The inhibition of rat liver chromatin protease by congeners of the phenylboronic acids.

A group of arylalkylboronic acids were synthesized in order to investigate the inhibitory potential of these compounds for rat liver chromatin protease (EC 3.4--). The effect of side chain length, side chain substitution and aromatic substitution on proteolytic activity in chromatin dissociated in salt and urea was assayed. It was determined that a side chain length two carbons long provided the greatest inhibitory effect with complete inhibition attainable at 20 mM concentration of phenylethylboronic acid. Aryl substitution in the ortho position proved to be the most potent structural change with complete inhibition attained by 1 mM concentration of 0-methylphenylethylboronic acid. The binding of these two inhibitors proved to be reversible.

Enhanced interleukin-1 production by human monocyte cell lines following treatment with 5-azacytidine.

Recent studies suggest that DNA modification, such as methylation of specific bases or substitution of altered bases, can play an important role in the control of eukaryotic gene expression. We chose to examine the effects of two DNA modifying agents, 5-azacytidine (AZA) and 5-bromodeoxyuridine (BUdR), on interleukin-1 (IL-1) gene expression in the human monocyte cell lines THP-1 and U937.1. THP-1 produced IL-1 upon stimulation with lipopolysaccharide (LPS), whereas U937.1 did not. Following treatment with AZA, U937.1 cells could be induced to produce IL-1 beta mRNA and release IL-1, but only if a stimulus such as LPS was present. In addition, the level of IL-1 beta mRNA produced and IL-1 released by THP-1 cells after induction could be doubled by treatment with AZA. In contrast, we were unable to alter IL-1 production by treatment of cells with BUdR in the absence or presence of inducers. These results suggest that the production of IL-1 may be, in part, regulated by methylation of DNA.

Localization of interleukin-1 receptor messenger RNA in murine hippocampus.

The cytokine interleukin-1 (IL-1) has numerous actions in brain, including pronounced neuroendocrine effects. Recent radioligand binding studies have identified high-affinity binding sites for 125I-recombinant human IL-1 alpha in the hippocampus with characteristics similar to those of IL-1 receptors in immune cells. The present study employed in situ hybridization histochemistry with 35S-labeled anti-sense cRNA probes derived from a full-length murine T-cell IL-1 receptor cDNA to identify cells producing IL-1 receptor mRNA in the murine hippocampus. An intense signal was observed over granule cells in the dentate gyrus. A weak to moderate signal was observed over the pyramidal cell layer of the hilus and CA3 region. Other aspects of the hippocampal formation, including the CA2 and CA1 regions, the subiculum, and the entorhinal area, displayed no signal above background. This distribution of IL-1 receptor mRNA was similar to that of 125I-IL-1 alpha binding sites and supports the growing body of evidence implicating IL-1 as a neurotransmitter/neuromodulator in brain.

Neuroprotective efficacy and mechanisms of novel pyrrolopyrimidine lipid peroxidation inhibitors in the gerbil forebrain ischemia model.

A brief period of bilateral carotid occlusion (BCO)-induced forebrain ischemia in gerbils triggers neuronal degeneration and the subsequent expression of amyloid precursor protein (APP), b-amyloid protein (b-AP), and apolipoprotein E (APO-E) in the selectively vulnerable CA1 region of the hippocampus. The increase in immunoreactivity is secondary to the postischemic degeneration of the CA1 neurons and is largely astrocyte-derived as evidenced by a simultaneous increase in glial fibrillary acidic protein (GFAP) staining. Oxygen radical-induced lipid peroxidation has been strongly suggested to play a role in postischemic neuronal damage and Alzheimer's disease. Recent literature suggests a possible link between early oxidative stress and APP overexpression. Therefore, the present investigation examined the effect of two novel brain-penetrating pyrrolopyrimidine lipid peroxidation inhibitors (PNU-101033E and PNU-104067F) on CA1 neurodegeneration and the subsequent increase in APP, b-AP, APO-E, and GFAP immunostaining at 4 days after a 5-minute episode of forebrain ischemia. Using an antibody for lipid peroxidation-derived malondialdehyde (MDA)-modified proteins, the authors also examined the effects of PNU-104067F on MDA immunostaining 2 days after ischemia, before completion of the neuronal loss. At 2 days, the authors also evaluated microglial activation using an antibody to surface major histocompatibility complex class II antigen expressed by activated microglia. Gerbils were treated at 30 mg/kg orally 30 minutes before the BCO and 2 hours after ischemia, followed by daily dosing for the next day (microglia and MDA) and the successive 3 days for APP, b-AP, APO-E, and GFAP immunostaining. APP and APO-E staining was significantly suppressed by 50% and 66%, respectively, with either compound. b-AP immunoreactivity was decreased 56% with both compounds, and GFAP expression was significantly decreased 53% (PNU-101033E) and 60.5% (PNU-104067F). There was a concomitant partial sparing of the CA1 hippocampal neurons by both PNU-101033E and PNU-104067F (P < .01) as determined by cresyl violet histochemistry. PNU-104067F significantly inhibited lipid peroxidation-derived MDA immunostaining and microglia activation (P < .05) at 48 hours after ischemia. Brain-penetrable lipid peroxidation inhibitors may provide attenuation of various glial response proteins after ischemic injury, probably secondary to neuronal protection.

Neuroprotective properties of the benzodiazepine receptor, partial agonist PNU-101017 in the gerbil forebrain ischemia model.

PNU-101017 is a novel, imidazoquinoline amide and benzodiazepine receptor partial agonist that has high affinity for the GABAA receptor subtypes containing the alpha 1 and alpha 3 or alpha 5 subunits. At each of these receptors, the compound is a partial agonist with approximately 50% of the intrinsic activity of the full agonist diazepam. In view of the previously demonstrated anti-ischemic effects of some GABA agonists, the purpose of this study was to determine the ability of PNU-101017 to salvage selectively vulnerable neuronal populations in the gerbil forebrain ischemia model. In an initial set of experiments, male gerbils were pretreated 30 minutes before ischemia induction (5 minutes) with PNU-101017 (3, 10, or 30 mg/kg intraperitoneally) and again 2 hours after reperfusion. In vehicle (0.05 N HC1)-treated gerbils, the loss of hippocampal CA1 neurons at 5 days was 80%. PNU-101017 was shown to produce a dose-related increase in CA1 neuronal survival; at either 10 or 30 mg/kg, the loss of CA1 neurons was only 21% (P < 0.005 versus vehicle). A second experiment, examined the therapeutic window for PNU-101017 using the dose level of 30 mg/kg intraperitoneally. Administration of the first of two doses (2 hours apart) at the time of reperfusion resulted in an identical decrease in CA1 damage at 5 days to that seen with preischemic treatment (P < 0.003 versus vehicle). Even with a delay of the initial dosing until 4 hours after reperfusion, PNU-101017 reduced CA1 neuronal loss to only 32% (P < 0.01 versus vehicle). In a third experiment in which the duration of the ischemic insult was increased to 10 minutes and the brains were not analyzed until 28 days after ischemia, daily PNU-101017 dosing for the full 28 days still significantly preserved CA1 neurons, although less effectively than in the milder 5 minute-ischemia model. The loss of dopaminergic nigrostriatal neurons was also reduced. The neuroprotective effect of PNU-101017 was not associated with any overt CNS depression and it did not correlate with hypothermia. This benzodiazepine-receptor partial agonist may have potential for the treatment of global cerebral ischemia.

A model for structure-dependent binding of Congo red to Alzheimer beta-amyloid fibrils.

The cytotoxic A beta fibril is a logical candidate for the entity causing the initiating damage to neurons in Alzheimer's disease and Down's syndrome. We have derived a model of binding for the dye molecule, Congo red (CR), to a beta-sheet structure of beta-amyloid (1-42). This model is based on the crystal coordinates of CR binding to porcine insulin fibrils from Turnell and Finch. Intact insulin is composed of protein dimers and X-ray diffraction studies show that CR intercalates between two insulin monomers at an interface formed by a pair of antiparallel beta-strands. The intercalation of CR has disrupted the four main-chain hydrogen bonds between the two beta-strands, but they are still tethered with each other through new hydrogen bonds with the CR nitrogen atoms. The CR molecule has been aligned along the homologous stretch of amino acids in Alzheimer beta peptide (two molecules in antiparallel distorted or pseudo beta-sheet conformation) using the crystal coordinates from the Turnell-Finch paper to arrive at a putative structure for CR binding to Alzheimer's amyloid fibrils.

Antagonist, partial agonist, and full agonist imidazo[1,5-a]quinoxaline amides and carbamates acting through the GABAA/benzodiazepine receptor.

(4RS)-1-(5-Cyclopropyl-1,2,4-oxadiazol-3-yl)-12,12a-dihyd roimidazo[1,5- a]pyrrolo[2,1-c]quinoxalin-10(11H)-one (1a), 5-benzoyl-3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-4,5- dihydroimidazo[1,5-a]quinoxaline (13b), and tert-butyl (4S)-12,12a-dihydroimidazo[1,5-a]pyrrolo[2,1- c]quinoxaline-1-carboxylate (1e), as well as other imidazo[1,5-a]quinoxaline amides and carbamates, represent a new series of compounds which bind with high affinity to the GABAA/benzodiazepine receptor. These compounds exhibit a wide range of intrinsic efficacies as measured by [35S]TBPS binding ratios. The synthesis of 1a begins with the addition of DL-glutamic acid to 1-fluoro-2-nitrobenzene, followed by reduction of the nitro group and subsequent ring closure to form 3-(carbethoxymethyl)-1,2,3,4-tetrahydroquinoxalin-2-one, followed by a second ring closure to afford (4RS)-1,5-dioxo-1,2,3,4,5,6-hexahydropyrrolo[1,2-a]quinoxali ne as the key intermediate. Appendage of a substituted imidazo ring via the anion of 5-cyclopropyl-1,2,4-oxadiazol-3-yl gives 1a. The (-)- and (+)-isomers of 1a were prepared from 1-fluoro-2-nitrobenzene and L- and D-glutamic acid, respectively. 1a and its enantiomers demonstrated affinity for the [3H]flunitrazepam binding site with Ki's of 0.87, 0.62, and 0.65 nM, respectively.

High-affinity partial agonist imidazo[1,5-a]quinoxaline amides, carbamates, and ureas at the gamma-aminobutyric acid A/benzodiazepine receptor complex.

A series of imidazo[1,5-a]quinoxaline amides, carbamates, and ureas which have high affinity for the gamma-aminobutyric acid A/benzodiazepine receptor complex was developed. Compounds within this class have varying efficacies ranging from antagonists to full agonists. However, most analogs were found to be partial agonists as indicated by [35S]TBPS and Cl- current ratios. Many of these compounds were also effective in antagonizing metrazole-induced seizures in accordance with anticonvulsant and possible anxiolytic activity. Selected quinoxalines displayed limited benzodiazepine-type side effects such as ethanol potentiation and physical dependence in animal models. Dimethylamino urea 41 emerged as the most interesting analog, having a partial agonist profile in vitro while possessing useful activity in animal models of anxiety such as the Vogel and Geller assays. In accordance with its partial agonist profile, 41 was devoid of typical benzodiazepine side effects.

3-Phenyl-substituted imidazo[1,5-alpha]quinoxalin-4-ones and imidazo[1,5-alpha]quinoxaline ureas that have high affinity at the GABAA/benzodiazepine receptor complex.

A series of imidazo[1,5-alpha]quinoxalin-4-ones and imidazo[1,5-alpha]quinoxaline ureas containing substituted phenyl groups at the 3-position was developed. Compounds within the imidazo[1,5-alpha]quinoxaline urea series had high affinity for the GABAA/benzodiazepine receptor complex with varying in vitro efficacy, although most analogs were partial agonists as indicated by [35S]TBPS and Cl- current ratios. Interestingly, a subseries of piperazine ureas was identified which had biphasic efficacy, becoming more antagonistic with increasing concentration. Analogs within the imidazo[1,5-alpha]quinoxalin-4-one series had substantially decreased binding affinity as compared to the quinoxaline urea series. These compounds ranged from antagonists to full agonists by in vitro analysis, with several derivatives having roughly 4-fold greater intrinsic activity than diazepam as indicated by Cl- current measurement. Numerous compounds from both series were effective in antagonizing metrazole-induced seizures, consistent with anti-convulsant properties and possible anxiolytic activity. Most of the quinoxaline ureas and quinoxalin-4-ones were active in an acute electroshock physical dependence side effect assay in mice precluding further development.

High-affinity alpha-aminobutyric acid A/benzodiazepine ligands: synthesis and structure-activity relationship studies of a new series of tetracyclic imidazoquinoxalines.

A series of tetracyclic imidazoquinoxaline analogs was developed which constrain the carbonyl group of the partial agonist 3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-[(dimethylamino)carbonyl] - 4,5-dihydroimidazo[1,5-alpha]quinoxaline (2, U-91571) away from the benzene ring. These analogs orient the carbonyl group in the opposite direction of the previously reported full agonist 1-(5- cyclopropyl-1,2,4-oxadiazol-3-yl)-12,12a-dihydroimidazo[1,5- alpha]pyrrolo [2,1-c]quinoxalin-10(11H)-one (3, U-89267). A number of approaches were utilized to form the "bottom" ring of this tetracyclic ring system including intramolecular cyclizations promoted by Lewis acids or base, as well as metal-carbenoid conditions. The size and substitution pattern of the additional ring was widely varied. Analogs within this series had high affinity for the benzodiazepine receptor on the alpha-aminobutyric acid A chloride ion channel complex. From TBPS shift and Cl- current assays, the in vitro efficacy of compounds within this class ranged from antagonists to partial agonists with only 18a identified as a full agonist. Additionally, several analogs were quite potent at antagonizing metrazole-induced seizures indicating possible anticonvulsant or anxiolytic activity. Unlike 3, analogs in this series did not have high affinity for the diazepam insensitive alpha 6 beta 2 delta 2 subtype. These results suggest that either constraining the carbonyl group away from the benzene ring or the greater planarity that results from the additional cyclic structure provides analogs with partial agonist properties and prevents effective interaction with the alpha 6 beta 2 delta 2 subtype.

Piperazine imidazo[1,5-a]quinoxaline ureas as high-affinity GABAA ligands of dual functionality.

A series of imidazo[1,5-a]quinoxaline piperazine ureas appended with a tert-butyl ester side chain at the 3-position was developed. Analogues within this series have high affinity for the gamma-aminobutyric acid A (GABAA)/benzodiazepine receptor complex with efficacies ranging from inverse agonists to full agonists. Many analogues were found to be partial agonists as indicated by [35S]TBPS and Cl- current ratios. Uniquely, a number of these analogues were found to have a bell-shaped dose-response profile in the alpha1 beta2 gamma2 subtype as determined by whole cell patch-clamp technique, where in vitro efficacy was found to decrease with increasing drug concentration. Many of the compounds from this series were effective in antagonizing metrazole-induced seizures, consistent with anticonvulsant and possibly anxiolytic activity. Additionally, several analogues were also effective in lowering cGMP levels (to control values) after applied stress, also consistent with anxiolytic-like properties. The most effective compounds in these screens were also active in animal models of anxiety such as the Vogel and Geller assays. The use of the piperazine substituent allowed for excellent drug levels and a long duration of action in the central nervous system for many of the quinoxalines, as determined by ex vivo assay. Pharmacokinetic analysis of several compounds indicated excellent oral bioavailability and a reasonable half-life in rats. From this series emerged two partial agonists (55, 91) which had good activity in anxiolytic models, acceptable pharmacokinetics, and minimal benzodiazepine-type side effects.

Enhancement by GABA of the association rate of picrotoxin and tert-butylbicyclophosphorothionate to the rat cloned alpha 1 beta 2 gamma 2 GABAA receptor subtype.

1. We examined how gamma-aminobutyric acid (GABA) influences interaction of picrotoxin and tert-butylbicyclophosphorothionate (TBPS) with recombinant rat alpha 1 beta 2 gamma 2 GABAA receptors stably expressed in human embryonic kidney cells (HEK293), as monitored with changes in Cl- currents measured by the whole-cell patch clamp technique. 2. During application of GABA (5 microM) for 15 s, picrotoxin and TBPS dose-dependently accelerated the decay of inward GABA-induced currents (a holding potential of -60 mV under a symmetrical Cl- gradient). The drugs, upon preincubation with the receptors, also reduced the initial current amplitude in a preincubation time and concentration-dependent manner. This indicates their interaction with both GABA-bound and resting receptors. 3. The half maximal inhibitory concentration for picrotoxin and TBPS at the beginning of a 15 s GABA (5 microM) pulse was several times greater than that obtained at the end of the pulse. GABA thus appears to enhance picrotoxin and TBPS potency, but only at concentrations leading to occupancy of both high and low affinity GABA sites, i.e., 5 microM. Preincubation of the receptors with the drugs in the presence of GABA at 200 nM, which leads to occupancy of only high affinity GABA sites in the alpha 1 beta 2 gamma 2 subtype, produced no appreciable change in potency of picrotoxin or TBPS. This indicates that they preferentially interact with multiliganded, but not monoliganded receptors, unlike U-93631, a novel ligand to the picrotoxin site, which has higher affinity to both mono- and multiliganded receptors than resting receptors. 4. The time-dependent decay and preincubation time-dependent reduction of initial amplitude of GABA-induced Cl- currents followed monoexponential time courses, and time constants thus obtained displayed a linear relationship with drug concentration. Analysis of the data using a kinetic model with a single drug site showed that GABA (5 microM) enhanced the association rate for picrotoxin and TBPS nearly 100 fold, but their dissociation rate only 10 fold. The dissociation rate obtained from current recovery from picrotoxin or TBPS block yielded nearly identical values to the above analysis.5. We conclude that picrotoxin and TBPS interact with both resting and GABA-bound receptors, but their affinity for the latter is about 10 times greater than that for the former, largely due to a markedly increased association rate to the multiliganded receptors (but not monoliganded ones). This and our earlier study with U-93631 improves our understanding of functional coupling between GABA and picrotoxin sites, which appears to be useful in characterizing the mode of interaction for various picrotoxin site ligands.

Interaction of beta-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-beta-carboline-3-carboxylate (DMCM), a beta-carboline inverse agonist for the benzodiazepine site, on gamma-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 microM) occupying only the benzodiazepine site decreased GABA-induced Cl currents in the alpha 1 beta 2 gamma 2 and alpha 3 beta 2 gamma 2 subtypes as expected from an inverse agonist, but produced no change in the alpha 6 beta 2 gamma 2 subtype (perhaps a neutral antagonist). The drug at higher concentrations (> 0.5 microM) enhanced Cl- currents in all the subtypes with a half maximal concentration of 6 to 20 microM, depending on the alpha isoform. In the alpha 1 beta 2 subtype, which is without the benzodiazepine site, DMCM monophasically increased Cl- currents with a half maximal concentration of 1.9 microM. 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 3 alpha, 21-dihydroxy-5 alpha-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 beta-carboline analogues, methyl-beta-carboline-3-carboxylate and propyl-beta-carboline-3-carboxylate markedly enhanced GABA-induced Cl currents in the alpha 1 beta 2 gamma 2 subtype, while N-methyl-beta-carboline-3-carboxamide and 1-methyl-7-methoxy-3,4-dihydro-beta-carboline did not. It appears that the 3-carboxyl ester moiety is necessary for beta-carbolines to interact with a novel site on GABAA receptors as agonists.

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 alpha 1 beta 2 gamma 2 receptor subtype as monitored with GABA-induced Cl- currents in the whole cell patch-clamp technique. At low concentrations (< 0.5 microM), U-97775 enhanced the currents with a maximal increase of 120% as normalized to 5 microM 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 microM, and the current amplitude was reduced to 40% below the control at 10 microM U-97775. The current inhibition by U-97775 (10 microM) 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 alpha beta upsilon receptor subtypes, U-97775 at low concentrations enhanced Cl- currents in the alpha 3 beta 2 upsilon 2,but not in the alpha 6 beta 2 upsilon 2 subtype. On the other hand, U-97775 at high concentrations reduced Cl- currents in all the receptor subtypes we examined, including those of two subunits, alpha 1 beta 2, beta 2 upsilon 2 and alpha 1 upsilon 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.

PNU-107484A with alpha isoform-dependent functional changes in alpha(x)beta2gamma2 subtypes of rat recombinant GABA(A) receptors.

1. We discovered a novel gamma-aminobutyric acidA (GABA(A)) receptor ligand displaying seemingly opposite functionalities, depending on the alpha isoform of the alpha(x)beta2gamma2 subtypes. PNU-107484A enhanced GABA-induced Cl- currents in the alpha1beta2gamma2 subtype, but inhibited the currents in the alpha3beta2gamma2 and alpha6beta2gamma2 subtypes, and its half-maximal concentrations in the subtypes were 3.1 +/- 0.5, 4.2 +/- 1, and 3.5 +/- 0.2 microM, respectively, without showing much dependency on alpha isoforms. 2. In the alpha1beta2 subtype, the drug at concentrations up to 40 microM showed no effect on GABA-induced Cl- currents, suggesting the requirement of the gamma subunit for its action. 3. PNU-107484A behaved like a positive allosteric modulator of the alpha1beta2gamma2 subtype with its binding site distinct from those for benzodiazepines, barbiturates and neurosteroids. With the alpha3beta2gamma2 subtype, the drug behaved like a non-competitive inhibitor of GABA, thus blocking Cl- currents by GABA alone or in the presence of pentobarbitone and neurosteroids. 4. It appears that PNU-107484A is a unique GABA(A) receptor ligand with alpha isoform-dependent functionalities, which may provide a basis for development of alpha isoform-selective ligands, and it could be useful as a probe to investigate the physiological roles of the various alpha isoform subtypes.

Differential affinity of dihydroimidazoquinoxalines and diimidazoquinazolines to the alpha 1 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 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 alpha 1 beta 2 gamma 2 and the alpha 6 beta 2 gamma 2 subtypes of cloned gamma-aminobutyric 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-d ihy dro [1,5-a]quinoxaline), 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 alpha 6 beta 2 gamma 2 subtype more pronouncedly than to the alpha 1 beta 2 gamma 2 subtype. The diimidazoquinazoline containing a 2-methyl group which projected below the plane of the rigid ring showed a markedly lower affinity to the alpha 6 beta 2 gamma 2 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 alpha 6 beta 2 gamma 2 subtype than the 5-acetyl counterpart. In particular, the 5-benzoyl analogue containing a 6-fluoro group showed no interaction with the alpha 6 beta 2 gamma 2 subtype even at the concentration of 10 microM, 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 alpha 6 beta 2 gamma 2 subtype shares overlapping regions with that of the alpha 1 beta 2 gamma 2 subtype, but has a sterically restricted out-of-plane region, which may be also incompatible with the 5-phenyl group of classical benzodiazepines.

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-oxid iazol-3yl)- 2,3-dihydro, is a ligand of high affinity for the benzodiazepine site of the GABAA receptor composed of alpha 1 beta 2 gamma 2 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.

Effect of spironolactone on androgen-dependent proteins in the ventral prostate of the rat.

The effect of spironolactone on five androgen-dependent proteins in the ventral prostate of the rat was investigated by two-dimensional gel electrophoresis. Spironolactone was given to intact male, castrated and androgen-stimulated castrated rats. It has been shown that spironolactone had no influence on the synthesis or accumulation of the androgen-dependent proteins in intact animals. However, spironolactone suppressed the restoration of the major androgen-dependent protein of low molecular weight in castrated rats given testosterone. The mechanism by which spironolactone exerts its anti-androgenic activity was shown to be unrelated to its capacity to inhibit the synthesis or accumulation of the five androgen-dependent proteins studied in this investigation.

Developmental aspects of androgen-dependent mRNA from rat ventral prostate using cloned cDNA.

The androgen-dependence of two mRNAs from rat ventral prostate coding for a 20 000 and an 11 000 dalton translation product has been investigated using complementary DNA cloned in the bacterial plasmid PBR322. One of the cloned insert DNAs from a recombinant plasmid, C-27, arrests the in vitro translation of C2 (Peeters et al. (1980) J. Biol. Chem. 255, 7017-7023). The other cloned insert DNA arrests the translation of a glycoprotein 20 000 daltons in size, with unknown function. The quantities of mRNA coding for the 20 000 and 11 000 dalton translation product were determined by hybridization of 32P-labeled inserts to filter-bound total RNA or poly(A+)-mRNA. Castration caused a decline in both mRNAs of 250-fold over 8 days. Stimulation with androgen of 5-week castrates restored the mRNA levels to 17% of intact for the 20 000 dalton translation product and 31% of intact for the 11 000 dalton translation product. The quantity of the two mRNAs found in the lateral poly(A+)-mRNA was about 1/10 that of the ventral level and the mRNAs were not detectable in the dorsal prostate, seminal vesicle or human prostate poly(A+)-mRNA populations. RNA from the ventral prostates of animals 10-21 days old contained mature levels of complementary sequences, suggesting a form of developmental posttranscriptional regulation for synthesis of the polypeptides which are not synthesized in mature quantities at this stage of development (Heyns et al. (1978) Endocrinology 103, 1090-1095; Kistler et al. (1981) Proc. Natl. Acad. Sci. (U.S.A.) 78, 737-741).

Uterine proteins influenced by estrogen exposure. Analysis by two-dimensional gel electrophoresis.

Two-dimensional gel electrophoresis has been utilized as a technique to investigate uterine protein patterns and protein distribution after estrogen administration. A combined in vivo stimulation, in vitro labeling technique was devised to investigate these questions. Animals were first primed in vivo with estrogen for 2 h to activate the normal early response to hormone and to stimulate protein synthesis. Tissue was removed and protein labeling occurred for 4 h with [35S]methionine. Tissue was then re-exposed in vitro to either estrogen or saline for an additional 1 h. Subfractionation of the uterine tissue and analysis of labeled proteins by two-dimensional gel electrophoresis has indicated the disappearance of some proteins from the cytoplasmic compartment and the appearance of other proteins in the nuclear fraction of estrogen-exposed samples. These findings suggest that in the mouse uterus, estrogen influences protein synthesis and also a redistribution of these proteins into various cellular fractions where they may act to elicit estrogen action.