An updated unified pharmacophore model of the benzodiazepine binding site on gamma-aminobutyric acid(a) receptors: correlation with comparative models.

A successful unified pharmacophore/receptor model which has guided the synthesis of subtype selective compounds is reviewed in light of recent developments both in ligand synthesis and structural studies of the binding site itself. The evaluation of experimental data in combination with a comparative model of the alpha1beta2gamma2 GABA(A) receptor leads to an orientation of the pharmacophore model within the Bz BS. Results not only are important for the rational design of selective ligands, but also for the identification and evaluation of possible roles which specific residues may have within the benzodiazepine binding pocket.

Alternate use of distinct intersubunit contacts controls GABAA receptor assembly and stoichiometry.

GABA(A) receptors are the major inhibitory transmitter receptors in the CNS. Recombinant GABA(A) receptors composed of alpha(1)beta(3)gamma(2) subunits have been demonstrated to assemble as pentamers consisting of two alpha(1), two beta(3), and one gamma(2) subunit. Using truncated and chimeric alpha(1) subunits, we identified the alpha(1)(80-100) sequence as a major binding site for gamma(2) subunits. In addition, we demonstrated its direct interaction with gamma(2)(91-104), a sequence that previously has been identified to form the contact to alpha(1) subunits. The observation that the amino acid residues alpha(1)P96 and alpha(1)H101, which can be photolabeled by [(3)H]flunitrazepam, are located within or adjacent to the alpha(1)(80-100) sequence, indicates that the benzodiazepine binding site of GABA(A) receptors is located close to this intersubunit contact. The observation that alpha(1)(80-100) interacts with gamma(2) but not with beta(3) subunits indicates the existence of an additional beta(3) binding site on alpha(1) subunits. The preferred alternate use of the gamma(2) and beta(3) binding sites in two different alpha(1) subunits of the same receptor ensures the incorporation of only a single gamma(2) subunit and thus, determines subunit stoichiometry of alpha(1)beta(3)gamma(2) receptors. Distinct binding sites and their alternate use can therefore explain how subunits of hetero-oligomeric transmembrane proteins assemble into a defined protein complex.

Use of bicuculline, a GABA antagonist, as a template for the development of a new class of ligands showing positive allosteric modulation of the GABA(A) receptor.

Analogues of bicuculline devoid of the benzo ring fused to the lactone moiety were prepared by reacting 2-(tert-butyl-dimethylsiloxy)furans with 3,4-dihydroisoquinolinium salts. Some of these compounds (e.g., ROD185, 8) acted as modulators of the GABAA receptor, displacing ligands of the benzodiazepine binding site. They also strongly stimulated GABA currents mediated by recombinant GABA(A) receptors expressed in Xenopus oocytes.

A novel positive allosteric modulator of the GABA(A) receptor: the action of (+)-ROD188.

(+)-ROD188 was synthesized in the search for novel ligands of the GABA binding site. It shares some structural similarity with bicuculline. (+)-ROD188 failed to displace [(3)H]-muscimol in binding studies and failed to induce channel opening in recombinant rat alpha1beta2gamma2 GABA(A) receptors functionally expressed in Xenopus oocytes. (+)-ROD188 allosterically stimulated GABA induced currents. Displacement of [(3)H]-Ro15-1788 indicated a low affinity action at the benzodiazepine binding site. In functional studies, stimulation by (+)-ROD188 was little sensitive to the presence of 1 microM of the benzodiazepine antagonist Ro 15-1788, and (+)-ROD188 also stimulated currents mediated by alpha1beta2, indicating a major mechanism of action different from that of benzodiazepines. Allosteric stimulation by (+)-ROD188 was similar in alpha1beta2N265S as in unmutated alpha1beta2, while that by loreclezole was strongly reduced. (+)-ROD188 also strongly stimulated currents elicited by either pentobarbital or 5alpha-pregnan-3alpha-ol-20-one (3alpha-OH-DHP), in line with a mode of action different from that of barbiturates or neurosteroids as channel agonists. Stimulation by (+)-ROD188 was largest in alpha6beta2gamma2 (alpha6beta2gamma2>>alpha1beta2gamma2=alpha5beta2gamma2++ +>alpha2beta2ga mma2= alpha3beta2gamma2), indicating a unique subunit isoform specificity. Miniature inhibitory postsynaptic currents (mIPSC) in cultures of rat hippocampal neurons, caused by spontaneous release of GABA showed a prolonged decay time in the presence of 30 microM (+)-ROD188, indicating an enhanced synaptic inhibitory transmission.

Synthesis and GABA(A) receptor activity of 6-oxa-analogs of neurosteroids.

The 6-oxasteroids 3alpha-hydroxy-6-oxa-5alpha-pregnan-20-one (3) and 3alpha-hydroxy-6-oxa-5beta-pregnan-20-one (4) were obtained from pregnenolone acetate via the corresponding (5alpha or 5beta) 3beta, 20beta-diacetoxy-6-oxa-pregnane. Both steroids showed ca. 100-fold reduced potency for modulating [(3)H]flunitrazepam, [(3)H]muscimol or [(35)S]TBPS binding to the GABA(A) receptor when compared to their natural carbon analogs 3alpha-hydroxy-5alpha-pregnan-20-one (1) and 3alpha-hydroxy-5beta-pregnan-20-one (2).

Chronic and acute effects of thiazolidinediones BM13.1258 and BM15.2054 on rat skeletal muscle glucose metabolism.

1 New thiazolidinediones BM13.1258 and BM15.2054 were studied with regard to their PPARgamma-agonistic activities and to their acute and chronic effects on glucose metabolism in soleus muscle strips from lean and genetically obese rats. 2 Both BM13.1258 and BM15.2054 revealed to be potent PPARgamma-activators in transient transfection assays in vitro. 3 In insulin-resistant obese rats, but not in lean rats, 10 days of oral treatment with either compound increased the stimulatory effect of insulin on muscle glycogen synthesis to a similar extent (insulin-induced increment in micromol glucose incorporated into glycogen g-1 h-1: control, +1.19+/-0.28; BM13.1258, +2.50+/-0.20; BM15.2054, +2.55+/-0.46; P<0.05 vs control each). 4 In parallel to insulin sensitization, mean glucose oxidation increased insulin-independently in response to BM13.1258 (to 191 and 183% of control in the absence and presence of insulin, respectively; P<0.01 each), which was hardly seen in response to BM15.2054 (to 137 and 124% of control, respectively; ns). 5 Comparable effects on PPARgamma activation and on amelioration of insulin resistance by BM13.1258 and BM15.2054 were therefore opposed by different effects on glucose oxidation. 6 In contrast to chronic oral treatment, acute exposure of muscles to BM13.1258 or BM15.2054 in vitro elicited a distinct catabolic response of glucose metabolism in specimens from both lean and obese rats. 7 The results provide evidence that BM13.1258 and BM15.2054 can affect muscle glucose metabolism via more than one mechanism of action. 8 Further efforts are required to clarify, to what extent other mechanisms besides insulin sensitization via the activation of PPARgamma are involved in the antidiabetic actions of thiazolidinediones.

EDPC: a novel high affinity ligand for the benzodiazepine site on rat GABA(A) receptors.

Rat recombinant alpha1beta2gamma2 gamma-aminobutyric acid type A (GABAA) receptors were functionally expressed in Xenopus laevis oocytes and analyzed for the action of EDPC (Ethyl 3-(1,3-dithian-2-yl)-1H-pyrrolo[2,3-c]pyridine-5-carboxylate) using electrophysiological techniques. EDPC inhibited GABA currents at low concentrations (IC50 approximately/= 2 nM). The inhibition by 100 nM EDPC could be reversed by 1 microM of the benzodiazepine antagonistflumazenil (Ro 15-1788), indicating a negative allosteric modulation via the benzodiazepine binding site. In line with this conclusion are radioactive ligand binding studies. EDPC inhibited the binding of 2 nM [3H]flunitrazepam to membranes from the cerebellum or the cortex with IC50 values of about 8 and 25 nM, respectively.

Failure of leptin to affect basal and insulin-stimulated glucose metabolism of rat skeletal muscle in vitro.

Studies on different isolated tissues have provided evidence that leptin may directly modulate cellular glucose handling. The present study was performed to elucidate leptin's action on basal and insulin-stimulated glucose metabolism in native muscle tissue, which under physiological circumstances is the quantitatively most important target tissue of insulin. Isolated rat soleus muscle strips were incubated for 1 h in the absence or presence of leptin (0, 1, 10, or 100 nmol/l) under basal or insulin-stimulated conditions (10 nmol/l). No effects of leptin were found on the rates of 3H-2-deoxy-glucose transport (basal: control, 314+/-14; 1 nmol/l leptin, 320+/-17; 10 nmol/l leptin, 314+/-13; 100 nmol/l leptin, 322+/-16; insulin-stimulated: control, 690+/-33; 1 nmol/l leptin, 691+/-29; 10 nmol/l leptin, 665+/-26; 100 nmol/l leptin, 664+/-27; cpm x mg(-1) x h(-1); NS vs respective control) and on net glucose incorporation into glycogen (basal: control, 1.75+/-0.18; 1 nmol/l leptin, 2.01+/-0.13; 10 nmol/l leptin, 1.92+/-0.11; 100 nmol/l leptin, 1.81+/-0.13; insulin-stimulated: control, 5.98+/-0.40; 1 nmol/l leptin, 5.93+/-0.30; 10 nmol/l leptin, 5.46+/-0.25; 100 nmol/l leptin, 5.85+/-0.30; micromol x g(-1) x h(-1); NS vs respective control). In parallel, leptin failed to affect rates of aerobic and anaerobic glycolysis as well as muscle glycogen content. Further experiments revealed that the inability of leptin to directly affect muscle glucose handling prevailed independently of muscle fiber type (soleus and epitrochlearis muscle), of ambient insulin concentrations (0-30 nmol/l), and of leptin exposure time (1 h or 6 h). Thus, our findings fail to support speculations about a physiological role of direct insulin-mimetic or insulin-desensitizing effects of leptin on skeletal muscle tissue.