2 IMIDAZOQUINOXALINE LIGANDS FOR THE BENZODIAZEPINE SITE SHARING A 2ND LOW-AFFINITY SITE ON RAT GABA(A) RECEPTORS BUT WITH THE OPPOSITE FUNCTIONALITY

1 Imidazoquinoxaline PNU-97775 and imidazoquinoline PNU-101017 are ben zodiazepine site ligands with a second low affinity binding site on GA BA(A) receptors, the occupancy of which at high drug concentrations re verses 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-10 0079 with a trifluoroethyl substituent produced a positive allosteric response. 3 These functional characteristics were monitored with Cl- c urrents measurements in cloned rat alpha x beta 2 gamma 2 subtypes of GABA(A) receptors expressed in human embryonic kidney 293 cells, and f urther confirmed in rat cerebrocortical membranes containing complex s ubtypes of GABA(A) receptors with binding of [S-35]-TBPS, which is a h igh affinity ligand specific for GABA(A) receptors with exquisite sens itivity to allosteric modulations. 4 This structure-functional relatio nship could be exploited to further our understanding of the second al losteric site of imidazoquinoxaline analogues, and to develop more eff ective benzodiazepine site ligands without typical side effects associ ated with those currently available on the market.