Addressing Selective Polypharmacology of Antipsychotic Drugs Targeting the Bioaminergic Receptors through the Receptor Dynamic Conformational Ensembles

Selective polypharmacology, where a drug acts on multiple rather than single molecular targets involved in a disease, emerges to develop a structure-based system biology approach to design drugs selectively targeting a disease-active protein network. We focus on the bioaminergic receptors that belong to the group of integral membrane signalling proteins coupled to the G protein and represent targets for therapeutic agents against schizophrenia and depression. Among them, it has been shown that the serotonin (5-HT2A and 5-HT6), dopamine (D2 and D3) receptors induce a cognition-enhancing effect (group 1), while the histamine (H1) and serotonin (5-HT2C) receptors lead to metabolic side effects and the 5-HT2B serotonin receptor causes pulmonary hypertension (group 2). Thus, the problem arises to develop an approach that allows identifying drugs targeting only the disease-active receptors, i.e. group 1. The recent release of several crystal structures of the bioaminergic receptors, involving the D3 and H1 receptors provides the possibility to model the structures of all receptors and initiate a study of the structural and dynamic context of selective polypharmacology. In this work, we use molecular dynamics simulations to generate a conformational space of the receptors and subsequently characterize its binding properties applying molecular probe mapping. All-against-all comparison of the generated probe maps of the selected diverse conformations of all receptors with the Tanimoto similarity coefficient (Tc) enable to separate the receptors of group 1 from group 2. The pharmacophore built based on the Tc-selected receptor conformations, using the multiple probe maps discovers structural features that can be used to design molecules selective towards the receptors of group 1. The importance of several predicted residues to ligand selectivity is supported by the available mutagenesis and ligand structure-activity relationships studies. In addition, the Tc-selected conformations of the receptors for group 1 show good performance in isolation of known ligands from a random decoy. Our computational structure-based protocol to tackle selective polypharmacology of antipsychotic drugs could be applied for other diseases involving multiple drug targets, such as oncologic and infectious disorders.

Insulin receptor substrate-2 is expressed in kidney epithelium and up-regulated in diabetic nephropathy

Diabetic nephropathy (DN) is a progressive fibrotic condition that may lead to end-stage renal disease and kidney failure. Transforming growth factor-ß1 and bone morphogenetic protein-7 (BMP7) have been shown to induce DN-like changes in the kidney and protect the kidney from such changes, respectively. Recent data identified insulin action at the level of the nephron as a crucial factor in the development and progression of DN. Insulin requires a family of insulin receptor substrate (IRS) proteins for its physiological effects, and many reports have highlighted the role of insulin and IRS proteins in kidney physiology and disease. Here, we observed IRS2 expression predominantly in the developing and adult kidney epithelium in mouse and human. BMP7 treatment of human kidney proximal tubule epithelial cells (HK-2 cells) increases IRS2 transcription. In addition, BMP7 treatment of HK-2 cells induces an electrophoretic shift in IRS2 migration on SDS/PAGE, and increased association with phosphatidylinositol-3-kinase, probably due to increased tyrosine/serine phosphorylation. In a cohort of DN patients with a range of chronic kidney disease severity, IRS2 mRNA levels were elevated approximately ninefold, with the majority of IRS2 staining evident in the kidney tubules in DN patients. These data show that IRS2 is expressed in the kidney epithelium and may play a role in the downstream protective events triggered by BMP7 in the kidney. The specific up-regulation of IRS2 in the kidney tubules of DN patients also indicates a novel role for IRS2 as a marker and/or mediator of human DN progression.

Dietary Restriction Induced Longevity is Mediated by Nuclear Receptor NHR-62 in Caenorhabditis elegans

Dietary restriction (DR) extends lifespan in a wide variety of species, yet the underlying mechanisms are not well understood. Here we show that the C. elegans HNF4a- related nuclear hormone receptor NHR-62 is required for metabolic and physiologic responses associated with DR-induced longevity. nhr-62 mediates the longevity of eat- 2 mutants, a genetic mimetic of dietary restriction, and blunts the longevity response of DR induced by bacterial food dilution at low nutrient levels. Metabolic changes associated with DR, including decreased Oil Red O staining, increased autophagy, and changes in fatty acid composition are partly reversed by mutation of nhr-62. Expression profiles reveal that several hundred genes induced by DR depend on the activity of NHR-62, including a putative lipase required for the DR response. This study provides critical evidence that nuclear hormone receptors regulate the DR response, suggesting hormonal and metabolic control of life span.

Simulations of Biased Agonists in the β2 Adrenergic Receptor with Accelerated Molecular Dynamics

The biased agonism of the G protein-coupled receptors (GPCRs), where in addition to a traditional G protein-signalling pathway a GPCR promotes intracellular signals though ß-arrestin, is a novel paradigm in pharmacology. Biochemical and biophysical studies have suggested that a GPCR forms a distinct ensemble of conformations signalling through the G protein and ß-arrestin. Here we report on the dynamics of the ß2 adrenergic receptor bound to the ß-arrestin and G protein biased agonists and the empty receptor to further characterize the receptor conformational changes caused by biased agonists. We use conventional and accelerated molecular dynamics (aMD) simulations to explore the conformational transitions of the GPCR from the active state to the inactive state. We found that aMD simulations enable monitoring the transition within the nanosecond timescale while capturing the known microscopic characteristics of the inactive states, such as the ionic lock, the inward position of F6.44, and water clusters. Distinct conformational states are shown to be stabilized by each biased agonist. In particular, in simulations of the receptor with the ß-arrestin biased agonist, N-cyclopentylbutanepherine we observe a different pattern of motions in helix 7 when compared to simulations with the G protein biased agonist, Salbutamol that involves perturbations of the network of interactions within the NPxxY motif. Understanding the network of interactions induced by biased ligands and the subsequent receptor conformational shifts will lead to development of more efficient drugs. © 2013 American Chemical Society

Rhinovirus upregulates transient receptor potential channels in a human neuronal cell line: Implications for respiratory virus-induced cough reflex sensitivity

ABSTRACT (250 words)
BACKGROUND: The mechanism underlying respiratory virus-induced cough hypersensitivity is unknown. Up-regulation of airway neuronal receptors responsible for sensing physical and chemical stimuli is one possibility and the transient receptor potential (TRP) channel family are potential candidates. We have used an in vitro model of sensory neurones and human rhinovirus (HRV-16) to study the effect of virus infection on TRP expression.
METHODS: IMR32 neuroblastoma cells were differentiated in culture to express three TRP channels, TRPV1, TRPA1 and TRPM8. Flow cytometry and qRT-PCR were used to measure TRP channel protein and mRNA levels following inoculation with live virus, inactivated virus, virus- induced soluble factors or pelleted virus particles. Multiplex bioassay was used to determine nerve growth factor (NGF), interleukin (IL)-1ß, IL-6 and IL-8 levels in response to infection.
RESULTS: Early up-regulation of TRPA1 and TRPV1 expression occurred 2 to4 hours post infection. This was independent of replicating virus as virus induced soluble factors alone were sufficient to increase channel expression 50 and 15 fold, respectively. NGF, IL-6 and IL-8 levels, increased in infected cell supernatants, represent possible candidates. In contrast, TRPM8 expression was maximal at 48 hours (9.6 fold) and required virus replication rather than soluble factors
CONCLUSIONS We show for the first time that rhinovirus can infect neuronal cells. Furthermore, infection causes up-regulation of TRP channels by channel specific mechanisms. Increase in TRPA1 and TRPV1 levels can be mediated by soluble factors induced by infection whereas TRPM8 requires replicating virus. TRP channels may be novel therapeutic targets for controlling virus-induced cough.

Dose-dependent effects of the once-daily GLP-1 receptor agonist lixisenatide in patients with Type 2 diabetes inadequately controlled with metformin:a randomized, double-blind, placebo-controlled trial

To evaluate the dose-response relationship of lixisenatide (AVE0010), a glucagon-like peptide-1 (GLP-1) receptor agonist, in metformin-treated patients with Type 2 diabetes.

Mifepristone, a glucocorticoid receptor antagonist, produces clinical and metabolic benefits in patients with Cushing's syndrome

Cushing's syndrome (CS) is a disorder associated with significant morbidity and mortality due to prolonged exposure to high cortisol concentrations.

Sirolimus Stimulates Vascular Stem/Progenitor Cell Migration and Differentiation Into Smooth Muscle Cells via Epidermal Growth Factor Receptor/Extracellular Signal-Regulated Kinase/β-Catenin Signaling Pathway

Sirolimus-eluting stent therapy has achieved considerable success in overcoming coronary artery restenosis. However, there remain a large number of patients presenting with restenosis after the treatment, and the source of its persistence remains unclarified. Although recent evidence supports the contribution of vascular stem/progenitor cells in restenosis formation, their functional and molecular responses to sirolimus are largely unknown.

Receptor-mediated endocytosis and intracellular trafficking of insulin and low-density lipoprotein by retinal vascular endothelial cells

PURPOSE: The authors investigated the receptor-mediated endocytosis (RME) and intracellular trafficking of insulin and low-density lipoprotein (LDL) in cultured retinal vascular endothelial cells (RVECs). METHODS: Low-density lipoprotein and insulin were conjugated to 10 nm colloidal gold, and these ligands were added to cultured bovine RVECs for 20 minutes at 4 degrees C. The cultures were then warmed to 37 degrees C and fixed after incubation times between 30 seconds and 1 hour. Control cells were incubated with unconjugated gold colloid at times and concentrations similar to those of the ligands. Additional control cells were exposed to several concentrations of anti-insulin receptor antibody or a saturating solution of unconjugated insulin before incubation with gold insulin. RESULTS: Using transmission electron microscopy, insulin gold and LDL gold were both observed at various stages of RME. Insulin-gold particles were first seen to bind to the apical plasma membrane (PM) before clustering in clathrin-coated pits and internalization in coated vesicles. Gold was later visualized in uncoated cytoplasmic vesicles, corresponding to early endosomes and multivesicular bodies (MVBs) or late endosomes. In several instances, localized regions of the limiting membrane of the MVBs appeared coated, a feature of endosomal membranes not previously described. After RME at the apical PM and passage through the endosomal system, the greater part of both insulin- and LDL-gold conjugates was seen to accumulate in large lysosome-like compartments. However, a small but significant proportion of the internalized ligands was transcytosed and released as discrete membrane-associated quanta at the basal cell surface. The uptake of LDL gold was greatly increased in highly vacuolated, late-passage RVECs. In controls, anti-insulin receptor antibody and excess unconjugated insulin caused up to 89% inhibition in gold-insulin binding and internalization. CONCLUSION: These results illustrate the internalization and intracellular trafficking by RVECs of insulin and LDL through highly efficient RME, and they provide evidence for at least two possible fates for the endocytosed ligands. This study outlines a route by which vital macromolecules may cross the inner blood-retinal barrier.

Increased expression of bronchial epithelial transient receptor potential vanilloid 1 channels in severe asthma

BACKGROUND: The airway epithelium is exposed to a range of physical and chemical irritants in the environment that are known to trigger asthma. Transient receptor potential (TRP) cation channels play a central role in sensory responses to noxious physical and chemical stimuli. Recent genetic evidence suggests an involvement of transient receptor potential vanilloid 1 (TRPV1), one member of the vanilloid subfamily of TRP channels, in the pathophysiology of asthma. The functional expression of TRPV1 on airway epithelium has yet to be elucidated.

OBJECTIVE: In this study we examined the molecular, functional, and immunohistochemical expression of TRPV1 in asthmatic and healthy airways.

METHODS: Bronchial biopsy specimens and bronchial brushings were obtained from healthy volunteers (n = 18), patients with mild-to-moderate asthma (n = 24), and patients with refractory asthma (n = 22). Cultured primary bronchial epithelial cells from patients with mild asthma (n = 4), nonasthmatic coughers (n = 4), and healthy subjects (n = 4) were studied to investigate the functional role of TRPV1.

RESULTS: Quantitative immunohistochemistry revealed significantly more TRPV1 expression in asthmatic patients compared with healthy subjects, with the greatest expression in patients with refractory asthma (P = .001). PCR and Western blotting analysis confirmed gene and protein expression of TRPV1 in cultured primary bronchial epithelial cells. Patch-clamp electrophysiology directly confirmed functional TRPV1 expression in all 3 groups. In functional assays the TRPV1 agonist capsaicin induced dose-dependent IL-8 release, which could be blocked by the antagonist capsazepine. Reduction of external pH from 7.4 to 6.4 activated a capsazepine-sensitive outwardly rectifying membrane current.

CONCLUSIONS: Functional TRPV1 channels are present in the human airway epithelium and overexpressed in the airways of patients with refractory asthma. These channels might represent a novel therapeutic target for the treatment of uncontrolled asthma.

Histamine H3 receptor-mediated inhibition of sympathetically evoked mydriasis in rats

This study was designed to determine if the histamine H3 receptor agonist R-alpha-methylhistamine would play a role in modulation of sympathetically evoked mydriasis in anesthetized rats, and if so, to ascertain the specific receptor subtype(s) involved. Reproducible frequency-response curves of pupillary dilation were generated by stimulation of the cervical preganglionic sympathetic nerve (1-32 Hz). Systemic administration of R-alpha-methylhistamine (0.3-3.0 mg kg(-1)) produced a dose-related inhibition of the evoked mydriasis. The greatest inhibition was seen at lower frequency levels, with about 43% depression observed at 2 Hz. The specific histamine H3 receptor antagonist, clobenpropit (3.0 mg kg(-1), i.v.), blocked the inhibitory effect of R-alpha-methylhistamine, whereas neither the histamine H2 receptor antagonist, cimetidine (5.0 mg kg(-1), i.v.), nor the histamine H1 receptor antagonist, chlorpheniramine (0.5 mg kg(-1), i.v.), was effective. The histamine H2 receptor agonist, dimaprit (10 mg kg(-1), i.v.), was also without effect on the evoked mydriasis. R-alpha-methylhistamine (3.0 mg kg(-1)) did not inhibit phenylephrine-induced mydriasis. These results support the conclusion that R-alpha-methylhistamine produces inhibition of sympathetically evoked mydriasis via histamine H3 receptor stimulation, presumably by an action on presynaptic histamine H3 receptors.

Exclusion of linkage between schizophrenia and the D2 dopamine receptor gene region of chromosome 11q in 112 Irish multiplex families

A leading theory hypothesizes that schizophrenia arises from dysregulation of the dopamine system in certain brain regions. As this dysregulation could arise from abnormal expression of D2 dopamine receptors, the D2 receptor gene (DRD2) on chromosome 11q is a candidate locus for schizophrenia. We tested whether allelic variation at DRD2 and five surrounding loci cosegregated with schizophrenia in 112 small- to moderate-size Irish families containing two or more members affected with schizophrenia or schizoaffective disorder, defined by DSM-III-R. Evidence of linkage was assessed using varying definitions of illness and modes of transmission. Assuming genetic homogeneity, linkage between schizophrenia and large regions of 11q around DRD2 could be strongly excluded. Assuming genetic heterogeneity, variation at the DRD2 locus could be rejected as a major risk factor for schizophrenia in more than 50% of these families for all models tested and in as few as 25% of the families for certain models. The DRD2 linkage in fewer than 25% of these families could not be excluded under any of the models tested. Our results suggest that the major component of genetic susceptibility to schizophrenia is not due to allelic variation at the DRD2 locus or other genes in the surrounding chromosomal region.