Establishment of CYP2D6 reference samples by multiple validated genotyping platforms.

Cytochrome P450 2D6 (cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6)), a highly polymorphic drug-metabolizing enzyme, is involved in the metabolism of one-quarter of the most commonly prescribed medications. Here we have applied multiple genotyping methods and Sanger sequencing to assign precise and reproducible CYP2D6 genotypes, including copy numbers, for 48 HapMap samples. Furthermore, by analyzing a set of 50 human liver microsomes using endoxifen formation from N-desmethyl-tamoxifen as the phenotype of interest, we observed a significant positive correlation between CYP2D6 genotype-assigned activity score and endoxifen formation rate (rs = 0.68 by rank correlation test, P = 5.3 × 10(-8)), which corroborated the genotype-phenotype prediction derived from our genotyping methodologies. In the future, these 48 publicly available HapMap samples characterized by multiple substantiated CYP2D6 genotyping platforms could serve as a reference resource for assay development, validation, quality control and proficiency testing for other CYP2D6 genotyping projects and for programs pursuing clinical pharmacogenomic testing implementation.

Genome-wide discovery of genetic variants affecting tamoxifen sensitivity and their clinical and functional validation.

BACKGROUND: Beyond estrogen receptor (ER), there are no validated predictors for tamoxifen (TAM) efficacy and toxicity. We utilized a genome-wide cell-based model to comprehensively evaluate genetic variants for their contribution to cellular sensitivity to TAM. DESIGN: Our discovery model incorporates multidimensional datasets, including genome-wide genotype, gene expression, and endoxifen-induced cellular growth inhibition in the International HapMap lymphoblastoid cell lines (LCLs). Genome-wide findings were further evaluated in NCI60 cancer cell lines. Gene knock-down experiments were performed in four breast cancer cell lines. Genetic variants identified in the cell-based model were examined in 245 Caucasian breast cancer patients who underwent TAM treatment. RESULTS: We identified seven novel single-nucleotide polymorphisms (SNPs) associated with endoxifen sensitivity through the expression of 10 genes using the genome-wide integrative analysis. All 10 genes identified in LCLs were associated with TAM sensitivity in NCI60 cancer cell lines, including USP7. USP7 knock-down resulted in increasing resistance to TAM in four breast cancer cell lines tested, which is consistent with the finding in LCLs and in the NCI60 cells. Furthermore, we identified SNPs that were associated with TAM-induced toxicities in breast cancer patients, after adjusting for other clinical factors. CONCLUSION: Our work demonstrates the utility of a cell-based model in genome-wide identification of pharmacogenomic markers.

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.

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.

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.

Thyroid hormonal modulation of the binding and activity of the GABAA receptor complex of brain.

Thyroid hormones, which are known to act by genomic mechanisms in peripheral tissues, were found to influence the binding and function of the GABAA receptor complex in brain membranes. Submicromolar concentrations of triiodothyronine and thyroxine stereospecifically stimulated the binding of [35S]t-butylbicyclophosphorothionate (a convulsant ligand for the GABAA receptor complex) to highly washed rat brain membranes, while higher concentrations of the hormones inhibited radioligand binding. GABA-stimulated 36Cl-flux in isolated brain membrane sacs was inhibited by L-triiodothyronine with a half-maximally inhibitory concentration (IC50) of 10(-7) M. Patch-clamp analysis of recombinant GABAA receptor subunits expressed in human embryonic kidney-293 cells showed an inhibition of chloride currents by thyroid hormones. This effect required only the alpha 1 beta 2 subunits, and was not blocked by the benzodiazepine antagonist flumazenil. Since thyroid hormones are known to be concentrated in nerve terminal preparations and subsequently released, the hormones may have non-genomic mechanisms of action as putative neurotransmitters or neuromodulators in brain and act through GABAA receptors.

Alterations of the benzodiazepine site of rat alpha 6 beta 2 gamma 2-GABAA receptor by replacement of several divergent amino-terminal regions with the alpha 1 counterparts.

1. The benzodiazepine site of the alpha 6 beta 2 gamma 2 subtype of gamma-aminobutyric acidA (GABAA) receptors is distinguishable from that of the alpha 1 beta 2 gamma 2 subtype by its inability to interact with classical benzodiazepines (i.e., diazepam) and its agonistic response to Ro 15-1788, which behaves as an antagonist in the alpha 1 beta 2 gamma 2 subtype. 2. The point mutation of Arg 100 of the alpha 6 subunit to histidine (the corresponding residue in alpha 1) has been shown to enable the alpha 6 beta 2 gamma 2 subtype to interact with diazepam but failed in this study to abolish the ability of Ro 15-1788 to enhance GABA-induced Cl- currents. 3. Here we identified the segment of P161 to L187 of alpha 6 to contain the functional region responsible for the agonistic action of Ro 15-1788. Its replacement with the corresponding alpha 1 sequence abolished the ability of Ro 15-1788 to enhance GABA currents without appreciable effects on its binding affinity to the benzodiazepine site or on the functionality of the other benzodiazepine site ligands such as diazepam, U-92330 and 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate. These data support the evidence that the functionality of a given ligand could arise from a single region of the benzodiazepine site, not shared by others. 4. In addition we have learned that several residues in the N-terminal region of alpha 6, such as R100, V142 and N143, have the ability to influence GABA-dependent Cl- current induction probably by allosterically modulating low affinity GABA sites.

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.

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.

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.

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.

Two imidazoquinoxaline ligands for the benzodiazepine site sharing a second low affinity site on rat GABA(A) 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 alphaxbeta2gamma2 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.

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.

Characterization of U-101017 as a GABA(A) receptor ligand of dual functionality.

Drugs acting on the benzodiazepine site of GABA(A) receptors are much safer than barbiturates, but are still liable to abuse. Recently, we have reported that a benzodiazepine site agonist, U-97775 (a dihydroimidazoquinoxaline analog), may have minimal abuse liability because of its interaction with a second, low-affinity site on GABA(A) receptors, the occupancy of which, at high drug concentrations, leads to a reversal of its agonistic activity on the benzodiazepine site and inhibition of GABA-induced Cl- currents [Br. J. Pharmacol. 115 (1995)19-24]. Here we report that U-101017 (7-chloro-5[(cis-3,5-dimethylpiperazine)carbonyl]imidazo[1,5a]quinoline- 3-carboxylate) is another similar benzodiazepine site agonist possessing the ability to reverse its agonistic activity at higher concentrations, but its ability to inhibit GABA currents is considerably milder than that of U-97775. In the alpha 6 beta 2 gamma 2 subtype where these drugs have no agonistic activity, for instance, U-101017 at concentrations up to 80 mu M, showed no appreciable effect on GABA currents, whereas U-97775 inhibited the currents with an IC(50) value of 10 mu M as measured with the whole cell patch clamp techniques in human embryonic kidney cells expressing recombinant receptors. Similar, milder inhibition of GABA currents by U-101017 was observed in the alpha 1 beta 2 gamma 2 and alpha 3 beta 2 gamma 2 subtypes. Furthermore, U-101017 was of higher efficacy in the alpha 1 beta 2 gamma 2 than alpha 3 beta 2 gamma 2 subtypes as compared to diazepam, although its binding affinity was not appreciably different in the two subtypes. We conclude that U-101017 is a partial benzodiazepine agonist, somewhat selective to the alpha 1 beta 2 gamma 2 subtype, and with the ability to limit its own agonistic activity over a wide range of doses through its interaction with the low affinity site, but without potential convulsant activity, inherent to agents which block GABA currents.

Stable expression of cloned rat GABAA receptor subunits in a human kidney cell line.

A predominant form of the GABAA/benzodiazepine receptor-Cl- channel complex is believed to consist of three different 48-55 kDa subunits (alpha, beta, gamma) with unknown stoichiometry. Plasmids containing the rat GABAA receptor cDNAs coding for alpha 1, beta 2, and gamma 2 were co-transfected, along with a plasmid encoding G418 resistance, into human embryonic kidney cells previously transformed with Adenovirus 5 (HEK-293) [J. Gen. Virol., 36 (1977) 59-72]. Four percent of the G418 resistant colonies were found to express mRNA for all three of the GABAA subunits constitutively. A single cell clone derived from one of the alpha 1 beta 2 gamma 2 expressors has demonstrated stable electrophysiological characteristics over 25 passages. The GABA-activated Cl- current in this cell line is blocked by picrotoxin and bicuculline, and is modulated by a variety of agonist and inverse agonist ligands including diazepam, Ro 154513, zolpidem, and beta-CCE. The cell line has been used successfully over a 12-month period as a screen for novel drugs modulating GABA-mediated polarization of neuronal cells.

Acceleration of GABA-dependent desensitization by mutating threonine 266 to alanine of the alpha 6 subunit of rat GABAA receptors.

Various GABAA receptor subunits share four highly homologous putative transmembrane domains (M1 to M4) and have been proposed to form an ion channel of pentameric structure with M2 lining the pore. The carboxyl terminal side of M2 contains three amino acid residues containing a hydroxyl group, which are Thr 265, 266 and serine 268 in the alpha 6 subunit. In order to study their functional role, we generated mutants of the alpha 6 subunit carrying a single point mutation of threonine 265 or 266 to alanine, or serine 268 to glycine. Co-expression of the mutants with beta 2 and gamma 2 subunits in human embryonic kidney cells produced functional receptors which are similar to the wild type in their sensitivity to a benzodiazepine agonist (U-92330), insensitivity to Zn, anion permeability, and GABA dose-response profiles as monitored with the whole cell patch clamp technique. Only in the alpha 6T266A beta 2 gamma 2 subtype, however, GABA-induced Cl- currents decayed much more rapidly than the wild type (about 10 times faster). Analysis of the GABA dependency of desensitization indicates that the T266A mutation enhanced the desensitization rate with little effect on the recovery rate from desensitization or on the half-maximal GABA concentration. We conclude that threonine 266 in the alpha 6 subunit plays a pivotal role in desensitization processes of GABAA receptors.

Functional expression of GABAA chloride channels and benzodiazepine binding sites in baculovirus infected insect cells.

We have employed the baculovirus expression system for the production of insect cell membranes having GABA/benzodiazepine binding sites. Three recombinant baculoviruses each harboring a different GABAA receptor cDNA were introduced into insect cells by simultaneous infection. Infected cells expressed GABA responsive Cl- channels and benzodiazepine binding sites with the same pharmacological specificity as animal cells expressing these receptor subunits.

Integrating cell-based and clinical genome-wide studies to identify genetic variants contributing to treatment failure in neuroblastoma patients.

High-risk neuroblastoma is an aggressive malignancy, with high rates of treatment failure. We evaluated genetic variants associated with in vitro sensitivity to two derivatives of cyclophosphamide for association with clinical response in a separate replication cohort of neuroblastoma patients (n = 2,709). To determine sensitivity, lymphoblastoid cell lines (LCLs) were exposed to increasing concentrations of 4-hydroperoxycyclophosphamide (4HC; n = 422) and phosphoramide mustard (PM; n = 428). Genome-wide association studies were performed to identify single-nucleotide polymorphisms (SNPs) associated with sensitivity to 4HC and PM. SNPs consistently associated with LCL sensitivity were analyzed for associations with event-free survival (EFS) in patients. Two linked SNPs, rs9908694 and rs1453560, were found to be associated with (i) sensitivity to PM in LCLs across populations and (ii) EFS in all patients (P = 0.01) and within the high-risk subset (P = 0.05). Our study highlights the value of cell-based models to identify candidate variants that may predict response to treatment in patients with cancer.

Block of voltage-gated potassium currents by anticonvulsant U-54494A in mouse neuroblastoma cells.

U-54494A [(+-)-cis-3,4-dichloro-N-methyl-N-[2-(1- pyrrolidinyl)-cyclohexyl]-benzamide], an anticonvulsant and Na channel blocker, was examined for its interaction with delayed rectifier K channels in mouse neuroblastoma cells (NIE-115) using whole-cell and inside-out configurations of the patch clamp techniques. U-54494A at anticonvulsant doses reversibly blocked the tetraethylammonium-sensitive outward K current in a time-dependent manner without affecting the current-voltage relationship. Analysis of the time-dependent block suggests interaction of U-54494A with the open state, but not with the resting state of the K channel. Furthermore, the drug showed no interaction with inactivated states of the K channel because it produced no noticeable effect on the steady-state inactivation curve or the recovery of the channel activity from inactivated states. The drug shifted the steady-state activation curve to a hyperpolarizing direction. This shift seems to result from underestimations of the maximal conductance of the K channel in the presence of U-54494A, because of its progressively increasing potency to block the K currents as the membrane potential became more depolarized. The potency of the drug was enhanced not only by depolarized membrane potentials, but also by raising the external pH. These results suggest that U-54494A interacts with a channel binding site which is located within the membrane electric field and approachable from the cytoplasmic side. The proposal was supported further by single channel recordings with inside-out patches in which U-54494A induced flickery blocks of single channel openings of the delayed rectifier.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective block of transient Ca channel current in mouse neuroblastoma cells by U-88779E.

Antioxidants and T-type Ca channel antagonists are neuroprotective during ischemia or other central nervous system traumas. U-88779E (1-[(4-chlorophenyl-phenyl)-methyl]-4-[(7-methoxy-5-isopropyl- 2,4,6-cycloheptatrien-1-one)-2-methyl]piperazine) has been shown to have both antioxidant activity and the ability to block Ca fluxes in cardiac microsomes. In this study, we examined the effect of U-88779E on Ca, Na and K channels in a neuronal cell line, N1E-115 cells. The drug blocked transient barium current (IBa) through low-threshold Ca channels (T-type) with little effect on other noninactivating IBa including the nifedipine-sensitive one. The drug at 20 microM reduced transient IBa at a constant rate, -7.2% of the control per min, and abolished the current within 15 min. This implies a continuous accumulation of the drug in cell membranes probably because of its high lipophilicity (log P = 7.003). U-88779E also blocked Na and K currents but at a rate about 8 times slower than that observed with transient IBa. Further studies on interactions of the drug with T-channels revealed that the drug had no effect on the shape of current-voltage curve, activation and inactivation kinetics, and steady-state activation curve. The drug, however, induced a hyperpolarizing shift in steady-state inactivation curve and became more potent under conditions where the channels in inactivated states prevail. These observations are consistent with the view that U-88779E has a higher affinity to T-type channels in inactivated states than in resting or open states.(ABSTRACT TRUNCATED AT 250 WORDS)