Ligand and structure-based methodologies for the prediction of the activity of G protein-coupled receptor ligands

Accurate in silico models for the quantitative prediction of the activity of G protein-coupled receptor (GPCR) ligands would greatly facilitate the process of drug discovery and development. Several methodologies have been developed based on the properties of the ligands, the direct study of the receptor-ligand interactions, or a combination of both approaches. Ligand-based three-dimensional quantitative structure-activity relationships (3D-QSAR) techniques, not requiring knowledge of the receptor structure, have been historically the first to be applied to the prediction of the activity of GPCR ligands. They are generally endowed with robustness and good ranking ability; however they are highly dependent on training sets. Structure-based techniques generally do not provide the level of accuracy necessary to yield meaningful rankings when applied to GPCR homology models. However, they are essentially independent from training sets and have a sufficient level of accuracy to allow an effective discrimination between binders and nonbinders, thus qualifying as viable lead discovery tools. The combination of ligand and structure-based methodologies in the form of receptor-based 3D-QSAR and ligand and structure-based consensus models results in robust and accurate quantitative predictions. The contribution of the structure-based component to these combined approaches is expected to become more substantial and effective in the future, as more sophisticated scoring functions are developed and more detailed structural information on GPCRs is gathered.

The respiratory syncytial virus small hydrophobic protein is phosphorylated via a mitogen-activated protein kinase p38-dependent tyrosine kinase activity during virus infection

The phosphorylation status of the small hydrophobic (SH) protein of respiratory syncytial virus (RSV) was examined in virus-infected Vero cells. The SH protein v.,as isolated from [S-35]methionine- and [P-33]orthophosphate-labelled IRSV-infected cells and analysed by SDS-PAGE. In each case, a protein product of the expected size for the SH protein was observed. Phosphoamino acid analysis and reactivity with the phosphotyrosine specific antibody PY20 showed that the SH protein was modified by tyrosine phosphorylation. The role or tyrosine kinase activity in SH protein phosphorylation was confirmed by the use of genistein, a broad-spectrum tyrosine kinase inhibitor, to inhibit SH protein phosphorylation. Further analysis showed that the different glycosylated forms of the SH protein were phosphorylated, as was the oligomeric form of the protein. Phosphorylation of the SH protein was specifically inhibited by the mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580, suggesting that SH protein phosphorylation occurs via a MAPK p38-dependent pathway. Analysis of virus-infected cells using fluorescence microscopy showed that, although the SH protein was distributed throughout the cytoplasm, it appeared to accumulate, at low levels, in the endoplasmic reticulum/Golgi complex, confirming recent observations. However, in the presence of SB203580. an increased accumulation of the SH protein in the Golgi complex was observed, although other virus structures, such as virus filaments and inclusion bodies, remained largely unaffected. These results showed that during RSV infection, the SH protein is modified by an MAPK p38-dependant tyrosine kinase activity and that this modification influences its cellular distribution.

Physical Activity, Gender, Weight Status, and Wellbeing in 9- to 11-Year-Old Children: A Cross-Sectional Survey

Background: The aim of this study was to examine the relationship between physical activity and wellbeing in children, and to further explore the extent to which this may vary by gender and weight status. Method: A representative sample of 1424 9- to 11-year-olds completed a self-report measure of physical activity, the Child Health and Illness Profile, KIDSCREEN, and a self-esteem scale. Body Mass Index (BMI) measurements were also obtained. Results: 24% of children achieved the recommended level of 60 minutes of moderate-tovigorous intensity physical activity (MVPA) per day, with more boys than girls achieving this level. Children achieving the recommended level of MVPA scored significantly higher on measures of the Child Health and Illness Profile (F(5, 1354) = 5.03; P < .001), KIDSCREEN (F(3, 1298) = 4.68; P = .003), and self-esteem (F(1,1271) = 18.73; P = .003) than less active children although the effect sizes were small (ηp2 ≈ .01). Substantial gender differences in wellbeing were found reflecting gender specific behaviors and socialization. Weight status had negligible influence on wellbeing. Conclusions: Children who meet the recommended guidelines of MVPA were more likely to have better wellbeing. When attempting to raise children’s physical activity levels consideration should be given to the specific relationships between wellbeing and physical activity.

Neuropeptide Y Y(1) receptor regulates protein turnover and constitutive gene expression in hypertrophying cardiomyocytes.

Increased levels of neuropeptide Y correlate with severity of left ventricular hypertrophy in vivo. At cardiomyocyte level, hypertrophy is characterised by increased mass and altered phenotype. The aims were to determine the contributions of increased synthesis and reduced degradation of protein to neuropeptide Y-mediated increase in mass, assess effects on gene expression, and characterise neuropeptide Y Y receptor subtype involvement. Neuropeptide Y (10 nM) increased protein mass of adult rat ventricular cardiomyocytes maintained in culture (24 h) (16%>basal) and de novo protein synthesis (incorporation of [14C]phenylalanine) (18%>basal). Neuropeptide Y (100 nM) prevented degradation of existing protein at 8 h. Actinomycin D (5 µM) attenuated increases in protein mass to neuropeptide Y (=1 nM) but not to neuropeptide Y (10 nM). [Leu31, Pro34]neuropeptide Y (10 nM), an agonist at neuropeptide Y Y1 receptors, increased protein mass (25%>basal) but did not stimulate protein synthesis. Neuropeptide Y-(3–36) (10 nM), an agonist at neuropeptide Y Y2 receptors, increased protein mass (29%>basal) and increased protein synthesis (13%>basal), respectively. Actinomycin D (5 µM) abolished the increase in protein mass elicited by neuropeptide Y-(3–36) but not that by [Leu31, Pro34]neuropeptide Y. BIBP3226 [(R)-N2-(diphenylacetyl)-N-(4-hydroxyphenylmethyl)-d-arginine amide] (1 µM), a neuropeptide Y Y1 receptor subtype-selective antagonist, and T4 [neuropeptide Y-(33–36)]4, a neuropeptide Y Y2 receptor subtype-selective antagonist, attenuated the increase in protein mass to 100 nM neuropeptide Y by 68% and 59%, respectively. Neuropeptide Y increased expression of the constitutive gene, myosin light chain-2 (MLC-2), maximally at 12 h (4.7-fold>basal) but did not induce (t=36 h) expression of foetal genes (atrial natriuretic peptide (ANP), skeletal-a-actin and myosin heavy chain-ß). This increase was attenuated by 86% and 51%, respectively, by BIBP3226 (1 µM) and T4 [neuropeptide Y-(33–36)]4 (100 nM). [Leu31, Pro34]neuropeptide Y (100 nM) (2.4-fold>basal) and peptide YY-(3–36) (100 nM) (2.3 fold>basal) increased expression of MLC-2 mRNA at 12 h. In conclusion, initiation of cardiomyocyte hypertrophy by neuropeptide Y requires activation of both neuropeptide Y Y1 and neuropeptide Y Y2 receptors and is associated with enhanced synthesis and attenuated degradation of protein together with increased expression of constitutive genes but not reinduction of foetal genes.

Specific growth rate plays a critical role in hydrogen peroxide resistance of the marine oligotrophic ultramicrobacterium Sphingomonas alaskensis strain RB2256

The marine oligotrophic ultramicrobacterium Sphingomonas alaskensis RB2256 has a physiology that is distinctly different from that of typical copiotrophic marine bacteria, such as Vibrio angustum S14. This includes a high level of inherent stress resistance and the absence of starvation-induced stress resistance to hydrogen peroxide. In addition to periods of starvation in the ocean, slow nutrient-limited growth is likely to be encountered by oligotrophic bacteria for substantial periods of time. In this study we examined the effects of growth rate on the resistance of S. alaskensis RB2256 to hydrogen peroxide under carbon or nitrogen limitation conditions in nutrient-limited chemostats. Glucose-limited cultures of S. alaskensis RB2256 at a specific growth rate of 0.02 to 0.13 h(-1) exhibited 10,000-fold-greater viability following 60 min of exposure to 25 mM hydrogen peroxide than tells growing at a rate of 0.14 h(-1) or higher. Growth rate control of stress resistance was found to be specific to carbon and energy limitation in this organism. In contrast, V. angustum S14 did not exhibit growth rate-dependent stress resistance. The dramatic switch in stress resistance that was observed under carbon and energy limitation conditions has not been described previously in bacteria and thus may be a characteristic of the oligotrophic ultramicrobacterium, Catalase activity varied marginally and did not correlate with the growth rate, indicating that hydrogen peroxide breakdown was not the primary mechanism of resistance. More than 1,000 spots were resolved on silver-stained protein gels for cultures growing at rates of 0.026, 0.076, and 0.18 h(-1). Twelve protein spots had intensities that varied by more than twofold between growth rates and hence are likely to be important for growth rate-dependent stress resistance. These studies demonstrated the crucial role that nutrient limitation plays in the physiology of S. alaskensis RB2256, especially under oxidative stress conditions.

Transmission of two novel mutations in a pedigree with familial lecithin:cholesterol acyltransferase deficiency:structure-function relationships and studies in a compound heterozygous proband

Two novel mutations were identified in a compound heterozygous male with lecithin:cholesterol acyltransferase (LCAT) deficiency. Exon sequence determination of the LCAT gene of the proband revealed two novel heterozygous mutations in exons one (C110T) and six (C991T) that predict non-conservative amino acid substitutions (Thr13Met and Pro307Ser, respectively). To assess the distinct functional impact of the separate mutant alleles, studies were conducted in the proband's 3-generation pedigree. The compound heterozygous proband had negligible HDL and severely reduced apolipoprotein A-I, LCAT mass, LCAT activity, and cholesterol esterification rate (CER). The proband's mother and two sisters were heterozygous for the Pro307Ser mutation and had low HDL, markedly reduced LCAT activity and CER, and the propensity for significant reductions in LCAT protein mass. The proband's father and two daughters were heterozygous for the Thr13Met mutation and also displayed low HDL, reduced LCAT activity and CER, and more modest decrements in LCAT mass. Mean LCAT specific activity was severely impaired in the compound heterozygous proband and was reduced by 50% in individuals heterozygous for either mutation, compared to wild type family members. It is also shown that the two mutations impair both catalytic activity and expression of the circulating protein.

Impaired activities of antioxidant enzymes elicit endothelial dysfunction in spontaneous hypertensive rats despite enhanced vascular nitric oxide generation

Objective: Enhanced oxidative stress is involved in mediating the endothelial dysfunction associated with hypertension. The aim of this study was to investigate the relative contributions of pro-oxidant and anti-oxidant enzymes to the pathogenesis of endothelial dysfunction in genetic hypertension. Methods: Dilator responses to endothelium-dependent and endothelium-independent agents such as acetylcholine (ACh) and sodium nitroprusside were measured in the thoracic aortas of 28-week-old spontaneously hypertensive rats (SHR) and their matched normotensive counterparts, Wistar Kyoto rats (WKY). The activity and expression (mRNA and protein levels) of endothelial nitric oxide synthase (eNOS), p22-phox, a membrane-bound component of NAD(P)H oxidase, and antioxidant enzymes, namely, superoxide dismutases (CuZn- and Mn-SOD), catalase and glutathione peroxidase (GPx), were also investigated in aortic rings. Results: Relaxant responses to ACh were attenuated in phenylephrine-precontracted SHR aortic rings, despite a 2-fold increase in eNOS expression and activity. Although the activity and/or expression of SODs, NAD(P)H oxidase (p22-phox) and GPx were elevated in SHR aorta, catalase activity and expression remained unchanged compared to WKY. Pretreatment of SHR aortic rings with the inhibitor of xanthine oxidase, allopurinol, and the inhibitor of cyclooxygenase, indomethacin, significantly potentiated ACh-induced relaxation. Pretreatment of SHR rings with catalase and Tiron, a superoxide anion (O) scavenger, increased the relaxant responses to the levels observed in WKY rings whereas pyrogallol, a O -generator, abolished relaxant responses to ACh. Conclusion: These data demonstrate that dysregulation of several enzymes, resulting in oxidative stress, contributes to the pathogenesis of endothelial dysfunction in SHR and indicate that the antioxidant enzyme catalase is of particular importance in the reversal of this defect. © 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Inhibition of Rho-kinase protects cerebral barrier from ischaemia-evoked injury through modulations of endothelial cell oxidative stress and tight junctions

Ischaemic strokes evoke blood-brain barrier (BBB) disruption and oedema formation through a series of mechanisms involving Rho-kinase activation. Using an animal model of human focal cerebral ischaemia, this study assessed and confirmed the therapeutic potential of Rho-kinase inhibition during the acute phase of stroke by displaying significantly improved functional outcome and reduced cerebral lesion and oedema volumes in fasudil- versus vehicle-treated animals. Analyses of ipsilateral and contralateral brain samples obtained from mice treated with vehicle or fasudil at the onset of reperfusion plus 4 h post-ischaemia or 4 h post-ischaemia alone revealed these benefits to be independent of changes in the activity and expressions of oxidative stress- and tight junction-related parameters. However, closer scrutiny of the same parameters in brain microvascular endothelial cells subjected to oxygen-glucose deprivation ± reperfusion revealed marked increases in prooxidant NADPH oxidase enzyme activity, superoxide anion release and in expressions of antioxidant enzyme catalase and tight junction protein claudin-5. Cotreatment of cells with Y-27632 prevented all of these changes and protected in vitro barrier integrity and function. These findings suggest that inhibition of Rho-kinase after acute ischaemic attacks improves cerebral integrity and function through regulation of endothelial cell oxidative stress and reorganization of intercellular junctions. Inhibition of Rho-kinase (ROCK) activity in a mouse model of human ischaemic stroke significantly improved functional outcome while reducing cerebral lesion and oedema volumes compared to vehicle-treated counterparts. Studies conducted with brain microvascular endothelial cells exposed to OGD ± R in the presence of Y-27632 revealed restoration of intercellular junctions and suppression of prooxidant NADPH oxidase activity as important factors in ROCK inhibition-mediated BBB protection.

Sedentary behaviour and physical activity in bronchiectasis: a cross-sectional study

BACKGROUND: The impact of bronchiectasis on sedentary behaviour and physical activity is unknown. It is important to explore this to identify the need for physical activity interventions and how to tailor interventions to this patient population. We aimed to explore the patterns and correlates of sedentary behaviour and physical activity in bronchiectasis.

METHODS: Physical activity was assessed in 63 patients with bronchiectasis using an ActiGraph GT3X+ accelerometer over seven days. Patients completed: questionnaires on health-related quality-of-life and attitudes to physical activity (questions based on an adaption of the transtheoretical model (TTM) of behaviour change); spirometry; and the modified shuttle test (MST). Multiple linear regression analysis using forward selection based on likelihood ratio statistics explored the correlates of sedentary behaviour and physical activity dimensions. Between-group analysis using independent sample t-tests were used to explore differences for selected variables.

RESULTS: Fifty-five patients had complete datasets. Average daily time, mean(standard deviation) spent in sedentary behaviour was 634(77)mins, light-lifestyle physical activity was 207(63)mins and moderate-vigorous physical activity (MVPA) was 25(20)mins. Only 11% of patients met recommended guidelines. Forced expiratory volume in one-second percentage predicted (FEV1% predicted) and disease severity were not correlates of sedentary behaviour or physical activity. For sedentary behaviour, decisional balance 'pros' score was the only correlate. Performance on the MST was the strongest correlate of physical activity. In addition to the MST, there were other important correlate variables for MVPA accumulated in ≥10-minute bouts (QOL-B Social Functioning) and for activity energy expenditure (Body Mass Index and QOL-B Respiratory Symptoms).

CONCLUSIONS: Patients with bronchiectasis demonstrated a largely inactive lifestyle and few met the recommended physical activity guidelines. Exercise capacity was the strongest correlate of physical activity, and dimensions of the QOL-B were also important. FEV1% predicted and disease severity were not correlates of sedentary behaviour or physical activity. The inclusion of a range of physical activity dimensions could facilitate in-depth exploration of patterns of physical activity. This study demonstrates the need for interventions targeted at reducing sedentary behaviour and increasing physical activity, and provides information to tailor interventions to the bronchiectasis population.

Exchange protein directly activated by cyclic AMP (EPAC) activation reverses neutrophil dysfunction induced by β2-agonists, corticosteroids, and critical illness

Background Neutrophils play a role in the pathogenesis of asthma, chronic obstructive pulmonary disease, and pulmonary infection. Impaired neutrophil phagocytosis predicts hospital-acquired infection. Despite this, remarkably few neutrophil-specific treatments exist. 

Objectives We sought to identify novel pathways for the restoration of effective neutrophil phagocytosis and to activate such pathways effectively in neutrophils from patients with impaired neutrophil phagocytosis. 

Methods Blood neutrophils were isolated from healthy volunteers and patients with impaired neutrophil function. In healthy neutrophils phagocytic impairment was induced experimentally by using β₂-agonists. Inhibitors and activators of cyclic AMP (cAMP)-dependent pathways were used to assess the influence on neutrophil phagocytosis in vitro. 

Results β₂-Agonists and corticosteroids inhibited neutrophil phagocytosis. Impairment of neutrophil phagocytosis by β₂-agonists was associated with significantly reduced RhoA activity. Inhibition of protein kinase A (PKA) restored phagocytosis and RhoA activity, suggesting that cAMP signals through PKA to drive phagocytic impairment. However, cAMP can signal through effectors other than PKA, such as exchange protein directly activated by cyclic AMP (EPAC). An EPAC-activating analog of cAMP (8CPT-2Me-cAMP) reversed neutrophil dysfunction induced by β₂-agonists or corticosteroids but did not increase RhoA activity. 8CPT-2Me-cAMP reversed phagocytic impairment induced by Rho kinase inhibition but was ineffective in the presence of Rap-1 GTPase inhibitors. 8CPT-2Me-cAMP restored function to neutrophils from patients with known acquired impairment of neutrophil phagocytosis. 

Conclusions EPAC activation consistently reverses clinical and experimental impairment of neutrophil phagocytosis. EPAC signals through Rap-1 and bypasses RhoA. EPAC activation represents a novel potential means by which to reverse impaired neutrophil phagocytosis.

Effects of roziglitazone and interactions with growth-regulating factors in ventricular cell hypertrophy

Chronic administration of thiazolidinediones might predispose to cardiac hypertrophy. The aim was to investigate direct effects of rosiglitazone in rat ventricular cardiomyocytes maintained in vitro (24 h). Rosiglitazone (=10-5 M) did not increase protein synthesis and produced small inconsistent increases in cellular protein. In the presence of serum (10% v/v), but not insulin-like growth factor (IGF-1, 10-8 M) or insulin (1 U/ml), an interaction with rosiglitazone to stimulate protein synthesis was observed. The hypertrophic responses to noradrenaline (5×10-6 M), PMA (10-7 M) and ET-1 (10-7 M) were not attenuated by rosiglitazone. Rosiglitazone (10-7 M) did not influence protein synthesis in response to insulin (1 U/ml) and elevated glucose (2.5×10-2 M) alone or in combination, but attenuated the increase in protein mass observed in response to elevated glucose alone. In re-differentiated cardiomyocytes, a model of established hypertrophy, rosiglitazone (10-8 M–10-6 M) increased protein synthesis. Together, these data indicate that rosiglitazone does not initiate cardiomyocyte hypertrophy directly in vitro. However, during chronic administration, the interaction of rosiglitazone with locally-derived growth-regulating factors may make a modest contribution to cardiac remodelling and influence the extent of compensatory hypertrophy of the compromised rat heart.

Induction of hypertrophic responsiveness of cardiomyocytes to neuropeptide Y in response to pressure overload.

To determine whether neuropeptide Y (NPY)-related mechanisms become activated with progression of cardiac hypertrophy in vivo, protein mass and de novo protein synthesis (incorporation of [(14)C]Phe, 0.1 muCi ml(-1)) were assessed in cardiomyocytes, obtained from spontaneously hypertensive rats (SHRs) and normotensive Wistar Kyoto rats (8, 12, 16, 20, and 24 weeks of age), and cultured for 24 h. NPY (10(-8) M) increased protein mass of cardiomyocytes from 16-week-old SHRs by 9.2 +/- 2.1% (n = 8, P

Temporal characteristics of cardiomyocyte hypertrophy in the spontaneously hypertensive rat.

Background The spontaneously hypertensive rat (SHR) is frequently used as model of cardiovascular disease, with considerable disparity in reported parameters of hypertrophy. The aim of this study was to assess the temporal changes occurring during the development and progression of cardiomyocyte hypertrophy in SHR, subsequent to pressure overload, compared to changes associated with normal aging using the normotensive Wistar–Kyoto (WKY) rat. Methods Ventricular cardiomyocytes were isolated from rats at 8, 12, 16, 20 and 24 weeks, and parameters of hypertrophy (cell dimensions, protein mass, de novo protein synthesis, and gene expression) and function (contraction and hypertrophic responsiveness in vitro) were assessed. Results Hypertension was evident at =7 weeks in SHRs. Heart:body mass ratio, cardiomyocyte protein mass and width were elevated (P

Connective tissue growth factor [CTGF]/CCN2 stimulates mesangial cell migration through integrated dissolution of focal adhesion complexes and activation of cell polarization

Connective tissue growth factor [CTGF]/CCN2 is a prototypic member of the CCN family of regulatory proteins. CTGF expression is up-regulated in a number of fibrotic diseases, including diabetic nephropathy, where it is believed to act as a downstream mediator of TGF-beta function; however, the exact mechanisms whereby CTGF mediates its effects remain unclear. Here, we describe the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The addition of CTGF to primary mesangial cells induced cell migration and cytoskeletal rearrangement but had no effect on cell proliferation. Cytoskeletal rearrangement was associated with a loss of focal adhesions, involving tyrosine dephosphorylation of focal adhesion kinase and paxillin, increased activity of the protein tyrosine phosphatase SHP-2, with a concomitant decrease in RhoA and Rac1 activity. Conversely, Cdc42 activity was increased by CTGF. These functional responses were associated with the phosphorylation and translocation of protein kinase C-zeta to the leading edge of migrating cells. Inhibition of CTGF-induced protein kinase C-zeta activity with a myristolated PKC-zeta inhibitor prevented cell migration. Moreover, transient transfection of human mesangial cells with a PKC-zeta kinase inactive mutant (dominant negative) expression vector also led to a decrease in CTGF-induced migration compared with wild-type. Furthermore, CTGF stimulated phosphorylation and activation of GSK-3beta. These data highlight for the first time an integrated mechanism whereby CTGF regulates cell migration through facilitative actin cytoskeleton disassembly, which is mediated by dephosphorylation of focal adhesion kinase and paxillin, loss of RhoA activity, activation of Cdc42, and phosphorylation of PKC-zeta and GSK-3beta. These changes indicate that the initial stages of CTGF mediated mesangial cell migration are similar to those involved in the process of cell polarization. These findings begin to shed mechanistic light on the renal diabetic milieu, where increased CTGF expression in the glomerulus contributes to cellular dysfunction.