An allosteric modulator binds to a conformational hub in the ß2 adrenergic receptor.

Most drugs acting on G-protein-coupled receptors target the orthosteric binding pocket where the native hormone or neurotransmitter binds. There is much interest in finding allosteric ligands for these targets because they modulate physiologic signaling and promise to be more selective than orthosteric ligands. Here we describe a newly developed allosteric modulator of the ß2-adrenergic receptor (ß2AR), AS408, that binds to the membrane-facing surface of transmembrane segments 3 and 5, as revealed by X-ray crystallography. AS408 disrupts a water-mediated polar network involving E1223.41 and the backbone carbonyls of V2065.45 and S2075.46. The AS408 binding site is adjacent to a previously identified molecular switch for ß2AR activation formed by I3.40, P5.50 and F6.44. The structure reveals how AS408 stabilizes the inactive conformation of this switch, thereby acting as a negative allosteric modulator for agonists and positive allosteric modulator for inverse agonists.

An Integrated Markov State Model and Path Metadynamics Approach To Characterize Drug Binding Processes.

Unveiling the mechanistic features of drug-target binding is of central interest in biophysics and drug discovery. Herein, we address this challenge by combining two major computational approaches, namely, Molecular Dynamics (MD) simulations and Markov State Models (MSM), with a Path Collective Variables (PCVs) description coupled with metadynamics. We apply our methodology to reconstruct the binding process of the antagonist alprenolol to the ß2-adrenergic receptor, a well-established pharmaceutical target. The devised protocol allowed us to estimate the binding free energy and identify the minimum free energy path leading to the protein-ligand complex. In summary, we show that MSM and PCVs can be efficiently integrated to shed light upon mechanistic and energetic details underlying complex recognition processes in biological systems.

Probing the Existence of a Metastable Binding Site at the ß2-Adrenergic Receptor with Homobivalent Bitopic Ligands.

Herein, we report the development of bitopic ligands aimed at targeting the orthosteric binding site (OBS) and a metastable binding site (MBS) within the same receptor unit. Previous molecular dynamics studies on ligand binding to the ß2-adrenergic receptor (ß2AR) suggested that ligands pause at transient, less-conserved MBSs. We envisioned that MBSs can be regarded as allosteric binding sites and targeted by homobivalent bitopic ligands linking two identical pharmacophores. Such ligands were designed based on docking of the antagonist (S)-alprenolol into the OBS and an MBS and synthesized. Pharmacological characterization revealed ligands with similar potency and affinity, slightly increased ß2/ß1AR-selectivity, and/or substantially slower ß2AR off-rates compared to (S)-alprenolol. Truncated bitopic ligands suggested the major contribution of the metastable pharmacophore to be a hydrophobic interaction with the ß2AR, while the linkers alone decreased the potency of the orthosteric fragment. Altogether, the study underlines the potential of targeting MBSs for improving the pharmacological profiles of ligands.

Identification of Alprenolol Hydrochloride as an Anti-prion Compound Using Surface Plasmon Resonance Imaging.

Prion diseases are transmissible neurodegenerative disorders of humans and animals, which are characterized by the aggregation of abnormal prion protein (PrPSc) in the central nervous system. Although several small compounds that bind to normal PrP (PrPC) have been shown to inhibit structural conversion of the protein, an effective therapy for human prion disease remains to be established. In this study, we screened 1200 existing drugs approved by the US Food and Drug Administration (FDA) for anti-prion activity using surface plasmon resonance imaging (SPRi). Of these drugs, 31 showed strong binding activity to recombinant human PrP, and three of these reduced the accumulation of PrPSc in prion-infected cells. One of the active compounds, alprenolol hydrochloride, which is used clinically as a ß-adrenergic blocker for hypertension, also reduced the accumulation of PrPSc in the brains of prion-infected mice at the middle stage of the disease when the drug was administered orally with their daily water from the day after infection. Docking simulation analysis suggested that alprenolol hydrochloride fitted into the hotspot within mouse PrPC, which is known as the most fragile structure within the protein. These findings provide evidence that SPRi is useful in identifying effective drug candidates for neurodegenerative diseases caused by abnormal protein aggregation, such as prion diseases.

ß-Arrestin-biased signaling mediates memory reconsolidation.

A long-standing hypothesis posits that a G protein-coupled signaling pathway mediates ß-adrenergic nervous system functions, including learning and memory. Here we report that memory retrieval (reactivation) induces the activation of ß1-adrenergic ß-arrestin signaling in the brain, which stimulates ERK signaling and protein synthesis, leading to postreactivation memory restabilization. ß-Arrestin2-deficient mice exhibit impaired memory reconsolidation in object recognition, Morris water maze, and cocaine-conditioned place preference paradigms. Postreactivation blockade of both brain ß-adrenergic Gs protein- and ß-arrestin-dependent pathways disrupts memory reconsolidation. Unexpectedly, selective blockade of the Gs/cAMP/PKA signaling but not the ß-arrestin/ERK signaling by the biased ß-adrenergic ligands does not inhibit reconsolidation. Moreover, the expression of ß-arrestin2 in the entorhinal cortex of ß-arrestin 2-deficient mice rescues ß1-adrenergic ERK signaling and reconsolidation in a G protein pathway-independent manner. We demonstrate that ß-arrestin-biased signaling regulates memory reconsolidation and reveal the potential for ß-arrestin-biased ligands in the treatment of memory-related disorders.

Identification of Radiodegradation Products of Acebutolol and Alprenolol by HPLC/MS/MS.

Two therapeutically active compounds from the group of ß-blockers, acebutolol (AC) and alprenolol (AL), in solid form were subjected to ionizing radiation emitted by a beam of high energy electrons from an accelerator with a standard sterilization dose of 25 kGy and in higher doses of 50-400 kGy. The effects of irradiation were detected by chromatographic methods (TLC, HPLC) and a hyphenated method (HPLC/MS/MS). No significant changes in the physicochemical properties of both compounds studied irradiated with 25 kGy were noted, but upon irradiation with the highest dose (400 kGy) the loss of AC and AL content determined by HPLC was 2.79 and 9.12%, respectively. The product of AC decomposition and the two products of AL decomposition were separated and identified by HPLC/MS/MS. It has been established that radiodegradation of AC and AL takes place by oxidation, leading to formation of the products of radiolysis, most probably alcohol derivatives of the ß-blockers studied. The additional product that appears on radiodegradation of AL is probably formed as a result of two simultaneous reactions: oxidation and CH2 group elimination.

Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates.

Optically pure epoxides are essential chiral precursors for the production of (S)-propranolol, (S)-alprenolol, and other ß-adrenergic receptor blocking drugs. Although the enzymatic production of these bulky epoxides has proven difficult, here we report a method to effectively improve the activity of BmEH, an epoxide hydrolase from Bacillus megaterium ECU1001 toward α-naphthyl glycidyl ether, the precursor of (S)-propranolol, by eliminating the steric hindrance near the potential product-release site. Using X-ray crystallography, mass spectrum, and molecular dynamics calculations, we have identified an active tunnel for substrate access and product release of this enzyme. The crystal structures revealed that there is an independent product-release site in BmEH that was not included in other reported epoxide hydrolase structures. By alanine scanning, two mutants, F128A and M145A, targeted to expand the potential product-release site displayed 42 and 25 times higher activities toward α-naphthyl glycidyl ether than the wild-type enzyme, respectively. These results show great promise for structure-based rational design in improving the catalytic efficiency of industrial enzymes for bulky substrates.

Computational study on the different ligands induced conformation change of ß2 adrenergic receptor-Gs protein complex.

ß2 adrenergic receptor (ß2AR) regulated many key physiological processes by activation of a heterotrimeric GTP binding protein (Gs protein). This process could be modulated by different types of ligands. But the details about this modulation process were still not depicted. Here, we performed molecular dynamics (MD) simulations on the structures of ß2AR-Gs protein in complex with different types of ligands. The simulation results demonstrated that the agonist BI-167107 could form hydrogen bonds with Ser203(5.42), Ser207(5.46) and Asn293(6.55) more than the inverse agonist ICI 118,551. The different binding modes of ligands further affected the conformation of ß2AR. The energy landscape profiled the energy contour map of the stable and dissociated conformation of Gαs and Gßγ when different types of ligands bound to ß2AR. It also showed the minimum energy pathway about the conformational change of Gαs and Gßγ along the reaction coordinates. By using interactive essential dynamics analysis, we found that Gαs and Gßγ domain of Gs protein had the tendency to separate when the inverse agonist ICI 118,551 bound to ß2AR. The α5-helix had a relatively quick movement with respect to transmembrane segments of ß2AR when the inverse agonist ICI 118,551 bound to ß2AR. Besides, the analysis of the centroid distance of Gαs and Gßγ showed that the Gαs was separated from Gßγ during the MD simulations. Our results not only could provide details about the different types of ligands that induced conformational change of ß2AR and Gs protein, but also supplied more information for different efficacies of drug design of ß2AR.

Influence of lipid composition on the structural stability of g-protein coupled receptor.

ß2 Adrenergic receptor (ß2AR) is a kind of G-protein coupled receptors (GPCRs) which transduce a wide range of extracellular signals into intracellular messages responsible for the regulation of diverse cell functions. Because of their functional ubiquity, GPCR is one of the most important drug targets in pharmaceutical industry. Although recent crystallographic studies provided both the active and the inactive states of some families of GPCRs, the influence of lipid composition of bilayer membrane on their activation is still poorly understood. In this work, we address the influence of lipid composition on the structural stability of GPCR, performing molecular dynamics simulations of three kinds of states: apo-, and agonist epinephrine-, or antagonist alprenolol-bound ß2AR. These three kinds of ß2ARs were embedded in four types of lipid membranes: (i) pure palmitoyl-oleoyl-phosphatidyl-choline (POPC), (ii) POPC/cholesterol (CHL), (iii) POPC/CHL/GM1 (GM1 ganglioside), (iv) POPC/palmitoyl-oleoyl-phosphatidyl-ethanolamine (POPE)/CHL/sphingomyeline (SM). The side chains of Lys267(6.29) and Asp331(7.58) showed different conformations among the three states in all types of lipid membranes. The distances between Lys267(6.29) and Asp331(7.58) of apo- and alprenolol-bound ß2ARs are smaller than that of the epinephrine-bound ß2AR. In contrast, ß2ARs in POPC/CHL bilayer were unstable in which the salt bridge; i.e., ionic lock, was not formed between Arg131(3.50) and Glu268(6.30). We have also examined the distribution of lipid molecules. A stable hydrophobic interaction between CHL and ß2AR was observed at transmembrane helix5 in POPC/CHL/GM1 and POPC/POPE/CHL/SM membranes. These results suggest that the lipid composition strongly affects the conformation of GPCR and essentially concerns the GPCR activation.

Sweeping of alprenolol enantiomers with an organic solvent and sulfated ß-cyclodextrin in capillary electrophoresis.

Sweeping, an on-line sample concentration technique in CE, is the picking and accumulation of analytes by the pseudostationary phase or complexing additive. In the presence of an electric field, the analytes concentrated at the additive front that initially penetrated the sample zone. Here, we describe the sweeping of cationic alprenolol enantiomers using sulfated ß-CD and organic solvent. The separation solution contained the anionic additive while ACN was in the sample solution. With fused silica capillaries, positive polarity, and solutions buffered at pH 3, the direction of the enantiomers' effective electrophoretic mobility was the same as the electrophoretic mobility (or electrophoretic mobility without additive). When the amount of ACN in the sample was increased (i.e. 60%), the interaction between the analytes and additive became negligible. This caused the sweeping boundary to shift from the electrophoretically moving ß-CD front to the zone between the sample and separation solution. The equation that described the narrowing of injected sample zone was derived. The performance of sweeping with 60% ACN in the sample was then studied under different operating conditions (e.g. type of injection, injection time, and CD concentration). The low interaction between enantiomers and additive gave only moderate increases in sensitivity (approximately tenfold), but was improved when field enhancement was used during electrokinetic injection. With a conductivity difference (separation/sample solution) of 70 and a short injection time of 30 s at 20 kV, peak improvements of >100-fold was easily achieved.

Enantioseparations in nonaqueous capillary electrophoresis using charged cyclodextrins.

The enantioseparation of acidic and basic compounds can be successfully achieved in nonaqueous capillary electrophoresis using single-isomer charged ß-cyclodextrin (ß-CD) derivatives of opposite charge to that of the analytes. This chapter describes how to separate the enantiomers of three basic substances selected as model compounds, i.e., alprenolol, bupranolol, and terbutaline, using the negatively charged heptakis(2,3-di-O-acetyl-6-O-sulfo)-ß-CD. The enantiomers of three acidic drugs (tiaprofenic acid, suprofen, and flurbiprofen) are resolved using a monosubstituted amino ß-CD derivative, namely, 6-monodeoxy-6-mono(3-hydroxy)propylamino-ß-CD.

Enantioselective biodegradation of pharmaceuticals, alprenolol and propranolol, by an activated sludge inoculum.

Biodegradation of chiral pharmaceuticals in the environment can be enantioselective. Thus quantification of enantiomeric fractions during the biodegradation process is crucial for assessing the fate of chiral pollutants. This work presents the biodegradation of alprenolol and propranolol using an activated sludge inoculum, monitored by a validated enantioselective HPLC method with fluorescence detection. The enantioseparation was optimized using a vancomycin-based chiral stationary phase under polar ionic mode. The method was validated using a minimal salts medium inoculated with activated sludge as matrix. The method was selective and linear in the range of 10-800 ng/ml, with a R²>0.99. The accuracy ranged from 85.0 percent to 103 percent, the recovery ranged from 79.9 percent to 103 percent, and the precision measured by the relative standard deviation (RSD) was <7.18 percent for intra-batch and <5.39 percent for inter-batch assays. The limits of quantification and detection for all enantiomers were 10 ng/ml and 2.5 ng/ml, respectively. The method was successfully applied to follow the biodegradation of the target pharmaceuticals using an activated sludge inoculum during a fifteen days assay. The results indicated slightly higher biodegradation rates for the S-enantiomeric forms of both beta-blockers. The presence of another carbon source maintained the enantioselective degradation pattern while enhancing biodegradation extent up to fourteen percent.

Ligand-specific interactions modulate kinetic, energetic, and mechanical properties of the human ß2 adrenergic receptor.

G protein-coupled receptors (GPCRs) are a class of versatile proteins that transduce signals across membranes. Extracellular stimuli induce inter- and intramolecular interactions that change the functional state of GPCRs and activate intracellular messenger molecules. How these interactions are established and how they modulate the functional state of GPCRs remain to be understood. We used dynamic single-molecule force spectroscopy to investigate how ligand binding modulates the energy landscape of the human ß2 adrenergic receptor (ß2 AR). Five different ligands representing either agonists, inverse agonists or neutral antagonists established a complex network of interactions that tuned the kinetic, energetic, and mechanical properties of functionally important structural regions of ß2 AR. These interactions were specific to the efficacy profile of the ligands investigated and suggest that the functional modulation of GPCRs follows structurally well-defined interaction patterns.

Kinetic capillary electrophoresis with mass-spectrometry detection (KCE-MS) facilitates label-free solution-based kinetic analysis of protein-small molecule binding.

Tandem tracker: Here we introduce a method for studying the kinetics of protein-small-molecule interactions based on kinetic capillary electrophoresis (KCE) separation and MS detection. Due to the variety of KCE methods and MS modes available, the KCE-MS tandem is a highly versatile platform for label-free, solution-based kinetic studies of affinity interactions.

Synthesis and characterization of high-affinity 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-labeled fluorescent ligands for human ß-adrenoceptors.

The growing practice of exploiting noninvasive fluorescence-based techniques to study G protein-coupled receptor pharmacology at the single cell and single molecule level demands the availability of high-quality fluorescent ligands. To this end, this study evaluated a new series of red-emitting ligands for the human ß-adrenoceptor family. Upon the basis of the orthosteric ligands propranolol, alprenolol, and pindolol, the synthesized linker-modified congeners were coupled to the commercially available fluorophore BODIPY 630/650-X. This yielded high-affinity ß-adrenoceptor fluorescent ligands for both the propranolol and alprenolol derivatives; however, the pindolol-based products displayed lower affinity. A fluorescent diethylene glycol linked propranolol derivative (18a) had the highest affinity (log K(D) of -9.53 and -8.46 as an antagonist of functional ß2- and ß1-mediated responses, respectively). Imaging studies with this compound further confirmed that it can be employed to selectively label the human ß2-adrenoceptor in single living cells, with receptor-associated binding prevented by preincubation with the nonfluorescent ß2-selective antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118551) ( J. Cardiovasc. Pharmacol.1983, 5, 430-437. ).

Pathway and mechanism of drug binding to G-protein-coupled receptors.

How drugs bind to their receptors--from initial association, through drug entry into the binding pocket, to adoption of the final bound conformation, or "pose"--has remained unknown, even for G-protein-coupled receptor modulators, which constitute one-third of all marketed drugs. We captured this pharmaceutically critical process in atomic detail using the first unbiased molecular dynamics simulations in which drug molecules spontaneously associate with G-protein-coupled receptors to achieve final poses matching those determined crystallographically. We found that several beta blockers and a beta agonist all traverse the same well-defined, dominant pathway as they bind to the ß(1)- and ß(2)-adrenergic receptors, initially making contact with a vestibule on each receptor's extracellular surface. Surprisingly, association with this vestibule, at a distance of 15 Å from the binding pocket, often presents the largest energetic barrier to binding, despite the fact that subsequent entry into the binding pocket requires the receptor to deform and the drug to squeeze through a narrow passage. The early barrier appears to reflect the substantial dehydration that takes place as the drug associates with the vestibule. Our atomic-level description of the binding process suggests opportunities for allosteric modulation and provides a structural foundation for future optimization of drug-receptor binding and unbinding rates.

1H and 13C NMR characteristics of ß-blockers.

The ß-blockers are important drugs and decades of clinical experience proved their high medical status. However, to the best of our knowledge, there is no complete assignment of (1)H and (13)C NMR resonances of popular representatives: acebutolol, alpenolol, pindolol, timolol and propranolol and the published NMR data on carvedilol and atenolol are incorrect. Therefore, (1)H and (13)C NMR spectroscopy was applied for the characterization of a series of ß-adrenolytics: carvedilol (1), pindolol (2), alprenolol (3), acebutolol (4), atenolol (5), propranolol (6) and timolol (7). Two-dimensional NMR experiments (COSY, HMQC, HMBC, NOESY) allowed the unequivocal assignment of (1)H and (13)C spectra for solution (DMSO-d(6) ). Salts and bases can be easily distinguished based on (13)C chemical shifts which are within 65.0-65.5 ppm (OC2) and 46.9-47.0 (NC3) for hydrochlorides and larger, ca. 68.4 ppm (OC2) and 50.3-52.6 (NC3) for bases. NMR data of 1-7 should be included in pharmacopoeias.

Labeling of alprenolol with fluorescent aryl iodide as a reagent based on Mizoroki-Heck coupling reaction.

A novel fluorescent labeling method for alprenolol was developed based on Mizoroki-Heck coupling reaction. We designed and synthesized fluorescent aryl iodide, 4-(4,5-diphenyl-1H-imidazol-2-yl)iodobenzene (DIBI) as a labeling reagent. DIBI has a lophine skeleton carrying an iodide atom acting as fluorophore and reactive center, respectively. In order to evaluate the usefulness of DIBI, a high-performance liquid chromatography (HPLC) with fluorescence detection method was developed for the determination of alprenolol as a model compound of terminal double bond. The fluorescent labeling of alprenolol with DIBI was achieved in the presence of palladium acetate as a catalyst, and the labeled alprenolol was detected fluorometrically. In addition, it was found that the fluorescence of DIBI derivative increased and red shifted when compared with that of DIBI. Furthermore, the proposed method could be applied to determine the alprenolol concentration in rat plasma after administration of alprenolol without interferences from biological components. The detection limit (S/N=3) for alprenolol in rat plasma was 0.74 ng/mL (30 fmol on column).

Approaches to estimate the time and height at the peak maximum in liquid chromatography based on a modified Gaussian model.

The time and height at the peak maximum are key parameters to describe a chromatographic peak with prediction or optimization purposes, or in the qualitative/quantitative analysis of samples. Three different approaches to estimate these parameters, using the experimental points in the peak maximum region, are here described and compared. The approaches are based on the reliable description of the peak profile using a modified Gaussian model with a parabolic variance (PVMG). In the first approach, non-linear fitting of the chromatographic data to the PVMG model is carried out to obtain the time and height at the peak maximum (Approach I). In the other two approaches, the PVMG model is linearized to carry out a linear fitting. In each case, the optimal number of processed points was assessed. The three approaches yielded highly satisfactory results, being Approach I the best in terms of accuracy and robustness. The assessment of the accuracy in the estimations was carried out using simulated peaks. These peaks were built with the parameters obtained from real peaks for several probe compounds eluted under reversed-phase liquid chromatographic (RPLC) conditions, to which noise was added.

Effect of radiation sterilization on alprenolol in the solid state studied by high-performance thin-layer chromatography.

The possibility of radiation sterilization of alprenolol (AL) has been studied. Irradiation of AL in solid form with a 25 kGy beam of electrons caused only an insignificant change in color that became more intense with increasing irradiation dose. Moreover, with increasing dose a decrease in pH, the content of water, and the degree of crystallinity were observed. AL in solid form was radiated with a high-energy electron beam (9.96 MeV) at doses from 25-400 kGy and analyzed by HPTLC using the mobile phase methanol-ammonia 25% (99 + 1, v/v). Densitometric analysis was carried out directly from chromatograms at 270 nm. The applied method was validated and characterized by good precision (RSD = 3.95%); good accuracy (80% level 100.15%, 100% level 99.99%, and 120% level 104.44%); and low LOD (LOD = 0.52 microg/zone and LOQ = 1.55 microg/zone). Chromatograms recorded for samples irradiated at the doses of 25 kGy were unchanged, but at higher doses (100-400 kGy) additional peaks corresponding to the radiodegradation products appeared (Rf = 0.24 and Rf = 0.40). The decrease in the concentration of AL was proportional to the applied radiation dose, and for 400 kGy the concentration of AL was 90.23%. The calculated radiolytic yield of the radiodegradation process was G(-AL) = 7.12 x 10(-7) mol/J.