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Receptor chromatography is an efficient analytical technique that combines the high separation ability of chromatography with the high specificity of receptors for drug recognition. In addition, this technique offers the advantages of active recognition, online separation, and convenient multidimensional target tracking. This strategy allows target active ingredients in complex systems, such as traditional Chinese medicines, to be efficiently screened and accurately identified. Furthermore, the interactions between ligands and immobilized proteins can be studied. To avoid a loss in function, receptor chromatography requires efficient, mild, and simple immobilization methods that do not damage the structure of the immobilized receptors. Improvements in the activity, stability, and ligand-recognition specificity of immobilized functional proteins can be achieved by selecting appropriate immobilization conditions. Notably, the protein immobilization method is not only closely related to the recognition ability of receptor chromatography but also determines the accuracy of the technique. Common methods for immobilizing functional proteins include physical adsorption, chemical reactions, biological affinity reactions, and click chemistry. Despite being easy to operate under mild reaction conditions, these methods have shortcomings, including poor reaction specificity and the necessity of using high-purity functional proteins to prepare chromatography columns. Maintaining the high activity of immobilized receptors and ensuring excellent identification and separation abilities are key challenges in the further development of receptor chromatography. In this work, these issues were addressed by introducing a specific bioorthogonal reaction involving haloalkane dehalogenase (Halo) and 6-chlorohexanoic acid for the immobilization of the α 1A -adrenergic receptor ( α 1A -AR). Specifically, Halo- α 1A -AR was immobilized on the surface of 6-chlorohexanoic acid-modified aminopropyl silica gel in one step. The stationary phase with immobilized Halo- α 1A -AR was characterized using scanning electron microscopy. Moreover, the activity of the Halo- α 1A -AR chromatographic column was evaluated using specific ligands (terazosin hydrochloride, phentolamine mesylate, tamsulosin hydrochloride, and urapidil) and nonspecific ligands (yohimbe and metoprolol) for α 1A -AR. Halo- α 1A -AR was successfully immobilized on the silica gel surface with good stability over 30 days, and the Halo- α 1A -AR chromatographic column exhibited good ligand-recognition activity. The nonlinear chromatography results indicated that prazosin hydrochloride, terazosin hydrochloride, and urapidil interacted with immobilized Halo- α 1A -AR through one type of binding site, with association constants of 3.85×10 5 , 5.00×10 5 , and 5.90×10 5 L/mol, respectively. In contrast, phentolamine mesylate and tamsulosin hydrochloride interacted with immobilized Halo- α 1A -AR through two types of binding site. The association constants with the high- and low-affinity binding sites were 3.12×10 6 and 6.01×10 5 L/mol, respectively, for phentolamine mesylate and 9.98×10 5 and 0.21×10 5 L/mol, respectively, for tamsulosin hydrochloride. Compared with the traditional carbonyldiimidazole method, the immobilization method developed in this work did not require receptor purification and thus minimized the loss of receptor activity. The affinity constants obtained with immobilized Halo- α 1A -AR were consistent with the values determined for receptor-ligand binding in solution, indicating that the Halo- α 1A -AR chromatography column is suitable for studying drug-protein interactions. This approach also provides a foundation for the efficient screening and accurate determination of target active ingredients in complex systems.

The β 3 -adrenoceptor agonist mirabegron is available for the treatment of storage symptoms of overactive bladder, including frequency, urgency, and incontinence. The off-target effects of mirabegron include binding to α 1 -adrenoceptors, which are central in the treatment of voiding symptoms. Here, we examined the structure-function relationships in the binding of mirabegron to a cryo-electron microscopy structure of α 1A . The binding was simulated by docking mirabegron to a 3D structure of a human α 1A -adrenoceptor (7YMH) using Autodock Vina. The simulations identified two binding states: slope orientation involving 10 positions and horizontal binding to the receptor surface involving 4 positions. No interactions occurred with positions constituting the α 1A binding pocket, including Asp-106, Ser-188, or Phe-312, despite the positioning of the phenylethanolamine moiety in transmembrane regions close to the binding pocket by contact with Phe-288, -289, and Val-107. Contact with the unique positions of α 1A included the transmembrane Met-292 during slope binding and exosite Phe-86 during horizontal binding. Exosite binding in slope orientation involved contact of the anilino part, rather than the aminothiazol end, to Ile-178, Ala-103, and Asn-179. In conclusion, contact with Met-292 and Phe-86, which are unique positions of α 1A , accounts for mirabegron binding to α 1A . Because of its lack of interactions with the binding pocket, mirabegron has lower affinity compared to α 1A -blockers and no effects on voiding symptoms.

α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α 1B AR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α 1B AR structure allows the identification of two unique secondary binding pockets. By structural comparison of α 1B AR with α 2 ARs, and by constructing α 1B AR-α 2C AR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α 1B AR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype., (© 2022. The Author(s).)

Several studies confirmed the reciprocal interactions between adrenergic and serotoninergic systems and the influence of these phenomena on the pathogenesis of anxiety. Hence, searching for chemical agents with a multifunctional pharmacodynamic profile may bring highly effective therapy for CNS disorders. This study presents a deep structural insight into the hydantoin-arylpiperazine group and their serotonin/α-adrenergic activity. The newly synthesized compounds were tested in the radioligand binding assay and the intrinsic activity was evaluated for the selected derivatives. The computer-aided SAR analysis enabled us to answer questions about the influence of particular structural fragments on selective vs. multifunctional activity. As a result of the performed investigations, there were two leading structures: (a) compound 12 with multifunctional adrenergic-serotonin activity, which is a promising candidate to be an effective anxiolytic agent; (b) compound 14 with high α 1A /α 1D affinity and selectivity towards α 1B , which is recommended due to the elimination of probable cardiotoxic effect . The structural conclusions of this work provide significant support for future lead optimization in order to achieve the desired pharmacodynamic profile in searching for new CNS-modulating agents.

Although class A seven-transmembrane helix (7TM) receptor hetero-oligomers have been proposed, information on the assembly and function of such higher-order hetero-oligomers is not available. Utilizing bioluminescence resonance energy transfer (BRET), bimolecular luminescence/fluorescence complementation (BiLC/BiFC), and BiLC/BiFC BRET in HEK293T cells, we provide evidence that chemokine (C-X-C motif) receptor 4, atypical chemokine receptor 3, α 1a -adrenoceptor, and arginine vasopressin receptor 1A form hetero-oligomers composed of 2-4 different protomers. We show that hetero-oligomerization per se and ligand binding to individual protomers regulate agonist-induced coupling to the signaling transducers of interacting receptor partners. Our findings support the concept that receptor hetero-oligomers form supramolecular machineries with molecular signaling properties distinct from the individual protomers. These findings provide a mechanism for the phenomenon of context-dependent receptor function., (© 2021 Federation of European Biochemical Societies.)

The C-terminal of G protein-coupled receptors is now recognized as being important for G protein activation and signaling function. To detect the role of C-terminal tail in receptor activation, we used the α1b-AR, which has a long C-terminal of 164 amino acids. We constructed the intramolecular FRET sensors, in which the C-terminal was truncated to 10 (∆C-10), 20 (∆C-20), 30 (∆C-30), 50 (∆C-50), 70 (∆C-70), or 90 (∆C-90). The truncated mutants of ∆C-10, ∆C-20, or ∆C-30 cannot induce FRET signal changes and downstream ERK1/2 phosphorylation. However, the truncated mutants of ∆C-50, ∆C-70, or ∆C-90 induce significant FRET signal changes and downstream ERK1/2 phosphorylation, especially ∆C-90. This is particularly true in the case of the ∆C-90, ∆C-70, or ∆C-50 which retained the potential phosphorylation sites (Ser401, Ser404, Ser408, or Ser410). The ∆C-90 showed an increase in agonist-induced FRET signal changes and ERK1/2 phosphorylation in PKC- or endocytosis-dependent and EGFR-, src-, or β-arrestin2-independent., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Background: Hypertension, one of the most common cardiovascular diseases that can cause coronary disease, stroke, myocardial infarction, and sudden death, it is the major contributor to cardiac failure as well as renal insufficiency., Objectives: As there are many cardio-active pyridazinone-base derivatives in clinical use, therefore, we aimed to synthesize a new series of pyridazin-3-ones and evaluate their vasorelaxant activity., Methods: A new series of synthesized compounds were carried out first by the synthesis of 6- flouroarylpyridazinones by cyclization of 3-(4-flourobenzoyl) propionic acid with hydrazine hydrate or arylhydrazines to provide the corresponding pyridazinone derivatives 2a-d. Mannich reaction was performed using morpholine or piperidine formaldehyde to obtain compounds 3a,b. On the other hand, reaction of 2a with various chloroacetamide intermediates, in dimethylformamide and potassium carbonate as a catalyst, afforded the target compounds 5a-c. The aromatic acid hydrazide intermediates 6a-g were prepared in 50-90% yield, by reacting to the prepared esters with hydrazine hydrate under reflux in ethanol. The two compounds 8a,b were prepared via condensation of 7a,b with ethyl chloroacetate in dry acetone. Finally, the target 2,4,6-trisubstituted pyridazinones 9a-c derivatives were obtained by the reaction of 7a with the appropriate aromatic aldehyde or substituted acetophenones. The new compounds were then evaluated for their vasorelaxant properties using isolated thoracic rat aortic rings. In addition, a homology model was built and molecular modeling simulation of these compounds into the active sites of the newly created α1a-adrenoceptor model was performed in order to predict and rationalize their affinities toward this receptor., Results: Among these compounds; 5a was the most potent, it exhibited approximately two-times the activity of prazosin (IC50 = 0.250, 0.487 mmol, respectively) also, fourteen compounds were more potent than prazosin., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Sample preparation for NMR studies of G protein-coupled receptors faces special requirements: Proteins need to be stable for prolonged measurements at elevated temperatures, they should ideally be uniformly labeled with the stable isotopes 13 C, 15 N, and all carbon-bound protons should be replaced by deuterons. In addition, certain NMR experiments require protonated methyl groups in the presence of a perdeuterated background. All these requirements are most easily satisfied when using Escherichia coli as the expression host. Here we describe a workflow, starting from a temperature-stabilized mutant of the α 1B -adrenergic receptor, obtained using the CHESS methodology, into an even more stable species, in which flexible parts from termini were removed and the intracellular loop 3 (ICL3) was stabilized against proteolytic cleavage. The yield after purification corresponds to 1-2 mg/L of D 2 O culture. The final purification step is ligand-affinity chromatography to ensure that only well-folded ligand-binding protein is isolated. Proper selection of detergent has a remarkable influence on the quality of NMR spectra. All optimization steps of sequence and detergent are monitored on a small scale by monitoring the melting temperature and long-term thermal stability to allow for screening of many conditions. The stabilized mutant of the α 1B -adrenergic receptor was additionally incorporated in nanodiscs, but displayed slightly inferior spectra compared to a sample in detergent micelles. Finally, both [ 15 N, 1 H]- as well as [ 13 C, 1 H]-HSQC spectra are shown highlighting the high quality of the final NMR sample. Importantly, the quality of [ 13 C, 1 H]-HSQC spectra indicates that the so prepared receptor could be used for studying side-chain dynamics., Competing Interests: Declaration of competing interest Herewith I declare that I have no competing interest concerning the paper entitled “Optimizing the α(1B)-Adrenergic Receptor for Solution NMR Studies” submitted to Biochimica et Biophysica Acta Biomembranes., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The structural poses of ligands that bind weakly to protein receptors are challenging to define. In this work we have studied ligand interactions with the adrenoreceptor (AR) subtypes, α 1A -AR and α 1B -AR, which belong to the G protein-coupled receptor (GPCR) superfamily, by employing the solution-based ligand-observed NMR method interligand NOEs for pharmacophore mapping (INPHARMA). A lack of receptor crystal structures and of subtype-selective drugs has hindered the definition of the physiological roles of each subtype and limited drug development. We determined the binding pose of the weakly binding α 1A -AR-selective agonist A-61603 relative to an endogenous agonist, epinephrine, at both α 1A -AR and α 1B -AR. The NMR experimental data were quantitatively compared, by using SpINPHARMA, to the back-calculated spectra based on ligand poses obtained from all-atom molecular dynamics simulations. The results helped mechanistically explain the selectivity of (R)-A-61603 towards α 1A -AR, thus demonstrating an approach for targeting subtype selectivity in ARs., (© 2020 Wiley-VCH GmbH.)

Phytol (PHY), a chlorophyll-derived diterpenoid, exhibits numerous pharmacological properties, including antioxidant, antimicrobial, and anticancer activities. This study evaluates the anti-diarrheal effect of phytol (PHY) along with its possible mechanism of action through in-vivo and in-silico models. The effect of PHY was investigated on castor oil-induced diarrhea in Swiss mice by using prazosin, propranolol, loperamide, and nifedipine as standards with or without PHY. PHY at 50 mg/kg (p.o.) and all other standards exhibit significant (p < 0.05) anti-diarrheal effect in mice. The effect was prominent in the loperamide and propranolol groups. PHY co-treated with prazosin and propranolol was found to increase in latent periods along with a significant reduction in diarrheal section during the observation period than other individual or combined groups. Furthermore, molecular docking studies also suggested that PHY showed better interactions with the α- and β-adrenergic receptors, especially with α-ADR1a and β-ADR1. In the former case, PHY showed interaction with hydroxyl group of Ser192 at a distance of 2.91Å, while in the latter it showed hydrogen bond interactions with Thr170 and Lys297 with a distance of 2.65 and 2.72Å, respectively. PHY exerted significant anti-diarrheal effect in Swiss mice, possibly through blocking α- and β-adrenergic receptors.

A positive feedback loop between inflammatory cytokines and alpha 1 -adrenoceptors (α 1 -AR) (a target of the sympathetic nervous system neurotransmitter norepinephrine) influences inflammatory responses in immune cells. This cross-talk between the sympathetic nervous system and immune system may play a role in promoting chronic inflammation. Emerging evidence shows that α 1 -AR interact with inflammatory cytokines in keratinocytes, and this epidermal adrenergic signalling may contribute to skin inflammatory responses following injury, disease or stress. In this study, utilizing an in vitro approach, we hypothesized that α 1 -AR interact in a positive feedback loop with inflammatory mediators in keratinocytes. The pro-inflammatory cytokine tumor necrosis factor α (TNFα) was used to induce an inflammatory state in cultured keratinocytes. TNFα increased interleukin (IL)-1β, IL-6, IL-8 and nerve growth factor (NGF) production and gene expression levels of α 1 -AR subtype B (α 1B -AR). Additional stimulation of α 1 -AR further increased IL-6 levels, while maintaining high levels of IL-8 and decreasing levels of IL-1β and NGF. Our results suggest that reciprocal influences between α 1 -ARs and inflammatory cytokines may play a role in normal inflammatory responses. However, if unchecked, this cycle could contribute to pathology (e.g. chronic inflammatory diseases, chronic pain conditions, and stress-induced cancer progression)., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

G protein-coupled receptors (GPCRs) use a series of conserved microswitches to transmit signals across the cell membrane via an allosteric network encompassing the ligand-binding site and the G protein-binding site. Crystal structures of GPCRs provide snapshots of their inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here, we analyzed the correlation between ligand binding and receptor conformation of the α 1A -adrenoreceptor, a GPCR that stimulates smooth muscle contraction in response to binding noradrenaline. NMR of [ 13 C ϵ H 3 ]methionine-labeled α 1A -adrenoreceptor variants, each exhibiting differing signaling capacities, revealed how different classes of ligands modulate the conformational equilibria of this receptor. [ 13 C ϵ H 3 ]Methionine residues near the microswitches exhibited distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. We propose that allosteric coupling among the microswitches controls the conformation of the α 1A -adrenoreceptor and underlies the mechanism of ligand modulation of GPCR signaling in cells., (© 2020 Wu et al.)

Purpose: Human eccrine sweat glands respond to α 1 -adrenergic receptor agonists. We recently reported that adrenergic mechanisms contribute to sweating in endurance-trained men during an incremental exercise to volitional fatigue. However, it remains unclear if this response is mediated by α 1 -adrenergic receptor activation., Methods: Twelve endurance-trained men performed an incremental cycling bout until exhaustion while wearing a water-perfused suit to clamp skin temperature at ~ 34 °C. Bilateral forearm sweat rates were measured wherein the distal area was treated with either 1% terazosin (α 1 -adrenergic receptor antagonist) or saline solution on the opposite limb (Control) via transdermal iontophoresis. We also measured proximal bilateral forearm sweat rate in untreated sites to confirm that no between-limb differences in forearm sweat rate occurred. Once sweat rate returned to pre-exercise resting levels at ~ 20 min postexercise, 0.25% phenylephrine (α 1 -adrenergic receptor agonist) was iontophoretically administered to skin to verify α 1 -adrenergic receptor blockade., Results: Sweat rates at the proximal untreated right and left forearm sites were similar during exercise (interaction, P = 0.581). Similarly, no effect of terazosin on sweat rate was measured relative to control site (interaction, P = 0.848). Postexercise administration of phenylephrine increased sweat rate at the control site (0.08 ± 0.09 mg cm -2  min -1 ), which was suppressed by ~ 90% at the terazosin-treated site (0.01 ± 0.02 mg cm -2  min -1 ) (P = 0.026), confirming that α 1 -adrenergic receptor blockade was intact., Conclusion: Our findings demonstrate that α 1 -adrenergic receptors located at eccrine sweat glands do not contribute to eccrine sweating during incremental exercise in young endurance-trained men.

Cell membrane-cloaked nanotechnology has attracted increasing attention owing to its unique bionic properties, such as specific recognition and biocompatibility conferred by the integrated membrane structure and receptors. However, this technology is limited by the dissociation of the cell membrane from its carrier. Here, we report a novel type of cell membrane-cloaked modified magnetic nanoparticle with good stability in drug discovery. High α1A-adrenergic receptor (α1A-AR) expressing HEK293 cell membrane-cloaked magnetic nanogrippers (α1A/MNGs) were used as a platform for the specific targeting and binding of α1A-AR antagonists as candidate bioactive compounds from traditional Chinese medicine (TCM). Furthermore, using a dynamic covalent bonding approach, α1A/MNGs showed great stability with positive control drug recoveries of α1A/MNGs showing almost no decline after use in five adsorption-desorption cycles. Moreover, the α1A/MNGs possessed a unilamellar membrane with magnetic features and exhibited good binding capacity and selectivity. Ultimately, TCM and pharmacological studies of the bioactivity of the screened compounds confirmed the considerable targeting and binding capability of α1A/MNGs. Application of aldehyde group modification in this drug-targeting concept further improved biomaterial stability and paves the way for the development of new drug discovery strategies. More importantly, the successful application of α1A/MNGs provides new insights into methodologies to improve the integration of cell membranes with the nanoparticle platform.

Rationale: Gq signaling in cardiac myocytes is classically considered toxic. Targeting Gq directly to test this is problematic, because cardiac myocytes have many Gq-coupled receptors., Objective: Test whether Gq coupling is required for the cardioprotective effects of an alpha-1A-AR (adrenergic receptor) agonist., Methods and Results: In recombinant cells, a mouse alpha-1A-AR with a 6-residue substitution in the third intracellular loop does not couple to Gq signaling. Here we studied a knockin mouse with this alpha-1A-AR mutation. Heart alpha-1A receptor levels and antagonist affinity in the knockin were identical to wild-type. In wild-type cardiac myocytes, the selective alpha-1A agonist A61603-stimulated phosphoinositide-phospholipase C and myocyte contraction. In myocytes with the alpha-1A knockin, both A61603 effects were absent, indicating that Gq coupling was absent. Surprisingly, A61603 activation of cardioprotective ERK (extracellular signal-regulated kinase) was markedly impaired in the KI mutant myocytes, and A61603 did not protect mutant myocytes from doxorubicin toxicity in vitro. Similarly, mice with the α1A KI mutation had increased mortality after transverse aortic constriction, and A61603 did not rescue cardiac function in mice with the Gq coupling-defective alpha-1A receptor., Conclusions: Gq coupling is required for cardioprotection by an alpha-1A-AR agonist. Gq signaling can be adaptive.

G protein-coupled receptors (GPCRs) convert extracellular stimuli to intracellular responses that regulate numerous physiological processes. Crystallographic and biophysical advances in GPCR structural analysis have aided investigations of structure-function relationships that clarify our understanding of these dynamic receptors, but the molecular mechanisms associated with activation and signaling for individual GPCRs may be more complex than was previously appreciated. Here, we investigated the proposed water-mediated, hydrogen-bonded activation switch between the conserved NPxxY motif on transmembrane helix 7 (TMH7) and a conserved tyrosine in TMH5, which contributes to α 1B -adrenoceptor (α 1B -AR) and β 2 -AR activation. Disrupting this bond by mutagenesis stabilized the α 1B -AR and the β 2 -AR in inactive-state conformations, which displayed decreased agonist potency for stimulating downstream IP 1 and cAMP signaling, respectively. Compared to that for wild-type receptors, agonist-mediated β-arrestin recruitment was substantially reduced or abolished for all α 1B -AR and β 2 -AR inactive-state mutants. However, the inactive-state β 2 -ARs exhibited decreased agonist affinity, whereas the inactive-state α 1B -ARs had enhanced agonist affinity. Conversely, antagonist affinity was unchanged for inactive-state conformations of both α 1B -AR and β 2 -AR. Removing the influence of agonist affinity on agonist potency gave a measure of signaling efficacy, which was markedly decreased for the α 1B -AR mutants but little altered for the β 2 -AR mutants. These findings highlight the pharmacological heterogeneity of inactive-state GPCR conformations, which may facilitate the rational design of drugs that target distinct conformational states of GPCRs., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Compounds with activity at serotonin (5-hydroxytryptamine) 5-HT 2 and α 1 adrenergic receptors have potential for the treatment of central nervous system disorders, drug addiction or overdose. Isolaureline, dicentrine and glaucine enantiomers were synthesized, and their in vitro functional activities at human 5-HT 2 and adrenergic α 1 receptor subtypes were evaluated. The enantiomers of isolaureline and dicentrine acted as antagonists at 5-HT 2 and α 1 receptors with (R)-isolaureline showing the greatest potency (pK b  = 8.14 at the 5-HT 2C receptor). Both (R)- and (S)-glaucine also antagonized α 1 receptors, but they behaved very differently to the other compounds at 5-HT 2 receptors: (S)-glaucine acted as a partial agonist at all three 5-HT 2 receptor subtypes, whereas (R)-glaucine appeared to act as a positive allosteric modulator at the 5-HT 2A receptor., (© 2018 John Wiley & Sons A/S.)

α 1A - and α 1B -adrenoceptors (ARs) are G protein-coupled receptors (GPCRs) that are activated by adrenaline and noradrenaline to modulate smooth muscle contraction in the periphery, and neuronal outputs in the central nervous system (CNS). α 1A - and α 1B -AR are clinically targeted with antagonists for hypertension and benign prostatic hyperplasia and are emerging CNS targets for treating neurodegenerative diseases. The benzodiazepines midazolam, diazepam, and lorazepam are proposed to be positive allosteric modulators (PAMs) of α 1 -ARs. Here, using thermostabilized, purified, α 1A - and α 1B -ARs, we sought to identify the benzodiazepine binding site and modulatory mechanism to inform the design of selective PAMs. However, using a combination of biophysical approaches no evidence was found for direct binding of several benzodiazepines to purified, stabilized α 1A - and α 1B -ARs. Similarly, in cell-based assays expressing unmodified α 1A - and α 1B -ARs, benzodiazepine treatment had no effect on fluorescent ligand binding, agonist-stimulated Ca 2+ release, or G protein activation. In contrast, several benzodiazepines positively modulated phenylephrine stimulation of a cAMP response element pathway by α 1A - and α 1B -ARs; however, this was shown to be caused by off-target inhibition of phosphodiesterases, known targets of diazepam. This study highlights how purified, stabilized GPCRs are useful for validating allosteric ligand binding and that care needs to be taken before assigning new targets to benzodiazepines.

(+)-Cyclazosin [(+)-1] is one of most selective antagonists of the α 1B -adrenoceptor subtype (selectivity ratios, α 1B /α 1A  = 13, α 1B /α 1D  = 38-39). To improve the selectivity, we synthesized and pharmacologically studied the blocking activity against α 1 -adrenoceptors of several homochiral analogues of (+)-cyclazosin featuring different substituents on the carbonyl or amine groups, namely (-)-2, (+)-3, (-)-4-(-)-8, (+)-9. Moreover, we studied the activity of some their opposite enantiomers, namely (-)-1, (-)-3, (+)-6, and (-)-9, to evaluate the influence of stereochemistry on selectivity. The benzyloxycarbonyl and methyl (4aS,8aR) analogues (+)-3 and (-)-6 improved in a significant way the α 1B selectivity of the progenitor compound: 4 and 14 time vs. the α 1D subtype and 35 and 77 times vs. the α 1A subtype, respectively. The study confirmed the importance of the hydrophobic cis-octahydroquinoxaline moiety of these molecules for the establishment of interactions with the α 1 -adrenoceptors as well that of their (4aS,8aR) stereochemistry to grant selectivity for the α 1B subtype. Hypotheses on the mode of interaction of these compounds were advanced on the basis of molecular modeling studies performed on compound (+)-3., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Key Points: Contraction-mediated blunting of postjunctional α-adrenergic vasoconstriction (functional sympatholysis) is attenuated in skeletal muscle of ageing males, brought on by altered postjunctional α 1 - and α 2 -adrenergic receptor sensitivity. The extent to which postjunctional α-adrenergic vasoconstriction occurs in the forearms at rest and during exercise in postmenopausal women remains unknown. The novel findings indicate that contraction-mediated blunting of α 1 - (via intra-arterial infusion of phenylephrine) but not α 2 -adrenergic (via intra-arterial infusion of dexmedetomidine) vasoconstriction was attenuated in postmenopausal women compared to young women. Additional important findings revealed that postjunctional α-adrenergic vasoconstrictor responsiveness at rest does not appear to be affected by age in women. Collectively, these results contribute to our understanding of local neurovascular control at rest and during exercise with age in women., Abstract: Contraction-mediated blunting of postjunctional α-adrenergic vasoconstriction (functional sympatholysis) is attenuated in older males; however, direct confirmation of this effect remains unknown in postmenopausal women (PMW). The present study examined whether PMW exhibit augmented postjunctional α-adrenergic receptor vasoconstriction at rest and during forearm exercise compared to young women (YW). Eight YW (24 ± 1 years) and eight PMW (65 ± 1 years) completed a series of randomized experimental trials: (1) at rest, (2) under high flow (adenosine infusion) conditions and (3) during 6 min of forearm exercise at relative (20% of maximum) and absolute (7 kg) intensities. Phenylephrine (α 1 -agonist) or dexmedetomidine (α 2 -agonist) was administered during the last 3 min of each trial to elicit α-adrenergic vasoconstriction. Forearm vascular conductance (FVC) was calculated from blood flow and blood pressure. Vasoconstrictor responsiveness was identified as the change in FVC (%) during α-adrenergic agonist infusions from baseline (resting trial) or from steady-state conditions (high flow and exercise trials). During resting and high flow trials, the %FVC during α 1 - and α 2 -agonist stimulation was similar between YW and PMW. During exercise, α 1 -mediated vasoconstriction was blunted in YW vs. PMW at relative (-6 ± 2% vs. -15 ± 3%) and absolute (-4 ± 2% vs. -14 ± 5%) workloads, such that blood flow and FVC were lower in PMW (P < 0.05 for all). Conversely, α 2 -mediated vasoconstriction was similar between YW and PMW at relative (-22 ± 3% vs. -22 ± 4%; P > 0.05) and absolute (-19 ± 3% vs. -18 ± 4%; P > 0.05) workloads. Collectively, these findings demonstrate that despite similar α-adrenergic vasoconstrictor responsiveness at rest, PMW have a decreased ability to attenuate α 1 -adrenergic vasoconstriction in contracting skeletal muscle., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

Purpose: Previous studies have shown that several components of the metabolic syndrome, such as hypertension, obesity or imbalanced lipid and carbohydrate homeostasis, are associated with the sympathetic nervous system overactivity. Therefore, the inhibition of the adrenergic nervous system seems to be a reasonable and appropriate therapeutic approach for the treatment of metabolic disturbances. It has been suggested that non-selective adrenoceptor antagonists could be particularly beneficial, since α 1 -adrenoceptor antagonists can improve disrupted lipid and carbohydrate profiles, while the inhibition of the α 2 -adrenoceptor may contribute to body weight reduction. The aim of the present study was to investigate the metabolic benefits deriving from administration of a non-selective α-adrenoceptor antagonist from the group of pyrrolidin-2-one derivatives. The aim of the present study was to investigate the potential metabolic benefits deriving from chronic administration of a non-selective α-adrenoceptor antagonist, from the group of pyrrolidin-2-one derivatives., Methods: The α 1 - and α 2 -adrenoreceptor affinities of the tested compound-1-(3-(4-(o-tolyl)piperazin-1-yl)propyl)pyrrolidin-2-one had been investigated previously by means of the radioligand binding assay. In the present study, we extended the pharmacological profile characteristics of the selected molecule by additional intrinistic activity assays. Next, we investigated the influence of the tested compound on body weight, hyperglycemia, hypertriglyceridemia, blood pressure in the animal model of obesity induced by a high-fat diet, and additionally we measured the spontaneous activity and body temperature., Results: The intrinistic activity studies revealed that the tested compound is a potent, non-selective antagonist of α 1B and α 2A -adrenoceptors. After the chronic administration of the tested compound, we observed reduced level of triglycerides and glucose in the rat plasma. Interestingly, the tested did not reduce the body weight and did not influence the blood pressure in normotensive animals. Additionally, the administration of the tested compound did not change the animals' spontaneous activity and body temperature., Conclusion: Non-selective α-adrenoceptor antagonist seems to carry potential benefits in the improvement of the reduction of elevated glucose and triglyceride level. The lack of influence on blood pressure suggests that compounds with such a pharmacological profile may be particulary beneficial for the patients with disturbed lipid and carbohydrate profile, who do not suffer from hypertension. These results are particulary valuable, since currently there are no safe α 2A -adrenoceptor antagonist drugs available in clinical use with the ability to modulate hyperglycemia that would not affect blood pressure.

Recently, we reported that chemokine (C-X-C motif) receptor (CXCR)4 and atypical chemokine receptor 3 regulate α 1 -adrenergic receptors (α 1 -AR) through the formation of hetero-oligomeric complexes. Whether α 1 -ARs also regulate chemokine receptor function within such heteromeric receptor complexes is unknown. We observed that activation of α 1b -AR within the α 1b -AR:CXCR4 heteromeric complex leads to cross-recruitment of β-arrestin2 to CXCR4, which could not be inhibited with AMD3100. Activation of CXCR4 did not cross-recruit β-arrestin2 to α 1b -AR. A peptide analogue of transmembrane domain 2 of CXCR4 interfered with α 1b -AR:CXCR4 heteromerization and inhibited α 1b -AR-mediated β-arrestin2 cross-recruitment. Phenylephrine (PE) induced internalization of CXCR4 in HEK293 cells co-expressing CXCR4 and α 1b -AR and of endogenous CXCR4 in human vascular smooth muscle cells (hVSMC). The latter was detectable despite blockade of CXCR4 with the neutralizing antibody 12G5. hVSMC migrated towards CXCL12 and PE, but not towards a combination of CXCL12 and PE. PE inhibited CXCL12-induced chemotaxis of hVSMC (IC 50 : 77 ± 30 nM). Phentolamine cross-inhibited CXCL12-induced chemotaxis of hVSMC, whereas AMD3100 did not cross-inhibit PE-induced chemotaxis. These data provide evidence for asymmetrical cross-regulation of CXCR4 by α 1 -adrenergic receptors within the heteromeric receptor complex. Our findings provide mechanistic insights into the function of α 1 -AR:CXCR4 heteromers and suggest alternative approaches to modulate CXCR4 in disease conditions.

Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists., Competing Interests: The authors declare that they have no conflicts of interest.

We have now cloned an alpha-1 adrenergic receptor (A1AR) from the cDNA library of oyster Crassostrea gigas, designating as CgA1AR-1. The full length of CgA1AR-1 was 1149 bp and it encodes a protein of 382 amino acids containing a 7 transmembrane domain, whose putative topology was similar to the A1ARs in higher organisms and shared similarity of 19% with mammalian A1ARs according to the phylogenic analysis. After cell transfection of CgA1AR-1 into HEK293T cells and the incubation with its specific agonist norepinephrine (NE), the concentration of second messenger Ca 2+ increased significantly (p < 0.05). But, this increasing of Ca 2+ could be inhibited by adding A1AR antagonist DOX. Tissue distribution assays using qRT-PCR suggested that CgA1AR-1 mRNA was ubiquitously expressed in all the major tissues of oyster. LPS stimulation could induce the up-regulation of CgA1AR-1 mRNA in haemocytes from 12 h to 24 h post stimulation. Moreover, the blocking of CgA1AR-1 by DOX before LPS stimulation affected the mRNA expression of oyster TNF (CGI&#95;10005109 and CGI&#95;10006440) in haemocytes, resulting in the rise of haemocyte phagocytic rate and apoptosis index. In addition to cellular immunity, CgA1AR-1 was also involved in humoral immunity of oyster. Inhibition of CgA1AR-1 with DOX could repress the up-regulation of LZY and SOD activities caused by LPS stimulation. These results suggested that CgA1AR-1 acted as an α-1 adrenergic receptor in cetacholaminergic neuroendocrine-immune network mediating both cellular and humoral immune response., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Arylpiperazines 2-11 were synthesized, and their biological profiles at α1-adrenergic receptors (α1-ARs) assessed by binding assays in CHO cells expressing human cloned subtypes and by functional experiments in isolated rat vas deferens (α1A), spleen (α1B), and aorta (α1D). Modifications at the 1,3-benzodioxole and phenyl phamacophoric units resulted in the identification of a number of potent compounds (moderately selective with respect to the α1b-AR), in binding experiments. Notably, compound 7 (LDT451) showed a subnanomolar pKi of 9.41 towards α1a-AR. An encouragingly lower α1B-potency was a general trend for all the series of compounds, which showed α1A/D over α1B selectivity in functional assays. If adequately optimized, such peculiar selectivity could have relevance for a potential LUTS/BPH therapeutic application., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Fluorescent ligands are gaining popularity as tools to aid GPCR research. Nonetheless, in vivo application of such tools is hampered due to their short excitation wavelengths in the visible range and lack of fluorogenic switch. Here we report the discovery of fluorescent ligands (3a-f) for α1-adrenergic receptors (α1-ARs) by conjugating the environment-sensitive fluorophore cyane 5 (Cy5) with the quinazoline pharmacophore. Among them, the conjugated compound 3a, with acylated piperazine and the shortest carbon chain spacer, exhibited potent binding and remarkable changes in fluorescence (10-fold) upon binding to α1-AR. Furthermore, it could be employed to selectively and specifically label α1-ARs with no washing procedures in single cells, prostate tissue slices, intact tumor xenografts and organs in living mice. Especially, the slice imaging results gave direct and visual evidence that there is a close relationship between α1-ARs and pathological prostate. It is anticipated that our fluorescent tools will find broad applications in the study of α1-AR pharmacology and physiology to aid development of novel therapeutics.

β-Adrenergic receptor antagonists are important therapeutics for the treatment of cardiovascular disorders. In the group of β-blockers, much attention is being paid to the third-generation drugs that possess important ancillary properties besides inhibiting β-adrenoceptors. Vasodilating activity of these drugs is produced through different mechanisms, such as nitric oxide (NO) release, β2 -agonistic action, α1 -blockade, antioxidant action, and Ca(2+) entry blockade. Here, a study on evaluation of the cardiovascular activity of five new compounds is presented. Compound 3a is a methyl and four of the tested compounds (3b-e) are dimethoxy derivatives of 1-(1H-indol-4-yloxy)-3-(2-(2-methoxyphenoxy)ethylamino)propan-2-ol. The obtained results confirmed that the methyl and dimethoxy derivatives of 1-(1H-indol-4-yloxy)-3-(2-(2-methoxyphenoxy)ethylamino)propan-2-ol and their enantiomers possess α1 - and β1 -adrenolytic activities and that the antiarrhythmic and hypotensive effects of the tested compounds are related to their adrenolytic properties., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The seminal human dopamine D3 receptor (hD3R) ligand BP 897 has shown interesting properties during clinical trials. However, its lack of selectivity towards human adrenergic receptor impedes further development. Two approaches were followed to increase hD3R selectivity. The lead optimisation succeeded, we disclose here ligands with subnanomolar potency for D3R, combined with a good selectivity for the closely related human dopamine D2 and human adrenergic alpha-1 receptors., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In rodents, kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3V) of the preoptic area are considered to provide a major stimulatory input to the GnRH neuronal network that is responsible for triggering the preovulatory LH surge. Noradrenaline (NA) is one of the main modulators of GnRH release, and NA fibers are found in close apposition to kisspeptin neurons in the RP3V. Our objective was to interrogate the role of NA signaling in the kisspeptin control of GnRH secretion during the estradiol induced LH surge in ovariectomized rats, using prazosin, an α1-adrenergic receptor antagonist. In control rats, the estradiol-induced LH surge at 17 hours was associated with a significant increase in GnRH and kisspeptin content in the median eminence with the increase in kisspeptin preceding that of GnRH and LH. Prazosin, administered 5 and 3 hours prior to the predicted time of the LH surge truncated the LH surge and abolished the rise in GnRH and kisspeptin in the median eminence. In the preoptic area, prazosin blocked the increases in Kiss1 gene expression and kisspeptin content in association with a disruption in the expression of the clock genes, Per1 and Bmal1. Together these findings demonstrate for the first time that NA modulates kisspeptin synthesis in the RP3V through the activation of α1-adrenergic receptors prior to the initiation of the LH surge and indicate a potential role of α1-adrenergic signaling in the circadian-controlled pathway timing of the preovulatory LH surge.

Indole-arylpiperazine derivatives have exhibited good selectivity for the α1A-adrenoceptor, but the structure-activity-binding mechanism relationship remains unclear. In the current study, three compounds (1, 2 and 3) were investigated through single-crystal X-ray diffraction analysis, density functional theory (DFT) calculations and molecular docking using a homology model of the α1A receptor. Compounds 1 and 3 form H-bonds networks to stabilize their three-dimensional structures, while C-H···π interactions play a significant role in the packing of 2. Based on DFT-optimized conformations, the HOMO-LUMO energy gaps and molecular electrostatic potential (MEP) were theoretically calculated at the B3LYP/6-311G (d, p) level of theory. Chemical reactivity increases in the order of 3 < 2 < 1, and the maximum positive region of the MEP maps is mainly localized over the NH group. The binding mechanisms of ligand-α1A-adrenoceptor complexes were illustrated by molecular docking. Binding to Gln177 of the second extracellular loop region via hydrogen bonds is likely to be essential for α1A-selective antagonists. The present work sheds light on the studies of structure-activity-binding mechanism and aids in the design of α1A antagonists with high selectivity.

Background: eIF3f is a multifunctional protein capable of interacting with proteins involved in different cellular processes, such as protein synthesis, DNA repair, and viral mRNA edition. In human cells, eIF3f is related to cell cycle and proliferation, and its deregulation compromises cell viability., Results: We here report that, in native conditions, eIF3f physically interacts with the alpha 1B-adrenergic receptor, a plasma membrane protein considered as a proto-oncogene, and involved in vasoconstriction and cell proliferation. The complex formed by eIF3f and alpha 1B-ADR was found in human and mouse cell lines. Upon catecholamine stimulation, eIF3f promotes adrenoceptor activity in vitro, independently of the eIF3f proline- and alanine-rich N-terminal region., Conclusions: The eIF3f/alpha adrenergic receptor interaction opens new insights regarding adrenoceptor-related transduction pathways and proliferation control in human cells. The eIf3f/alpha 1B-ADR complex is found in mammals and is not tissue specific.

Ligand binding and conformational changes that accompany signaling from G protein-coupled receptors (GPCRs) have mostly focused on the role of transmembrane helices and intracellular loop regions. However, recent studies, including several GPCRs cocrystallized with bound ligands, suggest that the extracellular surface (ECS) of GPCRs plays an important role in ligand recognition, selectivity, and binding, as well as potentially contributing to receptor activation and signaling. This study applied alanine-scanning mutagenesis to investigate the role of the complete ECS of the α1B-adrenoreceptor on norepinephrine (NE) potency, affinity, and efficacy. Half (24 of 48) of the ECS mutations significantly decreased NE potency in an inositol 1-phosphate assay. Most mutations reduced NE affinity (17) determined from [(3)H]prazosin displacement studies, whereas four mutations at the entrance to the NE binding pocket enhanced NE affinity. Removing the influence of NE affinity and receptor expression levels on NE potency gave a measure of NE efficacy, which was significantly decreased for 11 of 48 ECS mutants. These different effects tended to cluster to different regions of the ECS, which is consistent with different regions of the ECS playing discrete functional roles. Exposed ECS residues at the entrance to the NE binding pocket mostly affected NE affinity, whereas buried or structurally significant residues mostly affected NE efficacy. The broad potential for ECS mutations to affect GPCR function has relevance for the increasing number of nonsynonymous single nucleotide polymorphisms now being identified in GPCRs., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Despite the physiological and pharmacological importance of the α1A-adrenoreceptor, the mode of interactions of classical agonists and radioactive ligands with this receptor is not yet clearly defined. Here, we used mutagenesis studies and binding experiments to evaluate the importance of 11 receptor sites for the binding of (125)I-HEAT, (3)H-prazosin and epinephrine. Only one residue (F312) commonly interacts with the three molecules, and, surprisingly, D106 interacts only with epinephrine in a moderate way. Our docking model shows that prazosin and HEAT are almost superimposed into the orthosteric pocket with their tetralone and quinazoline rings close to the phenyl ring of the agonist., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Recently, 1-(1,4-dioxaspiro[4,5]dec-2-ylmethyl)-4-(2-methoxyphenyl)piperazine (1) was reported as a highly selective and potent 5-HT1AR ligand. In the present work we adopted an in-parallel synthetic strategy to rapidly explore a new set of arylpiperazine (7-32) that is structurally related to 1. The compounds were tested for binding affinity and functional activity at 5-HT1AR and α1-adrenoceptor subtypes and SAR studies were drawn. In particular, compounds 9, 27 and 30 emerged as promising α1 receptor antagonists, while compound 10 behaves as the most potent and efficacious 5-HT1AR agonist. All the compounds were docked into the 5HT1AR theoretical model and the results were in agreement with the biological experimental data. These findings may represent a new starting point for developing more selective α1 or 5-HT1AR ligands., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

A series of ring-substituted ethyl- and heptyl-linked 4-aminoquinoline dimers were synthesized and evaluated for their affinities at the 3 human α(1)-adrenoceptor (α(1)-AR) subtypes and the human serotonin 5-HT(1A)-receptor (5-HT(1A)-R). We find that the structure-specificity profiles are different for the two series at the α(1)-AR subtypes, which suggests that homobivalent 4-aminoquinolines can be developed with α(1)-AR subtype selectivity. The 8-methyl (8-Me) ethyl-linked analogue has the highest affinity for the α(1A)-AR, 7 nM, and the greatest capacity for discriminating between α(1A)-AR and α(1B)-AR (6-fold), α(1D)-AR (68-fold), and the 5-HT(1A)-R (168-fold). α(1B)-AR selectivity was observed with the 6-methyl (6-Me) derivative of the ethyl- and heptyl-linked 4-aminoquinoline dimers and the 7-methoxy (7-OMe) derivative of the heptyl-linked analogue. These substitutions result in 4- to 80-fold selectivity for α(1B)-AR over α(1A)-AR, α(1D)-AR, and 5-HT(1A)-R. In contrast, 4-aminoquinoline dimers with selectivity for α(1D)-AR are more elusive, since none studied to date has greater affinity for the α(1D)-AR over the other two α(1)-ARs. The selectivity of the 8-Me ethyl-linked 4-aminoquinoline dimer for the α(1A)-AR, and 6-Me ethyl-linked, and the 6-Me and 7-OMe heptyl-linked 4-aminoquinoline dimers for the α(1B)-AR, makes them promising leads for drug development of α(1A)-AR or α(1B)-AR subtype selective ligands with reduced 5-HT(1A)-R affinity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The role of naturally occurring human α1a-Adrenergic Receptor (α1aAR) genetic variants associated with cardiovascular disorders is poorly understood. Here, we present the novel findings that expression of human α1aAR-247R (247R) genetic variant in cardiomyoblasts leads to transition of cardiomyoblasts into a fibroblast-like phenotype, evidenced by morphology and distinct de novo expression of characteristic genes. These fibroblast-like cells exhibit constitutive, high proliferative capacity and agonist-induced hypertrophy compared with cells prior to transition. We demonstrate that constitutive, synergistic activation of EGFR, Src and ERK kinases is the potential molecular mechanism of this transition. We also demonstrate that 247R triggers two distinct EGFR transactivation-dependent signaling pathways: 1) constitutive Gq-independent β-arrestin-1/Src/MMP/EGFR/ERK-dependent hyperproliferation and 2) agonist-induced Gq- and EGFR/STAT-dependent hypertrophy. Interestingly, in cardiomyoblasts agonist-independent hyperproliferation is MMP-dependent, but in fibroblast-like cells it is MMP-independent, suggesting that expression of α1aAR genetic variant in cardiomyocytes may trigger extracellular matrix remodeling. Thus, these novel findings demonstrate that EGFR transactivation by α1aAR-247R leads to hyperproliferation, hypertrophy and alterations in cardiomyoblasts, suggesting that these unique genetically-mediated alterations in signaling pathways and cellular function may lead to myocardial fibrosis. Such extracellular matrix remodeling may contribute to the genesis of arrhythmias in certain types of heart failure., (Copyright © 2014 Elsevier Inc. All rights reserved.)

α1-Adrenoceptors (ARs; 1A, 1B, and 1D) have been determined to perform different prominent functions in the physiological responses of the sympathetic nervous system. A high-throughput screening assay (HTS) was set up to detect α1-AR subtype-selective agonists by a dual-luciferase reporter assay in HEK293 cells. Using the HTS assay, two novel compounds, CHE3 and CHK3, were discovered as α1-ARs agonists in α1-ARs expressed in HEK293 cells. These compounds also showed moderate/weak anti-proliferative activities against tested cancer cell lines. The HTS assay proposed in this study represents a potential method for discovering more α1-AR subtype-selective ligands.

The extracellular loops of the adrenoceptors present a potential therapeutic target in the design of highly selective adrenergic drugs. These regions are less conserved than the orthosteric binding site but have to date not been implicated in activation of adrenoceptors. A previously generated homology model identified an extracellular residue, D191, as a potential regulator of agonist binding. We have generated mutants of the α1B adrenoceptor replacing the charged aspartate, D191, as well as a potential interaction partner, K331, with uncharged alanines to observe effects on ligand binding and receptor activation. Significant 4-6 fold reductions in affinity for the endogenous agonists, epinephrine and norepinephrine were observed for receptors with the D191A mutation in the second extracellular loop. While changes in EC50 were observed, operational analysis yielded no apparent change in receptor activation. Based on these findings, we suggest that D191, in the second extracellular loop of the α1B adrenoceptor, acts as a 'point of first contact' for the receptor's endogenous agonists. Implication of the non-conserved extracellular regions of the receptor in agonist binding makes it a potential target for the design of highly selective drugs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Alpha(1D)-adrenergic receptor (α(1D)-AR) plays important roles in regulating physiological and pathological responses mediated by catecholamines, particularly in the cardiovascular and urinary systems. The present study was designed to investigate the expression profile of α(1D)-AR in the diabetic kidneys and its modulation by activation of peroxisome proliferator-activated receptors (PPARs). 12-week-old Zucker lean (ZL) and Zucker diabetic fatty (ZD) rats were treated with fenofibrate or rosiglitazone for 8-10 weeks. Gene microarray, real-time PCR, and confocal immunofluorescence microscopy were performed to assess mRNA and protein expression of α(1D)-AR in rat kidney tissue. Using microarray, we found that α(1D)-AR gene was dramatically upregulated in 22-week-old ZD rats compared to ZL controls. Quantitative PCR analysis verified a 16-fold increase in α(1D)-AR mRNA in renal cortex from ZD animals compared to normal controls. Chronic treatment with fenofibrate or rosiglitazone reduced renal cortical α(1D)-AR gene. Immunofluorescence staining confirmed that α(1D)-AR protein was induced in the glomeruli and tubules of diabetic rats. Moreover, dual immunostaining for α(1D)-AR and kidney injury molecule-1 indicated that α(1D)-AR was expressed in dedifferentiated proximal tubules of diabetic Zucker rats. Taken together, our results show that α(1D)-AR expression is upregulated in the diabetic kidneys. PPAR activation suppressed renal expression of α(1D)-AR in diabetic nephropathy.

Astrocyte Ca2+ signals in awake behaving mice are widespread, coordinated and differ fundamentally from the locally restricted Ca2+ transients observed ex vivo and in anesthetized animals. Here we show that the synchronized release of norepinephrine (NE) from locus coeruleus (LC) projections throughout the cerebral cortex mediate long-ranging Ca2+ signals by activation of astrocytic α1-adrenergic receptors. When LC output was triggered by either physiological sensory (whisker) stimulation or an air-puff startle response, astrocytes responded with fast Ca2+ transients that encompassed the entire imaged field (positioned over either frontal or parietal cortex). The application of adrenergic inhibitors, including α1-adrenergic antagonist prazosin, potently suppressed both evoked, as well as the frequently observed spontaneous astroglial Ca2+ signals. The LC-specific neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), which reduced cortical NE content by >90%, prevented nearly all astrocytic Ca2+ signals in awake mice. The observations indicate that in adult, unanesthetized mice, astrocytes do not respond directly to glutamatergic signaling evoked by sensory stimulation. Instead astrocytes appear to be the primary target for NE, with astrocytic Ca2+ signaling being triggered by the α1-adrenergic receptor. In turn, astrocytes may coordinate the broad effects of neuromodulators on neuronal activity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of (3)H-prazosin or (125)I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ρ-Da1a, prazosin and HEAT compete at the α1A-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of (3)H-prazosin or (125)I-HEAT, and the IC50 of ρ-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ρ-Da1a remained stable. The effect of ρ-Da1a on agonist-stimulated Ca(2+) release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α1A-adrenoceptor were evaluated for alterations in ρ-Da1a affinity. The D106(3.32)A and the S188(5.42)A/S192(5.46)A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86(2.64)A, F288(6.51)A and F312(7.39)A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86(2.64) was identified as a key interaction point for (125)I-HEAT, as the variant F86(2.64)A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ρ-Da1a interacts with the human α1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86(2.64), F288(6.51) and F312(7.39)) and agonist (F288(6.51) and F312(7.39)) ligands. Its selectivity for the α1A-adrenoceptor may result, at least partly, from its interaction with the residue F86(2.64), which appears to be important also for HEAT binding.

α₁-adrenoceptor (α₁-AR) subtype-selective ligands lacking off-target affinity for the 5-HT(1A) receptor (5-HT(1A)-R) will provide therapeutic benefits in the treatment of urogenital conditions such as benign prostatic hyperplasia. In this study we determined the affinity of 4-aminoquinoline and eleven homobivalent 4-aminoquinoline ligands (diquinolines) with alkane linkers of 2-12 atoms (C2-C12) for α(1A), α(1B) and α(1D)-ARs and the 5-HT(1A)-R. These ligands are α(1A)-AR antagonists with nanomolar affinity for α(1A) and α(1B)-ARs. They display linker-length dependent selectivity for α(1A/B)-ARs over α(1D)-AR and the 5-HT(1A)-R. The C2 diquinoline has the highest affinity for α1A-AR (pKi 7.60±0.26) and greater than 30-fold and 600-fold selectivity for α(1A)-AR over α(1D)-AR and 5-HT(1A)-R respectively. A decrease in affinity for α₁-ARs is observed as the linker length increases, reaching a nadir at 5 (α(1A/1B)-ARs) or 6 (α(1D)-AR) atoms; after which affinity increases as the linker is lengthened, peaking at 9 (α(1A/1B/1D)-ARs) or 8 (5-HT(1A)-R) atoms. Docking studies suggest that 4-aminoquinoline and C2 bind within the orthosteric binding site, while for C9 one end is situated within the orthosteric binding pocket, while the other 4-aminoquinoline moiety interacts with the extracellular surface. The limited α(1D)-AR and 5-HT(1A)-R affinity of these compounds makes them promising leads for future drug development of α(1A)-AR selective ligands without α(1D)-AR and the 5-HT(1A)-R off-target activity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β(1)-adrenergic receptor crystal structure, we modeled the α(1B)-adrenoceptor (α(1B)-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α(1B)-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α(1)-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs.

Demand on high-throughput methods for multi-target compounds screening continues to increase nowadays due to the decline of new drugs on the market. Two kinds of G-protein-coupled receptors, alpha1-adrenoceptor (α(1A)-AR) and beta2-adrenoceptor (β(2)-AR), were purified and immobilized on the surface of macroporous silica gel to prepare new chromatographic stationary phases. Control drugs (e.g., prazosin, terazosin, salbutamol, and terbutaline) were used to characterize the retention behavior of the obtained α(1A)-AR and β(2)-AR columns. This study also coupled both columns with a six-way switching valve to construct an automatic two-dimensional system for multi-target compounds screening in complex mixtures. Adrenaline hydrochloride was used as a representative drug to evaluate the chromatographic performance of the two dimensional system. The aqueous extracts from Salvia miltiorrhiza and Coptis chinensis were also analyzed by the automatic system. The compounds in S. miltiorrhiza had no binding to both α(1A)-AR and β(2)-AR columns. But berberine, palmatine and jatrorrhizine were screened as the bioactive compounds in C. chinensis, targeting both the receptors. The proposed method is an alternative for recognizing and separating the compounds targeting different proteins from a complex matrix., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The study is focused on a series of 5-arylidenehydantoin derivatives with a phenylpiperazine-hydroxypropyl fragment at N3 of the hydantoin ring. The compounds were assessed on their affinity for α(1)-adrenoceptors and evaluated in functional bioassays for their antagonistic properties. Crystal structures of (Z)-5-(4-chlorobenzylidene)-3-(3-(4-(2-ethoxyphenyl)piperazin-1-yl)-2-hydroxypropyl)imidazolidine-2,4-dione (7) and hydrochloride of (Z)-5-(4-chlorobenzylidene)-3-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)imidazolidine-2,4-dione (10a) were solved using the X-ray diffraction method. Classical molecular mechanics (MMFFs force field, MCMM, MacroModel) were used to predict 3D structure of compounds 5a-18a using a crystal structure of 7. SAR analysis was performed on the basis of Barbaro's pharmacophore model and structural properties of previously investigated α(1)-adrenoceptor antagonists possessing a hydantoin fragment. Most of the compounds exhibited significant affinities for α(1)-ARs in nanomolar range (40-290 nM). The highest activities (K(i)<75 nM) were observed for compounds possessing a 2-alkoxyphenylpiperazine fragment and two methoxy substituents at the benzylidene moiety. The results indicated that chemical properties, number and positions of substituents at the 5-arylidene fragment influenced the power of α(1)-affinities as follows: 3,4-di CH(3)O>2,4-di CH(3)O>4-Cl>2,3-di CH(3)O>H>4-N(CH(3))(2)., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We have provided the first evidence for specific heteromerization between the α(1A)-adrenoceptor (α(1A)AR) and CXC chemokine receptor 2 (CXCR2) in live cells. α(1A)AR and CXCR2 are both expressed in areas such as the stromal smooth muscle layer of the prostate. By utilizing the G protein-coupled receptor (GPCR) heteromer identification technology on the live cell-based bioluminescence resonance energy transfer (BRET) assay platform, our studies in human embryonic kidney 293 cells have identified norepinephrine-dependent β-arrestin recruitment that was in turn dependent upon co-expression of α(1A)AR with CXCR2. These findings have been supported by co-localization observed using confocal microscopy. This norepinephrine-dependent β-arrestin recruitment was inhibited not only by the α(1)AR antagonist Terazosin but also by the CXCR2-specific allosteric inverse agonist SB265610. Furthermore, Labetalol, which is marketed for hypertension as a nonselective β-adrenoceptor antagonist with α(1)AR antagonist properties, was identified as a heteromer-specific-biased agonist exhibiting partial agonism for inositol phosphate production but essentially full agonism for β-arrestin recruitment at the α(1A)AR-CXCR2 heteromer. Finally, bioluminescence resonance energy transfer studies with both receptors tagged suggest that α(1A)AR-CXCR2 heteromerization occurs constitutively and is not modulated by ligand. These findings support the concept of GPCR heteromer complexes exhibiting distinct pharmacology, thereby providing additional mechanisms through which GPCRs can potentially achieve their diverse biological functions. This has important implications for the use and future development of pharmaceuticals targeting these receptors.

An association between α(1)-adrenoceptor affinities, hERG K(+)-antagonistic properties and antiarrhythmic activities for a series of phenylpiperazine derivatives of hydantoin (2a-21a) was investigated. New compounds were synthesized and tested for their affinity for α(1)-adrenoceptors in radioligand binding assay using [(3)H]-prazosin as a selective radioligand. Antiarrhythmic activities in adrenaline- and barium chloride-induced arrhythmia models, an influence of the phenylpiperazine derivatives on the ECG-components and blood pressure were tested in vivo in normotensive rats. The hERG K(+)-antagonistic properties of the most potent antiarrhythmic agents were investigated in silico by the use of program QikProp. The highest α(1)-adrenoceptor affinity (K(i)=4.7 nM) and the strongest antiarrhythmic activity in adrenaline induced arrhythmia (ED(50)=0.1 mg/kg) was found for 1-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione hydrochloride (19a). The results indicated a significant correlation between α(1)-AR affinities (pK(i)) and antiarrhythmic activity (ED(50)) in adrenaline model (R(2)=0.92, p <0.005). Influence of the examined phenylpiperazine hydantoin derivatives on hERG K(+) channel, predicted by means of in silico methods, suggested their hERG K(+)-blocking properties., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Acquired resistance of cancer cells to various chemotherapeutic agents is known as multidrug resistance, and remains a critical factor in the success of cancer treatment. It is necessary to develop the inhibitors for multidrug resistance. The aim of this study was to examine the effects of eight α-adrenoceptor antagonists on ABCG2/BCRP-mediated resistance and transport. Previously established HeLa/SN100 cells, which overexpress ABCG2/BCRP but not ABCB1/MDR1, were used. The effects of the antagonists on sensitivity to mitoxantrone and the transport activity of Hoehst33342, both substrates for ABCG2/BCRP, were evaluated using the WST-1 assay and cellular kinetics, respectively. ABCG2/BCRP mRNA expression and the cell cycle were also examined by real-time RT-PCR and flow cytometry, respectively. Sensitivity to mitoxantrone was reversed by the α-adrenoceptor antagonists in a concentration-dependent manner, although such effects were also found in the parental HeLa cells. Levels of ABCG2/BCRP mRNA expression were not influenced by the antagonists. The transport activity of Hoechst33342 was decreased by doxazosin and prazosin, but unaffected by the other antagonists. In addition, doxazosin and prazosin increased the proportion of S phase cells in the cultures treated with mitoxantrone, whereas the other α-adrenoceptor antagonists increased the percentage of cells in G(2)/M phase. These findings suggested that doxazosin and prazosin reversed resistance mainly by inhibiting ABCG2/BCRP-mediated transport, but the others affected sensitivity to mitoxantrone via a different mechanism.

The present study explored the hypothesis that a prolonged 8 weeks exposure to a high fructose intake suppresses adrenergic and angiotensin II (Ang II)-mediated vasoconstriction and is associated with a higher contribution of α1D-adrenoceptors. A total of thirty-two Sprague-Dawley rats received either 20 % fructose solution (FFR) or tap water (control, C) to drink ad libitum for 8 weeks. Metabolic and haemodynamic parameters were assessed weekly. The renal cortical vasoconstrictor responses to noradrenaline (NA), phenylephrine (PE), methoxamine (ME) and Ang II were determined in the presence and absence of BMY7378 (α1D-adrenoceptor antagonist). FFR had increased blood pressure, plasma levels of glucose, TAG and insulin. FFR expressed reduced renal vascular responses to adrenergic agonists and Ang II (NA: 50 %, PE: 50 %, ME, 65 %, Ang II: 54 %). Furthermore in the C group, the magnitude of the renal cortical vasoconstriction to all agonists was blunted in the presence of the low or high dose of BMY7378 (NA: 30 and 31 %, PE: 23 and 33 %, ME: 19 and 44 %, Ang II: 53 and 77 %), respectively, while in the FFR, vasoconstriction was enhanced to adrenergic agonists and reduced to Ang II (NA: 8 and 83 %, PE: 55 %, ME, 2 and 177 %, Ang II: 61 and 31 %). Chronic high fructose intake blunts vascular sensitivity to adrenergic agonists and Ang II. Moreover, blocking of the α1D-adrenoceptor subtype results in enhancement of renal vasoconstriction to adrenergic agonists, suggesting an inhibitory action of α1D-adrenoceptors in the FFR. α1D-Adrenoceptors buffer the AT1-receptor response in the renal vasculature of normal rats and fructose feeding suppressed this interaction.

A series of 2-[(heteroaryl)methyl]imidazolines was synthesized and tested for their activities at α(1)- and α(2)-adrenoceptors and imidazoline I(1) and I(2) receptors. The most active 2-[(indazol-1-yl)methyl]imidazolines showed high or moderate affinities for α(1)- and α(2)-adrenoceptors. However, their intrinsic activities at α(2A)-adrenoceptors proved to be negligible. A selected 7-chloro derivative behaved as a potent α(1)-adrenoceptor antagonist and exhibited peripherally mediated hypotensive effects in rats., (Copyright © 2011 Elsevier Ltd. All rights reserved.)