Ligands of Adrenergic Receptors: A Structural Point of View.

Adrenergic receptors are G protein-coupled receptors for epinephrine and norepinephrine. They are targets of many drugs for various conditions, including treatment of hypertension, hypotension, and asthma. Adrenergic receptors are intensively studied in structural biology, displayed for binding poses of different types of ligands. Here, we summarized molecular mechanisms of ligand recognition and receptor activation exhibited by structure. We also reviewed recent advances in structure-based ligand discovery against adrenergic receptors.

Stereoselective cell uptake of adrenergic agonists and antagonists by organic cation transporters.

Stereoselectivity is well described for receptor binding and enzyme catalysis, but so far has only been scarcely investigated in carrier-mediated membrane transport. We thus studied transport kinetics of racemic (anti)adrenergic drugs by the organic cation transporters OCT1 (wild-type and allelic variants), OCT2, OCT3, MATE1, and MATE2-K with a focus on stereospecificity. OCT1 showed stereoselective uptake with up to 2-fold higher vmax over their corresponding counterpart enantiomers for (R,R)-fenoterol, (R,R)-formoterol, (S)-salbutamol, (S)-acebutolol, and (S)-atenolol. Orciprenaline and etilefrine were also transported stereoselectively. The Km was 2.1-fold and 1.5-fold lower for the (S,S)-enantiomers of fenoterol and formoterol, while no significant difference in Km was seen for the other aforementioned drugs. Common OCT1 variants showed similar enantiopreference to wild-type OCT1, with a few notable exceptions (e.g. a switch in enantiospecificity for fenoterol in OCT1*2 compared to the wild-type). Other cation transporters showed strong differences to OCT1 in stereoselectivity and transport activity: The closely related OCT2 displayed a 20-fold higher vmax for (S,S)-fenoterol compared to (R,R)-fenoterol and OCT2 and OCT3 showed 3.5-fold and 4.6-fold higher vmax for the pharmacologically active (R)-salbutamol over (S)-salbutamol. MATE1 and MATE2-K generally mediated transport with a higher capacity but lower affinity compared to OCT1, with moderate stereoselectivity. Our kinetic studies showed that significant stereoselectivity exists in solute carrier-mediated membrane transport of racemic beta-adrenergic drugs with surprising, and in some instances even opposing, preferences between closely related organic cation transporters. This may be relevant for drug therapy, given the strong involvement of these transporters in hepatic and renal drug elimination.

Microwave-Assisted Synthesis of Trazodone and Its Derivatives as New 5-HT1A Ligands: Binding and Docking Studies.

Trazodone, a well-known antidepressant drug widely used throughout the world, works as a 5-hydroxytryptamine (5-HT2) and α1-adrenergic receptor antagonist and a serotonin reuptake inhibitor. Our research aimed to develop a new method for the synthesis of trazodone and its derivatives. In the known methods of the synthesis of trazodone and its derivatives, organic and toxic solvents are used, and the synthesis time varies from several to several dozen hours. Our research shows that trazodone and its derivatives can be successfully obtained in the presence of potassium carbonate as a reaction medium in the microwave field in a few minutes. As a result of the research work, 17 derivatives of trazodone were obtained, including compounds that exhibit the characteristics of 5-HT1A receptor ligands. Molecular modeling studies were performed to understand the differences in the activity toward 5-HT1A and 5-HT2A receptors between ligand 10a (2-(6-(4-(3-chlorophenyl)piperazin-1-yl)hexyl)-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one) (5-HT1A Ki = 16 nM) and trazodone. The docking results indicate the lack of the binding of ligand 10a to 5-HT2AR, which is consistent with the in vitro studies. On the other hand, the docking results for the 5-HT1A receptor indicate two possible binding modes. Crystallographic studies support the hypothesis of an extended conformation.

The adrenergic receptor antagonist carvedilol interacts with serotonin 2A receptors both in vitro and in vivo.

There is increasing support for the potential clinical use of compounds that interact with serotonin 2A (5-HT2A) receptors. It is therefore of interest to discover novel compounds that interact with 5-HT2A receptors. In the present study, we used computational chemistry to identify critical ligand structural features of 5-HT2A receptor binding and function. Query of compound databases using those ligand features revealed the adrenergic receptor antagonist carvedilol as a high priority match. As carvedilol is used clinically for cardiovascular diseases, we conducted experiments to assess whether it has any interactions with 5-HT2A receptors. In vitro experiments demonstrated that carvedilol has high nanomolar affinity for 5-HT2A receptors. In vivo experiments demonstrated that carvedilol increases the ethanol-induced loss of the righting reflex and suppresses operant responding in mice, and that these effects are attenuated by pretreatment with the selective 5-HT2A receptor antagonist M100907. Moreover, carvedilol did not induce the head-twitch response in mice, suggesting a lack of psychedelic effects. However, carvedilol did not activate canonical 5-HT2A receptor signaling pathways and antagonized serotonin-mediated signaling. It also reduced the head-twitch response induced by 2,5-Dimethoxy-4-iodoamphetamine, suggesting potential in vivo antagonism, allosteric modulation, or functional bias. These data suggest that carvedilol has functionally relevant interactions with 5-HT2A receptors, providing a novel mechanism of action for a clinically used compound. However, our findings do not clearly delineate the precise mechanism of action of carvedilol at 5-HT2A receptors, and additional experiments are needed to elucidate the role of 5-HT2A receptors in the behavioral and clinical effects of carvedilol.

Design and synthesis of aryloxypropanolamine as ß3-adrenergic receptor antagonist in cancer and lipolysis.

ß-adrenergic receptors (ß-ARs) are broadly distributed in various tissues and regulate a panel of important physiological functions and disease states including cancer. Above all, ß3-adrenergic receptor (ß3-AR) plays a significant role in regulating lipolysis and thermogenesis in adipose tissue. In this study, we designed and synthesized a series of novel L-748,337 derivatives as selective human ß3-AR antagonists. Among all the tested L-748,337 analogs, compound 23d was found to display 23-fold more potent ß3-AR antagonist activity (EC50 = 0.5117 nM) than L-748,337 (EC50 = 11.91 nM). In vivo, compound 23d could alleviate weight loss and inhibit tumor growth in C26 tumor cachexia animal model.

Nanobody-Enabled Reverse Pharmacology on G-Protein-Coupled Receptors.

The conformational complexity of transmembrane signaling of G-protein-coupled receptors (GPCRs) is a central hurdle for the design of screens for receptor agonists. In their basal states, GPCRs have lower affinities for agonists compared to their G-protein-bound active state conformations. Moreover, different agonists can stabilize distinct active receptor conformations and do not uniformly activate all cellular signaling pathways linked to a given receptor (agonist bias). Comparative fragment screens were performed on a ß2 -adrenoreceptor-nanobody fusion locked in its active-state conformation by a G-protein-mimicking nanobody, and the same receptor in its basal-state conformation. This simple biophysical assay allowed the identification and ranking of multiple novel agonists and permitted classification of the efficacy of each hit in agonist, antagonist, or inverse agonist categories, thereby opening doors to nanobody-enabled reverse pharmacology.

Design, synthesis, crystal structure, biological evaluation and molecular docking studies of carbazole-arylpiperazine derivatives.

Subtype-selective α1-adrenoceptor (AR) antagonists display optimum therapeutic efficacies for the treatment of benign prostatic hyperplasia (BPH). In this study, we designed and synthesized novel carbazole-arylpiperazines derivatives (1 and 2) on the basis of the proposed pharmacophore model for α1-AR antagonists. Structural properties were investigated using single-crystal X-ray diffraction analysis. Comparison of crystal structures with ligand-based pharmacophore models revealed that the two agents may possess antagonistic effects on α1D subtype. Tissue functional assay in vitro showed that compound 2 exerted strong antagonistic activity on α1B-AR (pA2 7.13) with a poor selectivity for α1A and α1D subtypes. Compound 1 exhibited enhanced antagonistic effect on α1D subtype (pA2 7.06) and excellent selectivity for α1D over α1B (α1D/α1B ratio=79.4). To illustrate the relationship between antagonistic activity and chemical structure, molecular docking studies were performed using the homology models of α1 receptors. Binding mechanism indicated that small hydrophobic substituents attached to the arylpiperazine moiety were essential for rational design of α1D-selective antagonists.

GOMoDo: A GPCRs online modeling and docking webserver.

G-protein coupled receptors (GPCRs) are a superfamily of cell signaling membrane proteins that include >750 members in the human genome alone. They are the largest family of drug targets. The vast diversity and relevance of GPCRs contrasts with the paucity of structures available: only 21 unique GPCR structures have been experimentally determined as of the beginning of 2013. User-friendly modeling and small molecule docking tools are thus in great demand. While both GPCR structural predictions and docking servers exist separately, with GOMoDo (GPCR Online Modeling and Docking), we provide a web server to seamlessly model GPCR structures and dock ligands to the models in a single consistent pipeline. GOMoDo can automatically perform template choice, homology modeling and either blind or information-driven docking by combining together proven, state of the art bioinformatic tools. The web server gives the user the possibility of guiding the whole procedure. The GOMoDo server is freely accessible at http://molsim.sci.univr.it/gomodo.

A validated enantioselective LC-MS/MS assay for the simultaneous determination of carvedilol and its pharmacologically active 4'-hydroxyphenyl metabolite in human plasma: application to a clinical pharmacokinetic study.

Carvedilol is widely prescribed for the treatment of hypertension, heart failure and left ventricular dysfunction following myocardial infarction. A sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated to enable reliable and efficient bioanalysis of the (R)- and (S)-enantiomers of carvedilol and its pharmacologically active 4'-hydroxyphenyl metabolite in human plasma. Following plasma extraction using supported liquid extraction (SLE) in a 96-well plate format, extracted samples were derivatized with 2,3,4,6-tetra-O-acetyl-ß-D-glucopyranosyl isothiocyanate (GITC). Chromatographic separation was achieved by gradient elution on an ACQUITY UPLC HSS T3 analytical column. The impact of several potentially interfering isobaric metabolites on the quantification of the 4'-hydroxyphenyl metabolite (R)- and (S)-enantiomers was minimized by implementation of a combination of chromatographic and mass spectrometric techniques. Derivatized analytes and stable-labeled internal standards were detected by positive ion electrospray tandem mass spectrometry. The assay was validated over concentration ranges of 0.200-100 ng/mL for (R)- and (S)-carvedilol and 0.0200-10.0 ng/mL for (R)- and (S)-4'-hydroxyphenyl carvedilol. Intra- and inter-assay precision values for replicate quality control samples were within 11.9% for all analytes during the assay validation. Mean quality control accuracy values were within ±9.4% of nominal values for all analytes. Assay recoveries were high (>76%) and internal standard normalized matrix effects were minimal. The four analytes were stable in human plasma for at least 24 h at room temperature, 89 days at -20 °C and -70 °C, and following at least five freeze-thaw cycles. The validated assay was successfully applied to the quantification of the (R)- and (S)-enantiomers of both carvedilol and its pharmacologically active 4'-hydroxyphenyl metabolite in human plasma in support of a human pharmacokinetic study.

Enantioselective LC/ESI-MS/MS analysis and pharmacokinetic and tissue distribution study of (2RS)-1-(7-methoxy-1H-indol-4-yloxy)-3-(2-(2-methoxyphenoxy)ethylamino)-propan-2-ol in rats.

A sensitive and stereospecific liquid chromatography-tandem mass spectrometry method for the quantitative determination of TWo8 enantiomers ((2RS)-1-(7-methoxy-1H-indol-4-yloxy)-3-(2-(2-methoxyphenoxy)ethylamino)-propan-2-ol) was developed and validated in rat serum and some tissues. Racemic TWo8 is a new chemical entity, and it has been shown to possess pharmacological activity in vivo. The assay involved the diastereomeric derivatization of racemic TWo8 with 2,3,4,6-tetra-O-acetyl-beta-glucopyranosyl isothiocyanate. The TWo8 diastereoisomers quantification was performed on a triple quadrupole mass spectrometer employing an electrospray ionization technique. The precursor to the product ion transition for TWo8 derivatives and for the internal standard (carbamazepine) was m/z 776.4 → 387.2 and 237.4 → 194.4, respectively. The assay was validated with a linear range of 10-2000 ng/ml of racemic TWo8. The inter-day precisions for (-)-(S)-TWo8 and (+)-(R)-TWo8 were 2.1% to 14.9% and 1.3% to 14.8%, respectively. The inter-day accuracy for (-)-(S)-TWo8 and (+)-(R)-TWo8 was within 86% to 114% and 91% to 114%, respectively. A pilot pharmacokinetic study of this new ß-adrenolytic compound has shown that (-)-(S)-TWo8 is eliminated faster than its antipode. The terminal half-lives of (-)-(S)-TWo8 and (+)-(R)-TWo8 were 3.2 and 3.9 h, respectively. The compound distribution into different organs, evaluated in tissue homogenate samples following TWo8 intravenous administration, showed an enantioselective penetration of TWo8 enantiomers in the liver (p < 0.03), in the kidney (p < 0.001), and in the lungs (p < 0.05). The developed method using liquid chromatography-tandem mass spectrometry method with electrospray ionization could be employed for quantitative determination of compounds with similar structure.

Characterization of agglomerated carvedilol by hot-melt processes in a fluid bed and high shear granulator.

The purpose of this study was to prepare and characterize granulated carvedilol by melt-in and spray-on melt granulation in a fluid bed and a high shear granulator. Granulates having comparable particle size distribution and good flow properties were obtained with proper adjustment of process parameters for each binder (poloxamer 188, polyethylene glycol 4000, and gliceryl monosterate), procedure (spray-on and melt-in) and equipment (fluid bed and high shear granulator). In-line probes for particle size measurements proved to be a useful tool for determining the end point of melt granulation. The product temperature during melt granulation was found to be the critical process parameter for achieving appropriate granulate particle size distribution. The results showed that melt granulation using hydrophilic binders is an effective method to improve the dissolution rate of carvedilol. The method of binder addition to the powders (melt-in or spray-on procedure) was found to strongly influence the dissolution rate of carvedilol. The highest dissolution rates were obtained when the spray-on procedure is used, independently from the type of granulator used. The results also suggest that the most probable explanation for the increase in the dissolution rate of granulated carvedilol is improvement of the wettability through intimate contact between hydrophilic binder and hydrophobic drug.

Recent advance in the pharmacology of dihydropyrimidinone.

Dihydropyrimidinones (DHPMs) are a series of highly valuable small molecules possessing versatile pharmaceutical properties. Although the first one-pot synthesis of DHPMs had been reported more than 100 years ago, the fascinating achievement in DHPMs-based pharmacology during the past century promoted durative interests to the pharmacological and related studies of the scaffold, which lead to the discovery of many new biological functions of DHPMs. Recent pharmacological development on DHPMs-based molecules have been summarized in this review.

Molecular modeling study of chiral separation and recognition mechanism of ß-adrenergic antagonists by capillary electrophoresis.

Chiral separations of five ß-adrenergic antagonists (propranolol, esmolol, atenolol, metoprolol, and bisoprolol) were studied by capillary electrophoresis using six cyclodextrins (CDs) as the chiral selectors. Carboxymethylated-ß-cyclodextrin (CM-ß-CD) exhibited a higher enantioselectivity power compared to the other tested CDs. The influences of the concentration of CM-ß-CD, buffer pH, buffer concentration, temperature, and applied voltage were investigated. The good chiral separation of five ß-adrenergic antagonists was achieved using 50 mM Tris buffer at pH 4.0 containing 8 mM CM-ß-CD with an applied voltage of 24 kV at 20 °C. In order to understand possible chiral recognition mechanisms of these racemates with CM-ß-CD, host-guest binding procedures of CM-ß-CD and these racemates were studied using the molecular docking software Autodock. The binding free energy was calculated using the Autodock semi-empirical binding free energy function. The results showed that the phenyl or naphthyl ring inserted in the hydrophobic cavity of CM-ß-CD and the side chain was found to point out of the cyclodextrin rim. Hydrogen bonding between CM-ß-CD and these racemates played an important role in the process of enantionseparation and a model of the hydrogen bonding interaction positions was constructed. The difference in hydrogen bonding formed with the -OH next to the chiral center of the analytes may help to increase chiral discrimination and gave rise to a bigger separation factor. In addition, the longer side chain in the hydrophobic phenyl ring of the enantiomer was not beneficial for enantioseparation and the chiral selectivity factor was found to correspond to the difference in binding free energy.

Adenosine in the inflamed gut: a Janus faced compound.

The purine ribonucleoside adenosine (Ado) has been recognized for its regulatory functions in situations of cellular stress like ischemia, hypoxia and inflammation. The importance of extracellular Ado as a modulator in the immune system is a theme of great appreciation and the focus of recent increasing interest in the field of gastrointestinal inflammation. In this review, the different aspects of Ado signaling during inflammatory responses in the gut are discussed, considering the contribution of the four known Ado receptors (ARs; A(1), A(2A), A(2B), and A(3)), their mechanisms and expression patterns. Activation of these receptors in epithelial cells as well as in immune cells recruited to the inflamed intestinal mucosa determines the overall effect, ranging from a protective, anti-inflammatory modulation to a strong pro-inflammatory induction. Here we present the current advances in agonists and antagonists development and their potential therapeutic application studied in animal models of intestinal inflammation. In addition, alternative complementary approaches to manipulate such a complex signaling system are discussed, for example, the use of AR allosteric modulators or interference with Ado metabolism. Special features of the gut environment are taken into account: the contribution of diet components; the involvement of Ado in intestinal infections; the interactions with the gut microbiome, particularly, the recent exciting finding that an intestinal bacterium can directly produce extracellular Ado in response to host defense mechanisms in an inflammation scenario. Understanding each component of this dynamic system will broaden the possibilities for applying Ado signaling as a therapeutic target in gut inflammation.

Antiarrhythmic and antioxidant activity of novel pyrrolidin-2-one derivatives with adrenolytic properties.

A series of novel pyrrolidin-2-one derivatives (17 compounds) with adrenolytic properties was evaluated for antiarrhythmic, electrocardiographic and antioxidant activity. Some of them displayed antiarrhythmic activity in barium chloride-induced arrhythmia and in the rat coronary artery ligation-reperfusion model, and slightly decreased the heart rate, prolonged P-Q, Q-T intervals and QRS complex. Among them, compound EP-40 (1-[2-hydroxy-3-[4-[(2-hydroxyphenyl)piperazin-1-yl]propyl]pyrrolidin-2-one showed excellent antiarrhythmic activity. This compound had significantly antioxidant effect, too. The present results suggest that the antiarrhythmic effect of compound EP-40 is related to their adrenolytic and antioxidant properties. A biological activity prediction using the PASS software shows that compound EP-35 and EP-40 can be characterized by antiischemic activity; whereas, compound EP-68, EP-70, EP-71 could be good tachycardia agents.

Synthesis and adrenolytic activity of new propanolamines.

The synthesis of (2R,S)-1-(6-methoxy-4-(methoxymethyl)-1H-indol-5-yloxy)-3-(2-(2-methoxyphenoxy)ethylamino)propan-2-ol and (2R,S)-1-(4-methoxy-6-(methoxymethyl)-1H-indol-5-yloxy)-3-(2-(2-methoxyphenoxy)ethylamino)propan-2-ol is described. The compounds were tested for electrographic, antiarrhythmic, hypotensive, and spasmolytic activity, as well as for alpha(1)-, alpha(2)- and beta(1)-adrenoceptor binding affinity.

Novel delivery device for monolithical solid oral dosage forms for personalized medicine.

There is an evident need for solid oral dosage forms allowing patients' tailor-made dosing due to variations in metabolization or small therapeutic indexes of drug substances. The objective of this work is the development of a device equipped with a novel solid dosage form, containing carvedilol as model drug, for the delivery of monolithical drug carriers in individual doses. The device was developed and constructed enabling an exact feed rate and dose adjustment by a cutting mechanism. A twin-screw extruder was used for producing cylindrical solid dosage forms. Divided doses were characterized by mass variation, cutting behavior and drug dissolution in order to investigate their applicability for practical use. Different formulations could be extruded obtaining straight cylindrical rods, which are divisible in exact slices by using the novel device. Forces below 20 N were needed to divide doses which comply with pharmacopoeial specification "conformity of mass". The developed formulations exhibit a sustained release of carvedilol within a range from 7 up to 16 h. A novel system consisting of a device and a cylindrical dosage form was developed. Patients' individual doses can be applied as monolithical solid dosage forms for oral use.

Formulation and evaluation of a gastroretentive dosage form of labetalol hydrochloride.

Labetalol hydrochloride (LBT), 2-hydroxy-5-[1-hydroxy-2-[(1-methyl-3-phenylpropyl) amino] ethyl]-benzamide, a non-selective alpha, beta-adrenoceptor antagonist is used in the treatment of hypertension. It shows variable bioavailability ranging from 10-80% which may be attributed to its minimum solubility in pH range 6 to 10, the pH conditions prevailing at the major site of absorption i.e. small intestine. Also due to its half life of 3 to 6 hrs it is administered twice daily. In the present work non-effervescent sustained release gastroretentive floating tablets of labetalol hydrochloride have been developed using various grades of HPMC and Poloxamer M127 as wetting agent. The tablets were evaluated for in vitro drug release, floating time, floating lag time, swelling studies etc. The tablets formulated with HPMC K4M CR and HPMC K15M CR along with Poloxamer showed negligible floating lag time with a total floating time over 12 hrs with complete release. Formulation was optimized using Stat-Ease Design Expert 7.1 software. Optimized batch was evaluated for the effect of change of osmolarity and pH on drug release, floating and swelling behaviour.

Effects of synephrine and beta-phenethylamine on human alpha-adrenoceptor subtypes.

Synephrine and beta-phenethylamine, two naturally occurring compounds, are structurally related to ephedrine. In this study, the effects of synephrine and beta-phenethylamine on alpha-adrenergic receptor (alpha-AR) subtypes are investigated in human embryonic kidney (HEK293) or Chinese hamster ovary (CHO) cells, and compared to that of 1R,2S-norephedrine. The rank order of binding affinities was found to be the same for the subtypes tested (alpha(1A)-, alpha(2A)-, and alpha(2C)-AR) viz, 1R,2S-norephedrine > beta-phenethylamine > synephrine. Functional studies on the alpha(1A)-AR subtype showed that synephrine was a partial agonist giving a maximal response at 100 microM that was equal to 55.3 % of the L-phenylephrine maximum. In contrast, neither 1R,2S-norephedrine nor beta-phenethylamine exhibited agonist activity at the highest concentration tested (300 microM). beta-Phenethylamine was more potent as an antagonist than 1R,2S-norephedrine and synephrine on the alpha(1A)-AR subtype. Functional studies on the alpha(2A)- and alpha(2C)-AR subtypes indicated that synephrine and beta-phenethylamine did not act as agonists. Similar to 1R,2S-norephedrine, both of these analogs reversed the effect of medetomidine against forskolin-induced cAMP elevations at 300 microM, and the rank order of antagonist potency was: 1R,2S-norephedrine = beta-phenethylamine > synephrine; and beta-phenethylamine > 1R,2S-norephedrine > synephrine, respectively. These differences suggest that the presence of a 4-hydroxy group, as in synephrine, reduced the potency in these subtypes. In conclusion, at the alpha(1A)-AR, synephrine acted as a partial agonist, while beta-phenethylamine did not exhibit any direct agonist activity. Both, synephrine and beta-phenethylamine, may act as antagonists of pre-synaptic alpha(2A/2C)-ARs present in nerve terminals.