Molecular Determinants and Pharmacological Analysis for a Class of Competitive Non-transported Bicyclic Inhibitors of the Betaine/GABA Transporter BGT1.

The betaine/GABA transporter 1 (BGT1) is a member of the GABA transporter (GAT) family with still elusive function, largely due to a lack of potent and selective tool compounds. Based on modeling, we here present the design, synthesis and pharmacological evaluation of five novel conformationally restricted cyclic GABA analogs related to the previously reported highly potent and selective BGT1 inhibitor (1S,2S,5R)-5-aminobicyclo[3.1.0]hexane-2-carboxylic acid (bicyclo-GABA). Using [3H]GABA radioligand uptake assays at the four human GATs recombinantly expressed in mammalian cell lines, we identified bicyclo-GABA and its N-methylated analog (2) as the most potent and selective BGT1 inhibitors. Additional pharmacological characterization in a fluorescence-based membrane potential assay showed that bicyclo-GABA and 2 are competitive inhibitors, not substrates, at BGT1, which was validated by a Schild analysis for bicyclo-GABA (pK B value of 6.4). To further elaborate on the selectivity profile both compounds were tested at recombinant α1ß2γ2 GABAA receptors. Whereas bicyclo-GABA showed low micromolar agonistic activity, the N-methylated 2 was completely devoid of activity at GABAA receptors. To further reveal the binding mode of bicyclo-GABA and 2 binding hypotheses of the compounds were obtained from in silico-guided mutagenesis studies followed by pharmacological evaluation at selected BGT1 mutants. This identified the non-conserved BGT1 residues Q299 and E52 as the molecular determinants driving BGT1 activity and selectivity. The binding mode of bicyclo-GABA was further validated by the introduction of activity into the corresponding GAT3 mutant L314Q (38 times potency increase cf. wildtype). Altogether, our data reveal the molecular determinants for the activity of bicyclic GABA analogs, that despite their small size act as competitive inhibitors of BGT1. These compounds may serve as valuable tools to selectively and potently target BGT1 in order to decipher its elusive pharmacological role in the brain and periphery such as the liver and kidneys.

HOXA9-methylated DNA as a diagnostic biomarker of ovarian malignancy.

Aim: In ovarian cancer, methylated HOXA9 (meth-HOXA9) has been proposed as a relevant biomarker, however, its role in the carcinogenic development remains unknown. This study aimed at evaluating meth-HOXA9 as a diagnostic biomarker in ovarian cancer. Materials & methods: The meth-HOXA9 status was examined in 138 tissue specimens encompassing normal ovaries, benign- and borderline tumors, and ovarian cancer using droplet digital PCR. Results: Meth-HOXA9 was detected in 93% (82/88) and 88% (14/16) of ovarian cancer and borderline tumors, respectively. In patients with benign ovarian tumors meth-HOXA9 was detected in 17% (3/18). Using receiver operating characteristic (ROC) analysis meth-HOXA9 had a diagnostic accuracy of 98%. Conclusion: Meth-HOXA9 is highly cancer specific and could serve as a general diagnostic marker of ovarian malignancy.

Molecular Determinants Underlying Delta Selective Compound 2 Activity at δ-Containing GABAA Receptors.

Delta selective compound 2 (DS2; 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]benzamide) is one of the most widely used tools to study selective actions mediated by δ-subunit-containing GABAA receptors. DS2 was discovered over 10 years ago, but despite great efforts, the precise molecular site of action has remained elusive. Using a combination of computational modeling, site-directed mutagenesis, and cell-based pharmacological assays, we probed three potential binding sites for DS2 and analogs at α 4 ß 1 δ receptors: an α 4 (+) δ (-) interface site in the extracellular domain (ECD), equivalent to the diazepam binding site in αßγ 2 receptors, and two sites in the transmembrane domain (TMD) - one in the α 4 (+) ß 1 (-) and one in the α 4 (-) ß 1 (+) interface, with the α 4 (-) ß 1 (+) site corresponding to the binding site for etomidate and a recently disclosed low-affinity binding site for diazepam. We show that mutations in the ECD site did not abrogate DS2 modulation. However, mutations in the TMD α 4 (+) ß 1 (-) interface, either α 4(S303L) of the α 4 (+) side or ß 1(I289Q) of the ß 1 (-) side, convincingly disrupted the positive allosteric modulation by DS2. This was consistently demonstrated both in an assay measuring membrane potential changes and by whole-cell patch-clamp electrophysiology and rationalized by docking studies. Importantly, general sensitivity to modulators was not compromised in the mutated receptors. This study sheds important light on the long-sought molecular recognition site for DS2, refutes the misconception that the selectivity of DS2 for δ-containing receptors is caused by a direct interaction with the δ-subunit, and instead points toward a functional selectivity of DS2 and its analogs via a surprisingly well conserved binding pocket in the TMD. SIGNIFICANCE STATEMENT: δ-Containing GABAA receptors represent potential drug targets for the treatment of several neurological conditions with aberrant tonic inhibition, yet no drugs are currently in clinical use. With the identification of the molecular determinants responsible for positive modulation by the known compound delta selective compound 2, the ground is laid for design of ligands that selectively target δ-containing GABAA receptor subtypes, for better understanding of tonic inhibition, and ultimately, for rational development of novel drugs.

Structural Determinants for the Mode of Action of Imidazopyridine DS2 at δ-Containing γ-Aminobutyric Acid Type A Receptors.

Despite the therapeutic relevance of δ-containing γ-aminobutyric acid type A receptors (GABAARs) and the need for δ-selective compounds, the structural determinants for the mode and molecular site of action of δ-selective positive allosteric modulator imidazo[1,2-a]pyridine DS2 remain elusive. To guide the quest for insight, we synthesized a series of DS2 analogues guided by a structural receptor model. Using a fluorescence-based fluorometric imaging plate reader membrane potential assay, we found that the δ-selectivity and the pharmacological profile are severely affected by substituents in the 5-position of the imidazopyridine core scaffold. Interestingly, the 5-methyl, 5-bromo, and 5-chloro DS2 analogues, 30, 35, and 36, were shown to be superior to DS2 at α4ß1δ as mid-high nanomolar potency δ-selective allosteric modulators, displaying 6-16 times higher potency than DS2. Of these, 30 also displayed at least 60-fold selectivity for α4ß1δ over α4ß1γ2 receptor subtypes representing a potential tool for the selective characterization of δ-containing GABAARs in general.

Self-rated physical fitness and measured cardiorespiratory fitness, muscular strength, and body composition.

The purpose was to examine the correlation and association between a single-item question of self-rated physical fitness and objective measures of fitness and cardiometabolic risk factors in a large population-based study. Participants were 3441 men and women aged 18-85 years who filled in a questionnaire and participated in a clinical health examination in the Danish Health Examination Survey 2007-2008. Cardiorespiratory fitness was estimated by an indirect maximal exercise test. Muscle strength was measured by (a) sit-to-stand test, (b) handgrip strength, and (c) bent arm strength. Body mass index and fat percentage were used as measures for body composition. Associations were derived from regression analyses, correlations were calculated using Spearman's correlation test, and agreement was tested by kappa statistics. Within categories of self-rated physical fitness moving from lowest to highest, objectively measured cardiorespiratory fitness increased. Self-rated physical fitness was strongly correlated to cardiorespiratory fitness (rmen  = 0.69 and rwomen  = 0.65) and moderately correlated to the sit-to-stand test (rmen  = 0.49 and rwomen  = 0.48), bent arm strength (r = 0.45) and fat percentage (r = 0.46) among men, and handgrip strength among women (r =0.41). Mutually adjusted analysis showed a significant association between self-reported physical fitness and cardiorespiratory fitness for men and women and bent arm strength for women. The single-item question of physical fitness was correlated to cardiorespiratory fitness, muscle strength, and body composition. However, this study suggests that it mainly captures cardiorespiratory fitness. At the population level, the single-item question could be a useful tool to identify and monitor variation in fitness levels.

Should leisure-time sedentary behavior be replaced with sleep or physical activity for prevention of diabetes?

The aim was to examine the effects of replacing self-reported leisure-time sedentary behavior with sleep, light-to-moderate physical activity, or vigorous physical activity on incident diabetes among Danish adults using isotemporal substitution modeling. Participants ≥25 years from the Danish Capital Region Health Survey 2007 (N = 69 800, response rate 52.3%), 2010 (N = 95 150, response rate 52.3%), and 2013 (N = 95 150, response rate 43.5%) were included. Information on daily sleep duration, leisure-time sedentary behavior, and movement behaviors was collected by questionnaire. Information on incident diabetes was obtained from National registers. Analyses included Cox proportional hazards regression models and isotemporal substitution analyses, with time (in years) from baseline to incident diabetes or censoring December 31, 2017. Potential confounders, sex, age, BMI, ethnicity, education, smoking, inflammatory joint disease, perceived stress, physical and mental component scale and work status, were included. Out of N = 87 339 in the final study sample, n = 3007 had incident diabetes during a mean follow-up time of 7.4 years. Adults with incident diabetes included more men, higher mean age, and higher BMI, compared to respondents without incident diabetes. Theoretically substituting 30 minutes of leisure-time sedentary behavior with light-to-moderate PA (HR: 0.96; 95% CI: 0.94; 0.98) or with vigorous PA (HR: 0.82; 95% CI: 0.72; 0.94) decreased the risk of incident diabetes. We found no change in incident diabetes risk of substituting sedentary time with sleep (HR: 1.00; 95% CI: 0.97; 1.02). Substituting 30 minutes per day of leisure-time sedentary behavior with light-to-moderate or vigorous PA may significantly reduce the risk of incident diabetes among adults.

Discovery of 2-(Imidazo[1,2- b]pyridazin-2-yl)acetic Acid as a New Class of Ligands Selective for the γ-Hydroxybutyric Acid (GHB) High-Affinity Binding Sites.

Gabazine, a γ-aminobutyric acid type A (GABAA) receptor antagonist, has previously been reported to inhibit the binding of [3H]NCS-382, a representative ligand of the high-affinity binding site for the neuroactive substance γ-hydroxybutyric acid (GHB). We herein report a study on the structural determinants of gabazine for binding to (i) the orthosteric binding site of the GABAA receptor and (ii) the high-affinity GHB binding site. Expanding the structural diversity of available ligands for the high-affinity GHB binding sites, this study identified 2-(imidazo[1,2- b]pyridazin-2-yl)acetic acid as a novel ligand-scaffold leading to analogues with relatively high affinity ( Ki 0.19-2.19 µM) and >50 times selectivity for the [3H]NCS-382 over [3H]muscimol binding sites. These results highlight that gabazine interacts with the high-affinity GHB and orthosteric GABAA receptor binding sites differently and that distinct analogues can be generated to select between them. To facilitate further in vivo studies, a promising prodrug candidate for brain delivery was identified.

Total sitting time, leisure time physical activity and risk of hospitalization due to low back pain: The Danish Health Examination Survey cohort 2007-2008.

AIMS: This study aimed to test the hypotheses that a high total sitting time and vigorous physical activity in leisure time increase the risk of low back pain and herniated lumbar disc disease. METHODS: A total of 76,438 adults answered questions regarding their total sitting time and physical activity during leisure time in the Danish Health Examination Survey 2007-2008. Information on low back pain diagnoses up to 10 September 2015 was obtained from The National Patient Register. The mean follow-up time was 7.4 years. Data were analysed using Cox regression analysis with adjustment for potential confounders. Multiple imputations were performed for missing values. RESULTS: During the follow-up period, 1796 individuals were diagnosed with low back pain, of whom 479 were diagnosed with herniated lumbar disc disease. Total sitting time was not associated with low back pain or herniated lumbar disc disease. However, moderate or vigorous physical activity, as compared to light physical activity, was associated with increased risk of low back pain (HR = 1.16, 95% CI: 1.03-1.30 and HR = 1.45, 95% CI: 1.15-1.83). Moderate, but not vigorous physical activity was associated with increased risk of herniated lumbar disc disease. CONCLUSIONS: The results suggest that total sitting time is not associated with low back pain, but moderate and vigorous physical activity is associated with increased risk of low back pain compared with light physical activity.

Development of a Robust Mammalian Cell-based Assay for Studying Recombinant α4 ß1/3 δ GABAA Receptor Subtypes.

δ-Containing GABAA receptors are located extrasynaptically and mediate tonic inhibition. Their involvement in brain physiology positions them as interesting drug targets. There is thus a continued interest in establishing reliable recombinant expression systems for δ-containing GABAA receptors. Inconveniently, the recombinant expression of especially α4 ß1/3 δ receptors has been found to be notoriously difficult, resulting in mixed receptor populations and/or stoichiometries and differential pharmacology depending on the expression system used. With the aim of developing a facile and robust 96-well format cell-based assay for extrasynaptic α4 ß1/3 δ receptors, we have engineered and validated a HEK293 Flp-In™ cell line stably expressing the human GABAA δ-subunit. Upon co-transfection of α4 and ß1/3 subunits, at optimized ratios, we have established a well-defined system for expressing α4 ß1/3 δ receptors and used the fluorescence-based FLIPR Membrane Potential (FMP) assay to evaluate their pharmacology. Using the known reference compounds GABA and THIP, ternary α4 ß1/3 δ and binary α4 ß1/3 receptors could be distinguished based on potency and kinetic profiles but not efficacy. As expected, DS2 was able to potentiate only δ-containing receptors, whereas Zn2+ had an inhibitory effect only at binary receptors. By contrast, the hitherto reported δ-selective compounds, AA29504 and 3-OH-2'MeO6MF, were non-selective. The expression system was further validated using patch clamp electrophysiology, in which the superagonism of THIP was confirmed. The established FMP assay set-up, based on transient expression of human α4 and ß1/3 subunits into a δ-subunit stable HEK293 Flp-In™ cell line, portrays a simple 96-well format assay as a useful supplement to electrophysiological recordings on δ-containing GABAA receptors.

A comparison of pediatric basic life support self-led and instructor-led training among nurses.

OBJECTIVE: Pediatric cardiac arrest carries a poor prognosis. Basic life support improves survival. Studies on pediatric basic life support (PBLS) training are sparse. The aim of our study was to investigate the effect of self-training in PBLS. PARTICIPANTS AND METHODS: We conducted a prospective controlled trial enrolling nurses from pediatric and maternity wards (n=29 in each group). Self-training, including a manikin and access to a web-based video on PBLS, was compared with a 2-h instructor-led course. Two weeks after training, all participants were tested in a mock scenario of pediatric cardiac arrest. Fifteen parameters equivalent to the steps in the PBLS algorithm - for example, effective ventilations, effective chest compressions, calling for help, and correct sequence of actions, were evaluated and rated dichotomously (1=approved or 0=not approved). RESULTS: No difference was observed in the baseline demographics between the self-training group and the instructor-led group. The participants in the self-training group accessed the website 2±1.5 times (mean±SD) and spent 41±25 min on the site. There was no significant difference between the two groups in the overall average score (10.5 in the self-training group vs. 10.0 in the instructor-led group, P=0.51) or in any of the 15 parameters. After the study, all participants felt that they had improved their skills and felt capable of performing PBLS. CONCLUSION: Self-training is not statistically different to instructor-led training in teaching PBLS. Self-evaluated confidence improved, but showed no difference between groups. PBLS may be disseminated through self-training.

Does High Tobacco Consumption Cause Psychological Distress? A Mendelian Randomization Study.

BACKGROUND: Increasing evidence suggests that smoking influences mental health negatively. This study investigated whether high tobacco consumption is causally related to psychological distress in a Mendelian randomization design, using a variant in the nicotine acetylcholine receptor gene CHRNA3-known to influence individual tobacco consumption-as instrumental variable for tobacco consumption. METHODS: Data from 90 108 participants in the Copenhagen General Population Study was used. Exposures included self-reported cigarettes/day and pack years and the CHRNA3 rs1051730 genotype as instrumental variable for tobacco consumption. Three dimensions of psychological distress were studied: Stress, fatigue, and hopelessness. Analyses with the CHRNA3 genotype were stratified by smoking status. RESULTS: Self-reported amount of smoking was associated with all three dimensions of psychological distress. For instance among participants smoking 30 cigarettes/day or more, the odds ratio (OR) for stress was 1.67 (95% confidence interval [CI] 1.47-1.89) compared to never-smokers. Corresponding ORs for fatigue and hopelessness were 2.18 (95% CI 1.92-2.47) and 3.08 (95% CI 2.62-3.62). Among current smokers, homozygotes and heterozygotes for the CHRNA3 genotype had higher tobacco consumption than noncarriers. Nevertheless, the CHRNA3 genotype was not associated with psychological distress neither in current nor in former or never-smokers. For instance among current smokers, the OR for stress was 1.02 (95% CI 0.91-1.15) among homozygotes compared to noncarriers of the CHRNA3 genotype. CONCLUSIONS: Though a strong association between tobacco consumption and psychological distress was found, there was no clear evidence that high tobacco consumption was causally related to psychological distress. IMPLICATIONS: Smoking is associated with several mental health outcomes and smoking cessation is associated with improved mental health. Causality in the association between smoking and mental health is difficult to establish using observational data. Using a genotype known to influence tobacco consumption as instrumental variable for amount of smoking, we found no clear evidence of a direct causal path between high tobacco consumption and psychological distress. Whatever causes the strong association between tobacco consumption and psychological distress, the co-occurrence is important to consider both in interventions for smoking prevention and cessation.

Total sitting time and the risk of incident diabetes in Danish adults (the DANHES cohort) over 5†years: a prospective study.

AIMS: To test the hypothesis that total sitting time is associated with incident diabetes, after adjustment for physical activity and obesity. METHODS: 72 608 Danish adults from the DANHES cohort reported their total sitting time in 2007-2008 and were followed-up for 5 years, in relation to register-based incident diabetes mellitus. Cox regression analyses were used, and the effect-modifying influence of obesity and physical activity assessed. RESULTS: The age-sex adjusted HR for developing diabetes among those who sat 10+ h/day as compared to <6 h/day was 1.35 (95% CI 1.17 to 1.57). The relative risks were similar by gender, but were largely attenuated by adjustment for potential confounding factors including physical activity, and statistically non-significant for all categories of body mass index except the obese. CONCLUSIONS: The association between total sitting time and incident diabetes is substantially moderated by physical activity and obesity. Total sitting time remains a risk factor for diabetes only in inactive and obese populations.

Occupational heavy lifting and risk of ischemic heart disease and all-cause mortality.

BACKGROUND: Occupational heavy lifting is known to impose a high cardiovascular strain, but the risk of ischemic heart disease (IHD) from occupational heavy lifting is unknown. The objective was to investigate the association between occupational heavy lifting and risk of IHD and all-cause mortality, and the influence of occupational and leisure time physical activity on this association. METHODS: Data were analyzed from 1987, 1994, and 2000 from the Danish National Health Interview Surveys providing a sample of 6,692 working men and 5,921 working women aged 16-85 years without cardiovascular disease at baseline. Conventional risk factors for the outcomes IHD and all-cause mortality were controlled for in Cox analyses. RESULTS: Among men, heavy lifting was associated with increased risk for IHD (hazard ratio (HR): 1.52, 95% Confidence interval (95% CI): 1.15, 2.02), while a decreased risk was associated with occupational (HR: 0.50, 95% CI: 0.37, 0.68) and leisure time (HR: 0.73, 95% CI: 0.56, 0.95) physical activity. Referencing men with high occupational physical activity and no heavy lifting, men with high occupational physical activity and heavy lifting did not have an increased risk (HR: 1.11, 95% CI:0.68, 1.82), while men with low occupational physical activity and heavy lifting had a substantial increased risk (HR: 2.56, 95% CI:1.52, 4.32). No significant associations were found for all-cause mortality or for females. CONCLUSION: These findings indicate an excessive risk for IHD from occupational heavy lifting among men, particularly among those with low occupational and leisure time physical activity.

Socio-economic inequality in preterm birth: a comparative study of the Nordic countries from 1981 to 2000.

During the 1980s and 1990s, there were large social and structural changes within the Nordic countries. Here we examine time changes in risks of preterm birth by maternal educational attainment in Denmark, Finland, Norway and Sweden. Information on gestational age and maternal socio-economic position was obtained from the NorCHASE database, which includes comparable population-based register data of births from Denmark, Finland, Sweden and Norway from 1981 to 2000. The risks of very preterm birth (<32 gestational weeks) and moderately preterm birth (32-36 gestational weeks) were calculated by maternal educational attainment and analysed in 5-year intervals from 1981 to 2000. Compared with mothers with >12 years of education, mothers with <10 years of education had similarly increased risks of very, and to a lesser extent moderately, preterm birth in all four countries. The educational gradient increased slightly over time in very preterm births in Denmark, while there was a slight narrowing of the gap in Sweden. In moderately preterm births, the educational inequality gap was constant over the study period in Denmark, Norway and Sweden, but narrowed in Finland. The educational gradient in preterm birth remained broadly stable from 1981 to 2000 in all four countries. Consequently, the socio-economic inequalities in preterm birth were not strongly influenced by structural changes during the period.