Proximity-accelerated chemical coupling reaction in the benzodiazepine-binding site of gamma-aminobutyric acid type A receptors: superposition of different allosteric modulators

Benzodiazepines are widely used drugs. They exert sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects and act through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the gamma-aminobutyric acid type A (GABA(A)) receptor. Ligands of the benzodiazepine-binding site are classified into three groups depending on their mode of action: positive and negative allosteric modulators and antagonists. To rationally design ligands of the benzodiazepine site in different isoforms of the GABA(A) receptor, we need to understand the relative positioning and overlap of modulators of different allosteric properties. To solve these questions, we used a proximity-accelerated irreversible chemical coupling reaction. GABA(A) receptor residues thought to reside in the benzodiazepine-binding site were individually mutated to cysteine and combined with a cysteine-reactive benzodiazepine site ligand. Direct apposition of reaction partners is expected to lead to a covalent reaction. We describe here such a reaction of predominantly alpha(1)H101C and also three other mutants (alpha(1)G157C, alpha(1)V202C, and alpha(1)V211C) with an Imid-NCS derivative in which a reactive isothiocyanate group (-NCS) replaces the azide group (-N(3)) in the partial negative allosteric modulator Ro15-4513. Our results show four contact points of imidazobenzodiazepines with the receptor, alpha(1)H101C being shared by classical benzodiazepines. Taken together with previous data, a similar orientation of these ligands within the benzodiazepine-binding pocket may be proposed.

11beta-Hydroxysteroid dehydrogenase type 2 in pregnancy and preeclampsia

Cortisol availability is controlled by 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which inactivates cortisol in cortisone, unable to bind to the glucocorticoid receptor. The 11beta-HSD2 enzyme activity limits either intracellular cortisol concentrations or within the uteroplacental compartment the transfer of cortisol into the fetal circulation. Mechanisms, by which 11beta-HSD2 activity is controlled, include transcriptional control, posttranscriptional modifications of 11beta-HSD2 transcript half-life, epigenetic regulation via methylation of genomic DNA and direct inhibition of enzymatic activity. The 11beta-HSD2 expression and activity is reduced in preeclampsia and the enzyme activity correlates with factors associated with increased vasoconstriction, such as an increased angiotensin II receptor subtype 1 expression, and notably fetal growth. Numerous signals such as proinflammatory cytokines known to be present and/or elevated in preeclampsia regulate 11beta-HSD2 activity. Shallow trophoblast invasion with the resulting hypoxemia seems to critically reduce available 11beta-HSD2 activity. A positive feedback exists as activated glucocorticoid receptors do enhance 11beta-HSD2 mRNA transcription and mRNA stability. No data are currently available on pregnancy and either epigenetic or direct effects on the activity of the translated enzyme.

Human adrenal corticocarcinoma NCI-H295R cells produce more androgens than NCI-H295A cells and differ in 3beta-hydroxysteroid dehydrogenase type 2 and 17,20 lyase activities

The human adrenal cortex produces mineralocorticoids, glucocorticoids, and androgens in a species-specific, hormonally regulated, zone-specific, and developmentally characteristic fashion. Most molecular studies of adrenal steroidogenesis use human adrenocortical NCI-H295A and NCI-H295R cells as a model because appropriate animal models do not exist. NCI-H295A and NCI-H295R cells originate from the same adrenocortical carcinoma which produced predominantly androgens but also smaller amounts of mineralocorticoids and glucocorticoids. Research data obtained from either NCI-H295A or NCI-H295R cells are generally compared, although for the same experiments no direct comparison between the two cell lines has been performed. Therefore, we compared the steroid profile and the expression pattern of important genes involved in steroidogenesis in both cell lines. We found that steroidogenesis differs profoundly. NCI-H295A cells produce more mineralocorticoids, whereas NCI-H295R cells produce more androgens. Expression of the 3beta-hydroxysteroid dehydrogenase (HSD3B2), cytochrome b5, and sulfonyltransferase genes is higher in NCI-H295A cells, whereas expression of the cytochrome P450c17 (CYP17), 21-hydroxylase (CYP21), and P450 oxidoreductase genes does not differ between the cell lines. We found lower 3beta-hydroxysteroid dehydrogenase type 2 but higher 17,20-lyase activity in NCI-H295R cells explaining the 'androgenic' steroid profile for these cells and resembling the zona reticularis of the human adrenal cortex. Both cell lines were found to express the ACTH receptor at low levels consistent with low stimulation by ACTH. By contrast, both cell lines were readily stimulated by 8Br-cAMP. The angiotensin type 1 receptor was highly expressed in NCI-H295R than NCI-H295A cells and angiotensin II stimulated steroidogenesis in NCI-H295R but not NCI-H295A cells. Our data suggest that comparative studies between NCI-H295A and NCI-H295R cells may help find important regulators of mineralocorticoid or androgen biosynthesis.

Novel, potent, and radio-iodinatable somatostatin receptor 1 (sst1) selective analogues

The proposed sst(1) pharmacophore (J. Med. Chem. 2005, 48, 523-533) derived from the NMR structures of a family of mono- and dicyclic undecamers was used to design octa-, hepta-, and hexamers with high affinity and selectivity for the somatostatin sst(1) receptor. These compounds were tested for their in vitro binding properties to all five somatostatin (SRIF) receptors using receptor autoradiography; those with high SRIF receptor subtype 1 (sst(1)) affinity and selectivity were shown to be agonists when tested functionally in a luciferase reporter gene assay. Des-AA(1,4-6,10,12,13)-[DTyr(2),DAgl(NMe,2naphthoyl)(8),IAmp(9)]-SRIF-Thr-NH(2) (25) was radio-iodinated ((125)I-25) and specifically labeled sst(1)-expressing cells and tissues. 3D NMR structures were calculated for des-AA(1,4-6,10,12,13)-[DPhe(2),DTrp(8),IAmp(9)]-SRIF-Thr-NH(2) (16), des-AA(1,2,4-6,10,12,13)-[DAgl(NMe,2naphthoyl)(8),IAmp(9)]-SRIF-Thr-NH(2) (23), and des-AA(1,2,4-6,10,12,13)-[DAgl(NMe,2naphthoyl)(8),IAmp(9),Tyr(11)]-SRIF-NH(2) (27) in DMSO. Though the analogues have the sst(1) pharmacophore residues at the previously determined distances from each other, the positioning of the aromatic residues in 16, 23, and 27 is different from that described earlier, suggesting an induced fit mechanism for sst(1) binding of these novel, less constrained sst(1)-selective family members.

Discovery of novel CB2 receptor ligands by a pharmacophore-based virtual screening workflow

Cannabinoid receptor 2 (CB(2) receptor) ligands are potential candidates for the therapy of chronic pain, inflammatory disorders, atherosclerosis, and osteoporosis. We describe the development of pharmacophore models for CB(2) receptor ligands, as well as a pharmacophore-based virtual screening workflow, which resulted in 14 hits for experimental follow-up. Seven compounds were identified with K(i) values below 25 microM. The CB(2) receptor-selective pyridine tetrahydrocannabinol analogue 8 (K(i) = 1.78 microM) was identified as a CB(2) partial agonist. Acetamides 12 (K(i) = 1.35 microM) and 18 (K(i) = 2.1 microM) represent new scaffolds for CB(2) receptor-selective antagonists and inverse agonists, respectively. Overall, our pharmacophore-based workflow yielded three novel scaffolds for the chemical development of CB(2) receptor ligands.

The RYR2-encoded ryanodine receptor/calcium release channel in patients diagnosed previously with either catecholaminergic polymorphic ventricular tachycardia or genotype negative, exercise-induced long QT syndrome: a comprehensive open reading frame mutational analysis.

OBJECTIVES This study was undertaken to determine the spectrum and prevalence of mutations in the RYR2-encoded cardiac ryanodine receptor in cases with exertional syncope and normal corrected QT interval (QTc). BACKGROUND Mutations in RYR2 cause type 1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a cardiac channelopathy with increased propensity for lethal ventricular dysrhythmias. Most RYR2 mutational analyses target 3 canonical domains encoded by <40% of the translated exons. The extent of CPVT1-associated mutations localizing outside of these domains remains unknown as RYR2 has not been examined comprehensively in most patient cohorts. METHODS Mutational analysis of all RYR2 exons was performed using polymerase chain reaction, high-performance liquid chromatography, and deoxyribonucleic acid sequencing on 155 unrelated patients (49% females, 96% Caucasian, age at diagnosis 20 +/- 15 years, mean QTc 428 +/- 29 ms), with either clinical diagnosis of CPVT (n = 110) or an initial diagnosis of exercise-induced long QT syndrome but with QTc <480 ms and a subsequent negative long QT syndrome genetic test (n = 45). RESULTS Sixty-three (34 novel) possible CPVT1-associated mutations, absent in 400 reference alleles, were detected in 73 unrelated patients (47%). Thirteen new mutation-containing exons were identified. Two-thirds of the CPVT1-positive patients had mutations that localized to 1 of 16 exons. CONCLUSIONS Possible CPVT1 mutations in RYR2 were identified in nearly one-half of this cohort; 45 of the 105 translated exons are now known to host possible mutations. Considering that approximately 65% of CPVT1-positive cases would be discovered by selective analysis of 16 exons, a tiered targeting strategy for CPVT genetic testing should be considered.

Beta 1-integrins are required for hippocampal AMPA receptor-dependent synaptic transmission, synaptic plasticity, and working memory.

Integrins comprise a large family of cell adhesion receptors that mediate diverse biological events through cell-cell and cell-extracellular matrix interactions. Recent studies have shown that several integrins are localized to synapses with suggested roles in synaptic plasticity and memory formation. We generated a postnatal forebrain and excitatory neuron-specific knock-out of beta1-integrin in the mouse. Electrophysiological studies demonstrated that these mutants have impaired synaptic transmission through AMPA receptors and diminished NMDA receptor-dependent long-term potentiation. Despite the impairment in hippocampal synaptic transmission, the mutants displayed normal hippocampal-dependent spatial and contextual memory but were impaired in a hippocampal-dependent, nonmatching-to-place working memory task. These phenotypes parallel those observed in animals carrying knock-outs of the GluR1 (glutamate receptor subunit 1) subunit of the AMPA receptor. These observations suggest a new function of beta1-integrins as regulators of synaptic glutamate receptor function and working memory.

Angiotensin receptor agonistic autoantibody is highly prevalent in preeclampsia: correlation with disease severity.

Preeclampsia (PE), a syndrome affecting 5% of pregnancies, characterized by hypertension and proteinuria, is a leading cause of maternal and fetal morbidity and mortality. The condition is often accompanied by the presence of a circulating maternal autoantibody, the angiotensin II type I receptor agonistic autoantibody (AT(1)-AA). However, the prevalence of AT(1)-AA in PE remains unknown, and the correlation of AT(1)-AA titers with the severity of the disease remains undetermined. We used a sensitive and high-throughput luciferase bioassay to detect AT(1)-AA levels in the serum of 30 normal, 37 preeclamptic (10 mild and 27 severe), and 23 gestational hypertensive individuals. Here we report that AT(1)-AA is highly prevalent in PE ( approximately 95%). Next, by comparing the levels of AT(1)-AA among women with mild and severe PE, we found that the titer of AT(1)-AA is proportional to the severity of the disease. Intriguingly, among severe preeclamptic patients, we discovered that the titer of AT(1)-AA is significantly correlated with the clinical features of PE: systolic blood pressure (r=0.56), proteinuria (r=0.70), and soluble fms-like tyrosine kinase-1 level (r=0.71), respectively. Notably, only AT(1)-AA, and not soluble fms-like tyrosine kinase-1, levels are elevated in gestational hypertensive patients. These data serve as compelling clinical evidence that AT(1)-AA is highly prevalent in PE, and its titer is strongly correlated to the severity of the disease.

The detrimental role of angiotensin receptor agonistic autoantibodies in intrauterine growth restriction seen in preeclampsia.

Growth-restricted fetuses are at risk for a variety of lifelong medical conditions. Preeclampsia, a life-threatening hypertensive disorder of pregnancy, is associated with fetuses who suffer from intrauterine growth restriction (IUGR). Recently, emerging evidence indicates that preeclamptic women harbor AT(1) receptor agonistic autoantibodies (AT(1)-AAs) that contribute to the disease features. However, the exact role of AT(1)-AAs in IUGR and the underlying mechanisms have not been identified. We report that these autoantibodies are present in the cord blood of women with preeclampsia and retain the ability to activate AT(1) receptors. Using an autoantibody-induced animal model of preeclampsia, we show that AT(1)-AAs cross the mouse placenta, enter fetal circulation, and lead to small fetuses with organ growth retardation. AT(1)-AAs also induce apoptosis in the placentas of pregnant mice, human villous explants, and human trophoblast cells. Finally, autoantibody-induced IUGR and placental apoptosis are diminished by either losartan or an autoantibody-neutralizing peptide. Thus, these studies identify AT(1)-AA as a novel causative factor of preeclampsia-associated IUGR and offer two possible underlying mechanisms: a direct detrimental effect on fetal development by crossing the placenta and entering fetal circulation, and indirectly through AT(1)-AA-induced placental damage. Our findings highlight AT(1)-AAs as important therapeutic targets.

Human TNF receptor p75/80 gene structure and promoter characterization

Tumor necrosis factor receptor p75/80 ((TNF-R p75/80) is a 75 kDa type 1 transmembrane protein expressed predominately on cells of hematopoietic lineage. TNF-R p75/80 belongs to the TNF receptor superfamily characterized by cysteine-rich extracellular regions composed of three to six disulfide-linked domains. In the present report, we have characterized, for the first time, the complete gene structure for human TNF-R p75/80 which spans approximately 43 kbp. The gene consists of 10 exons (ranging from 34 bp to 2.5 kbp) and 9 introns (343 bp to 19 kbp). Consensus elements for transcription factors involved in T cell development and activation were noted in the 5$\sp\prime$ flanking region including TCF-1, Ikaros, AP-1, CK-2, IL-6RE, ISRE, GAS, NF-$\kappa$B and SP1, as well as an unusually high GC content and CpG frequency that appears characteristic of some TNF-R family members. The unusual (GATA)$\sb{\rm n}$ and (GAA)(GGA) repeats found within intron 1 may prove useful for further genome analysis within the 1p36 chromosomal locus. The human TNF-R p75/80 gene structure will permit further assessment of its involvement in normal hematopoietic cell development and function, autoimmune disease, and non-random translocations in hematopoietic malignancies. The region 1.8 kb 5$\sp\prime$ of the ATG was able to drive luciferase expression when transfected into cell lines expressing TNF-R p75/80. Further characterization of the 5$\sp\prime$-regulatory region will aid in determining factors and signal transduction pathways involved in regulating TNF-R p75/80 expression. ^

Effects of colostrum versus formula feeding on hepatic glucocorticoid and α1- and β2-adrenergic receptors in neonatal calves and their effect on glucose and lipid metabolism

Neonatal energy metabolism in calves has to adapt to extrauterine life and depends on colostrum feeding. The adrenergic and glucocorticoid systems are involved in postnatal maturation of pathways related to energy metabolism and calves show elevated plasma concentrations of cortisol and catecholamines during perinatal life. We tested the hypothesis that hepatic glucocorticoid receptors (GR) and α₁- and β₂-adrenergic receptors (AR) in neonatal calves are involved in adaptation of postnatal energy metabolism and that respective binding capacities depend on colostrum feeding. Calves were fed colostrum (CF; n=7) or a milk-based formula (FF; n=7) with similar nutrient content up to d 4 of life. Blood samples were taken daily before feeding and 2h after feeding on d 4 of life to measure metabolites and hormones related to energy metabolism in blood plasma. Liver tissue was obtained 2 h after feeding on d 4 to measure hepatic fat content and binding capacity of AR and GR. Maximal binding capacity and binding affinity were calculated by saturation binding assays using [(3)H]-prazosin and [(3)H]-CGP-12177 for determination of α₁- and β₂-AR and [(3)H]-dexamethasone for determination of GR in liver. Additional liver samples were taken to measure mRNA abundance of AR and GR, and of key enzymes related to hepatic glucose and lipid metabolism. Plasma concentrations of albumin, triacylglycerides, insulin-like growth factor I, leptin, and thyroid hormones changed until d 4 and all these variables except leptin and thyroid hormones responded to feed intake on d 4. Diet effects were determined for albumin, insulin-like growth factor I, leptin, and thyroid hormones. Binding capacity for GR was greater and for α₁-AR tended to be greater in CF than in FF calves. Binding affinities were in the same range for each receptor type. Gene expression of α₁-AR (ADRA1) tended to be lower in CF than FF calves. Binding capacity of GR was related to parameters of glucose and lipid metabolism, whereas β₂-AR binding capacity was negatively associated with glucose metabolism. In conclusion, our results indicate a dependence of GR and α₁-AR on milk feeding immediately after birth and point to an involvement of hepatic GR and AR in postnatal adaptation of glucose and lipid metabolism in calves.

Association of long-term nicotine abstinence with normal metabotropic glutamate receptor-5 binding

BACKGROUND Nicotine addiction is a major public health problem and is associated with primary glutamatergic dysfunction. We recently showed marked global reductions in metabotropic glutamate receptor type 5 (mGluR5) binding in smokers and recent ex-smokers (average abstinence duration of 25 weeks). The goal of this study was to examine the role of mGluR5 downregulation in nicotine addiction by investigating a group of long-term ex-smokers (abstinence >1.5 years), and to explore associations between mGluR5 binding and relapse in recent ex-smokers. METHODS Images of mGluR5 receptor binding were acquired in 14 long-term ex-smokers, using positron emission tomography with radiolabeled [11C]ABP688, which binds to an allosteric site with high specificity. RESULTS Long-term ex-smokers and individuals who had never smoked showed no differences in mGluR5 binding in any of the brain regions examined. Long-term ex-smokers showed significantly higher mGluR5 binding than recent ex-smokers, most prominently in the frontal cortex (42%) and thalamus (57%). CONCLUSIONS Our findings suggest that downregulation of mGluR5 is a pathogenetic mechanism underlying nicotine dependence and the high relapse rate in individuals previously exposed to nicotine. Therefore, mGluR5 receptor binding appears to be an effective biomarker in smoking and a promising target for the discovery of novel medication for nicotine dependence and other substance-related disorders.

Behavioral Profile of the Novel Cannabinoid Agonist AM4054

Although cannabinoid drugs have been used for thousands of years both recreationally and therapeutically, little has been known about their mechanisms of action until recently. Since the discovery of the endogenous cannabinoid CB1 receptor in 1988, the behavioral profile of cannabinoid receptor ligands has been much more thoroughly defined. Cannabinoid CB1 agonists have been shown to produce a variety of behavioral effects including suppression of locomotion, catalepsy, hypothermia, and analgesia. Research has also demonstrated that these behavioral effects can be inhibited by CB1 receptor antagonists including SR 141716 and AM 251. Although behavioral indicators of anxiety including thigmotaxis have been observed in several different paradigms, there is inconclusive and often times contradictory evidence to define the role of anxiety in CB1 receptor activation. The present study addressed the behavioral profile of AM 4054, a novel full agonist at the CB1 receptor, as well as the ability of the CB1 antagonist AM 251 to reverse these effects. To further identify and expand research on the suppression of locomotion and induction of thigmotaxis with the administration of a CB1 agonist, experiment 1 was conducted in the open field. In this experiment, each rat (n=40) was randomly assigned one of the five treatments: vehicle, 0.16, 0.32, 0.64, or 1.25 mg/kg AM 4054. After a 30 minute pre-treatment, each subject was tested in the open field for 18 minutes. Results indicated that AM 4054 produced a dose-related suppression of locomotion as well as the subtle presence of thigmotaxis in two out of four doses. In experiment 2, subjects (n=40) received either vehicle or 2.0 or 4.0 mg/kg AM 251 60 minutes prior to testing. After 30 minutes, the subjects were given either a 0.3 mg/kg dose of AM 4054 or vehicle. After a total pretreatment duration of 60 minutes, the animals were tested on a battery of tasks including an 18 minute session in locomotor boxes. Experiment 2 was a continuation of a previous study conducted in the same lab, which confirmed the effects of AM 4054 on this tetrad of tasks as being consistent with other cannabinoid agonists. In this experiment the effects of AM 4054 were reversed by the administration of the CB1 antagonist AM 251. Past studies have shown that AM 4054 is a highly potent drug with behavioral actions similar to other cannabinoid CB1 agonists. Furthermore, AM 4054 can be a useful drug in future studies, and has potential therapeutic value for the treatment of various conditions.

Binding of the HIV-1 gp120 -V3 to cellular glycosphingolipids is necessary for CXCR4 recruitment and infection

Infection by human immunodeficiency virus type 1 (HIV-1) is a multi-step process, and detailed analyses of the various events critical for productive infection are necessary to clearly understanding the infection process and identifying novel targets for therapeutic interventions. Evidence from this study reveals binding of the viral envelope protein to host cell glycosphingolipids (GSLs) as a novel event necessary for the orderly progression of the host cell-entry and productive infection by HIV-1. Data obtained from co-immunoprecipitation analyses and confocal microscopy showed that the ability of viral envelope to interact with the co-receptor CXCR4 and productive infection of HIV-1 were inhibited in cells rendered GSL-deficient, while both these activities were restored after reconstitution of the cells with specific GSLs like GM3. Furthermore, evidence was obtained using peptide-inhibitors of HIV-1 infection to show that binding of a specific region within the V3-loop of the envelope protein gp120 to the host cell GSLs is the trigger necessary for the CD4-bound gp120 to recruit the CXCR4 co-receptor. Infection-inhibitory activity of the V3 peptides was compromised in GSL-deficient cells, but could be restored by reconstitution of GSLs. Based on these findings, a revised model for HIV-1 infection is proposed that accounts for the established interactions between the viral envelope and host cell receptors while enumerating the importance of the new findings that fill the gap in the current knowledge of the sequential events for the HIV-1 entry. According to this model, post-CD4 binding of the HIV-1 envelope surface protein gp120 to host cell GSLs, mediated by the gp120-V3 region, enables formation of the gp120-CD4-GSL-CXCR4 immune-complex and productive infection. The identification of cellular GSLs as an additional class of co-factors necessary for HIV-1 infection is important for enhancing the basic knowledge of the HIV-1 entry that can be exploited for developing novel antiviral therapeutic strategies. ^

RAC3, a steroid/nuclear receptor-associated coactivator that is related to SRC-1 and TIF2

Steroids, thyroid hormones, vitamin D3, and retinoids are lipophilic small molecules that regulate diverse biological effects such as cell differentiation, development, and homeostasis. The actions of these hormones are mediated by steroid/nuclear receptors which function as ligand-dependent transcriptional regulators. Transcriptional activation by ligand-bound receptors is a complex process requiring dissociation and recruitment of several additional cofactors. We report here the cloning and characterization of receptor-associated coactivator 3 (RAC3), a human transcriptional coactivator for steroid/nuclear receptors. RAC3 interacts with several liganded receptors through a mechanism which requires their respective ligand-dependent activation domains. RAC3 can activate transcription when tethered to a heterologous DNA-binding domain. Overexpression of RAC3 enhances the ligand-dependent transcriptional activation by the receptors in mammalian cells. Sequence analysis reveals that RAC3 is related to steroid receptor coactivator 1 (SRC-1) and transcriptional intermediate factor 2 (TIF2), two of the most potent coactivators for steroid/nuclear receptors. Thus, RAC3 is a member of a growing coactivator network that should be useful as a tool for understanding hormone action and as a target for developing new therapeutic agents that can block hormone-dependent neoplasia.