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.

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.

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.

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.

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.

Bitopic Ligands Support the Presence of a Metastable Binding Site at the ß2 Adrenergic Receptor.

Metastable binding sites (MBS) have been observed in a multitude of molecular dynamics simulations and can be considered low affinity allosteric binding sites (ABS) that function as stepping stones as the ligand moves toward the orthosteric binding site (OBS). Herein, we show that MBS can be utilized as ABS in ligand design, resulting in ligands with improved binding kinetics. Four homobivalent bitopic ligands (1-4) were designed by molecular docking of (S)-alprenolol ((S)-ALP) in the cocrystal structure of the ß2 adrenergic receptor (ß2AR) bound to the antagonist ALP. Ligand 4 displayed a potency and affinity similar to (S)-ALP, but with a >4-fold increase in residence time. The proposed binding mode was confirmed by X-ray crystallography of ligand 4 in complex with the ß2AR. This ligand design principle can find applications beyond the ß2AR and G protein-coupled receptors (GPCRs) as a general approach for improving the pharmacological profile of orthosteric ligands by targeting the OBS and an MBS simultaneously.

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.

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.

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.

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.

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.

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.

Preparation and evaluation of propranolol molecularly imprinted solid-phase microextraction fiber for trace analysis of beta-blockers in urine and plasma samples.

An improved multiple co-polymerization technique was developed to prepare a novel molecularly imprinted polymer (MIP)-coated solid-phase microextraction (SPME) fiber with propranolol as template. Investigation was performed for the characteristics and application of the fibers. The MIP coating was highly crosslinked and porous with the average thickness of only 25.0 microm. Consequently, the adsorption and desorption of beta-blockers within the MIP coating could be achieved quickly. The specific selectivity was discovered with the MIP-coated fibers to propranolol and its structural analogues such as atenolol, pindolol, and alprenolol. In contrast, only non-specific adsorption could be shown with the non-imprinted polymer (NIP)-coated fibers, and the extraction efficiencies of propranolol and pindolol with the MIP-coated fibers were higher markedly than that with the commercial SPME fibers. A MIP-coated SPME coupled with high-performance liquid chromatography (HPLC) method for propranolol and pindolol determination was developed under the optimized extraction conditions. Linear ranges for propranolol and pindolol were 20-1000 microg L(-1) and detection limits were 3.8 and 6.9 microg L(-1), respectively. Propranolol and pindolol in the spiked human urine and plasma samples, extracted with organic solvent firstly, could be simultaneous monitored with satisfactory recoveries through this method.

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.

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.

Effects of a strongly adsorbed additive on process performance in chiral preparative chromatography.

The shapes of elution profiles are often significantly influenced by the presence of strongly adsorbed additives in the mobile phase. This aspect needs to be considered in quantitative optimization of preparative chromatography. The theoretical study carried out here is based on available thermodynamic information for the enantiomers of three beta-blockers, alprenolol, propranolol, and atenolol, on a teicoplanin chiral stationary phase (Chirobiotic T) using methanol/acetonitrile as the mobile phase and acetic acid/triethylamine as the additive. The properties of this strong additive made it possible to tune the binary elution profiles in any combination of the following apparent band shapes: anti-Langmuir/anti-Langmuir, anti-Langmuir/Langmuir and Langmuir/Langmuir. Optimization of the productivity and yield, when performing repetitive batch injections, was investigated using the equilibrium dispersive model. We show that it is important to consider the invisible additive perturbation peak when defining the cycle time and therefore a model-based optimization needs to take this into account. Furthermore, both productivity and yield could be improved for the two unusual shape combinations in comparison to the traditional Langmuir/Langmuir case.

Effects of alcohols on CE enantioseparation of basic drugs with native gamma-CD as chiral selector.

The effects of alcohol on the CE enantioseparation of selected basic drugs with gamma-CD as the chiral selector was investigated. The enantioseparation behavior of the analytes with gamma-CD in the absence and presence of different alcohols specifically methanol, ethanol, 2-propanol (IPA), and 2-methyl-2-propanol (TBA), the relationship of enantiomeric resolution (R(s)) values with either hydrophobicity or bulkiness of the alcohols, as well as the effect of these alcohols on interaction of the analytes with gamma-CD were studied. Results showed that hydrophobicity and/or bulkiness of alcohols have an influence on the enantioresolution of most of the analytes based on the relatively high correlation coefficients (R) obtained between R(s) versus log P and between R(s) versus ovality (i.e., parameter to indicate bulkiness of a molecule). Comparison of the values of the average binding constants obtained for each enantiomeric pair in the presence and absence of 5% IPA showed that alcohols can increase, decrease, or give a minimal effect on the analyte-gamma-CD interaction depending on the analyte. Furthermore, the significant enhancement in the enantioresolution of both propranolol and pindolol in the presence of either IPA or TBA led to the baseline enantioresolution of both drugs using 35 mM gamma-CD.

Metabolite characterization in drug discovery utilizing robotic liquid-handling, quadruple time-of-flight mass spectrometry and in-silico prediction.

An assay method for identification of metabolites from in vitro microsomal incubations was developed for use in the early stage of drug discovery. We have developed a practical approach which involves integrated sample generation, sample preparation, bioanalysis, and data handling to maximize sample throughput and speed up the process for identification of metabolites. The assay system consisted of a robotic liquid handler (Genesis workstation) to generate and process samples, PALLAS MetabolExpert software to predict possible metabolites, exact mass measurement via a tandem quadrupole time-of-flight mass spectrometer (QTOF-MS) coupled with liquid chromatography to analyze samples, MetaboLynx software to find potential metabolites and Advanced Chemistry Development/MS (ACD/MS) software to provide guidance to the most likely hypothetical metabolite chemical structures. For purposes of evaluating this new method, dextromethorphan, alprenolol, and propranolol were incubated separately for up to 60 minutes with rat and human hepatic microsomes. The incubation and sample preparation were carried out in 96-well plates using the Genesis workstation. The bioanalysis was performed by LC-MS/MS using QTOF with MetaboLynx software to find metabolites. Metabolic products formed in vitro by rat and human microsomes were separated using an analytical column C18 with gradient elution at flow rate of 250 micro l/min. The internal mass calibration was performed by continuous postcolumn infusion of Haloperidol. The mass spectra from incubations containing NADPH were compared to those without NADPH (control) using the MetaboLynx software to find potential metabolites. Finally, the MS/MS spectra were processed by the ACD/MS software to predict the chemical structure. MetaboLynx software successfully identified metabolites for each of the drugs studied by automatically discerning expected metabolites. Exact differences in masses between each metabolite and parent drug were measured from five replicate sample injections. All measured values are accurate to less than 0.001Da or 3.8 ppm with the standard deviation within 0.0015 Da, which allowed good prediction/confirmation of empirical formulae. Hypothetical chemical structures were achieved by the ACD/MS software and provided a useful tool to assist in prediction of the metabolic pathways of the drugs. The metabolites identified were in good agreement with previously published results for all three compounds. This new method will greatly enhance throughput, which in turn will facilitate our ability to rapidly provide this guidance to the synthetic chemist.

Functional properties of atypical beta-adrenoceptors on the guinea pig duodenum.

In this study, we attempted to further characterize atypical beta-adrenoceptors on the guinea pig duodenum. (-)-Enantiomers of isoprenaline and noradrenaline were more potent than its (+)-enantiomers. The isomeric activity ratios ((+)/(-)) were less than those obtained in the guinea pig atria and trachea. The concentration-response curves to catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline), to the selective beta(3)-adrenoceptor agonist, BRL37344 ((R*, R*)-(+/-)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid sodium), and to the non-conventional partial beta(3)-adrenoceptor agonist, (+/-)-CGP12177A ((+/-)-[4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride), were resistant to blockade by (+/-)-pindobind, the beta-adrenoceptor alkylating agent. (-)-Noradrenaline and (-)-adrenaline were more potent than dopamine and (-)-phenylephrine, respectively. Selective beta(2)-adrenoceptor agonists possess agonistic activities at atypical beta-adrenoceptors. (+/-)-Propranolol and (+/-)-bupranolol had no agonistic effect, whereas (+/-)-alprenolol, (+/-)-pindolol, (+/-)-nadolol, (+/-)-CGP12177A and (+/-)-carteolol exhibited agonistic activities at atypical beta-adrenoceptors. These results suggest that pharmacological properties of atypical beta-adrenoceptors differ from those of conventional beta(1)- and beta(2)-adrenoceptors on the guinea pig.