes of GABA_A receptors expressed in human embryonic kidney 293 cells, and further confirmed in rat cerebrocortical membranes containing complex subtypes of GABA_A receptors with binding of [³⁵S]-TBPS, which is a high affinity ligand specific for GABA_A receptors with exquisite sensitivity to allosteric modulations.
4. 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.