[Establishment and evaluation of haloalkane dehalogenase tagged α1A-adrenergic receptor chromatography].

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 α1A-adrenergic receptor (α1A-AR). Specifically, Halo-α1A-AR was immobilized on the surface of 6-chlorohexanoic acid-modified aminopropyl silica gel in one step. The stationary phase with immobilized Halo-α1A-AR was characterized using scanning electron microscopy. Moreover, the activity of the Halo-α1A-AR chromatographic column was evaluated using specific ligands (terazosin hydrochloride, phentolamine mesylate, tamsulosin hydrochloride, and urapidil) and nonspecific ligands (yohimbe and metoprolol) for α1A-AR. Halo-α1A-AR was successfully immobilized on the silica gel surface with good stability over 30 days, and the Halo-α1A-AR chromatographic column exhibited good ligand-recognition activity. The nonlinear chromatography results indicated that prazosin hydrochloride, terazosin hydrochloride, and urapidil interacted with immobilized Halo-α1A-AR through one type of binding site, with association constants of 3.85×105, 5.00×105, and 5.90×105L/mol, respectively. In contrast, phentolamine mesylate and tamsulosin hydrochloride interacted with immobilized Halo-α1A-AR through two types of binding site. The association constants with the high- and low-affinity binding sites were 3.12×106 and 6.01×105L/mol, respectively, for phentolamine mesylate and 9.98×105 and 0.21×105L/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-α1A-AR were consistent with the values determined for receptor-ligand binding in solution, indicating that the Halo-α1A-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.

Ligand-Receptor Interactions and Structure-Function Relationships in Off-Target Binding of the ß3-Adrenergic Agonist Mirabegron to α1A-Adrenergic Receptors.

The ß3-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 α1-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 α1A. The binding was simulated by docking mirabegron to a 3D structure of a human α1A-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 α1A 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 α1A 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 α1A, accounts for mirabegron binding to α1A. Because of its lack of interactions with the binding pocket, mirabegron has lower affinity compared to α1A-blockers and no effects on voiding symptoms.

Crystal structure of the α1B-adrenergic receptor reveals molecular determinants of selective ligand recognition.

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 α1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α1BAR with α2ARs, and by constructing α1BAR-α2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α1BAR-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.

The Structural Determinants for α1-Adrenergic/Serotonin Receptors Activity among Phenylpiperazine-Hydantoin Derivatives.

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 α1A/α1D affinity and selectivity towards α1B, 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.

Class A G protein-coupled receptors assemble into functional higher-order hetero-oligomers.

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, α1a -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.

The C-terminal of the α1b-adreneroceptor is a key determinant for its structure integrity and biological functions.

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 ß-arrestin2-independent.

Synthesis, Molecular Modeling of Novel Substituted Pyridazinones and their Vasorelaxant Activities.

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.

Anti-diarrheal activities of phytol along with its possible mechanism of action through in-vivo and in-silico models.

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 ß-adrenergic receptors, especially with α-ADR1a and ß-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 ß-adrenergic receptors.

Optimizing the α1B-adrenergic receptor for solution NMR studies.

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 13C, 15N, 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 α1B-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 D2O 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 α1B-adrenergic receptor was additionally incorporated in nanodiscs, but displayed slightly inferior spectra compared to a sample in detergent micelles. Finally, both [15N,1H]- as well as [13C,1H]-HSQC spectra are shown highlighting the high quality of the final NMR sample. Importantly, the quality of [13C,1H]-HSQC spectra indicates that the so prepared receptor could be used for studying side-chain dynamics.

INPHARMA-Based Determination of Ligand Binding Modes at α1 -Adrenergic Receptors Explains the Molecular Basis of Subtype Selectivity.

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, α1A -AR and α1B -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 α1A -AR-selective agonist A-61603 relative to an endogenous agonist, epinephrine, at both α1A -AR and α1B -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 α1A -AR, thus demonstrating an approach for targeting subtype selectivity in ARs.

A positive feedback loop between alpha1-adrenoceptors and inflammatory cytokines in keratinocytes.

A positive feedback loop between inflammatory cytokines and alpha1-adrenoceptors (α1-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 α1-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 α1-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)-1ß, IL-6, IL-8 and nerve growth factor (NGF) production and gene expression levels of α1-AR subtype B (α1B-AR). Additional stimulation of α1-AR further increased IL-6 levels, while maintaining high levels of IL-8 and decreasing levels of IL-1ß and NGF. Our results suggest that reciprocal influences between α1-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).

Probing the correlation between ligand efficacy and conformational diversity at the α1A-adrenoreceptor reveals allosteric coupling of its microswitches.

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 α1A-adrenoreceptor, a GPCR that stimulates smooth muscle contraction in response to binding noradrenaline. NMR of [13CϵH3]methionine-labeled α1A-adrenoreceptor variants, each exhibiting differing signaling capacities, revealed how different classes of ligands modulate the conformational equilibria of this receptor. [13CϵH3]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 α1A-adrenoreceptor and underlies the mechanism of ligand modulation of GPCR signaling in cells.

Does α1-adrenergic receptor blockade modulate sweating during incremental exercise in young endurance-trained men?

PURPOSE: Human eccrine sweat glands respond to α1-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 α1-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 (α1-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 (α1-adrenergic receptor agonist) was iontophoretically administered to skin to verify α1-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 α1-adrenergic receptor blockade was intact. CONCLUSION: Our findings demonstrate that α1-adrenergic receptors located at eccrine sweat glands do not contribute to eccrine sweating during incremental exercise in young endurance-trained men.

A novel cell membrane-cloaked magnetic nanogripper with enhanced stability for drug discovery.

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.

Coupling to Gq Signaling Is Required for Cardioprotection by an Alpha-1A-Adrenergic Receptor Agonist.

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.

Mutations in the NPxxY motif stabilize pharmacologically distinct conformational states of the α1B- and ß2-adrenoceptors.

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 α1B-adrenoceptor (α1B-AR) and ß2-AR activation. Disrupting this bond by mutagenesis stabilized the α1B-AR and the ß2-AR in inactive-state conformations, which displayed decreased agonist potency for stimulating downstream IP1 and cAMP signaling, respectively. Compared to that for wild-type receptors, agonist-mediated ß-arrestin recruitment was substantially reduced or abolished for all α1B-AR and ß2-AR inactive-state mutants. However, the inactive-state ß2-ARs exhibited decreased agonist affinity, whereas the inactive-state α1B-ARs had enhanced agonist affinity. Conversely, antagonist affinity was unchanged for inactive-state conformations of both α1B-AR and ß2-AR. Removing the influence of agonist affinity on agonist potency gave a measure of signaling efficacy, which was markedly decreased for the α1B-AR mutants but little altered for the ß2-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.

Diazepam is not a direct allosteric modulator of α1-adrenoceptors, but modulates receptor signaling by inhibiting phosphodiesterase-4.

α1A- and α1B-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). α1A- and α1B-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 α1-ARs. Here, using thermostabilized, purified, α1A- and α1B-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 α1A- and α1B-ARs. Similarly, in cell-based assays expressing unmodified α1A- and α1B-ARs, benzodiazepine treatment had no effect on fluorescent ligand binding, agonist-stimulated Ca2+ release, or G protein activation. In contrast, several benzodiazepines positively modulated phenylephrine stimulation of a cAMP response element pathway by α1A- and α1B-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.

In vitro functional evaluation of isolaureline, dicentrine and glaucine enantiomers at 5-HT2 and α1 receptors.

Compounds with activity at serotonin (5-hydroxytryptamine) 5-HT2 and α1 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-HT2 and adrenergic α1 receptor subtypes were evaluated. The enantiomers of isolaureline and dicentrine acted as antagonists at 5-HT2 and α1 receptors with (R)-isolaureline showing the greatest potency (pKb  = 8.14 at the 5-HT2C receptor). Both (R)- and (S)-glaucine also antagonized α1 receptors, but they behaved very differently to the other compounds at 5-HT2 receptors: (S)-glaucine acted as a partial agonist at all three 5-HT2 receptor subtypes, whereas (R)-glaucine appeared to act as a positive allosteric modulator at the 5-HT2A receptor.

Impaired modulation of postjunctional α1 - but not α2 -adrenergic vasoconstriction in contracting forearm muscle of postmenopausal women.

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 α1 - and α2 -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 α1 - (via intra-arterial infusion of phenylephrine) but not α2 -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 (α1 -agonist) or dexmedetomidine (α2 -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 α1 - and α2 -agonist stimulation was similar between YW and PMW. During exercise, α1 -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, α2 -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 α1 -adrenergic vasoconstriction in contracting skeletal muscle.

Chiral analogues of (+)-cyclazosin as potent α1B-adrenoceptor selective antagonist.

(+)-Cyclazosin [(+)-1] is one of most selective antagonists of the α1B-adrenoceptor subtype (selectivity ratios, α1B/α1A = 13, α1B/α1D = 38-39). To improve the selectivity, we synthesized and pharmacologically studied the blocking activity against α1-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 α1B selectivity of the progenitor compound: 4 and 14 time vs. the α1D subtype and 35 and 77 times vs. the α1A subtype, respectively. The study confirmed the importance of the hydrophobic cis-octahydroquinoxaline moiety of these molecules for the establishment of interactions with the α1-adrenoceptors as well that of their (4aS,8aR) stereochemistry to grant selectivity for the α1B subtype. Hypotheses on the mode of interaction of these compounds were advanced on the basis of molecular modeling studies performed on compound (+)-3.