Heterocyclic acetamide and benzamide derivatives as potent and selective β3-adrenergic receptor agonists with improved rodent pharmacokinetic profiles

A series of amide derived β₃-adrenergic receptor (AR) agonists is described. The discovery and optimization of several series of compounds derived from 1, is used to lay the SAR foundation for second generation β₃-AR agonists for the treatment of overactive bladder.     

The role of β-adrenergic receptor signaling in the proliferation of hemangioma-derived endothelial cells

Background

Infantile hemangioma (IH) is a benign vascular neoplasm that arises from the abnormal proliferation of endothelial cells and enhanced angiogenesis. Recently, propranolol has been found to be effective in the management of IH, suggesting that β-adrenergic receptors (β-ARs) may play an important role in the pathogenesis of IH.

Results

In the present study, we investigated the β-adrenergic signaling that is associated with hemangioma-derived endothelial cell (HemEC) proliferation. The results showed that both β₁- and β₂-ARs were expressed in HemECs. Stimulation of the β-ARs by isoprenaline induced cell proliferation and elevation of second messenger cAMP levels. The proliferation-promoting action of isoprenaline was abolished by a β₁-selective antagonist and was more effectively abolished by a β₂-selective antagonist; the mechanism for the action of the antagonists was a G₀/G₁ phase cell cycle arrest which was associated with decreased cyclin D1, CDK-4, CDK-6 and phospho-Rb expression. Pre-treatment of the cells with VEGFR-2 or ERK inhibitors also prevented the isoprenaline-mediated proliferation of cells. In agreement with the involvement of β-ARs and VEGFR-2 in the HemEC response, β-AR antagonists and the VEGFR-2 inhibitor significantly attenuated isoprenaline-induced ERK phosphorylation. Moreover, treating the cells with isoprenaline markedly increased VEGF-A expression and VEGFR-2 activity in a β₂-AR-dependent manner.

Conclusions

We have demonstrated that the activation of the β-ARs in the ERK pathway may be important mechanisms in promoting HemEC growth. Furthermore, stimulation of the β-AR may transactivate VEGFR-2 signaling and further increase HemEC proliferation.     

Molecular dynamics simulations of the effect of the G-protein and diffusible ligands on the β2-adrenergic receptor

G-protein-coupled receptors have extraordinary therapeutic potential as targets for a broad spectrum of diseases. Understanding their function at the molecular level is therefore essential. A variety of crystal structures have made the investigation of the inactive receptor state possible. Recently released X-ray structures of opsin and the β₂-adrenergic receptor (β₂AR) have provided insight into the active receptor state. In addition, we have contributed to the crystal structure of an irreversible agonist-β₂ adrenoceptor complex. These extensive studies and biophysical investigations have revealed that agonist binding leads to a low-affinity conformation of the active state that is suggested to facilitate G-protein binding. The high-affinity receptor state, which promotes signal transduction, is only formed in the presence of both agonist and G-protein. Despite numerous crystal structures, it is not yet clear how ligands tune receptor dynamics and G-protein binding. We have now used molecular dynamics simulations to elucidate the distinct impact of agonist and inverse agonist on receptor conformation and G-protein binding by investigating the influence of the ligands on the structure and dynamics of a complex composed of β₂AR and the C-terminal end of the Gα_s subunit (GαCT). The simulations clearly showed that the agonist isoprenaline and the inverse agonist carazolol influence the ligand-binding site and the interaction between β₂AR and GαCT differently. Isoprenaline induced an inward motion of helix 5, whereas carazolol blocked the rearrangement of the extracellular part of the receptor. Moreover, in the presence of isoprenaline, β₂AR and GαCT form a stable interaction that is destabilized by carazolol.     

Zebrafish β-adrenergic receptor mRNA expression and control of pigmentation

Beta adrenergic receptors (β-ARs) are members of the G-protein-coupled receptor superfamily and mediate various physiological processes in many species. The expression patterns and functions of β-ARs in zebrafish are, however, largely unknown. We have identified zebrafish β-AR orthologs, which we have designated as adrb1, adrb2a, adrb2b, adrb3a and adrb3b. adrb1 was found to be expressed in the heart and brain. Expression of adrb2a predominated in the brain and skin, whereas adrb2b was found to be highly expressed in muscle, pancreas and liver. Both adrb3a and adrb3b were exclusively expressed in blood. Knock-down of these β-ARs by morpholino oligonucleotides revealed a functional importance of adrb2a in pigmentation. Expression of atp5a1 and atp5b, genes that encode subunits of F1F0-ATPase, which is known to be involved in pigmentation, was significantly increased by knock-down of adrb2a. Our data suggest that adrb2a may regulate pigmentation, partly by modulating F1F0-ATPase.     

Regulation of contractility and metabolic signaling by the β2-adrenergic receptor in rat ventricular muscle

AimsCardiac function is modulated by the sympathetic nervous system through β-adrenergic receptor (β-AR) activity and this represents the main regulatory mechanism for cardiac performance. To date, however, the metabolic and molecular responses to β₂-agonists are not well characterized. Therefore, we studied the inotropic effect and signaling response to selective β₂-AR activation by tulobuterol.Main methodsStrips of rat right ventricle were electrically stimulated (1 Hz) in standard Tyrode solution (95% O₂, 5% CO₂) in the presence of the β₁-antagonist CGP-20712A (1 μM). A cumulative dose–response curve for tulobuterol (0.1–10 μM), in the presence or absence of the phosphodiesterase (PDE) inhibitor IBMX (30 μM), or 10 min incubation (1 μM) with the β₂-agonist tulobuterol was performed.Key findingsβ₂-AR stimulation induced a positive inotropic effect (maximal effect = 33 ± 3.3%) and a decrease in the time required for half relaxation (from 45 ± 0.6 to 31 ± 1.8 ms, ? 30%, p < 0.001) after the inhibition of PDEs. After 10 min of β₂-AR stimulation, p-AMPKα^T172 (54%), p-PKB^T308 (38%), p-AS160^T642 (46%) and p-CREB^S133 (63%) increased, without any change in p-PKA^T197.SignificanceThese results suggest that the regulation of ventricular contractility is not the primary function of the β₂-AR. Rather, β₂-AR could function to activate PKB and AMPK signaling, thereby modulating muscle mass and energetic metabolism of rat ventricular muscle.     

Nuclear GPCRs in cardiomyocytes: an insider's view of β-adrenergic receptor signaling

In recent years, we have come to appreciate the complexity of G protein-coupled receptor signaling in general and β-adrenergic receptor (β-AR) signaling in particular. Starting originally from three β-AR subtypes expressed in cardiomyocytes with relatively simple, linear signaling cascades, it is now clear that there are large receptor-based networks which provide a rich and diverse set of responses depending on their complement of signaling partners and the physiological state. More recently, it has become clear that subcellular localization of these signaling complexes also enriches the diversity of phenotypic outcomes. Here, we review our understanding of the signaling repertoire controlled by nuclear β-AR subtypes as well our understanding of the novel roles for G proteins themselves in the nucleus, with a special focus, where possible, on their effects in cardiomyocytes. Finally, we discuss the potential pathological implications of alterations in nuclear β-AR signaling.     

Receptor specific crosstalk and modulation of signaling upon heterodimerization between β1-adrenergic receptor and somatostatin receptor-5

In the present study we describe heterodimerization, trafficking, coupling to adenylyl cyclase and signaling in HEK-293 cells cotransfected with human-somatostatin receptor 5 (hSSTR5) and β₁-adrenergic receptor (β₁AR). hSSTR5/β₁AR exists as heterodimers in basal conditions which was further enhanced upon synergistic activation of both receptors. Activation of either β₁AR or hSSTR5 displayed dissociation of heterodimerization. In cotransfectants, β₁AR effect on cAMP was predominant; however, blocking β₁AR with antagonist resulted in 60% inhibition of forskolin-stimulated cAMP in the presence of hSSTR5 agonists. cAMP/PKA pathway in cotransfected cells was regulated in receptor-specific manner, in contrast, the status of pERK1/2 and pPI3K/AKT was predominantly regulated by hSSTR5. The expression levels of phosphorylated NFAT remained unchanged indicating blockade of calcineurin-mediated dephosphorylation and nuclear translocation of NFAT, the process predominantly regulated by pJNK in SSTR5 dependent manner. Taken together, the functional consequences of results described here might have relevance in the cardiovascular system where SSTR and AR subtypes play important roles.     

Genetic and phenotypic targeting of β-adrenergic signaling in heart failure

Heart failure is a leading cause of hospitalization worldwide. No major significant improvements in prognosis have been achieved for heart failure over the last several decades despite advances in disease management. Heart failure itself represents a final common endpoint for several disease entities, including hypertension and coronary artery disease. On a molecular level, certain biochemical features remain common to failing myocardium. Among these are alterations in the -adrenergic receptor (-AR) signaling cascade. Recent advances in transgenic and gene therapy techniques have presented novel therapeutic strategies for management of heart failure via genetic manipulation of -AR signaling including the targeted inhibition of the -AR kinase (ARK1 or GRK2). In this review, we will discuss the -AR signaling changes that accompany heart failure as well as corresponding therapeutic strategies. We will then review the evidence from transgenic mouse work supporting the use of -AR manipulation in the failing heart and more recent in vivo applications of gene therapy directed at reversing or preventing heart failure. (Mol Cell Biochem 263: 5–9, 2004)     

Adenosine A1 receptors heterodimerize with β1- and β2-adrenergic receptors creating novel receptor complexes with altered G protein coupling and signaling

G protein coupled receptors play crucial roles in mediating cellular responses to external stimuli, and increasing evidence suggests that they function as multiple units comprising homo/heterodimers and hetero-oligomers. Adenosine and β-adrenergic receptors are co-expressed in numerous tissues and mediate important cellular responses to the autocoid adenosine and sympathetic stimulation, respectively. The present study was undertaken to examine whether adenosine A₁ARs heterodimerize with β₁- and/or β₂-adrenergic receptors (β₁R and β₂R), and whether such interactions lead to functional consequences. Co-immunoprecipitation and co-localization studies with differentially epitope-tagged A₁, β₁, and β₂ receptors transiently co-expressed in HEK-293 cells indicate that A₁AR forms constitutive heterodimers with both β₁R and β₂R. This heterodimerization significantly influenced orthosteric ligand binding affinity of both β₁R and β₂R without altering ligand binding properties of A₁AR. Receptor-mediated ERK1/2 phosphorylation significantly increased in cells expressing A₁AR/β₁R and A₁AR/β₂R heteromers. β-Receptor-mediated cAMP production was not altered in A₁AR/β₁R expressing cells, but was significantly reduced in the A₁AR/β₂R cells. The inhibitory effect of the A₁AR on cAMP production was abrogated in both A₁AR/β₁R and A₁AR/β₂R expressing cells in response to the A₁AR agonist CCPA. Co-immunoprecipitation studies conducted with human heart tissue lysates indicate that endogenous A₁AR, β₁R, and β₂R also form heterodimers. Taken together, our data suggest that heterodimerization between A₁ and β receptors leads to altered receptor pharmacology, functional coupling, and intracellular signaling pathways. Unique and differential receptor cross-talk between these two important receptor families may offer the opportunity to fine-tune crucial signaling responses and development of more specific therapeutic interventions.     

Agonist-directed desensitization of the β2-adrenergic receptor

The β(2)-adrenergic receptor (β(2)AR) agonists with reduced tachyphylaxis may offer new therapeutic agents with improved tolerance profile. However, receptor desensitization assays are often inferred at the single signaling molecule level, thus ligand-directed desensitization is poorly understood. Here we report a label-free biosensor whole cell assay with microfluidics to determine ligand-directed desensitization of the β(2)AR. Together with mechanistic deconvolution using small molecule inhibitors, the receptor desensitization and resensitization patterns under the short-term agonist exposure manifested the long-acting agonism of salmeterol, and differentiated the mechanisms of agonist-directed desensitization between a full agonist epinephrine and a partial agonist pindolol. This study reveals the cellular mechanisms of agonist-selective β(2)AR desensitization at the whole cell level.     

Computer modeling of δ opioid receptor and interaction with ligands

Summary Human σ opioid receptor (σOR), a G-protein-coupled receptor, has been modeled using the helix axes as revealed by the crystallographic structure of bacteriorhodopsin and ligand binding profiles of single-point mutants of σOR. The model revealed feasibility of existence of a second disulfide bridge between the transmembrane helices (TMHs) 6 and 7, Cys²⁷³-Cys³⁰³. A common binding site has been suggested for high-affinity selective agonists DPDPE, DPLPE, DTLET, BW373U86 and antagonist Naltrindole. Docking calculations have shown that the amino group of the ligands forms a hydrogen bond with the imidazole ring of His³⁰¹ (TMH7) rather than with Asp¹²⁸ (TMH3) and is not a cation counterpart of this highly conserved aspartyl residue. All the findings and the model shed light on the putative structure and functioning of opioid receptors and can be used for designing further mutagenesis experiments.     

Cell contact-dependent functional selectivity of β2-adrenergic receptor ligands in stimulating cAMP accumulation and extracellular signal-regulated kinase phosphorylation

Activation of β(2)-adrenegic receptor (β(2)-AR) leads to an increase in intracellular cAMP and activation of ERK. These two signals are activated by the interaction of the receptor with different transducer partners. We showed that the intrinsic activities of β(2)-AR ligands for stimulating cAMP production and ERK phosphorylation responses in HEK-293 cells were not correlated. The lack of correlation resulted mainly from the discrepancy between the intrinsic activities of two groups of ligands for these two responses: The first group consisted of clenbuterol, cimaterol, procaterol, and terbutaline which acted as full agonists for cAMP production but displayed very weak effect on ERK phosphorylation. The second group comprised adrenaline and noradrenaline which displayed higher intrinsic activity for the ERK phosphorylation than for the cAMP response. Thus, both groups behaved as functionally selective ligands. The functional selectivity of the first group was observable only in adherent cells when confluence was approximately 100%. When cell-cell contact was minimized either by decreasing the density of the adherent cells or by bringing the cells into suspension, the first group of ligands gained the ability to stimulate ERK phosphorylation without a change in their effect on cAMP production. In contrast, selectivity of the second group was independent of the adherence state of the cells. Our results show that the inherent "bias" of ligands in coupling a G protein-coupled receptor to different transducers may not always be revealed as functional selectivity when there is a "cross-talk" between the signaling pathways activated by the same receptor.     

Analysis of L-DOPA and droxidopa binding to human β2-adrenergic receptor

Over the last two decades, an increasing number of studies has been devoted to a deeper understanding of the molecular process involved in the binding of various agonists and antagonists to active and inactive conformations of β₂-adrenergic receptor (β₂AR). The 3.2 Å x-ray crystal structure of human β₂AR active state in combination with the endogenous low affinity agonist adrenaline offers an ideal starting structure for studying the binding of various catecholamines to adrenergic receptors. We show that molecular docking of levodopa (L-DOPA) and droxidopa into rigid and flexible β₂AR models leads for both ligands to binding anchor sites comparable to those experimentally reported for adrenaline, namely D113/N312 and S203/S204/S207 side chains. Both ligands have a hydrogen bond network that is extremely similar to those of noradrenaline and dopamine. Interestingly, redocking neutral and protonated versions of adrenaline to rigid and flexible β₂AR models results in binding poses that are more energetically stable and distinct from the x-ray crystal structure. Similarly, lowest energy conformations of noradrenaline and dopamine generated by docking into flexible β₂AR models had binding free energies lower than those of best poses in rigid receptor models. Furthermore, our findings show that L-DOPA and droxidopa molecules have binding affinities comparable to those predicted for adrenaline, noradrenaline, and dopamine, which are consistent with previous experimental and computational findings and supported by the molecular dynamics simulations of β₂AR-ligand complexes performed here.     

Serotonin receptor signaling and regulation via β-arrestins

Serotonin receptors are the product of 15 distinct genes, 14 of which are G protein-coupled receptors. These receptors are expressed in a wide range of cell types, including distinct neuronal populations, and promote diverse functional responses in multiple organ systems. These receptors are important for mediating the in vivo effects of their cognate neurotransmitter, serotonin, as well as the endogenous tryptamines. In addition, the actions of many drugs are mediated, either directly or indirectly, through serotonin receptors, including antidepressants, antipsychotics, anxiolytics, sleep aids, migraine therapies, gastrointestinal therapeutics and hallucinogenic drugs. It is becoming increasingly evident that serotonin receptors can engage in differential signaling that is determined by the chemical nature of the ligand and that ligands that demonstrate a predilection for inducing a particular signaling cascade are considered to have “functional selectivity”. The elucidation of the cellular signaling pathways that mediate the physiological responses to serotonin and other agonists is an active area of investigation and will be an onward-looking focal point for determining how to effectively and selectively promote beneficial serotonergic mimicry while avoiding unwanted clinical side effects. This review highlights the modulation of serotonin 2A, 2C, and four receptors by β-arrestins, which may represent a fulcrum for biasing receptor responsiveness in vivo.     

Serotonin receptor signaling and regulation via β-arrestins

Serotonin receptors are the product of 15 distinct genes, 14 of which are G protein-coupled receptors. These receptors are expressed in a wide range of cell types, including distinct neuronal populations, and promote diverse functional responses in multiple organ systems. These receptors are important for mediating the in vivo effects of their cognate neurotransmitter, serotonin, as well as the endogenous tryptamines. In addition, the actions of many drugs are mediated, either directly or indirectly, through serotonin receptors, including antidepressants, antipsychotics, anxiolytics, sleep aids, migraine therapies, gastrointestinal therapeutics and hallucinogenic drugs. It is becoming increasingly evident that serotonin receptors can engage in differential signaling that is determined by the chemical nature of the ligand and that ligands that demonstrate a predilection for inducing a particular signaling cascade are considered to have "functional selectivity". The elucidation of the cellular signaling pathways that mediate the physiological responses to serotonin and other agonists is an active area of investigation and will be an onward-looking focal point for determining how to effectively and selectively promote beneficial serotonergic mimicry while avoiding unwanted clinical side effects. This review highlights the modulation of serotonin 2A, 2C, and four receptors by β-arrestins, which may represent a fulcrum for biasing receptor responsiveness in vivo.     

Multiple ligand-specific conformations of the β2-adrenergic receptor

Seven-transmembrane receptors (7TMRs), also called G protein-coupled receptors (GPCRs), represent the largest class of drug targets, and they can signal through several distinct mechanisms including those mediated by G proteins and the multifunctional adaptor proteins β-arrestins. Moreover, several receptor ligands with differential efficacies toward these distinct signaling pathways have been identified. However, the structural basis and mechanism underlying this 'biased agonism' remains largely unknown. Here, we develop a quantitative mass spectrometry strategy that measures specific reactivities of individual side chains to investigate dynamic conformational changes in the β(2)-adrenergic receptor occupied by nine functionally distinct ligands. Unexpectedly, only a minority of residues showed reactivity patterns consistent with classical agonism, whereas the majority showed distinct patterns of reactivity even between functionally similar ligands. These findings demonstrate, contrary to two-state models for receptor activity, that there is significant variability in receptor conformations induced by different ligands, which has significant implications for the design of new therapeutic agents.     

Signaling diversity of mu- and delta- opioid receptor ligands: Re-evaluating the benefits of β-arrestin/G protein signaling bias

Opioid analgesics are elective for treating moderate to severe pain but their use is restricted by severe side effects. Signaling bias has been proposed as a viable means for improving this situation. To exploit this opportunity, continuous efforts are devoted to understand how ligand-specific modulations of receptor functions could mediate the different in vivo effects of opioids. Advances in the field have led to the development of biased agonists based on hypotheses that allocated desired and undesired effects to specific signaling pathways. However, the prevalent hypothesis associating β-arrestin to opioid side effects was recently challenged and multiple of the newly developed biased drugs may not display the superior side effects profile that was sought. Moreover, biased agonism at opioid receptors is now known to be time- and cell-dependent, which adds a new layer of complexity for bias estimation. Here, we first review the signaling mechanisms underlying desired and undesired effects of opioids. We then describe biased agonism at opioid receptors and discuss the different perspectives that support the desired and undesired effects of opioids in view of exploiting biased signaling for therapeutic purposes. Finally, we explore how signaling kinetics and cellular background can influence the magnitude and directionality of bias at those receptors.     

Adrenergic regulation of HCN4 channel requires protein association with β2-adrenergic receptor

β(1)- and β(2)-adrenergic receptors utilize different signaling mechanisms to control cardiac function. Recent studies demonstrated that β(2)-adrenergic receptors (β(2)ARs) colocalize with some ion channels that are critical for proper cardiac function. Here, we demonstrate that β(2)ARs form protein complexes with the pacemaker HCN4 channel, as well as with other subtypes of HCN channels. The adrenergic receptor-binding site was identified at a proximal region of the N-terminal tail of the HCN4 channel. A synthetic peptide derived from the β(2)AR-binding domain of the HCN4 channel disrupted interaction between HCN4 and β(2)AR. In addition, treatment with this peptide prevented adrenergic augmentation of pacemaker currents and spontaneous contraction rates but did not affect adrenergic regulation of voltage-gated calcium currents. These results suggest that the ion channel-receptor complex is a critical mechanism in ion channel regulation.     

Discovery of highly potent and selective biphenylacylsulfonamide-based β3-adrenergic receptor agonists and molecular modeling based on the solved X-ray structure of the β2-adrenergic receptor: Part 6

As an extension of research, we have investigated modification of left-hand side (LHS) of biphenyl analogues containing an acylsulfonamide moiety in the development of potent and selective human β₃-adrenergic receptor (AR) agonists. Result of structure–activity relationships (SAR) and cassette-dosing evaluation in dogs showed that the hydroxynorephedrine analogue 16 had an excellent balance of in vitro and in vivo potency with pharmacokinetic profiles. In addition, to facilitate structure-based drug design (SBDD), we also have performed a docking study of biphenyl analogues based on the X-ray structure of the β₂-adrenergic receptor.