Exploring the molecular basis of selectivity in A1 adenosine receptors agonists: a case study

Adenosine is a naturally occurring purine nucleoside that has a wide variety of well-documented regulatory functions and physiological roles. Selective activation of the adenosine A₁ receptor has drawn attention in drug discovery for the therapeutic effects on neural and cardiovascular disorders. We have developed a model of the human A₁ adenosine receptor using bovine rhodopsin as a template. A flexible docking approach has been subsequently carried out for evaluating the molecular interactions of twenty-one selective A₁ agonists with the receptor model. The results of these studies are consistent with mutational and biochemical data. In particular, they highlight a wide hydrogen-bonding network between the nucleoside portion of the ligands and the A₁ receptor as well as key amino acids for hydrophobic interactions with the different N⁶-groups of the agonists. The models presented here provide a detailed molecular map for the selective stimulation of the adenosine A₁ receptor subtype and a steady basis for the rational design of new A₁ selective ligands.     

A case study on biological activity in a surface-bound multicomponent system: the biotin-streptavidin-peroxidase system

The adsorption of multiple protein layers on biotinylated organic surfaces has been characterized using surface plasmon resonance (SPR) and atomic force microscopy (AFM). Diffusion-limited loading of the biotinylated self-assembled monolayers (SAMs) ensures a precise control of the streptavidin surface density. For the subsequent interaction with biotinylated peroxidase, SPR data hint at a streptavidin density dependent orientation during peroxidase adsorption. Microcontact printed well-defined two-dimensional patterned surfaces of biotinylated organothiols and protein-resistant OEG-thiols allow an in-situ differentiation of specific and nonspecific adsorption (e.g., mono- vs multilayer adsorption). Additionally, the very important issue of biological activity of surface-bound enzymes is addressed by comparing the enzyme activities in solution with that for surface-bound species.     

In search of novel ligands using a structure-based approach: a case study on the adenosine A<Subscript>2A</Subscript> receptor

In this work, we present a case study to explore the challenges associated with finding novel molecules for a receptor that has been studied in depth and has a wealth of chemical information available. Specifically, we apply a previously described protocol that incorporates explicit water molecules in the ligand binding site to prospectively screen over 2.5 million drug-like and lead-like compounds from the commercially available eMolecules database in search of novel binders to the adenosine A_2A receptor (A_2AAR). A total of seventy-one compounds were selected for purchase and biochemical assaying based on high ligand efficiency and high novelty (Tanimoto coefficient ≤0.25 to any A_2AAR tested compound). These molecules were then tested for their affinity to the adenosine A_2A receptor in a radioligand binding assay. We identified two hits that fulfilled the criterion of ~50 % radioligand displacement at a concentration of 10 μM. Next we selected an additional eight novel molecules that were predicted to make a bidentate interaction with Asn253^6.55, a key interacting residue in the binding pocket of the A_2AAR. None of these eight molecules were found to be active. Based on these results we discuss the advantages of structure-based methods and the challenges associated with finding chemically novel molecules for well-explored targets.     

Frontal analysis microchip capillary electrophoresis to study the binding of ligands to receptors derivatized on magnetic beads

The model binding of the glycopeptide antibiotic teicoplanin (Teic) from Actinoplanes teichomyceticus, immobilized on magnetic microspheres, to d-Ala-d-Ala terminus peptides was assessed using microchip capillary electrophoresis (MCE) with continuous frontal analysis (FA). Teic is closely related to vancomycin (Van), historically, the drug of last resort used to treat many Gram-positive bacterial infections. Glycopeptide antibiotics inhibit bacterial growth by binding to the d-Ala-d-Ala terminus on the cell wall of Gram-positive bacteria via hydrogen bonds, thereby preventing the enzyme-mediated cross-linking of peptidoglycan and eventual cell death. In this work direct and competitive bead-based assays in a microfluidic chip are demonstrated. The binding constants obtained using the technique are comparable with values reported in the literature.     

Nanoindentation of high performance semicrystalline polymers: A case study on PEEK

Semicrystalline high performance polymers (HPPs) exhibit desirable thermomechanical properties which make them suitable materials in several industries. However, when their mechanical characteristics are investigated at nanoscale, the inhomogeneous nature of semicrystalline HPPs results in measurement of localized properties which might not represent the overall material response. In this paper, the nanomechanics of poly(ether-ether-ketone) (PEEK) as a tough HPP is scrutinized using the nanoindentation technique. It is demonstrated how surface properties of this polymer can affect the nanoindentation measurements and reveal significant anomalies compared to the nanoindentation of fully crystalline or amorphous solids. In addition, polishing and annealing procedures are introduced as simple techniques that can be used for eliminating the inhomogeneity of the mechanical response of PEEK. Lastly, the capability of depth sensing indentation for determining the distribution of crystalline and amorphous sub-regions within semicrystalline solids is scrutinized and critically discussed.     

A study on the heat transfer geometry in MBDTA

This paper presents the quantitative application of the Metal Blocked Differential Thermal Analysis (MBDTA). Calibrations have been electrically carried out by using three types of calibration heaters (pen, ring and spiral), which are placed inside the sample. By means of this arrangement, thermophysical properties of the samples make the quantitative evaluation of the results easy. The influence of the size and sample diameter, the types of heater, the space between heater and thermocouple, in other words, the factors affecting calibration and the effect of the heat transfer geometry on the determination of the fusion heat have been investigated.

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Zusammenfassung Vorliegende Arbeit stellt eine quantitative Anwendung der Metallblock-DTA (MBDTA) dar. Es wurden elektrisch Kalibrationen mit verschiedenen, innerhalb der Probe befindlichen Kalibrationserhitzern (Stift, Ring, Spirale) durchgeführt. Mit Hilfe dieser Anordnung wird die Auswertung der Ergebnisse durch die thermophysischen Eigenschaften der Probe erleichtert. Es wurde der Einfluß von Größe und Probendurchmesser, von Erhitzertyp, von Abstand zwischen Erhitzer und Thermoelement, mit anderen Worten von denjenigen Faktoren, die die Kalibration beeinflussen sowie der Einfluß der Wärmetransportgeometrie auf die Bestimmung der Schmelzwärme untersucht.

     

Comparison of three strategies to evaluate uncertainty from in-house validation data. A case study: mercury determination in sediments

In the present paper, three approaches are compared for the evaluation of the combined uncertainty in the determination of mercury in aquatic sediments by an aqua regia extraction procedure. For this, the data obtained in validation studies from five certified reference materials (CRMs), covering a range of concentrations from 0.8 to 130 mg kg⁻¹ of mercury and analysed by three atomic spectroscopic techniques (cold vapour generation atomic fluorescence spectrometry, CV-AFS, cold vapour generation atomic absorption spectroscopy, and inductively coupled plasma mass spectroscopy), were considered. The combined uncertainty was firstly assessed by considering separately the data obtained for each CRM analysed (approach A). Moreover, this assessment was also performed with two other calculation approaches (B and C) based on the pooled data obtained from the validation step. The comparison of the results obtained for the different techniques showed a clear bias effect when using CV-AFS with nitric acid as a diluent. In relation to the strategies tested for the combined uncertainty assessment, approach C proved to be the easiest and friendliest method for uncertainty assessment.      

Relative binding orientations of adenosine A1 receptor ligands — A test case for Distributed Multipole Analysis in medicinal chemistry

Summary The electrostatic properties of adenosine-based agonists and xanthine-based antagonists for the adenosine A₁ receptor were used to assess various proposals for their relative orientation in the unknown binding site. The electrostatic properties were calculated from distributed multipole representations of SCF wavefunctions. A range of methods of assessing the electrostatic similarity of the ligands were used in the comparison. One of the methods, comparing the sign of the potential around the two molecules, gave inconclusive results. The other approaches, however, provided a mutually complementary and consistent picture of the electrostatic similarity and dissimilarity of the molecules in the three proposed relative orientations. This was significantly different from the results obtained previously with MOPAC AM1 point charges. In the standard model overlay, where the aromatic nitrogen atoms of both agonists and antagonists are in the same position relative to the binding site, the electrostatic potentials are so dissimilar that binding to the same receptor site is highly unlikely. Overlaying the N⁶-region of adenosine with that near C8 of theophylline (the N⁶-C8 model) produces the greatest similarity in electrostatic properties for these ligands. However, N⁶-cyclopentyladenosine (CPA) and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) show greater electrostatic similarity when the aromatic rings are superimposed according to the flipped model, in which the xanthine ring is rotated around its horizontal axis. This difference is mainly attributed to the change in conformation of N⁶-substituted adenosines and could result in a different orientation for theophylline and DPCPX within the receptor binding site. However, it is more likely that DPCPX also binds according to the N⁶-C8 model, as this model gives the best steric overlay and would be favoured by the lipophilic forces, provided that the binding site residues could accommodate the different electrostatic properties in the N⁶/N7-region. Finally, we have shown that Distributed Multipole Analysis (DMA) offers a new, feasible tool for the medicinal chemist, because it provides the use of reliable electrostatic models to determine plausible relative binding orientations.Abbreviations DMA distributed multipole analysis - SCF self-consistent field - CPA N⁶-cyclopentyladenosine - DPCPX 1,3-dipropyl-8-cyclopentylxanthine - R-PIA R-1-phenyl-2-propyladenosine - S-PIA S-1-phenyl-2-propyladenosine     

A study on the utilization of the Youden plot to evaluate proficiency test results

Methods of utilizing the Youden plot obtained from interlaboratory comparison tests are proposed. The Youden plot is a powerful tool for visually evaluating between- or within-laboratory errors. However, there are no generally applicable methods to indicate the anomalies in the between- and the within-laboratory errors in qualitative terms. An evaluation frame was therefore developed in this study as an indication method. Moreover, the Youden plot is considered to be useful for visually assessing the comprehensive performance of laboratories using two types of results. Although the confidence ellipse has been proposed in ISO 13528:2005 for this purpose, robust determination of the parameters has not yet been implemented. In the present study, robust determination of one of the parameters, the correlation coefficient, was developed. Our proposals were analytically validated using two types of statistical models. In addition, the properties were evaluated through computational simulations. From the results obtained, it can be concluded that our proposals are practically applicable to actual interlaboratory comparison tests.     

A simple flow cell system to evaluate in vitro bacterial adhesion on solids

A simple flow cell system was developed for studies of bacterial adhesion on hydrophobic or hydrophilic surfaces in the presence or absence of saliva. The system consists of flow cells made of borosilicate glass tubes (flow cell volume, 0.45 ml) that are connected to syringes mounted on an infusion pump. Hydrophilic or hydrophobic surfaces were obtained by treating the flow cells with acid or dichlorodimethylsilane, respectively. Liquids and bacterial suspensions were withdrawn directly into the flow cells and the flow rate was monitored through the infusion pump. The adhesion of ³H-labeled Streptococcus mutans cells to bare or saliva-coated surfaces was studied in this system by determining the radioactivity retained in the flow cells after rinsing with buffer at different flow rates. The results indicate that factors like the bacterial cell density, the surface properties, the presence of saliva pellicle and the rinsing flow rate significantly affect the adhesion of S. mutans. These results are in agreement with previous findings obtained with other flow cell systems. The present system has the advantages of using small volumes of liquids and allowing defined laminar flow conditions. Moreover, the entire cell, not only selected area spots, are used for the evaluation of bacterial adhesion. No part of the flow cell is re-used, thus avoiding the risk of contamination with other components. The system may therefore be suitable for assays testing the effect of different compounds on bacterial adhesion.     

双核V配合物中金属-金属键的INDO定域化方法研究

本文利用INDO和E_R定域化方法,对Ⅴ~2(μ-η^2-S~2)~2(SCCH~2)~4和[V~2O~2Cl~6]^2-双核配合物进行了量子化学计算研究,讨论了它们的电子结构和化学键性质,并讨论了利用mulliken键序判别金属原子间成键的可靠.进而基于过渡金属原子主要是以d亚层轨道参与成键,建议用金属原子d亚层的键序讨论键强度的新方法.     

A brief review of the methods used to evaluate vapour pressures and sublimation enthalpies

A brief review of the experimental methods used to evaluate vapour pressures and sublimation enthalpies is presented. The methods discussed have been used for determining the results of several substituted benzenes that were collected in a database, with the main purpose of developing new estimation methods of these thermodynamic properties. A critical evaluation of the two most used calorimetric techniques for determining enthalpies of sublimation is also addressed.     

The ability of capping phenylphosphinidene ligands to confer the trigonal prismatic geometry on hexaruthenium frameworks

Reaction of [Ru₃(CO)₁₂] with a three-fold molar amount of PPhH₂ in toluene under reflux affords a range of products including two hexaruthenium species, [Ru₆(μ₄-PPh)₂(μ₃-PPh)₂(CO)₁₂] and [Ru₆(μ₄-PPh)₃(μ₃-PPh)₂(CO)₁₂], established X-ray crystallographically to have distorted trigonal prismatic skeletal geometries.     

On the performance of quantum chemical methods to predict solvatochromic effects: the case of acrolein in aqueous solution

The performance of the Hartree-Fock method and the three density functionals B3LYP, PBE0, and CAM-B3LYP is compared to results based on the coupled cluster singles and doubles model in predictions of the solvatochromic effects on the vertical n-->pi* and pi-->pi* electronic excitation energies of acrolein. All electronic structure methods employed the same solvent model, which is based on the combined quantum mechanics/molecular mechanics approach together with a dynamical averaging scheme. In addition to the predicted solvatochromic effects, we have also performed spectroscopic UV measurements of acrolein in vapor phase and aqueous solution. The gas-to-aqueous solution shift of the n-->pi* excitation energy is well reproduced by using all density functional methods considered. However, the B3LYP and PBE0 functionals completely fail to describe the pi-->pi* electronic transition in solution, whereas the recent CAM-B3LYP functional performs well also in this case. The pi-->pi* excitation energy of acrolein in water solution is found to be very dependent on intermolecular induction and nonelectrostatic interactions. The computed excitation energies of acrolein in vacuum and solution compare well to experimental data.     

Synthesis of coumarin derivatives in a microfluidic flow system employing the Pechmann condensation: A case study

For the synthesis of coumarin derivatives using the Pechmann condensation scheme, an acidic ionic liquid catalyst, abbreviated as [EBsImH][HSO ₄ ] , was prepared from the ring opening of 1,4-butanesultone by 1-ethylimidazole, followed by the addition of 1 equiv. H₂SO₄(c). The [EBsImH][HSO ₄ ] -catalyzed Pechmann condensation reactions proceeded smoothly in a batch setup, with recyclable [EBsImH][HSO ₄ ] showing great catalytic activity. The acidic ionic liquid catalyst [EBsImH][HSO ₄ ] was recovered from EtOAc/H₂O extraction of the product mixture, where the H₂O layer was worked up and dried for reuse in consecutive runs of the Pechmann condensation reactions, maintaining >85% conversion for four times. The catalytic reactions were also carried out in a microfluidic flow setup. The flow parameters, the reactant molar amounts, and the additional H₂SO₄ as a modifying acid catalyst were optimized in the current case study. A minimum conversion rate of 2.8 g/hr of coumarin derivatives was demonstrated.     

A high performance system to study the influence of temperature in on-line solid-phase extraction capillary electrophoresis

A novel high performance system to control the temperature of the microcartridge in on-line solid phase extraction capillary electrophoresis (SPE–CE) is introduced. The mini-device consists in a thermostatic bath that fits inside of the cassette of any commercial CE instrument, while its temperature is controlled from an external circuit of liquid connecting three different water baths. The circuits are controlled from a switchboard connected to an array of electrovalves that allow to rapidly alternate the water circulation through the mini-thermostatic-bath between temperatures from 5 to 90 °C. The combination of the mini-device and the forced-air thermostatization system of the commercial CE instrument allows to optimize independently the temperature of the sample loading, the clean-up, the analyte elution and the electrophoretic separation steps.