Human Β3-adrenoceptor Research Articles

Transgenic Rat Model Overexpressing Endothelial β 3 ‐adrenoceptor: a New Model for Heart Failure with Preserved Ejection Fraction

Heart failure (HF) with preserved ejection fraction (pEF) is a growing health burden affecting the elderly. Patients classically express impaired cardiac relaxation and filling. Due to the lack of patient biopsies and accurate animal models, its physiopathology remains unclear and no specific treatment is currently available. In this context, our team developed a transgenic rat model (Tgβ3) overexpressing human β3-adrenoceptor at endothelial level. At 45 weeks of age, males presented an increase in left ventricle end diastolic pressure (LVEDP; WT: 5.6±1.2; Tgβ3: 11.7±1.1; p<0.01) and dP/dtmax, a contractility parameter (WT: 6333±370; Tgβ3: 7808±191; p<0.01), characteristic of an increase in LV stiffness and in peripheral resistances. Echographic study showed an altered filling pattern with an increase in early-to-late filling (E/A) ratio (WT:1.14±0.01; Tgβ3: 1.32±0.04; p<0.01), with a pEF but without macroscopic cardiac remodeling. These results showed that Tgβ3 rats presented a restrictive cardiac filling, representative of the HFpEF phenotype. Further studies, such as fibrosis quantification and calcium cycle study, are needed to determine the underlying mechanisms of the diastolic impairment, as well as to identify molecular targets altered in this disease in order to develop a new therapeutic strategy for HFpEF treatment.

Association between polymorphism of β3-adrenoceptor gene and overactive bladder: a meta-analysis.

Genetic variations in the human β3-adrenoceptor (β3-AR) gene are known to be involved in insufficient relaxation of the bladder muscle during urine storage. The Trp64Arg polymorphism in the β3-AR gene has been found to be an important regulator of the development of overactive bladder (OAB). However, the association between this polymorphism and OAB remains controversial. Therefore, we conducted a meta-analysis to explore the association between the Trp64Arg polymorphism and OAB risk. We examined 2 case-control studies, including a total of 149 OAB cases and 270 healthy controls. The meta-analysis results showed that the Arg allele of the β3-AR gene is positively associated with OAB susceptibility, while Arg allele carriers (Trp64Arg + Arg64Arg) showed positive associations with OAB. These results also demonstrated that the Trp64Arg polymorphism in the β3-AR gene is involved in the pathogenesis of OAB.

Interhelical Interaction and Receptor Phosphorylation Regulate the Activation Kinetics of Different Human β1-Adrenoceptor Variants

G protein-coupled receptors represent the largest class of drug targets, but genetic variation within G protein-coupled receptors leads to variable drug responses and, thereby, compromises their therapeutic application. One of the most intensely studied examples is a hyperfunctional variant of the human β1-adrenoceptor that carries an arginine at position 389 in helix 8 (Arg-389-ADRB1). However, the mechanism underlying the higher efficacy of the Arg-389 variant remained unclear to date. Despite its hyperfunctionality, we found the Arg-389 variant of ADRB1 to be hyperphosphorylated upon continuous stimulation with norepinephrine compared with the Gly-389 variant. Using ADRB1 sensors to monitor activation kinetics by fluorescence resonance energy transfer, Arg-389-ADRB1 exerted faster activation speed and arrestin recruitment than the Gly-389 variant. Both activation speed and arrestin recruitment depended on phosphorylation of the receptor, as shown by knockdown of G protein-coupled receptor kinases and phosphorylation-deficient ADRB1 mutants. Structural modeling of the human β1-adrenoceptor suggested interaction of the side chain of Arg-389 with opposing amino acid residues in helix 1. Site-directed mutagenesis of Lys-85 and Thr-86 in helix 1 revealed that this interaction indeed determined ADRB1 activation kinetics. Taken together, these findings indicate that differences in interhelical interaction regulate the different activation speed and efficacy of ADRB1 variants.

Detection of the secondary, low-affinity β1 -adrenoceptor site in living cells using the fluorescent CGP 12177 derivative BODIPY-TMR-CGP.

Background and PurposeCGP 12177 not only inhibits agonist effects mediated through the catecholamine site of the β1-adrenoceptor with high affinity, but also exhibits agonist effects of its own at higher concentrations through a secondary, low-affinity β1-adrenoceptor site or conformation. β-blocker affinities for this ‘CGP 12177’ site of the human β1-adrenoceptor have thus far only been characterized in functional studies. Here, we used the fluorescent CGP 12177 analogue BODIPY-TMR-CGP to directly investigate receptor–ligand interactions at the secondary binding site of the β1-adrenoceptor.Experimental ApproachThe human β1-adrenoceptor was stably expressed in CHO cells containing a cAMP response element (CRE)-secreted placental alkaline phosphatase (SPAP) reporter gene construct. Functional responses of BODIPY-TMR-CGP were determined in the CRE-SPAP reporter gene assay, and manual and automated confocal microscopy platforms used to investigate the binding properties of BODIPY-TMR-CGP.Key ResultsBODIPY-TMR-CGP displayed a pharmacological profile similar to that of CGP 12177, retaining agonist activity at the secondary β1-adrenoceptor site. In confocal microscopy studies, specific BODIPY-TMR-CGP binding allowed clear visualization of β1-adrenoceptors in live cells. Using a wider concentration range of labelled ligand in a high-content fluorescence-based binding assay than is possible in radioligand binding assays, two-site inhibition binding curves of β-adrenoceptor antagonists were revealed in CHO cells expressing the human β1-adrenoceptor, but not the β2-adrenoceptor.Conclusions and ImplicationsThe fluorescent CGP 12177 analogue allowed the detection of the β1-adrenoceptor secondary site in both functional and binding studies. This suggests that BODIPY-TMR-CGP presents an important and novel fluorescent tool to investigate the nature of the secondary β1-adrenoceptor site.

Salmeterol’s Extremeβ2 Selectivity Is Due to Residues in Both Extracellular Loops and Transmembrane Domains

Salmeterol is a long-acting β2-agonist, widely used as an inhaled treatment of asthma and chronic obstructive pulmonary disease. It has very high β2-affinity (log KD -8.95) and is very selective for the β2-adrenoceptor (1000-fold selectivity over the β1-adrenoceptor). This study used a mutagenesis approach to determine the exact amino acids in the human β2-adrenoceptor responsible for this very high selectivity. Wild-type β2- and β1-adrenoceptors, chimeric β2/β1-adrenoceptors, and receptors with single-point mutations were transfected into Chinese hamster ovary-K1 cells, and affinity and function were studied using [(3)H]CGP 12177 [(-)-4-(3-tert-butylamino-2-hydroxypropoxy)-benzimidazol-2-one] whole-cell binding and [(3)H]cAMP accumulation. Extracellular loop 3 (and specifically amino acid K305) had the largest single effect by reducing salmeterol's affinity for the β2-adrenoceptor by 31-fold. H296 in transmembrane 6 also had a major effect (18-fold reduction in salmeterol affinity). Combining these, in the double mutant β2-H296K-K305D, reduced salmeterol's affinity by 275-fold, to within 4-fold of that of the β1-adrenoceptor, without affecting the affinity or selectivity of other β2-agonists (salbutamol, formoterol, fenoterol, clenbuterol, or adrenaline). Another important amino acid was Y308 in transmembrane 7, although this also affected the affinity and selectivity of other agonists. F194 in extracellular loop 2 and R304 in extracellular loop 3 also had minor effects. None of these mutations (including the double mutant β2-H296K-K305D) affected the efficacy or duration of action of salmeterol. This suggests that the high affinity and selectivity of salmeterol are due to specific amino acids within the receptor itself, but that the duration of action is at least in part due to other factors, for example lipophilicity.

The Odd Sibling: Features ofβ3-Adrenoceptor Pharmacology

β3-Adrenoceptor agonists have recently been introduced for the treatment of overactive urinary bladder syndrome. Their target, the β3-adrenoceptor, was discovered much later than β1- and β2-adrenoceptors and exhibits unique properties which make extrapolation of findings from the other two subtypes difficult and the β3-adrenoceptor a less-understood subtype. This article discusses three aspects of β3-adrenoceptor pharmacology. First, the ligand-recognition profile of β3-adrenoceptors differs considerably from that of the other two subtypes, i.e., many antagonists considered as nonselective actually are β3-sparing, including propranolol or nadolol. Many agonists and antagonists classically considered as being β3-selective actually are not, including BRL 37,344 ((±)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy] acetic acid sodium hydrate) or SR 59,230 (3-(2-ethylphenoxy)-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol oxalate). Moreover, the binding pocket apparently differs between the human and rodent β3-adrenoceptor, yielding considerable species differences in potency. Second, the expression pattern of β3-adrenoceptors is more restricted than that of other subtypes, particularly in humans; this makes extrapolation of rodent findings to the human situation difficult, but it may result in a smaller potential for side effects. The role of β3-adrenoceptor gene polymorphisms has insufficiently been explored and may differ even between primate species. Third, β3-adrenoceptors lack the phosphorylation sites involved in agonist-induced desensitization of the other two subtypes. Thus, they exhibit downregulation and/or desensitization in some, but not other, cell types and tissues. When desensitization occurs, it most often is at the level of mRNA or signaling molecule expression. All three of these factors have implications for future studies to better understand the β3-adrenoceptor as a novel pharmacological target.

Multivalent design of long-acting β2-adrenoceptor agonists incorporating biarylamines

A series of potent β2-adrenoceptor agonists incorporating a biarylamine secondary binding group was identified. The previously reported milveterol (5), identified by a multivalent approach and containing a typical β2-agonist primary binding group linked via a phenethylamine linker to a hydrophilic secondary binding group, served as an initiation point. A more hydrophobic set of secondary binding groups was explored, prepared rapidly from a common intermediate by Buchwald–Hartwig amination. TD-5471 (25), a potent and selective full agonist of the human β2-adrenoceptor, was identified as the most promising agent. It is potent, with slow onset in an in vitro guinea pig trachea model and shows a dose-dependent and long duration of action in an in vivo guinea pig model of bronchoprotection. TD-5471 is structurally differentiated from milveterol and its long duration of action is consistent with a correlation with hydrophobicity observed in other long-acting β2-agonist discovery programs.

Exploring drug–protein interactions using the relationship between injection volume and capacity factor

Affinity chromatography is the most widespread and widely accepted methodology for exploring drug–protein and protein–protein interactions. Despite the successful application of frontal analysis and zonal elution in affinity chromatography, research into the creation of new mathematical tools for data processing is encouraged due to these two methods’ drawbacks of long analysis times and high ligand consumption. In this work, we created a novel mathematical model using the relationship between the molar amount of an injected solute and its capacity factor. We validated the method by analyzing the binding of drugs to human serum albumin (HSA) and β2-adrenoceptor (β2-AR). The association constants of omeprazole, propranolol and promethazine binding to HSA were determined to be (4.10±0.24)×104, (2.30±0.12)×104 and (1.24±0.14)×104M−1, respectively. These constants agreed with previously reported literature results of 4.60×104, 2.30×104 and 1.40×104M−1. Salbutamol, norepinephrine, isoprenaline, bamethane and methoxyphenamine were found to bind to β2-AR with association constants of (1.11±0.06)×103, (0.95±0.03)×103, (1.66±0.12)×103, (0.47±0.04)×103 and (0.43±0.02)×103M−1, respectively, which positively correlated to the negative logarithm of the dissociation constants obtained via radio-ligand binding assays. The proposed model is relatively fast and conserves ligand, and it has the potential to serve as an alternative method for rapidly revealing drug–protein and protein–protein interactions.

Identification of key residues in transmembrane 4 responsible for the secondary, low-affinity conformation of the human β1-adrenoceptor.

The β1-adrenoceptor exists in two agonist conformations/states: 1) a high-affinity state where responses to catecholamines and other agonists (e.g., cimaterol) are potently inhibited by β1-adrenoceptor antagonists, and 2) a low-affinity secondary conformation where agonist responses, particularly CGP12177 [(-)-4-(3-tert-butylamino-2-hydroxypropoxy)-benzimidazol-2-one] are relatively resistant to inhibition by β1-adrenoceptor antagonists. Although both states have been demonstrated in many species (including human), the precise nature of the secondary state is unknown and does not occur in the closely related β2-adrenoceptor. Here, using site-directed mutagenesis and functional measurements of production of a cyclic AMP response element upstream of a secreted placental alkaline phosphatase reporter gene and accumulation of (3)H-cAMP, we examined the pharmacological consequences of swapping transmembrane (TM) regions of the human β1- and β2-adrenoceptors, followed by single point mutations, to determine the key residues involved in the β1-adrenoceptor secondary conformation. We found that TM4 (particularly amino acids L195 and W199) had a major role in the generation of the secondary β1-adrenoceptor conformation. Thus, unlike at the human β1-wild-type adrenoceptor, at β1-TM4 mutant receptors, cimaterol and CGP12177 responses were both potently inhibited by antagonists. CGP12177 acted as a simple partial agonist with similar KB and EC50 values in the β1-TM4 but not β1-wild-type receptors. Furthermore pindolol switched from a biphasic concentration response at human β1-wild-type adrenoceptors to a monophasic concentration response in the β1-TM4 mutant receptors. Mutation of these amino acids to those found in the β2-adrenoceptor (L195Q and W199Y), or mutation of a single residue (W199D) in the human β1-adrenoceptor thus abolished this secondary conformation and created a β1-adrenoceptor with only one high-affinity agonist conformation.

Molecular dynamics simulations to explore the active/inactive conformers of guinea pig β2 adrenoceptor for the selective design of agonists or antagonists

It is well known that guinea pig β2 adrenoceptors (Gβ2ARs) and human β2 adrenoceptors (Hβ2ARs) have structural similarity. However, only one conformational state of Gβ2ARs has been studied – the putative inactive state. As adrenoceptors have a repertoire of conformations, and there is evidence that a certain conformation is stabilised as a ligand approaches, the aim of this study was to build four models of Gβ2ARs by using putative active/inactive Hβ2AR conformers as a template. We evaluated the accuracy of these models in regard to the binding mode and affinity values of a set of known β2AR ligands through docking and molecular dynamics simulations. During docking simulations, ligands reached Gβ2AR sites similar to those reported for Hβ2ARs. The greatest differences between conformational states were found in the domains (TM5 and TM6) previously suggested as being key to ligand recognition. The coefficients of determination between experimental and calculated affinity values were near to but less than 0.66 in all cases. The highest values were for agonists on the active models and antagonists on the inactive model. The four Gβ2AR models proved useful for analysing agonist/antagonist activity. The results suggest that the selection of an adequate model is dependent on the intrinsic activity of a given ligand.

Molecular dynamics simulations to explore the active/inactive conformers of guinea pig β2 adrenoceptor for the selective design of agonists or antagonists

It is well known that guinea pig β2 adrenoceptors (Gβ2ARs) and human β2 adrenoceptors (Hβ2ARs) have structural similarity. However, only one conformational state of Gβ2ARs has been studied – the putative inactive state. As adrenoceptors have a repertoire of conformations, and there is evidence that a certain conformation is stabilised as a ligand approaches, the aim of this study was to build four models of Gβ2ARs by using putative active/inactive Hβ2AR conformers as a template. We evaluated the accuracy of these models in regard to the binding mode and affinity values of a set of known β2AR ligands through docking and molecular dynamics simulations. During docking simulations, ligands reached Gβ2AR sites similar to those reported for Hβ2ARs. The greatest differences between conformational states were found in the domains (TM5 and TM6) previously suggested as being key to ligand recognition. The coefficients of determination between experimental and calculated affinity values were near to but less than 0....

Discovery of a Rapidly Metabolized, Long-Acting β2 Adrenergic Receptor Agonist with a Short Onset Time Incorporating a Sulfone Group Suitable for Once-Daily Dosing

A series of novel, potent, and selective human β2 adrenoceptor agonists incorporating a sulfone moiety on the terminal right-hand-side phenyl ring of (R)-salmeterol is presented. Sulfone 10b had salmeterol-like potency and selectivity profile, long duration of action on guinea pig trachea, and longer than salmeterol duration of action in vivo, suitable for once-daily dosing. It had lower than salmeterol oral absorption in rat, lower bioavailability in rat and dog, and a high turnover in human hepatocytes. It was metabolized in human hepatocytes by hydroxylation, oxidation, cleavage, and conjugation; most of the metabolites would be expected to have reduced or no β2 activity. The 4-biphenylsulfonic acid was identified as a crystalline, non-hygroscopic salt of 10b, suitable for inhaled delivery. Furthermore, it was free of any genetic toxicity issues and was considered as a backup to vilanterol.

Impact of polymorphic variants on the molecular pharmacology of the two-agonist conformations of the human β1-adrenoceptor.

β-blockers are widely used to improve symptoms and prolong life in heart disease primarily by inhibiting the actions of endogenous catecholamines at the β1-adrenoceptor. There are two common naturally occurring polymorphisms within the human β1-adrenoceptor sequence: Ser or Gly at position 49 in the N-terminus and Gly or Arg at position 389 in the C-terminus and some clinical studies have suggested that expression of certain variants may be associated with disease and affect response to treatment with β-blockers. The β1-adrenoceptor also exists in two agonist conformations - a high affinity catecholamine conformation and a low affinity secondary agonist conformation. Receptor-effector coupling and intracellular signalling from the different conformations may be affected by the polymorphic variants.Here, we examine in detail the molecular pharmacology of the β1-adrenoceptor polymorphic variants with respect to ligand affinity, efficacy, activation of the different agonist conformations and signal transduction and determine whether the polymorphic variants do indeed affect this secondary conformation. Stable cell lines expressing the wildtype and polymorphic variants were constructed and receptor pharmacology examined using whole cell binding and intracellular secondary messenger techniques.There was no difference in affinity for agonists and antagonists at the human wildtype β1-adrenoceptor (Ser49/Gly389) and the polymorphic variants Gly49/Gly389 and Ser49/Arg389. Furthermore, the polymorphic variant receptors both have two active agonist conformations with pharmacological properties similar to the wildtype receptor. Although the polymorphism at position 389 is thought to occur in an intracellular domain important for Gs-coupling, the two agonist conformations of the polymorphic variants stimulate intracellular signalling pathways, including Gs-cAMP intracellular signalling, in a manner very similar to that of the wildtype receptor.

Pharmacokinetics of Mirabegron, a β3-Adrenoceptor Agonist for Treatment of Overactive Bladder, in Healthy Japanese Male Subjects: Results from Single- and Multiple-Dose Studies

BackgroundMirabegron is a human β3-adrenoceptor agonist for the treatment of overactive bladder. The pharmacokinetic profile of mirabegron has been extensively characterized in healthy Caucasian subjects.ObjectiveThe objective of this study was to evaluate the pharmacokinetics, dose-proportionality, and tolerability of mirabegron following single and multiple oral doses in healthy Japanese male subjects. The results were compared with those reported in non-Japanese (primarily Caucasian) subjects.MethodsTwo studies were conducted. In a single-blind, randomized, placebo-controlled, parallel-group, single- and multiple-ascending dose study (Study 1), mirabegron oral controlled absorption system (OCAS) tablets were administered at single doses of 50, 100, 200, 300, and 400 mg, with eight subjects (six active, two placebo) per dose group (Part I), and once daily for 7 days at 100 and 200 mg with 12 subjects (eight active, four placebo) per group (Part II). In an open-label, three-period, single-ascending dose study (Study 2), mirabegron OCAS was administered to 12 subjects at 25, 50, and 100 mg in an intra-subject dose-escalation design. Plasma and/or urine samples were collected up to 72 h after the first and last dose and analyzed for mirabegron. Pharmacokinetic parameters were determined using non-compartmental methods. Tolerability assessments included physical examinations, vital signs, 12-lead electrocardiogram, clinical laboratory tests (biochemistry, hematology, and urinalysis), and adverse event (AE) monitoring.ResultsForty and 24 young male subjects completed Part I and II, respectively, of Study 1. Twelve young males completed Study 2. After single oral doses (25–400 mg), maximum plasma concentrations (C max) were reached at approximately 2.8–4.0 h postdose. Plasma exposure (C max and area under the plasma concentration–time curve) of mirabegron increased more than dose proportionally at single doses of 25–100 mg and approximately dose proportionally at high doses of 300 and 400 mg. A more than dose proportional increase in plasma exposure was noted in the body of the same individual. Mirabegron accumulated twofold upon once-daily dosing relative to single-dose data. Steady state was reached within 7 days. Mirabegron was generally well-tolerated at single doses up to 400 mg and multiple doses up to 200 mg. The AE with the highest incidence was increased pulse rate at 400 mg in Study 1.ConclusionsMirabegron OCAS exhibits similar single- and multiple-dose pharmacokinetic characteristics and deviations from dose proportionality in healthy Japanese male subjects compared with those observed in non-Japanese (primarily Caucasian) subjects in previous studies.

The new radioligand [3H]-L 748,337 differentially labels human and rat β3-adrenoceptors

As no suitable radioligand exists for the detection of β3-adrenoceptors, we have explored the radioligand binding properties of a tritiated version of the selective β3-adrenoceptor antagonist L 748,337. Kinetic and equilibrium saturation and competition binding experiments were performed with [3H]-L 748,337 on membrane fractions of HEK and CHO cells stably transfected with human and rat β-adrenoceptor subtypes. Based on both association/dissociation kinetic and equilibrium saturation binding studies in transfected HEK cells, [3H]-L 748,337 exhibited an affinity of approximately 2nM for human β3-adrenoceptors. Competition studies with agonists and subtype-selective antagonists validated its binding to β3-adrenoceptors. In CHO cells transfected with human β3-adrenoceptors similar saturable high-affinity of [3H]-L 748,337 was observed. While some isoprenaline-sensitive [3H]-L 748,337 binding was also observed in CHO cells transfected with human β1- or β2-adrenoceptors, this was not saturable in a similar concentration range and/or not sensitive to the antagonists propranolol and SR 59,230, indicating that it did not primarily involve β-adrenoceptors. In CHO cells transfected with rat β3-adrenoceptors [3H]-L 748,337 exhibited a considerably lower affinity than with the human subtype (12–95nM). Low affinity for the rat β3-adrenoceptor was also found with unlabelled L 748,337 in rat bladder strip relaxation experiments. We conclude that L 748,337 apparently has lower affinity for the rat than the human β3-adrenoceptors and that [3H]-L 748,337 can bind to a low-affinity site distinct from the orthosteric pocket of β-adrenoceptors. Nevertheless, [3H]-L 748,337 appears to be the most promising radioligand for the selective labelling of human β3-adrenoceptors reported to date.

TRK-380, a Novel Selective Human β3-Adrenoceptor Agonist, Ameliorates Formalin-induced Pollakiuria in Rats and Carbachol-induced Bladder Contraction in Dogs

To investigate the effects of TRK-380, a selective β3-adrenoceptor (β3-AR) agonist, on voiding behavior in rats with pollakiuria and on carbachol (CCh)-induced bladder contraction in dogs. The voiding behavior of female Sprague Dawley rats was recorded continuously with a balance. Rats were intravesically pretreated with 2.5% formalin under isoflurane anesthesia. The next day, the effect of TRK-380 (7.5-30 mg/kg, orally) or tolterodine, an antimuscarinic drug (3.75-15 mg/kg, orally), on the voiding frequency was evaluated. In another experiment, male beagle dogs were anesthetized with pentobarbital, CCh (3 μg/kg, intravenously) was administered tothem, and the effect of TRK-380 (0.1 or 0.3 μg/kg/minute, intravenously infusion) on CCh-induced bladder contraction was evaluated. Rats treated with formalin showed a significant increase in the voiding frequency compared with the sham group, and the increase in it was significantly and dose-dependently suppressed by TRK-380 at doses of ≥15 mg/kg. In contrast, tolterodine did not lead to a significant change in the voiding frequency even at the highest dose. In dogs, CCh-induced bladder contraction was dose-dependently suppressed by TRK-380; the plasma concentration required for 30% suppression of the CCh-induced bladder contraction (30% relaxation) was 4.90 ng/mL. This study indicated that TRK-380 ameliorated pollakiuria, which was resistant to an antimuscarinic drug, and that it also suppressed the bladder contraction induced by cholinergic stimulation in dogs, whose bladder relaxation is known to be predominantly mediated by β3-ARs, as in humans. These data strengthen the therapeutic potential of β3-AR for the treatment of overactive bladder.

Discovery of β2 Adrenergic Receptor Ligands Using Biosensor Fragment Screening of Tagged Wild-Type Receptor

G-protein coupled receptors (GPCRs) are the primary target class of currently marketed drugs, accounting for about a quarter of all drug targets of approved medicines. However, almost all the screening efforts for novel ligand discovery rely exclusively on cellular systems overexpressing the receptors. An alternative ligand discovery strategy is a fragment-based drug discovery, where low molecular weight compounds, known as fragments, are screened as initial starting points for optimization. However, the screening of fragment libraries usually employs biophysical screening methods, and as such, it has not been routinely applied to membrane proteins. We present here a surface plasmon resonance biosensor approach that enables, cell-free, label-free, fragment screening that directly measures fragment interactions with wild-type GPCRs. We exemplify the method by the discovery of novel, selective, high affinity antagonists of human β2 adrenoceptor.

Agonist-induced desensitization of human β3-adrenoceptors expressed in human embryonic kidney cells

β3-Adrenoceptors are resistant to agonist-induced desensitization in some cell types but susceptible in others including transfected human embryonic kidney (HEK) cells. Therefore, we have studied cellular and molecular changes involved in agonist-induced β3-adrenoceptor desensitization in HEK cells. Cells were treated with isoprenaline or forskolin, and following wash-out, cyclic adenosine monophosphate (cAMP) accumulation in response to freshly added agonist was quantified. Receptor and G protein expression were quantified by radioligand binding and immunoblot experiments, respectively. Treatment with isoprenaline induced a concentration- and time-dependent desensitization of cAMP accumulation in response to freshly added isoprenaline. This functional desensitization primarily consisted of reduced maximum responses with little change of agonist potency. Maximum desensitization was achieved by pre-treatment with 10 μM isoprenaline for 24h. It was not accompanied by changes in β3-adrenoceptor density as assessed in saturation radioligand-binding studies. The desensitization was associated with a small reduction in immunoreactivity for α-subunits for Gs and Gi1, whereas that for Gi2, Gi3, and Gq/11 was not significantly altered. In cells treated with pertussis toxin, isoprenaline-induced cAMP accumulation as well as desensitization by isoprenaline pre-treatment remained unchanged. Isoprenaline pre-treatment also reduced forskolin-induced cAMP accumulation; conversely, pre-treatment with forskolin caused a similar desensitization of isoprenaline-induced cAMP accumulation. We conclude that agonist-induced β3-adrenoceptor desensitization in HEK cells does not involve reduced receptor numbers and small, if any, reduction of Gs expression; changes at the level of adenylyl cyclase function can fully explain this desensitization.

Synthesis and in vitro and in vivo characterization of highly β1-selective β-adrenoceptor partial agonists.

β-Adrenoceptor antagonists boast a 50-year use for symptomatic control in numerous cardiovascular diseases. One might expect highly selective antagonists are available for the human β-adrenoceptor subtype involved in these diseases, yet few truly β1-selective molecules exist. To address this clinical need, we re-evaluated LK 204-545 (1),1 a selective β1-adrenoceptor antagonist, and discovered it possessed significant partial agonism. Removal of 1’s aromatic nitrile afforded 19, a ligand with similar β1-adrenoceptor selectivity and partial agonism (log KD of −7.75 and −5.15 as an antagonist of functional β1- and β2-mediated responses, respectively, and 34% of the maximal response of isoprenaline (β1)). In vitro β-adrenoceptor selectivity and partial agonism of 19 were mirrored in vivo. We designed analogues of 19 to improve affinity, selectivity, and partial agonism. Although partial agonism could not be fully attenuated, SAR suggests that an extended alkoxyalkoxy side chain, alongside substituents at the meta- or para-positions of the phenylurea, increases ligand affinity and β1-selectivity.

Pro-apoptotic effect of anti-β1-adrenergic receptor antibodies in periodontitis patients

An anti-β1-adrenergic antibody from the sera of periodontitis patients (anti-β1-AR IgG) against the second extracellular loop of the human β1-adrenoceptor (β1-AR) has been shown to cause rat atria apoptosis. The anti-β1-AR IgG binds and activates atria β1-AR, increasing the intracellular calcium concentration, which, in turn, activates caspases-3, -8, and -9. The β1-AR and the post-receptor activation of calcium/calmodulin (CaM) lead to increased inducible nitric oxide synthase (iNOS) activity, with an increase in cyclic GMP (cGMP) accumulation as well as increased JNK phosphorylation and cyclic AMP (cAMP) production. We also observed an apoptotic effect of anti-β1-AR IgG, with increased generation of PGE2. Comparatively, xamoterol, an authentic β1-AR agonist, mimicked the autoantibody effect on rat atria β1-AR apoptosis. Our results suggest that autoantibodies from the sera of periodontitis patients bind and interact with rat atria β1-AR, provoking apoptosis. This implicates a series of modulatory cardiac signaling events that could alter normal heart function and may occur with chronic stimulation of the atria β1-AR, which could lead to heart failure. These results suggest an important link between periodontitis and cardiovascular disease.