Identification of the first-in-class dual inhibitors of human DNA topoisomerase IIα and indoleamine-2,3-dioxygenase 1 (IDO 1) with strong anticancer properties.

Molecular docking of a large set of thiosemicarbazide-based ligands resulted in obtaining compounds that inhibited both human DNA topoisomerase IIα and indoleamine-2,3-dioxygenase-1 (IDO1). To the best of our knowledge, these compounds are the first dual inhibitors targeting these two enzymes. As both of them participate in the anticancer response, the effect of the compounds on a panel of cancer cell lines was examined. Among the cell lines tested, lung cancer (A549) and melanoma (A375) cells were the most sensitive to compounds 1 (IC50=0.23 µg/ml), 2 (IC50=0.83 µg/ml) and 3 (IC50=0.25 µg/ml). The observed activity was even 90-fold higher than that of etoposide, with selectivity index values reaching 125. In-silico simulations showed that contact between 1-3 and human DNA topoisomerase II was maintained through aromatic moieties located at limiting edges of ligand molecules and intensive interactions of the thiosemicarbazide core with the DNA fragments present in the catalytic site of the enzyme.

Synthesis of Novel 2-(Cyclopentylamino)thiazol-4(5H)-one Derivatives with Potential Anticancer, Antioxidant, and 11ß-HSD Inhibitory Activities.

In this study, a series of nine new 2-(cyclopentylamino)thiazol-4(5H)-one derivatives were synthesized, and their anticancer, antioxidant, and 11ß-hydroxysteroid dehydrogenase (11ß-HSD) inhibitory activities were tested. Anticancer activity has been assessed using the MTS (MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay against human colon carcinoma (Caco-2), human pancreatic carcinoma (PANC-1), glioma (U-118 MG), human breast carcinoma (MDA-MB-231), and skin melanoma (SK-MEL-30) cancer cell lines. Cell viability reductions, especially in the case of Caco-2, MDA-MB-231, and SK-MEL-30 lines, were observed for most compounds. In addition, the redox status was investigated and oxidative, but nitrosative stress was not noted at a concentration of 500 µM compounds tested. At the same time, a low level of reduced glutathione was observed in all cell lines when treated with compound 3g (5-(4-bromophenyl)-2-(cyclopentylamino)thiazol-4(5H)-one) that most inhibited tumor cell proliferation. However, the most interesting results were obtained in the study of inhibitory activity towards two 11ß-HSD isoforms. Many compounds at a concentration of 10 µM showed significant inhibitory activity against 11ß-HSD1 (11ß-hydroxysteroid dehydrogenase type 1). The compound 3h (2-(cyclopentylamino)-1-thia-3-azaspiro[4.5]dec-2-en-4-one) showed the strongest 11ß-HSD1 inhibitory effect (IC50 = 0.07 µM) and was more selective than carbenoxolone. Therefore, it was selected as a candidate for further research.

Evaluation of ß-adrenergic ligands for development of pharmacological heart failure and transparency models in zebrafish.

Cardiovascular toxicity represents one of the most common reasons for clinical trial failure. Consequently, early identification of novel cardioprotective strategies could prevent the later-stage drug-induced cardiac side effects. The use of zebrafish (Danio rerio) in preclinical studies has greatly increased. High-throughput and low-cost of assays make zebrafish model ideal for initial drug discovery. A common strategy to induce heart failure is a chronic ß-adrenergic (ßAR) stimulation. Herein, we set out to test a panel of ßAR agonists to develop a pharmacological heart failure model in zebrafish. We assessed ßAR agonists with respect to the elicited mortality, changes in heart rate, and morphological alterations in zebrafish larvae according to Fish Embryo Acute Toxicity Test. Among the tested ßAR agonists, epinephrine elicited the most potent onset of heart stimulation (EC50 = 0.05 mM), which corresponds with its physiological role as catecholamine. However, when used at ten-fold higher dose (0.5 mM), the same compound caused severe heart rate inhibition (-28.70 beats/min), which can be attributed to its cardiotoxicity. Further studies revealed that isoetharine abolished body pigmentation at the sublethal dose of 7.50 mM. Additionally, as a proof of concept that zebrafish can mimic human cardiac physiology, we tested ßAR antagonists (propranolol, carvedilol, metoprolol, and labetalol) and verified that they inhibited fish heart rate in a similar fashion as in humans. In conclusion, we proposed two novel pharmacological models in zebrafish; i.e., epinephrine-dependent heart failure and isoetharine-dependent transparent zebrafish. We provided strong evidence that the zebrafish model constitutes a valuable tool for cardiovascular research.

The Val34Met, Thr164Ile and Ser220Cys Polymorphisms of the ß2-Adrenergic Receptor and Their Consequences on the Receptor Conformational Features: A Molecular Dynamics Simulation Study.

The gene encoding the ß2-adrenergic receptor (ß2-AR) is polymorphic, which results in possible differences in a primary structure of this protein. It has been shown that certain types of polymorphisms are correlated with some clinical features of asthma, including airways reactivity, whereas the influence of other is not yet understood. Among polymorphisms affecting amino acids at positions 16, 27, 34, 164 and 220, the latter three are present in the crystal structure of ß2-AR, which facilitates studying them by means of molecular dynamics simulations. The current study was focused on investigating to what extent the three polymorphisms of ß2-AR (i.e., Val34Met, Thr164Ile and Ser220Cys) affect the interaction of ß2-AR with its natural molecular environment which includes: lipid bilayer (in the case of all three polymorphs) and Gs protein (which participates in ß2-AR-mediated signaling; in the case of Ser220Cys). We have designed and carried out a series of molecular dynamics simulations at different level of resolution (i.e., either coarse-grained or atomistic simulations), accompanied by thermodynamic integration protocol, in order to identify potential polymorphism-induced alterations in structural, conformational or energetic features of ß2-AR. The results indicate the lack of significant differences in the case of energies involved in the ß2-AR-lipid bilayer interactions. Some differences have been observed when considering the polymorphism-induced alterations in ß2-AR-Gs protein binding, but their magnitude is also negligible in relation to the absolute free energy difference correlated with the ß2-AR-Gs affinity. The Val34Met and Thr164Ile polymorphisms are weakly correlated with alteration of the conformational features of the receptor around polymorphic sites. On the contrary, it has been concluded that the Ser220Cys polymorphism is correlated with several structural alterations located in the intracellular region of ß2-AR, which can induce G-protein binding and, subsequently, the polymorphism-correlated therapeutic responses. More precisely, these alterations involve vicinity of intracellular loops and, in part, are the direct consequence of disturbed interactions of Ser/Cys220 sidechain within 5th transmembrane domain. Structurally, the dynamic structure exhibited by the ß2-ARSer220 polymorph is closer to the Gs-compatible structure of ß2-AR.

Amorphous Inclusion Complexes: Molecular Interactions of Hesperidin and Hesperetin with HP-Β-CD and Their Biological Effects.

This study aimed at obtaining hesperidin (Hed) and hesperetin (Het) systems with HP-ß-CD by means of the solvent evaporation method. The produced systems were identified using infrared spectroscopy (FT-IR), X-ray powder diffraction (XRPD), and differential scanning calorimetry (DSC). Moreover, in silico docking and molecular dynamics studies were performed to assess the most preferable site of interactions between tested compounds and HP-ß-CD. The changes of physicochemical properties (solubility, dissolution rate, and permeability) were determined chromatographically. The impact of modification on biological activity was tested in an antioxidant study as well as with regards to inhibition of enzymes important in pathogenesis of neurodegenerative diseases. The results indicated improvement in solubility over 1000 and 2000 times for Hed and Het, respectively. Permeability studies revealed that Hed has difficulties in crossing biological membranes, in contrast with Het, which can be considered to be well absorbed. The improved physicochemical properties influenced the biological activity in a positive manner by the increase in inhibitory activity on the DPPH radical and cholinoesterases. To conclude the use of HP-ß-CD as a carrier in the formation of an amorphous inclusion complex seems to be a promising approach to improve the biological activity and bioavailability of Hed and Het.

The Systems of Naringenin with Solubilizers Expand Its Capability to Prevent Neurodegenerative Diseases.

BACKGROUND: Naringenin (NAR) is a flavonoid with excellent antioxidant and neuroprotective potential that is limited by its low solubility. Thus, solid dispersions with ß-cyclodextrin (ß-CD), hydroxypropyl-ß-cyclodextrin (HP-ß-CD), hydroxypropylmethylcellulose (HPMC), and microenvironmental pH modifiers were prepared. METHODS: The systems formation analysis was performed by X-Ray Powder Diffraction (XRPD) and Fourier-transform infrared spectroscopy (FT-IR). Water solubility and dissolution rates were studied with a pH of 1.2 and 6.8. In vitro permeability through the gastrointestinal tract (GIT) and the blood-brain barrier (BBB) was assessed with the parallel artificial membrane permeability assay (PAMPA) assay. The antioxidant activity was studied with the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and cupric ion reducing antioxidant capacity (CUPRAC) assays, while in vitro enzymes studies involved the inhibition of acetylcholinesterase, butyrylcholinesterase, and tyrosinase. For the most promising system, in silico studies were conducted. RESULTS: NAR solubility was increased 458-fold by the solid dispersion NAR:HP-ß-CD:NaHCO3 in a mass ratio of 1:3:1. The dissolution rate was elevated from 8.216% to 88.712% in a pH of 1.2 and from 11.644% to 88.843% in a pH of 6.8 (within 3 h). NAR GIT permeability, described as the apparent permeability coefficient, was increased from 2.789 × 10-6 cm s-1 to 2.909 × 10-5 cm s-1 in an acidic pH and from 1.197 × 10-6 cm s-1 to 2.145 × 10-5 cm s-1 in a basic pH. NAR BBB permeability was established as 4.275 × 10-6 cm s-1. The antioxidant activity and enzyme inhibition were also increased. Computational studies confirmed NAR:HP-ß-CD inclusion complex formation. CONCLUSIONS: A significant improvement in NAR solubility was associated with an increase in its biological activity.

Molecular Structure of Cefuroxime Axetil Complexes with α-, ß-, γ-, and 2-Hydroxypropyl-ß-Cyclodextrins: Molecular Simulations and Raman Spectroscopic and Imaging Studies.

The formation of cefuroxime axetil+cyclodextrin (CA+CD) complexes increases the aqueous solubility of CA, improves its physico-chemical properties, and facilitates a biomembrane-mediated drug delivery process. In CD-based tablet formulations, it is crucial to investigate the molecular details of complexes in final pharmaceutical preparation. In this study, Raman spectroscopy and mapping were applied for the detection and identification of chemical groups involved in α-, ß-, γ-, and 2-hydroxypropyl-ß-CD (2-HP- ß-CD)+CA complexation process. The experimental studies have been complemented by molecular dynamics-based investigations, providing additional molecular details of CA+CD interactions. It has been demonstrated that CA forms the guest-host type inclusion complexes with all studied CDs; however, the nature of the interactions is slightly different. It seems that both α- and ß-CD interact with furanyl and methoxy moieties of CA, γ-CD forms a more diverse pattern of interactions with CA, which are not observed in other CDs, whereas 2HP-ß-CD binds CA with the contribution of hydrogen bonding. Apart from supporting this interpretation of the experimental data, molecular dynamics simulations allowed for ordering the CA+CD binding affinities. The obtained results proved that the molecular details of the host-guest complexation can be successfully predicted from the combination of Raman spectroscopy and molecular modeling.

Combinations of Piperine with Hydroxypropyl-ß-Cyclodextrin as a Multifunctional System.

Piperine is an alkaloid that has extensive pharmacological activity and impacts other active substances bioavailability due to inhibition of CYP450 enzymes, stimulation of amino acid transporters and P-glycoprotein inhibition. Low solubility and the associated low bioavailability of piperine limit its potential. The combination of piperine with 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) causes a significant increase in its solubility and, consequently, an increase in permeability through gastrointestinal tract membranes and the blood-brain barrier. X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR) were used to characterize interactions between piperine and HP-ß-CD. The observed physicochemical changes should be combined with the process of piperine and CD system formation. Importantly, with an increase in solubility and permeability of piperine as a result of interaction with CD, it was proven to maintain its biological activity concerning the antioxidant potential (2,2-diphenyl-1-picryl-hydrazyl-hydrate assay), inhibition of enzymes essential for the inflammatory process and for neurodegenerative changes (hyaluronidase, acetylcholinesterase, butyrylcholinesterase).

Structural Insights into Ligand-Receptor Interactions Involved in Biased Agonism of G-Protein Coupled Receptors.

G protein-coupled receptors (GPCRs) are versatile signaling proteins that mediate complex cellular responses to hormones and neurotransmitters. Ligand directed signaling is observed when agonists, upon binding to the same receptor, trigger significantly different configuration of intracellular events. The current work reviews the structurally defined ligand - receptor interactions that can be related to specific molecular mechanisms of ligand directed signaling across different receptors belonging to class A of GPCRs. Recent advances in GPCR structural biology allow for mapping receptors' binding sites with residues particularly important in recognition of ligands' structural features that are responsible for biased signaling. Various studies show particular role of specific residues lining the extended ligand binding domains, biased agonists may alternatively affect their interhelical interactions and flexibility what can be translated into intracellular loop rearrangements. Studies on opioid and angiotensin receptors indicate importance of residues located deeper within the binding cavity and direct interactions with receptor residues linking the ortosteric ligand binding site with the intracellular transducer binding domain. Collection of results across different receptors may suggest elements of common molecular mechanisms which are responsible for passing alternative signals from biased agonists.

Chirality Effects in Biomolecular Systems: Calculation of the Relative Free Energies by Molecular Dynamics Simulations.

Chirality plays an essential role in chemical and biological sciences. At the molecular level, the effects associated with this phenomenon can be studied by using the well-established technique of molecular dynamics simulations. In this work, we present several approaches suited for the molecular dynamics-based free energy calculation in chiral systems. In particular, we have proposed and tested the following strategies relying on the application of general, enhanced sampling methods: (i) biased sampling in the two-dimensional space, along the coordinates defined by the values of the selected torsional angles; (ii) biased sampling in the one- or two-dimensional space, along the path-based coordinate(s); (iii) rational alteration of the system's Hamiltonian in order to enable the interconversion between stereoisomers and reweighting the biased distribution of configurations; (iv) using the free energy landscape generated within approaches (i) or (ii) as time-independent bias in order to further improve sampling efficiency and simultaneously account for multiple chiral centers. All approaches have been tested on a set of model compounds (fenoterol, fructofuranose, and bromochlorofluoromethane), demonstrating the good performance but also some differences in the range of their applicabilities.

Efficient sampling of high-energy states by machine learning force fields.

Regarding their application in the field of molecular sciences, machine learning (ML) methods are capable of combining the high accuracy of ab initio potentials with an efficiency closer to that of classical molecular mechanics. By relying on the reference data (e.g., atomic configurations and corresponding energies), the ML algorithms can reconstruct the potential energy surface for simple molecular systems, which may subsequently serve as a computationally inexpensive force field. The accuracy of such an ML force field is highly dependent on the character of the dataset that was used for its training. In this work, we show that omitting the high-energy states, which results from following the Boltzmann distribution, may lead to a catastrophic loss of accuracy in certain regions of the configurational phase space. To overcome this challenge, we have proposed an alternative solution for generating the ML input data. The most essential step is the biased subsampling of the configurations, aimed at increasing the population of hardly accessible states, usually located on energy barriers. The applicability of the proposed procedure is demonstrated on the example of conformational rearrangements in the two flexible, heterocyclic molecules. This approach provides an essential component required to obtain the ML force fields, accurate within the whole configurational phase space of the system.

Tedizolid-Cyclodextrin System as Delayed-Release Drug Delivery with Antibacterial Activity.

Progressive increase in bacterial resistance has caused an urgent need to introduce new antibiotics, one of them being oxazolidinones with their representative tedizolid. Despite the broad spectrum of activity of the parent tedizolid, it is characterized by low water solubility, which limits its use. The combination of the active molecule with a multifunctional excipient, which is cyclodextrins, allows preservation of its pharmacological activity and modification of its physicochemical properties. Therefore, the aim of the study was to change the dissolution rate and permeability through the model membrane of tedizolid by formation of solid dispersions with a cyclodextrin. The research included identification of tedizolid-hydroxypropyl-ß-cyclodextrin (tedizolid/HP-ß-CD) inclusion complex by thermal method (Differential Scanning Colorimetry), spectroscopic methods (powder X-ray diffraction, Fourier-Transform Infrared spectroscopy), and molecular docking. The second part of the research concerned the physicochemical properties (dissolution and permeability) and the biological properties of the system in terms of its microbiological activity. An increase in the dissolution rate was observed in the presence of cyclodextrin, while maintaining a high permeation coefficient and high microbiological activity. The proposed approach is an opportunity to develop drug delivery systems used in the treatment of resistant bacterial infections, in which, in addition to modifying the physicochemical properties caused by cyclodextrin, we observe a favorable change in the pharmacological potential of the bioactives.

Thermodynamic study of new antiepileptic compounds by combining chromatography on the phosphatidylcholine biomimetic stationary phase and differential scanning calorimetry.

Liquid chromatography coupled to spectrophotometric detection of new antiepileptic compounds, 1,2,4-triazole-3-thione derivatives, on immobilized artificial membrane phosphatidylcholine is reported. The curves representing the relationship between ln k versus 1/T generated under isocratic conditions by the use of methanol and acetonitrile-containing eluent systems have been constructed in order to determine the thermodynamic parameters: the enthalpies, entropies and the relative free energies. The hydrocarbon chains of analytes significantly influenced the membrane behavior of the whole molecules. Excellent correlations of the theoretical lipophilicity with the experimental thermodynamic descriptors, have confirmed contribution of the hydrophobic interactions in the retention process. However, presence of sulfur or oxygen as heteroatoms at R1 substituents in the 1,2,4-triazole ring appears to be responsible for more pronounced selectivity of these compounds on the phosphatidylcholine stationary phase. Molecular dynamics simulations revealed the selective preferences of the phosphatidylcholine with respect to the compounds with either ether of sulfide moieties. Experimental and theoretical set-ups resulted in corresponding outcomes.

Thiazolo[3,2-a]pyrimidin-5-one derivatives as a novel class of 11ß-hydroxysteroid dehydrogenase inhibitors.

11ß-hydroxysteroid type 1 dehydrogenase (11ß-HSD1) is an enzyme that increases tissue concentrations of cortisol. Selective inhibitors of this enzyme regulate the level of cortisol and thus play a key role in the treatment of Cushing's syndrome, metabolic syndrome and type 2 diabetes. In this study the inhibitory activity of 29 thiazolo[3,2-a]pyrimidin-5-one derivatives on 11ß-HSD1 were investigated. Studies were carried out with pooled human liver microsomes. A lot of analyzed compounds show activity for inhibiting 11ß-HSD1 (up to 59.15% at concentration 10 µmol/l). Molecular docking simulation show that the molecule of the most active compound: 7-(cyclohexylmethyl)-2-iodomethyl-2,3-dihydrothiazolo[3,2-a]pyrimidin-5-one forms hydrogen bonds with Ala172, Leu171, Leu215 or Tyr177. In addition, the cycloalkane moiety can create the hydrophobic contacts with NADP+. For this compound also the most favourable Docking Score value was obtained. The most active compound only in the slight degree inhibits 11ß-HSD2 activity and is a selective inhibitor of 11ß-hydroxysteroid dehydrogenase type 1. Consequently it can have a real effect on the regulation of the cortisol level in the body.

Solid-phase extraction using octadecyl-bonded silica modified with photosynthetic pigments from Spinacia oleracea L. for the preconcentration of lead(II) ions from aqueous samples.

Spinacia oleracea L. extract was immobilized on an octadecyl-bonded silica surface to produce a new sorbent for the solid-phase extraction of trace amounts of metal ions from aqueous neutral samples. A measurement of the metal content has been performed by using graphite furnace atomic absorption spectroscopy. The affinity of the investigated bivalent metal cations for the modified sorbent are in the order: Pb(II) > Cu(II) > Ni(II) > Zn(II) ≈ Cd(II) ≈ Co(II). The quantum-chemically calculated chlorophyll-a-metal ion binding energies were consistent with the measured affinities of the corresponding metal ions to the investigated sorbent. The maximum sorption capacity obtained for Pb(II) was equal to 1.44 µmol/g. The value of lead uptake was significantly higher in comparison to the one reported for other sorbents and biosorbents. Immobilized chlorophyll a is responsible for a chelation process with stoichiometry 1:1 owing to the porphyrin rings, which was confirmed by the quantitative analysis performed by reversed-phase high-performance liquid chromatography with diode array detection. The Toth adsorption isotherm model was applicable to the description of the adsorption process of either chlorophyll a or Pb(II). The structural analysis of sorbent was done using Fourier-transform Raman spectroscopy and scanning electron microscopy with an energy dispersive X-ray detector.

Ligand-induced action of the W2866.48 rotamer toggle switch in the ß2-adrenergic receptor.

Studies focused on GPCRs, particularly on the ß2-adrenergic receptor (ß2-AR), have demonstrated the relationship between ligand structure, receptor conformational changes and the corresponding pharmacological outcomes. Herein, we studied the molecular details of the rotameric flip of the W2866.48 sidechain, i.e. a presumed action switch that has not been reported in native ß2-AR thus far. It is believed that although both the 'active' and 'inactive' conformers of ß2-AR exhibit similar conformations of this switch, it may still play a substantial role in the ligand-induced activation of the receptor. By using both experimental methods (time-resolved fluorescence spectroscopy) and molecular modeling techniques (enhanced-sampling molecular dynamics), we characterized the conformational rearrangements of W2866.48 in relation to the type of ligand present in the binding cavity and to the conformation of the receptor ('active' vs. 'inactive' ß2-AR). We found that the conformational behaviour of W2866.48 is correlated with the pharmacological character of the ligand present in the binding cavity but not with the instantaneous conformation of the receptor. Namely, agonists promote the W2866.48 conformations that facilitate the increase of the solvation within the inner receptor channel. In contrast, antagonists and inverse agonists act toward the decrease of the solvation in the inner channel. This creates an opportunity for using computational methodologies in determining the pharmacological properties of various ligands. The combination of the time-resolved fluorescence spectroscopy technique with the enhanced-sampling molecular dynamics simulations is shown to be a powerful tool for studying the ligand-induced conformational rearrangements in GPCRs.

Natural terpene derivatives as new structural task-specific ionic liquids to enhance the enantiorecognition of acidic enantiomers on teicoplanin-based stationary phase by high-performance liquid chromatography.

We present the specific cooperative effect of a semisynthetic glycopeptide antibiotic teicoplanin and chiral ionic liquids containing the (1R,2S,5R)-(-)-menthol moiety on the chiral recognition of enantiomers of mandelic acid, vanilmandelic acid, and phenyllactic acid. Experiments were performed chromatographically on an Astec Chirobiotic T chiral stationary phase applying the mobile phase with the addition of the chiral ionic liquids. The stereoselective binding of enantiomers to teicoplanin in presence of new chiral ionic liquids were evaluated applying thermodynamic measurements and the docking simulations. Both the experimental and theoretical methods revealed that the chiral recognition of enantiomers in the presence of new chiral ionic liquids was enthalpy driven. The changes of the teicoplanin conformation occurring upon binding of the chiral ionic liquids are responsible for the differences in the standard changes in Gibbs energy (ΔG0 ) values obtained for complexes formed by the R and S enantiomers and teicoplanin. Docking simulations revealed the steric adjustment between the chiral ionic liquids cyclohexane ring (chair conformation) and the ß-d-glucosamine ring of teicoplanin and additionally hydrophobic interactions between the decanoic aliphatic chain of teicoplanin and the alkyl group of the tested salts. The obtained terpene derivatives can be considered as "structural task-specific ionic liquids" responsible for enhancing the chiral resolution in synergistic systems with two chiral selectors.

Acyclic forms of aldohexoses and ketohexoses in aqueous and DMSO solutions: conformational features studied using molecular dynamics simulations.

The molecular properties of aldohexoses and ketohexoses are usually studied in the context of their cyclic, furanose or pyranose structures which is due to the abundance of related tautomeric forms in aqueous solution. We studied the conformational features of a complete series of D-aldohexoses (D-allose, D-altrose, D-glucose, D-mannose, D-gulose, d-idose, D-galactose and D-talose) and D-ketohexoses (D-psicose, D-fructose, D-sorbose and D-tagatose) as well as of L-psicose by using microsecond-timescale molecular dynamics in explicit water and DMSO with the use of enhanced sampling methods. In each of the studied cases the preferred conformation corresponded to an extended chain structure; the less populated conformers included the quasi-cyclic structures, close to furanose rings and common for both aldo- and ketohexoses. The orientational preferences of the aldehyde or ketone groups are correlated with the relative populations of anomers characteristic of cyclic aldo- and ketohexoses, respectively, thus indicating that basic features of anomeric equilibria are preserved even if hexose molecules are not in their cyclic forms. No analogous relationship is observed in the case of other structural characteristics, such as the preferences of acyclic molecules to form either the furanose-or pyranose-like structures or maintaining the chair-like geometry of pseudo-pyranose rings.

Tyrosine 308 is necessary for ligand-directed Gs protein-biased signaling of ß2-adrenoceptor.

Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, ß2-adrenoceptor (ß2-AR) couples dually to Gs and Gi proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of ß2-AR causes a switch in receptor coupling from Gs to Gi. More recent studies have demonstrated that phosphorylation of ß2-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the Gi coupling. We have previously shown that although most ß2-AR agonists cause both Gs and Gi activation, (R,R')-fenoterol preferentially activates ß2-AR-Gs signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on ß2-AR essential for Gs-biased signaling. Following stimulation with a ß2-AR-Gs-biased agonist (R,R')-4'-aminofenoterol, the Gi disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type ß2-AR. Interestingly, Y308F substitution on ß2-AR enabled (R,R')-4'-aminofenoterol to activate Gi and to produce these responses in a pertussis toxin-sensitive manner without altering ß2-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of ß2-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of ß2-AR stabilize receptor conformations favoring the receptor-Gs protein coupling and subsequently result in Gs-biased agonism.

Agonist binding by the ß2-adrenergic receptor: an effect of receptor conformation on ligand association-dissociation characteristics.

The ß2-adrenergic receptor (ß2-AR), a G protein-coupled receptor (GPCR), is a physiologically important transmembrane protein that is a target for drugs used for treatment of asthma and cardiovascular diseases. Study of the first steps of ligand recognition and the molecular basis of ligand binding to the orthosteric site is essential for understanding the pharmacological properties of the receptor. In this work we investigated the characteristic features of the agonist association-dissociation process to and from the different conformational forms of ß2-AR by use of advanced molecular modeling techniques. The investigation was focused on estimating the free energy profiles (FEPs) corresponding to the process of a full agonist ((R,R)-fenoterol) and an inverse agonist (carazolol) binding and unbinding to and from ß2-AR. The two different conformational forms of ß2-AR, i.e. active ß2-AR-PDB: 3P0G and inactive ß2-AR-PDB: 2RH1 were included in this stage of the study. We revealed several significant qualitative differences between FEPs characteristic of both conformational forms. Both FEPs suggest the existence of three transient binding sites in the extracellular domain of ß2-AR. Comparison of the residues surrounding these transient binding sites in both ß2-AR states revealed the importance of the aromatic residues F194, H93(2.64), H296(6.58), and H178 (extracellular part of ß2-AR) in the early stages of the binding process. In addition, slightly different exit and entry paths are preferred by the ligand molecule in the extracellular part of ß2-AR, depending on the conformation of the receptor.