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This study investigates the structural distinctiveness of orthosteric ligand-binding sites of several human beta(2) adrenergic receptor (beta(2)-AR) conformations that have been obtained from a set of independent molecular dynamics (MD) simulations in the presence of intracellular loop 3 (ICL3). A docking protocol was established in order to classify each receptor conformation via its binding affinity to selected ligands with known efficacy. This work's main goal was to reveal many subtle features of the ligand-binding site, presenting alternative conformations, which might be considered as either active- or inactive-like but mostly specific for that ligand. Agonists, inverse agonists, and antagonists were docked to each MD conformer with distinct binding pockets, using different docking tools and scoring functions. Mostly favored receptor conformation persistently observed in all docking/scoring evaluations was classified as active or inactive based on the type of ligand's biological effect. Classified MD conformers were further tested for their ability to discriminate agonists from inverse agonists/antagonists, and several conformers were proposed as important targets to be used in virtual screening experiments that were often limited to a single X-ray structure.

G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome and are important therapeutic targets. During the last decade, the number of atomic -resolution structures of GPCRs has increased rapidly, providing insights into drug binding at the molecular level. These breakthroughs have created excitement regarding the potential of using structural information in ligand design and initiated a new era of rational drug discovery for GPCRs. The molecular docking method is now widely applied to model the threedimensional structures of GPCR-ligand complexes and screen for chemical probes in large compound libraries. In this review article, we first summarize the current structural coverage of the GPCR superfamily and the understanding of receptor-ligand interactions at atomic resolution. We then present the general workflow of structure-based virtual screening and strategies to discover GPCR ligands in chemical libraries. We assess the state of the art of this research field by summarizing prospective applications of virtual screening based on experimental structures. Strategies to identify compounds with specific efficacy and selectivity profiles are discussed, illustrating the opportunities and limitations of the molecular docking method. Our overview shows that structure-based virtual screening can discover novel leads and will be essential in pursuing the next generation of GPCR drugs. Significance Statement--Extraordinary advances in the structural biology of G protein-coupled receptors have revealed the molecular details of ligand recognition by this large family of therapeutic targets, providing novel avenues for rational drug design. Structure-based docking is an efficient computational approach to identify novel chemical probes from large compound libraries, which has the potential to accelerate the development of drug candidates.

Amphiphilic agents, called detergents, are invaluable tools for studying membrane proteins. However, membrane proteins encapsulated by conventional head-to-tail detergents tend to denature or aggregate, necessitating the development of structurally distinct molecules with improved efficacy. Here, a novel class of diastereomeric detergents with a cyclopentane core unit, designated cyclopentane-based maltosides (CPMs), were prepared and evaluated for their ability to solubilize and stabilize several model membrane proteins. A couple of CPMs displayed enhanced behavior compared with the benchmark conventional detergent, n-dodecyl-β-d-maltoside (DDM), for all the tested membrane proteins including two G-protein-coupled receptors (GPCRs). Furthermore, CPM-C12 was notable for its ability to confer enhanced membrane protein stability compared with the previously developed conformationally rigid NBMs [J. Am. Chem. Soc. 2017, 139, 3072] and LMNG. The effect of the individual CPMs on protein stability varied depending on both the detergent configuration (cis/trans) and alkyl chain length, allowing us draw conclusions on the detergent structure-property-efficacy relationship. Thus, this study not only provides novel detergent tools useful for membrane protein research but also reports on structural features of the detergents critical for detergent efficacy in stabilizing membrane proteins.

Membrane proteins play critical roles in a variety of cellular processes. For a detailed molecular level understanding of their biological functions and roles in disease, it is necessary to extract them from the native membranes. While the amphipathic nature of these bio-macromolecules presents technical challenges, amphiphilic assistants such as detergents serve as useful tools for membrane protein structural and functional studies. Conventional detergents are limited in their ability to maintain the structural integrity of membrane proteins and thus it is essential to develop novel agents with enhanced properties. Here, we designed and characterized a novel class of amphiphiles with vitamin E (i.e., α-tocopherol) as the hydrophobic tail group and saccharide units as the hydrophilic head group. Designated vitamin E-based glycosides (VEGs), these agents were evaluated for their ability to solubilize and stabilize a set of membrane proteins. VEG representatives not only conferred markedly enhanced stability to a diverse range of membrane proteins compared to conventional detergents, but VEG-3 also showed notable efficacy toward stabilization and visualization of a membrane protein complex. In addition to hydrophile–lipophile balance (HLB) of detergent molecules, the chain length and molecular geometry of the detergent hydrophobic group seem key factors in determining detergent efficacy for membrane protein (complex) stability.

Background Patients with severe asthma have increased airway remodelling and elevated numbers of circulating fibrocytes with enhanced myofibroblastic differentiation capacity, despite being treated with high doses of corticosteroids, and long acting β2-adrenergic receptor (AR) agonists (LABAs). We determined the effect of β2-AR agonists, alone or in combination with corticosteroids, on fibrocyte function. Methods Non-adherent non-T cells from peripheral blood mononuclear cells isolated from healthy subjects and patients with non-severe or severe asthma were treated with the β2-AR agonist, salmeterol, in the presence or absence of the corticosteroid dexamethasone. The number of fibrocytes (collagen I+/CD45+ cells) and differentiating fibrocytes (α-smooth muscle actin+ cells), and the expression of CC chemokine receptor 7 and of β2-AR were determined using flow cytometry. The role of cyclic adenosine monophosphate (cAMP) was elucidated using the cAMP analogue 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) and the phosphodiesterase type IV (PDE4) inhibitor, rolipram. Results Salmeterol reduced the proliferation, myofibroblastic differentiation and CCR7 expression of fibrocytes from healthy subjects and non-severe asthma patients. Fibrocytes from severe asthma patients had a lower baseline surface β2-AR expression and were relatively insensitive to salmeterol but not to 8-Br-cAMP or rolipram. Dexamethasone increased β2-AR expression and enhanced the inhibitory effect of salmeterol on severe asthma fibrocyte differentiation. Conclusions Fibrocytes from patients with severe asthma are relatively insensitive to the inhibitory effects of salmeterol, an effect which is reversed by combination with corticosteroids.

Despite their importance in biology and medicinal chemistry, structural and functional studies of membrane proteins present major challenges. To study diverse membrane proteins, it is crucial to have the correct detergent to efficiently extract and stabilize the proteins from the native membranes for biochemical/biophysical downstream analyses. But many membrane proteins, particularly eukaryotic ones, are recalcitrant to stabilization and/or crystallization with currently available detergents and thus there are major efforts to develop novel detergents with enhanced properties. Here, a novel class of trehalose-cored amphiphiles are introduced, with multiple alkyl chains and carbohydrates projecting from the trehalose core unit are introduced. A few members displayed enhanced protein stabilization behavior compared to the benchmark conventional detergent, n-dodecyl-β-D-maltoside (DDM), for multiple tested membrane proteins: (i) a bacterial leucine transporter (LeuT), (ii) the R. capsulatus photosynthetic superassembly, and (iii) the human β2 adrenergic receptor (β2AR). Due to synthetic convenience and their favourable behaviors for a range of membrane proteins, these agents have potential for membrane protein research. In addition, the detergent property–efficacy relationship discussed here will guide future design of novel detergents.

We prepared an amphiphile with a penta-phenylene lipophilic group and a branched trimaltoside head group. This new agent, designated penta-phenylene maltoside (PPM), showed a marked tendency to self-assembly into micelles via strong aromatic-aromatic interactions in aqueous media, as evidenced by 1 H NMR spectroscopy and fluorescence studies. When utilized for membrane protein studies, this new agent was superior to DDM, a gold standard conventional detergent, in stabilizing multiple proteins long term. The ability of this agent to form aromatic-aromatic interactions is likely responsible for enhanced protein stabilization when associated with a target membrane protein.

G protein-coupled receptors (GPCRs) are central to many fundamental cellular signaling pathways. They transduce signals from the outside to the inside of cells in physiological processes ranging from vision to immune response. It is extremely challenging to look at them individually using conventional experimental techniques. Recently, a pseudo atomistic molecular model has emerged as a valuable tool to access information on GPCRs, more specifically on their interactions with their environment in their native cell membrane and the consequences on their supramolecular organization. This approach uses the Martini coarse grain (CG) model to describe the receptors, lipids, and solvent in molecular dynamics (MD) simulations and in enough detail to allow conserving the chemical specificity of the different molecules. The elimination of unnecessary degrees of freedom has opened up large-scale simulations of the lipid mediated supramolecular organization of GPCRs. Here, after introducing the Martini CGMD method, we review these studies carried out on various members of the GPCR family, including rhodopsin (visual receptor), opioid receptors, adrenergic receptors, adenosine receptors, dopamine receptor, and sphingosine 1-phosphate receptor. These studies have brought to light an interesting set of novel biophysical principles. The insights range from revealing localized and heterogeneous deformations of the membrane bilayer at the surface of the protein, specific interactions of lipid molecules with individual GPCRs, to the effect of the membrane matrix on global GPCR self-assembly. The review ends with an overview of the lessons learned from the use of the CGMD method, the biophysical chemical findings on lipid-protein interplay.

Amphipathic agents are widely used in various fields including biomedical sciences. Micelle-forming detergents are particularly useful for in vitro membrane-protein characterization. As many conventional detergents are limited in their ability to stabilize membrane proteins, it is necessary to develop novel detergents to facilitate membrane-protein research. In the current study, we developed novel trimaltoside detergents with an alkyl pendant-bearing terphenyl unit as a hydrophobic group, designated terphenyl-cored maltosides (TPMs). We found that the geometry of the detergent hydrophobic group substantially impacts detergent self-assembly behavior, as well as detergent efficacy for membrane-protein stabilization. TPM-Vs, with a bent terphenyl group, were superior to the linear counterparts (TPM-Ls) at stabilizing multiple membrane proteins. The favorable protein stabilization efficacy of these bent TPMs is likely associated with a binding mode with membrane proteins distinct from conventional detergents and facial amphiphiles. When compared to n-dodecyl-β-d-maltoside (DDM), most TPMs were superior or comparable to this gold standard detergent at stabilizing membrane proteins. Notably, TPM-L3 was particularly effective at stabilizing the human β2 adrenergic receptor (β2 AR), a G-protein coupled receptor, and its complex with Gs protein. Thus, the current study not only provides novel detergent tools that are useful for membrane-protein study, but also suggests a critical role for detergent hydrophobic group geometry in governing detergent efficacy.

This study aimed to functionally characterize β2-adrenergic (β2AR) and insulin receptor (IR) heteromers in regard to β-arrestin 2 (βarr2) recruitment and cAMP signaling and to examine the involvement of the cytoplasmic portion of the IR β chain in heteromerization with β2AR. Evidence for β2AR:IR:βarr2 complex formation and the specificity of the IR:βarr2 interaction was first provided by bioinfomatics analysis. Receptor-heteromer investigation technology (HIT) then provided functional evidence of β2AR:IR heterodimerization by showing isoproterenol-induced but not insulin-induced GFP2-βarr2 recruitment to the β2AR:IR complex; the IR:βarr2 interaction was found to only be constitutive. The constitutive IR:βarr2 BRET signal (BRETconst) was significantly smaller in cells coexpressing IR-RLuc8 and a GFP2-βarr2 1-185 mutant lacking the proposed IR binding domain. β2AR:IR heteromerization also influenced the pharmacological phenotype of β2AR, i.e., its efficacy in recruiting βarr2 and activating cAMP signaling. Evidence suggesting involvement of the cytoplasmic portion of the IR β chain in the interaction with β2AR was provided by BRET2 saturation and HIT assays using an IR 1-1271 stop mutant lacking the IR C-terminal tail region. For the complex consisting of IR 1-1271-RLuc8:β2AR-GFP2, saturation was not reached, most likely reflecting random collisions between IR 1-1271 and β2AR. Furthermore, in the HIT assay, no substantial agonist-induced increase in the BRET2 signal was detected that would be indicative of βarr2 recruitment to the IR 1-1271:β2AR heteromer. Complementary 3D visualization of β2AR:IR provided supporting evidence for stability of the heterotetramer complex and identified amino acid residues involved in β2AR:IR heteromerization. This article is part of the Special Issue entitled 'Receptor heteromers and their allosteric receptor-receptor interactions'.

Amphiphiles are critical tools for the structural and functional study of membrane proteins. Membrane proteins encapsulated by conventional head-to-tail detergents tend to undergo structural degradation, necessitating the development of structurally novel agents with improved efficacy. In recent years, facial amphiphiles have yielded encouraging results in terms of membrane protein stability. Herein, we report a new facial detergent (i.e., LFA-C4) that confers greater stability to tested membrane proteins than the bola form analogue. Owing to the increased facial property and the adaptability of the detergent micelles in complex with different membrane proteins, LFA-C4 yields increased stability compared to n-dodecyl-β-d-maltoside (DDM). Thus, this study not only describes a novel maltoside detergent with enhanced protein-stabilizing properties, but also shows that the customizable nature of a detergent plays an important role in the stabilization of membrane proteins. Owing to both synthetic convenience and enhanced stabilization efficacy for a range of membrane proteins, the new agent has major potential in membrane protein research.

Rationale: [beta(2)-Agonists are the most common form of treatment of asthma, but there is significant variability in response to these medications. A significant proportion of this responsiveness may be heritable. Objectives: To investigate whether a genome-wide association study (GWAS) could identify novel pharmacogenetic loci in asthma. Methods: We performed a GWAS of acute bronchodilator response (BDR) to inhaled beta(2)-agonists. A total of 444,088 single-nucleotide polymorphisms (SNPs) were examined in 724 individuals from the SNP Health Association Resource (SHARe) Asthma Resource Project (SHARP). The top 50 SNPs were carried forward to replication in a population of 444 individuals. Measurements and Main Results: The combined P value for four SNPs reached statistical genome-wide significance after correcting for multiple comparisons. Combined P values for rs350729, rs1840321, rs1384918, and rs1319797 were 2:21 X 10(-10), 5.75 X 10(-8), 9.3 X 10(-8), 3.95 X 10(-8), respectively. The significant variants all map to a novel genetic region on chromosome 2 neat the ASB3 gene, a region associated with smooth muscle proliferation. As compared with the wild type, the presence of the minor alleles reduced the degree of BDR by 20% in the original population and by a similar percentage in the confirmatory population. Conclusions: These GWAS findings for BDR in subjects with asthma suggest that a gene associated with smooth muscle proliferation may influence a proportion of the smooth muscle relaxation that occurs in asthma.

The critical contribution of membrane proteins in normal cellular function makes their detailed structure and functional analysis essential. Detergents, amphipathic agents with the ability to maintain membrane proteins in a soluble state in aqueous solution, have key roles in membrane protein manipulation. Structural and functional stability is a prerequisite for biophysical characterization. However, many conventional detergents are limited in their ability to stabilize membrane proteins, making development of novel detergents for membrane protein manipulation an important research area. The architecture of a detergent hydrophobic group, that directly interacts with the hydrophobic segment of membrane proteins, is a key factor in dictating their efficacy for both membrane protein solubilization and stabilization. In the current study, we developed two sets of maltoside-based detergents with four alkyl chains by introducing dendronic hydrophobic groups connected to a trimaltoside head group, designated dendronic trimaltosides (DTMs). Representative DTMs conferred enhanced stabilization to multiple membrane proteins compared to the benchmark conventional detergent, DDM. One DTM (i.e., DTM-A6) clearly outperformed DDM in stabilizing human β2 adrenergic receptor (β2AR) and its complex with Gs protein. A further evaluation of this DTM led to a clear visualization of β2AR-Gs complex via electron microscopic analysis. Thus, the current study not only provides novel detergent tools useful for membrane protein study, but also suggests that the dendronic architecture has a role in governing detergent efficacy for membrane protein stabilization.

Detergents are essential tools for functional and structural studies of membrane proteins. However, conventional detergents are limited in their scope and utility, particularly for eukaryotic membrane proteins. Thus, there are major efforts to develop new amphipathic agents with enhanced properties. Here, a novel class of diastereomeric agents with a preorganized conformation, designated norbornane-based maltosides (NBMs), were prepared and evaluated for their ability to solubilize and stabilize membrane proteins. Representative NBMs displayed enhanced behaviors compared to n-dodecyl-β-d-maltoside (DDM) for all membrane proteins tested. Efficacy of the individual NBMs varied depending on the overall detergent shape and alkyl chain length. Specifically, NBMs with no kink in the lipophilic region conferred greater stability to the proteins than NBMs with a kink. In addition, long alkyl chain NBMs were generally better at stabilizing membrane proteins than short alkyl chain agents. Furthermore, use of one well-behaving NBM enabled us to attain a marked stabilization and clear visualization of a challenging membrane protein complex using electron microscopy. Thus, this study not only describes novel maltoside detergents with enhanced protein-stabilizing properties but also suggests that overall detergent geometry has an important role in determining membrane protein stability. Notably, this is the first systematic study on the effect of detergent kinking on micellar properties and associated membrane protein stability.

L-Lype Ca2+ channels (LTCC) play a crucial role in cardiac excitation-contraction coupling. We previously found that in failing ventricular myocytes of mice chronically treated with isoproterenol, basal t-tubular (TT) LTCC activity was halved by activation of protein phosphatase (PP)2A whereas basal surface sarcolemmal (SS) LTCC activity was doubled by inhibition of PP1. Interestingly, chronic treatment of these mice with pertussis toxin almost completely normalized TT and SS LTCC densities and cardiac contractility. In the present study, we therefore sought to identify the G(i/o) protein coupled receptors in cardiac myocytes (i.e. beta(2)-adrenergic, M-2-muscarinic and A(1)-adenosine receptors) that are responsible for these abnormalities in heart failure by chronically administrating mice a selective antagonist of each receptor (ICI118,551, atropine and 8-cyclopentyl-1,3-dipropilxanthine (DPCPX), respectively) with isoproterenol. Compared with mice treated with isoproterenol alone, mice treated with isoproterenol plus ICI118,551 or atropine, but not DPCPX showed significantly lower lung weight/tibial length, higher fractional shortening, lower left ventricular end-diastolic pressure and higher dP/dt(max) and dP/dt(min). In addition, ventricular myocytes of mice treated with isoproterenol plus ICI118,551 or atropine, but not DPCPX exhibited significantly higher TT and lower SS LTCC current densities than those of mice treated with isoproterenol alone due to normalization of the PP activities. These results indicate that beta(2)-adrenergic, M-2-muscarinic, but not A(1)-adenosine receptors contribute to reduced ventricular contractility at least partially by decreasing basal TT LTCC activity in heart failure. Therefore, antagonists of beta(2)-alrenergic and/or M-2-muscarinic receptors can be good adjuncts to beta(1)-adrenergic receptor antagonists in the treatment of heart failure., Article, EUROPEAN JOURNAL OF PHARMACOLOGY. 724:122-131 (2014)

G protein-coupled receptors (GPCRs) are central to many fundamental cellular signaling pathways. They transduce signals from the outside to the inside of cells in physiological processes ranging from vision to immune response. It is extremely challenging to look at them individually using conventional experimental techniques. Recently, a pseudo atomistic molecular model has emerged as a valuable tool to access information on GPCRs, more specifically on their interactions with their environment in their native cell membrane and the consequences on their supramolecular organization. This approach uses the Martini coarse grain (CG) model to describe the receptors, lipids, and solvent in molecular dynamics (MD) simulations and in enough detail to allow conserving the chemical specificity of the different molecules. The elimination of unnecessary degrees of freedom has opened up large-scale simulations of the lipidmediated supramolecular organization of GPCRs. Here, after introducing the Martini CGMD method, we review these studies carried out on various members of the GPCR family, including rhodopsin (visual receptor), opioid receptors, adrenergic receptors, adenosine receptors, dopamine receptor, and sphingosine 1-phosphate receptor. These studies have brought to light an interesting set of novel biophysical principles. The insights range from revealing localized and heterogeneous deformations of the membrane bilayer at the surface of the protein, specific interactions of lipid molecules with individual GPCRs, to the effect of the membrane matrix on global GPCR self-assembly. The review ends with an overview of the lessons learned from the use of the CGMD method, the biophysical-chemical findings on lipid-protein interplay.

Amphiphile selection is a crucial step in membrane protein structural and functional study. As conventional detergents have limited scope and utility, novel agents with enhanced efficacy need to be developed. Although a large number of novel agents have been reported, so far there has been no systematically designed comparative study of the protein stabilization efficacy of stereo-isomeric amphiphiles. Here we designed and prepared a novel class of stereo-isomeric amphiphiles, designated butane-1,2,3,4-tetraol-based maltosides (BTMs). These stereoisomers showed markedly different behaviour for most of the targeted membrane proteins depending on the chirality of the linker region. These findings indicate an important role for detergent stereochemistry in membrane protein stabilization. In addition, we generally observed enhanced detergent efficacy with increasing alkyl chain length, reinforcing the importance of the balance between hydrophobicity and hydrophilicity in detergent design. The stereo-isomeric difference in detergent efficacy observed provides an important design principle for the development of novel amphiphiles for membrane protein manipulation. Amphiphile selection is a crucial step in membrane protein structural and functional study. As conventional detergents have limited scope and utility, novel agents with enhanced efficacy need to be developed. Although a large number of novel agents have been reported, so far there has been no systematically designed comparative study of the protein stabilization efficacy of stereo-isomeric amphiphiles. Here we designed and prepared a novel class of stereo-isomeric amphiphiles, designated butane-1,2,3,4-tetraol-based maltosides (BTMs). These stereoisomers showed markedly different behaviour for most of the targeted membrane proteins depending on the chirality of the linker region. These findings indicate an important role for detergent stereochemistry in membrane protein stabilization. In addition, we generally observed enhanced detergent efficacy with increasing alkyl chain length, reinforcing the importance of the balance between hydrophobicity and hydrophilicity in detergent design. The stereo-isomeric difference in detergent efficacy observed provides an important design principle for the development of novel amphiphiles for membrane protein manipulation.

Septic shock remains a global health challenge with millions of cases every year, high rates of mortality and morbidity, impaired quality of life among survivors and relatives, and high resource use both in developed and developing nations. Care and outcomes are improving through organisational initiatives and updated clinical practice guidelines based on clinical research mainly carried out by large collaborative networks. This progress is likely to continue through the collaborative work of the established and merging trials groups in many parts of the world and through refined trial methodology and translational work. In this review, international experts summarize the current position of clinical research in septic shock and propose a research agenda to advance this field.

Detergents are essential tools for membrane protein manipulation. Micelles formed by detergent molecules have the ability to encapsulate the hydrophobic domains of membrane proteins. The resulting protein-detergent complexes (PDCs) are compatible with the polar environments of aqueous media, making structural and functional analysis feasible. Although a number of novel agents have been developed to overcome the limitations of conventional detergents, most of them have traditional head groups such as glucoside or maltoside. In this study, we introduce a class of amphiphiles, the PSA’Es with a novel highly branched penta-saccharide hydrophilic group. The PSA’Es conferred markedly increased stability to a diverse range of membrane proteins compared to conventional detergents, indicating a positive role for the new hydrophilic group in maintaining the native protein integrity. In addition, PDCs formed by PSA’Es were smaller and more suitable for electron microscopic analysis than those formed by DDM, indicating that the new agents have significant potential for the structure-function studies of membrane proteins.

Catecholamines exert their physiological actions through α and β adrenergic receptors (ARs). As ARs are not exclusively expressed on neuroendocrine cells, but also on leukocytes, they may facilitate neuroendocrine modulation of immune responses. We sequenced the β(2a)-AR in common carp, and studied its expression profile and involvement in the regulation of teleost innate immune responses. β(2a)-AR messenger RNA was found to be constitutively expressed in brain areas, especially in the preoptic nucleus (NPO, homologous to the mammalian hypothalamus), and in immune organs. During the active phase of an in vivo inflammatory response, induced by i.p. zymosan treatment, β(2a)-AR gene expression was up-regulated in the peritoneal leukocytes. Additionally, adrenaline in vitro reduced the synthesis of oxygen radical species and nitric oxide, while it enhanced arginase activity in fish phagocytes. Furthermore, in vitro adrenaline administration inhibited expression of pro-inflammatory cytokines, chemokines and their receptors. It is therefore hypothesized that adrenaline will down-regulate phagocyte skewing toward classical/innate polarization.

Background This study investigates the allosteric coupling that exists between the intra- and extracellular parts of human β2-adrenergic receptor (β2-AR), in the presence of the intracellular loop 3 (ICL3), which is missing in all crystallographic experiments and most of the simulation studies reported so far. Our recent 1 μs long MD run has revealed a transition to the so-called very inactive state of the receptor, in which ICL3 packed under the G protein’s binding cavity and completely blocked its accessibility to G protein. Simultaneously, an outward tilt of transmembrane helix 5 (TM5) caused an expansion of the extracellular ligand-binding site. In the current study, we performed independent runs with a total duration of 4 μs to further investigate the very inactive state with packed ICL3 and the allosteric coupling event (three unrestrained runs and five runs with bond restraints at the ligand-binding site). Results In all three independent unrestrained runs (each 500 ns long), ICL3 preserved its initially packed/closed conformation within the studied time frame, suggesting an inhibition of the receptor’s activity. Specific bond restraints were later imposed between some key residues at the ligand-binding site, which have been experimentally determined to interact with the ligand. Restraining the binding site region to an open state facilitated ICL3 closure, whereas a relatively constrained/closed binding site hindered ICL3 packing. However, the reverse operation, i.e. opening of the packed ICL3, could not be realized by restraining the binding site region to a closed state. Thus, any attempt failed to free the ICL3 from its locked state due to the presence of persistent hydrogen bonds. Conclusions Overall, our simulations indicated that starting with very inactive states, the receptor stayed almost irreversibly inhibited, which in turn decreased the overall mobility of the receptor. Bond restraints which represented the geometric restrictions caused by ligands of various sizes when bound at the ligand-binding site, induced the expected conformational changes in TM5, TM6 and consequently, ICL3. Still, once ICL3 was packed, the allosteric coupling became ineffective due to strong hydrogen bonds connecting ICL3 to the core of the receptor. Electronic supplementary material The online version of this article (doi:10.1186/s12900-016-0061-9) contains supplementary material, which is available to authorized users.

Background New evidence has suggested that people with asthma who are homozygous for arginine at aminoacid 16 of the beta(2)-adrenergic receptor (ADRB2) might not benefit from longacting beta(2)-agonist therapy. We, therefore, investigated whether ADRB2 polymorphisms affect response to longacting beta(2)-agonists in combination with inhaled corticosteroids.Methods Asthmatics were stratified by ADRB2 genotype in two studies to assess the effects of inhaled corticosteroids plus longacting beta(2)-agonists on asthma exacerbations. In study 1 (double-blind), 2250 asthmatics were randomly assigned to budesonide plus formoterol maintenance and reliever therapy, fixed-dose budesonide plus formoterol, or fixed-dose fluticasone plus salmeterol for 6 months. Study 2 (open-label) consisted of 405 asthmatics and compared an adjustable regimen of budesonide plus formoterol with fixed-dose budesonide plus formoterol and fixed-dose fluticasone plus salmeterol for 7 months. The relation between ADRB2 polymorphism, severe asthma exacerbations, and other asthma outcomes was analysed. Primary endpoints for studies 1 and 2 were severe asthma exacerbation and asthma control as assessed by measures of exacerbations, respectively.Findings In study 1, Gly16Arg genotype had no effect on the percentage of participants with severe exacerbations across all treatment groups (99 [12%] of 833 Gly/Gly, 110 [11%] of 1028 Gly/Arg, and 32 [9%] of 361 Arg/Arg participants). Secondary endpoints, including forced expiratory volume in 1 s, peak expiratory flow, use of as-needed medication, and number of nights with awakenings were similar between genotype groups. No relation was recorded between ADRB2 haplotype and primary and secondary endpoints. In study 2, the frequency of asthma exacerbations (15 [9%] of 168 Gly/Gly, 13 [8%] of 169 Gly/Arg, and 6 [9%] of 67 Arg/Arg participants) and other study endpoints were closely similar for all ADRB2 genotypes.Interpretation Since we showed no pharmacogenetic effect of ADRB2 variation on therapeutic response in asthma, patients, irrespective of their genotype, can continue to receive inhaled corticosteroids plus longacting beta(2)-agonists.

The primary molecular target for clinically used opioids is the μ-opioid receptor (MOR). Besides the major seven-transmembrane (7TM) receptors, the MOR gene codes for alternatively spliced six-transmembrane (6TM) isoforms, the biological and clinical significance of which remains unclear. Here, we show that the otherwise exclusively intracellular localized 6TM-MOR translocates to the plasma membrane upon coexpression with β2-adrenergic receptors (β2-ARs) through an interaction with the fifth and sixth helices of β2-AR. Coexpression of the two receptors in BE(2)-C neuroblastoma cells potentiates calcium responses to a 6TM-MOR ligand and this calcium response is completely blocked by a selective β2-antagonist in BE(2)-C cells and in trigeminal and dorsal root ganglia. Co-administration of 6TM-MOR and β2-AR ligands leads to substantial analgesic synergy and completely reverses opioid-induced hyperalgesia in rodent behavioral models. Together, our results provide evidence that the heterodimerization of 6TM-MOR with β2-AR underlies a molecular mechanism for 6TM cellular signaling, presenting a unique functional responses to opioids. This signaling pathway may contribute to the hyperalgesic effects of opioids that can be efficiently blocked by β2-AR antagonists, providing a new avenue for opioid therapy.

Rationale: [beta(2)-Agonists are the most common form of treatment of asthma, but there is significant variability in response to these medications. A significant proportion of this responsiveness may be heritable. Objectives: To investigate whether a genome-wide association study (GWAS) could identify novel pharmacogenetic loci in asthma. Methods: We performed a GWAS of acute bronchodilator response (BDR) to inhaled beta(2)-agonists. A total of 444,088 single-nucleotide polymorphisms (SNPs) were examined in 724 individuals from the SNP Health Association Resource (SHARe) Asthma Resource Project (SHARP). The top 50 SNPs were carried forward to replication in a population of 444 individuals. Measurements and Main Results: The combined P value for four SNPs reached statistical genome-wide significance after correcting for multiple comparisons. Combined P values for rs350729, rs1840321, rs1384918, and rs1319797 were 2:21 X 10(-10), 5.75 X 10(-8), 9.3 X 10(-8), 3.95 X 10(-8), respectively. The significant variants all map to a novel genetic region on chromosome 2 neat the ASB3 gene, a region associated with smooth muscle proliferation. As compared with the wild type, the presence of the minor alleles reduced the degree of BDR by 20% in the original population and by a similar percentage in the confirmatory population. Conclusions: These GWAS findings for BDR in subjects with asthma suggest that a gene associated with smooth muscle proliferation may influence a proportion of the smooth muscle relaxation that occurs in asthma.

Adenylyl cyclase (AC) mediates signalling following activation of G(alphas)-coupled receptors such as the beta(2)- adrenergic receptor (PAR). Genetic variation in the receptor component of this pathway can alter signal transduction and the response to beta-agonists; in asthma, but little is known about downstream effectors. Here, we characterize the population genomics and signalling effects of a polymorphism within the coding region of the AC9 gene that results in an lie to Met substitution at amino acid 772 within the C1b region of the enzyme. Allele frequencies were 0.300 and 0.375 in Caucasians and Asians but were lower in African-Americans (0.163). The functional effects were studied in stably transfected HEK293 cells recombinantly expressing equivalent levels of wild-type (Ile(772)) and polymorphic (Met(772)) AC9. The polymorphic substitution results in a loss of function compared to wild-type AC9. Met(772) AC9 has lower basal and beta(2)AR-mediated adenylyl cyclase activities compared to Ile(772) AC9, as well as reduced activity following stimulation of G(alphas) by NaF. Direct stimulation of AC9 activity by Mn2+/- was also depressed in Met(772) membranes, indicating decreased catalytic function, consistent with the location of residue 772. AC9 mRNA and protein were expressed in multiple human lung cell-types, including airway smooth muscle and airway epithelium. In the treatment of asthma, there is marked heterogeneity in the response to inhaled beta-agonists which is associated with polymorphisms of the beta(2)AR. Identification of a common AC9 variant that confers reduced enzyme activity reveals an additional polymorphism that should be considered in pharmacogenetic studies of beta-agonist therapy of asthma. (C) 2003 Lippincott Williams Wilkins.

In this paper we propose a multivariate extension of family-based association tests based on generalized estimating equations. The test can be applied to multiple phenotypes and to phenotypic data obtained in longitudinal studies without making any distributional assumptions for the phenotypic observations. Methods for handling missing phenotypic information are discussed. Further, we compare the power of the multivariate test with permutation tests and with using separate tests for each outcome which are adjusted for multiple testing. Application of the proposed test to an asthma study illustrates the power of the approach.

We introduce a new powerful nonparametric testing strategy for family-based association studies in which multiple quantitative traits are recorded and the phenotype with the strongest genetic component is not known prior to the analysis. In the first stage, using a population-based test based on the generalized estimating equation approach, we test all recorded phenotypes for association with the marker locus without biasing the nominal significance level of the later family-based analysis. In the second stage the phenotype with the smallest p value is selected and tested by a family-based association test for association with the marker locus. This strategy is robust against population admixture and stratification and does not require any adjustment for multiple testing. We demonstrate the advantages of this testing strategy over standard methodology in a simulation study. The practical importance of our testing strategy is illustrated by applications to the Childhood Asthma Management Program asthma data sets. Copyright (C) 2003 S. Karger AG, Basel.

Peptide- and protein-protein dockings were carried out on beta(2)-adrenergic receptor (beta(2)AR) to confirm the presence of transmembrane helix 6 (TM6) at the interface region between two beta(2)AR monomers, thereby its possible role in dimerization as suggested in numerous experimental and computational studies. Initially, a portion of TM6 was modeled as a peptide consisting of 23 residues and blindly docked to beta(2)AR monomer using a rigid body approach. Interestingly, all highest score conformations preferred to be near TM5 and TM6 regions of the receptor. Furthermore, longer peptides generated from a whole TM region were blindly docked to beta(2)AR using the same rigid body approach. This yielded a total of seven docked peptides, each derived from one TM helix. Most interestingly, for each peptide, TM6 was among the most preferred binding site region in the receptor. Besides the peptide dockings, two beta(2)AR monomers were blindly docked to each other using a full rigid-body search of docking orientations, which yielded a total of 16,000 dimer conformations. Each dimer was then filtered according to a fitness value based on the membrane topology. Among 149 complexes that met the topology requirements, 102 conformers were composed of two monomers oriented in opposite directions, whereas in the remaining 47, the monomers were arranged in parallel. Lastly, all 149 conformers were clustered based on a root mean-squared distance value of 6 angstrom. In agreement with the peptide results, the clustering yielded the largest population of conformers with the highest Z-score value having TM6 at the interface region.

International audience; Olfactory receptors (ORs) play a crucial role in detecting the odorant molecules present in the surrounding environment. These receptors, which belong to class A G-protein-coupled receptors, constitute the largest transmembrane protein family in the human genome. Functional studies showed that the OR family includes members that are able to respond to a large set of odorants and members that are activated by a relatively small number of related odorants. To understand the molecular mechanisms that govern the receptor-ligand interactions, we overexpressed the human OR hOR1A1 in a stable tetracycline-inducible HEK293S cell line. This receptor was engineered by inserting a C-terminal rholD4 epitope tag and an N-terminal FLAG epitope tag to allow its purification and detection. The functional activity of the FLAG-rholD4-tagged hOR1A1 in heterologous HEK293S cells was analysed using a real-time cAMP assay. A two-step purification using monoclonal anti-FLAG immunoaffinity purification and gel filtration was then employed to purify the detergent-solubilized receptor. A size exclusion chromatography-multi-angle light scattering analysis showed the presence of monomeric and dimeric forms of FLAG-rholD4-tagged hOR1A1. The amounts of the monomeric and dimeric forms purified from sixty T175 flasks were approximately 1.6 and 1.1 mg, respectively. The circular dichroism analysis showed that the purified receptor was properly folded. Ligand binding was quantified using an intrinsic tryptophan fluorescence assay and revealed that the detergent-solubilized FLAG-rholD4-tagged hOR1A1 bound its cognate odorant, dihydrojasmone, with an affinity in the micromolar range. These results pave the way for future crystallographic and NMR studies.

Novel high affinity compounds for human beta(2)-adrenergic receptor (beta(2)-AR) were searched among the clean drug-like subset of ZINC database consisting of 9,928,465 molecules that satisfy the Lipinski's rule of five. The screening protocol consisted of a high-throughput pharmacophore screening followed by an extensive amount of docking and rescoring. The pharmacophore model was composed of key features shared by all five inactive states of beta(2)-AR in complex with inverse agonists and antagonists. To test the discriminatory power of the pharmacophore model, a small-scale screening was initially performed on a database consisting of 117 compounds of which 53 antagonists were taken as active inhibitors and 64 agonists as inactive inhibitors. Accordingly, 7.3% of the ZINC database subset (729,413 compounds) satisfied the pharmacophore requirements, along with 44 antagonists and 17 agonists. Afterwards, all these hit compounds were docked to the inactive apo form of the receptor using various docking and scoring protocols. Following each docking experiment, the best pose was further evaluated based on the existence of key residues for antagonist binding in its vicinity. After final evaluations based on the human intestinal absorption (HIA) and the blood brain barrier (BBB) penetration properties, 62 hit compounds have been clustered based on their structural similarity and as a result four scaffolds were revealed. Two of these scaffolds were also observed in three high affinity compounds with experimentally known K-i values. Moreover, novel chemical compounds with distinct structures have been determined as potential beta(2)-AR drug candidates. (C) 2014 Elsevier Inc. All rights reserved.

Prolonged (18 h) incubation of isolated bovine tracheal smooth muscle with the beta (2)-adrenoceptor agonist fenoterol (10 muM) induced desensitization of isoprenaline-induced adenylyl cyclase activity in bovine tracheal smooth muscle membranes, characterized by a 25% decrease in maximal effect (E-max) (P

1 According to the two state receptor model, the beta (2)-adrenergic receptor (beta (2)-AR) isomerizes between an inactive state and a constitutively active state, which couples to the stimulatory G-protein in the absence of agonist. In bovine tracheal smooth muscle (BTSM), we investigated the effect of short and long term beta (2)-AR activation by fenoterol on constitutive receptor activity.2 Preincubation of the BTSM strips for 5 min, 30 min and 18 h with 10 muM fenoterol, followed by extensive washout (3 h, 37 degreesC), caused a rapid and time-dependent inhibition of KCl-induced contraction, reaching 68 +/- 10, 51 +/- 6 and 46 +/- 4% of control, respectively, at 40 mM KCI (P 3 Preincubation of BTSM with 0.1, 1.0 and 10 M fenoterol during 18 h caused a concentration-dependent decrease of the 40 mM KCI response to 70 +/- 5, 47 +/- 12 and 43 +/- 9% of control, respectively (P 4 The reduced KCI contractions were reversed in the presence of 1 M timolol. Moreover, the sensitivity to KCl in the presence of timolol was enhanced after fenoterol incubation. Inverse agonism was also found for other beta -blockers, with a rank order of efficacy of pindolol greater than or equal to timolol = propranolol > alprenolol greater than or equal to sotalol > labetalol.5 At 25 mM KCI-induced tone, the contraction induced by cumulative timolol administration was competitively antagonized by the less efficacious inverse agonist labetalol, indicating that the fenoterol-induced effects cannot be explained by residual beta -agonist binding.6 In conclusion, fenoterol treatment of BTSM causes a time- and concentration-dependent development of constitutive beta (2)-AR activity, which can be reversed by various inverse agonists. The beta -agonist-induced changes could represent a novel regulation mechanism of beta (2)-AR activity.

There is a major interest in investigating the genetic components of allergy and asthma. Four different areas are involved in the study of complex genetic diseases: family studies, assessment of phenotype, segregation analysis and gene mapping. initial assessment of phenotype must be practical, reproducible and relatively independent of compounding variables. Phenotypes important in allergy and asthma include atopic parameters such as total serum IgE, bronchial hyper-responsiveness and the presence/absence of clinical asthma. Numerous family and twin studies have suggested the presence of a heritable component for allergy, bronchial hyper-responsiveness and asthma. The number of genes involved in these complex genetic disorders and their mode of inheritance have not been fully determined. Our group has been involved in a collaborative US-Dutch study in which 92 families with over 500 individuals have been phenotyped and DNA has been obtained for genotyping. initial results of the classification of family members show that approximately 26% of the offspring of families ascertained through a parent with asthma have an asthmatic phenotype. A large number of these offspring with clinical evidence of asthma do not have a prior physician diagnosis of asthma, suggesting that there is a spectrum which ranges from preclinical to symptomatic asthma. The familial aggregation of asthma and other obstructive airway diseases in these families is consistent with a significant genetic component. Initial linkage studies have been performed on two characteristics of the allergic and asthmatic phenotype. Total serum IEE was analysed because this measure correlates with the clinical repression of allergy, bronchial hyper-responsiveness and asthma. Segregation analysis of total serum IgE provided evidence for a recessive mode of inheritance. Sib pair analyses and maximum Likelihood scores suggest that a gene regulating IgE production maps to chromosome 5q. Bronchial hyper-responsivenss and total serum IgE are related to asthma in population-based studies. Sib pair analyses for bronchial responsiveness showed significant Linkage to markers on chromosome 5q.

Carvedilol, a nonselective β-blocker, may be more effective than the selective β-blocker metoprolol in reducing the risk of thromboembolic events in heart failure. The aim of this study was, first, to assess whether there is a differential response in cardiac sympathetic activity by123I-meta-iodobenzylguanidine (123I-MIBG) imaging when either β-blocker is used. Second, we assessed whether that response correlates with levels of various serum factors that serve as markers for coagulability. Methods: In this prospective, randomized, open-label crossover study with masked outcome assessments, stable heart failure patients (left ventricular ejection fraction

Bronchodilator response (BDR) is an important asthma phenotype that measures reversibility of airway obstruction by comparing lung function (i.e. FEV1) before and after the administration of a short-acting β2-agonist, the most common rescue medications used for the treatment of asthma. BDR also serves as a test of β2-agonist efficacy. BDR is a complex trait that is partly under genetic control. A genome-wide association study (GWAS) of BDR, quantified as percent change in baseline FEV1 after administration of a β2-agonist, was performed with 1,644 non-Hispanic white asthmatic subjects from six drug clinical trials: CAMP, LOCCS, LODO, a medication trial conducted by Sepracor, CARE, and ACRN. Data for 469,884 single-nucleotide polymorphisms (SNPs) were used to measure the association of SNPs with BDR using a linear regression model, while adjusting for age, sex, and height. Replication of primary P-values was attempted in 501 white subjects from SARP and 550 white subjects from DAG. Experimental evidence supporting the top gene was obtained via siRNA knockdown and Western blotting analyses. The lowest overall combined P-value was 9.7E-07 for SNP rs295137, near the SPATS2L gene. Among subjects in the primary analysis, those with rs295137 TT genotype had a median BDR of 16.0 (IQR = [6.2, 32.4]), while those with CC or TC genotypes had a median BDR of 10.9 (IQR = [5.0, 22.2]). SPATS2L mRNA knockdown resulted in increased β2-adrenergic receptor levels. Our results suggest that SPATS2L may be an important regulator of β2-adrenergic receptor down-regulation and that there is promise in gaining a better understanding of the biological mechanisms of differential response to β2-agonists through GWAS., Author Summary Bronchodilator response (BDR) is an important asthma phenotype that measures reversibility of airway obstruction by comparing lung function before and after the administration of short-acting β2-agonists, common medications used for asthma treatment. We performed a genome-wide association study of BDR with 1,644 white asthmatic subjects from six drug clinical trials and attempted to replicate these findings in 1,051 white subjects from two independent cohorts. The most significant associated variant was near the SPATS2L gene. We knocked down SPATS2L mRNA in human airway smooth muscle cells and found that β2-adrenergic receptor levels increased, suggesting that SPATS2L may be a regulator of BDR. Our results highlight the promise of pursuing GWAS results that do not necessarily reach genome-wide significance and are an example of how results from pharmacogenetic GWAS can be studied functionally.

Ligand-receptor interactions are at the basis of the mediation of our physiological responses to a large variety of ligands, such as hormones, neurotransmitters and environmental stimulants, and their tuning represents the goal of a large variety of therapies. Several molecular details of these interactions are still largely unknown. In an effort to shed some light on this important issue, we performed a computational study on the interaction of two related compounds differing by a single methyl group (clozapine and desmethylclozapine) with a [Formula: see text]-opioid receptor. According to experiments, desmethylclozapine is more active than clozapine, providing a system well suited for a comparative study. We investigated stable configurations of the two drugs inside the receptor by simulating their escape routes by molecular dynamics simulations. Our results point out that the action of the compounds might be related to the spatial and temporal distribution of the affinity sites they visit during their permanency. Moreover, no particularly pronounced structural perturbations of the receptor were detected during the simulations, reinforcing the idea of a strong dynamical character of the interaction process, with an important role played by the solvent in addition.

G protein coupled receptors (GPCRs) are a large eukaryotic protein family of transmembrane receptors that react to a signal coming from the extracellular environment to generate an intracellular response through the activation of a signal transduction pathway mediated by a heterotrimeric G protein. Their diversity, dictated by the multiplicity of stimuli to which they respond and by the variety of intracellular signalling pathways they activate, make them one of the most prominent families of validated pharmacological targets in biomedicine. In recent years, major breakthroughs in structure determination of GPCRs have given new stimuli to the exploration of the biology of these proteins, providing a structural basis to understand the molecular origin of GPCR mechanisms of action. Based on the information coming from these structural studies, a number of recent in silico investigations used molecular dynamics (MD) simulations to contribute to our knowledge of GPCRs. In this review, we will focus on investigations that, taking advantage of the tremendous progress in both hardware and software, made testable hypotheses that have been validated by subsequent structural studies. These state-of-the-art molecular simulations highlight the potential of microsecond MD simulations as a valuable tool in GPCR structural biology and biophysics.

Tsianos GI, Evangelou E, Boot A, Zillikens MC, van Meurs JB, Uitterlinden AG, Ioannidis JP. Associations of polymorphisms of eight muscle-or metabolism-related genes with performance in Mount Olympus marathon runners. J Appl Physiol 108: 567-574, 2010. First published December 31, 2009; doi:10.1152/japplphysiol.00780.2009.-Athletic endurance performance is probably partly under genetic control, but genetic association studies have yielded inconclusive results. The objective of the present study was to evaluate the association of polymorphisms in eight muscle-or metabolism-related genes with endurance performance in participants of the Olympus Marathon running race. We recruited 438 athletes who participated in the 2007 and 2008 annual running events of the Olympus Marathon: a 43.8-km race with an ascent from sea level to 2,690-m altitude and then a descent to 300 m. Phenotypes of interest were the competitive event time at the specific Olympus Marathon where the athlete was enrolled, the fastest reported timing ever achieved in an Olympus Marathon, and how many kilometers per week the athlete ran during the previous year. Eleven polymorphisms in alpha(3)-actinin (ACTN3), AMP deaminase-1 (AMPD1), bradykinin B(2) receptor (BDKRB2), beta(2)-adrenergic receptor (ADRB2), peroxisome proliferator-activated receptor (PPAR)-gamma coactivator-1 alpha (PPARGC1A), PPAR-gamma (PPARA), PPAR-gamma (PPARD), and apoliprotein E (APOE) were evaluated. Hardy-Weinberg equilibrium testing on the overall cohort of male athletes showed a significant deviation for BDKRB2 rs1799722 (P = 0.018; P = 0.006 when limited to 316 habitual male runners) with an excess of the TT genotype. Across all athletes, no associations showed nominal statistical significance for any of the three phenotypes, and the same was true when analyses were limited to men (n = 417). When limited to 316 male athletes who identified running as their preferred sport, ADRB2 rs1042713 had nominally significant associations with faster times for the minor (A) allele for the fastest time ever (P = 0.01). The direction of effect was identical as previously postulated only for BDKRB2 rs1799722 and ADRB2 rs1042713, indicating consistency. BDKRB2 rs1799722 and ADRB2 rs1042713 have some support for being implicated in endurance performance among habitual runners and require further investigation. Journal of Applied Physiology

Background New evidence has suggested that people with asthma who are homozygous for arginine at aminoacid 16 of the beta(2)-adrenergic receptor (ADRB2) might not benefit from longacting beta(2)-agonist therapy. We, therefore, investigated whether ADRB2 polymorphisms affect response to longacting beta(2)-agonists in combination with inhaled corticosteroids. Methods Asthmatics were stratified by ADRB2 genotype in two studies to assess the effects of inhaled corticosteroids plus longacting beta(2)-agonists on asthma exacerbations. In study 1 (double-blind), 2250 asthmatics were randomly assigned to budesonide plus formoterol maintenance and reliever therapy, fixed-dose budesonide plus formoterol, or fixed-dose fluticasone plus salmeterol for 6 months. Study 2 (open-label) consisted of 405 asthmatics and compared an adjustable regimen of budesonide plus formoterol with fixed-dose budesonide plus formoterol and fixed-dose fluticasone plus salmeterol for 7 months. The relation between ADRB2 polymorphism, severe asthma exacerbations, and other asthma outcomes was analysed. Primary endpoints for studies 1 and 2 were severe asthma exacerbation and asthma control as assessed by measures of exacerbations, respectively. Findings In study 1, Gly16Arg genotype had no effect on the percentage of participants with severe exacerbations across all treatment groups (99 [12%] of 833 Gly/Gly, 110 [11%] of 1028 Gly/Arg, and 32 [9%] of 361 Arg/Arg participants). Secondary endpoints, including forced expiratory volume in 1 s, peak expiratory flow, use of as-needed medication, and number of nights with awakenings were similar between genotype groups. No relation was recorded between ADRB2 haplotype and primary and secondary endpoints. In study 2, the frequency of asthma exacerbations (15 [9%] of 168 Gly/Gly, 13 [8%] of 169 Gly/Arg, and 6 [9%] of 67 Arg/Arg participants) and other study endpoints were closely similar for all ADRB2 genotypes. Interpretation Since we showed no pharmacogenetic effect of ADRB2 variation on therapeutic response in asthma, patients, irrespective of their genotype, can continue to receive inhaled corticosteroids plus longacting beta(2)-agonists.