Gopalan V. Pillai

α4β3δ GABAAReceptors Characterized by Fluorescence Resonance Energy Transfer-derived Measurements of Membrane Potential

Selective modulators of γ-aminobutyric acid, type A (GABAA) receptors containing α4subunits may provide new treatments for epilepsy and premenstrual syndrome. Using mouse L(−tk) cells, we stably expressed the native GABAA receptor subunit combinations α3β3γ2,α4β3γ2, and, for the first time, α4β3δ and characterized their properties using a novel fluorescence resonance energy transfer assay of GABA-evoked depolarizations. GABA evoked concentration-dependent decreases in fluorescence resonance energy transfer that were blocked by GABAA receptor antagonists and, for α3β3γ2and α4β3γ2 receptors, modulated by benzodiazepines with the expected subtype specificity. When combined with α4 and β3, δ subunits, compared with γ2, conferred greater sensitivity to the agonists GABA, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), and muscimol and greater maximal efficacy to THIP. α4β3δ responses were markedly modulated by steroids and anesthetics. Alphaxalone, pentobarbital, and pregnanolone were all 3–7-fold more efficacious at α4β3δ compared with α4β3γ2. The fluorescence technique used in this study has proven valuable for extensive characterization of a novel GABAA receptor. For GABAA receptors containing α4 subunits, our experiments reveal that inclusion of δ instead of γ2subunits can increase the affinity and in some cases the efficacy of agonists and can increase the efficacy of allosteric modulators. Pregnanolone was a particularly efficacious modulator of α4β3δ receptors, consistent with a central role for this subunit combination in premenstrual syndrome.

Identification of amino acid residues responsible for the α5 subunit binding selectivity of L‐655,708, a benzodiazepine binding site ligand at the GABAA receptor

L‐655,708 is a ligand for the benzodiazepine site of the γ‐aminobutyric acid type A (GABAA) receptor that exhibits a 100‐fold higher affinity for α5‐containing receptors compared with α1‐containing receptors. Molecular biology approaches have been used to determine which residues in the α5 subunit are responsible for this selectivity. Two amino acids have been identified, α5Thr208 and α5Ile215, 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 α5‐containing receptors. L‐655,708 is a partial inverse agonist at the GABAA receptor which exhibited no functional selectivity between α1‐ and α5‐containing receptors and showed no change in efficacy at receptors containing α1 subunits where amino acids at both of the sites had been altered to their α5 counterparts (α1ΔSer205‐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 GABAA receptor, and further delineate a region just N‐terminal to the first transmembrane domain of the receptor α subunit that contributes to this binding site.

Human D2 and D4 dopamine receptors couple through βγ G-protein subunits to inwardly rectifying K+ channels (GIRK1) in a Xenopus oocyte expression system : Selective antagonism by L-741,626 and L-745,870 respectively

To examine the effects of a novel selective D4 receptor ligand, L-745,870 (3-[4-(4-chlorophenyl)piperazin-1-yl]methyl-1H-pyrrolo[2,3-b]pyrid ine), on human dopamine receptor function, the ability of this ligand to antagonise G-protein gated inwardly rectifying K+ (GIRK/Kir3) currents activated by cloned human D2 and D4 receptors expressed in Xenopus oocytes was examined using voltage-clamp recording. Its effects were also contrasted with that of a selective D2 receptor antagonist L-741,626. L-745,870 had no detectable agonist activity on human D4 receptors and selectively blocked currents activated by D4 but not D2 receptors. The role of G-protein subunits in dopamine receptor modulation of GIRK currents was also examined by co-expression of beta1 and/or gamma2 subunits on spontaneously active and receptor-activated currents. Currents activated by both D2 and D4 receptors were occluded by direct activation of GIRK currents following co-transfection with the cDNA encoding G-protein betagamma subunits. These data demonstrate that L-745,870 and L-741,626 act as antagonists on human D4 and D2 receptors respectively, and that activation of GIRK channels by these dopamine receptors can be disrupted by direct stimulation of K+ currents by G-protein betagamma subunits.

Salicylidene salicylhydrazide, a selective inhibitor of β1-containing GABAA receptors

A high‐throughput assay utilizing the voltage/ion probe reader (VIPR) technology identified salicylidene salicylhydrazide (SCS) as being a potent selective inhibitor of α2β1γ1θ GABAA receptors with a maximum inhibition of 56±5% and an IC50 of 32 (23, 45) nM. Evaluation of this compound using patch‐clamp electrophysiological techniques demonstrated that the compound behaved in a manner selective for receptors containing the β1 subunit (e.g. maximum inhibition of 68.1±2.7% and IC50 value of 5.3 (4.4, 6.5) nM on α2β1γ1 receptors). The presence of a β1 subunit was paramount for the inhibition with changes between α1 and α2, γ1 and γ2, and the presence of a θ subunit having little effect. On all subtypes, SCS produced incomplete inhibition with the greatest level of inhibition at α1β1γ1θ receptors (74.3±1.4%). SCS displayed no use or voltage dependence, suggesting that it does not bind within the channel region. Concentration – response curves to GABA in the presence of SCS revealed a reduction in the maximum response with no change in the EC50 or Hill coefficient. In addition, SCS inhibited pentobarbitone‐induced currents. Threonine 255, located within transmembrane domain (TM) 1, and isoleucine 308, located extracellularly just prior to TM3, were required for inhibition by SCS. SCS did not compete with the known allosteric modulators, picrotoxin, pregnenolone sulphate, dehydroepiandrosterone 3‐sulphate, bicuculline, loreclezole or mefenamic acid. Neither was the inhibition by SCS influenced by the benzodiazepine site antagonist flumazenil. In conclusion, SCS is unique in selectively inhibiting GABAA receptors containing the β1 subunit via an allosteric mechanism. The importance of threonine 255 and isoleucine 308 within the β1 subunit and the lack of interaction with a range of GABAA receptor modulators suggests that SCS is interacting at a previously unidentified site.

Identification of an Orally Bioavailable, Potent, and Selective Inhibitor of GlyT1.

Amalgamation of the structure-activity relationship of two series of GlyT1 inhibitors developed at Merck led to the discovery of a clinical candidate, compound 16 (DCCCyB), which demonstrated excellent in vivo occupancy of GlyT1 transporters in rhesus monkey as determined by displacement of a PET tracer ligand.

Optimisation of a series of potent, selective and orally bioavailable GlyT1 inhibitors.

A series of heterocyclic sulfonamides have been developed which are potent and selective inhibitors of hGlyT1. SAR studies to optimise the in vitro and in vivo properties are described. Optimisation of the central scaffold resulted in cyclohexane sulfones 28 and 29, which have good PK properties and show promise for further development.