Binding Networks Identify Targetable Protein Pockets for Mechanism-Based Drug Design

The human genome codes only a few thousand druggable proteins, mainly receptors and enzymes. While this pool of available drug targets is limited, there is an untapped potential for discovering new drug-binding mechanisms and modes. For example, enzymes with long binding cavities offer numerous prerequisite binding sites that may be visited by an inhibitor during migration from a bulk solution to the destination site. Drug design can use these prerequisite sites as new structural targets. However, identifying these ephemeral sites is challenging. Here, we introduce a new method called NetBinder for the systematic identification and classification of prerequisite binding sites at atomic resolution. NetBinder is based on atomistic simulations of the full inhibitor binding process and provides a networking framework on which to select the most important binding modes and uncover the entire binding mechanism, including previously undiscovered events. NetBinder was validated by a study of the binding mechanism of blebbistatin (a potent inhibitor) to myosin 2 (a promising target for cancer chemotherapy). Myosin 2 is a good test enzyme because, like other potential targets, it has a long internal binding cavity that provides blebbistatin with numerous potential prerequisite binding sites. The mechanism proposed by NetBinder of myosin 2 structural changes during blebbistatin binding shows excellent agreement with experimentally determined binding sites and structural changes. While NetBinder was tested on myosin 2, it may easily be adopted to other proteins with long internal cavities, such as G-protein-coupled receptors or ion channels, the most popular current drug targets. NetBinder provides a new paradigm for drug design by a network-based elucidation of binding mechanisms at an atomic resolution.     

Ultrastructure of oocytes of the Urostreptus atrobrunneus (Diplopoda,Spirostreptida, Spirostreptidae): A potential urban centers plague

The knowledge of the process of egg formation is indispensable for understanding the mechanisms involved in the reproduction of different species. In this context, the objective of this work was to describe the ultrastructure of the oocytes of Urostreptus atrobrunneus (Spirostreptida), a potential plague of urban centers in different locations of São Paulo State. The lack of knowledge about the morphology, physiology, and the reproductive behavior of the species have hindered an effective control of it. The oocytes of U. atrobrunneus presented three development stages: young oocyte or type I; intermediary oocyte or type II; and mature oocyte or type III. During the oocyte development, the cytoplasm become filled with several globules of protein, drops of lipids, and sphaerocrystals, and it was not observed in many organelles in the oocytes with exception of mitochondria, abundant, principally in young oocytes. The vitelline membrane is also deposited in a discontinuous form and the chorion does not present differentiation of layers. The follicular epithelium alters its shape according to the development phase of the oocyte. Part of the vitellus is from exogenous origin and part is endogenous. Before this, only two studies about the ultrastructural analysis of the female germ cells of diplopods were published. Microsc. Res. Tech. © 2012 Wiley Periodicals, Inc.     

LC-MS method development to evaluate major triterpenes in skins and cuticular waxes of grape berries

Predominant triterpenes (free oleanolic acid, β-sitosterol and β-sitosterol-3-O-β- d -glucoside) in skin and cuticular wax of grape berries have been analysed by liquid chromatography–mass spectrometry (LC-APCI-MS). The validity of the developed method was established by determining linearity, recovery, precision, accuracy, limit of detection and quantification. Detection limits were in the range of 0.12–0.25 mg L⁻¹, and linearity values ranged up to 105 mg L⁻¹. The repeatability of the method was good. High variability was found among the measured grape varieties based on triterpene content, quantitites of oleanolic acid of cuticular wax ranged from 31.53 mg kg⁻¹ to 162.01 mg kg⁻¹ of the twelve analysed samples. The highest sitosterol content was measured in the sample 'Othello' (73.12 mg kg⁻¹), while maximum sitosterol glucoside content was also found in 'Othello' variety (13.68 mg kg⁻¹). The results showed that the study on triterpenes could be an informative tool to characterise grape varieties.     

Preparation, Characterization and Catalytic Activities of Immobilized Enzyme Mimics

In order to find highly active and selective oxygen-transfer catalysts with appreciable durability, Cu(II)–histidine complexes were covalently grafted onto a chlorinated polystyrene resin as copper-containing enzyme mimics. The Cu(II)-histidine complexes and the mobile polymer were to resemble the active center and the proteomic skeleton of the enzymes, respectively. The resulting heterogenized complexes were expected to be nearly so active and more durable catalysts that are easier to recycle than their homogeneous counterparts. The substances were tested in a superoxide radical anion dismutation reaction. Control for the syntheses was exerted by protecting either the N-terminal or the C-terminal of the covalently grafted l-histidine molecules. During the preparative work generally applied methods of synthetic organic chemistry (alkylation or esterification) were used. Various anchored complexes were prepared and characterized by classical analytical methods, different forms of spectroscopy as well as molecular modeling. The covalently grafted complexes having the protected amino acids as ligands displayed remarkably high activities in the superoxide dismutase (SOD) test reaction.     

Properties of naphthalene oligomers polyalkylated with linear 1-alkenes

Star-shaped alkylaromatic hydrocarbons consisting of oligomeric naphthalene cores and long straight-chain alkyl arms have been synthesised using a one-pot method: Where 1-alkene is 1-hexene, 1-octene, 1-decene, 1-dodecene, 1-tetradecene, 1-hexadecene, and 1-octadecene, respectively. Different linear 1-alkenes with increasing chain length have been reacted with naphthalene oligomers in an identical way, and the properties of the total products compared and related to the chain length of the alkylating normal 1-alkene. Consequent to this, increases in M̌_n, (= 550–1900), viscosity indexes (90–161), pour points (—24 to + 17°C), flash points (210–330°C), and oxidation resistance, could be observed. These products exhibited properties good enough to be suitable for use as lubricant base stocks and/or additives.     

Coherences of Instrumental and Sensory Characteristics: Case Study on Cherry Tomatoes

The aim of this study was to investigate 6 cherry tomato varieties in terms of morphological, instrumental, and sensory attributes. Hungarian cherry tomato landraces have not been investigated in comparison with new commercial varieties for these traits. Parameters investigated were water‐soluble antioxidant capacity (FRAP, DPPH, and TEAC), and total polyphenol, vitamin C, β‐carotene, lycopene, total soluble solids, and acid contents. Colorimetric measurements as well as sensory analyses were conducted. It was concluded that varied antioxidant assays should be used in parallel to overcome the selectivity of any 1 method. Total phenolic content significantly contributed to results of antioxidant assays for the investigated varieties. The sensory profiles of the 6 cherry tomato varieties have been created. The differences between the products based on the 18 attributes were analyzed by Tukey post hoc test. The biplot of the principal component analysis showed that the sensory panel could discriminate the samples along the principal components. No correlation was found between colorimetric data a* and b* measured from pulp and lycopene, but a negative connection of β‐carotene and hue was noted. Total polyphenol content showed correlations with colorimetric results, except for b*. The influence of tomato skin color on color perception is significant as in the present study instrumental data measured from pulp did not match that of the panelists evaluating intact fruit. Instrumental results of sugar content were supported by the ratings of the sensory panel.     

Rotor-stator molecular crystals of fullerenes with cubane

Cubane (C8H8) and fullerene (C60) are famous cage molecules with shapes of platonic or archimedean solids. Their remarkable chemical and solid-state properties have induced great scientific interest. Both materials form polymorphic crystals of molecules with variable orientational ordering. The idea of intercalating fullerene with cubane was raised several years ago but no attempts at preparation have been reported. Here we show that C60 and similarly C70 form high-symmetry molecular crystals with cubane owing to topological molecular recognition between the convex surface of fullerenes and the concave cubane. Static cubane occupies the octahedral voids of the face-centred-cubic structures and acts as a bearing between the rotating fullerene molecules. The smooth contact of the rotor and stator molecules decreases significantly the temperature of orientational ordering. These materials have great topochemical importance: at elevated temperatures they transform to high-stability covalent derivatives although preserving their crystalline appearance. The size-dependent molecular recognition promises selective formation of related structures with higher fullerenes and/or substituted cubanes.     

Abiotic Stress Response of Near-Isogenic Spring Durum Wheat Lines under Different Sowing Densities

A detailed study was made of changes in the plant development, morphology, physiology and yield biology of near-isogenic lines of spring durum wheat sown in the field with different plant densities in two consecutive years (2013–2014). An analysis was made of the drought tolerance of isogenic lines selected for yield QTLs (QYld.idw-2B and QYld.idw-3B), and the presence of QTL effects was examined in spring sowings. Comparisons were made of the traits of the isogenic pairs QYld.idw-3B++ and QYld.idw-3B−− both within and between the pairs. Changes in the polyamine content, antioxidant enzyme activity, chlorophyll content of the flag leaf and the normalized difference vegetation index (NDVI) of the plot were monitored in response to drought stress, and the relationship between these components and the yield was analyzed. In the case of moderate stress, differences between the NIL++ and NIL−− pairs appeared in the early dough stage, indicating that the QYld.idw-3B++ QTL region was able to maintain photosynthetic activity for a longer period, resulting in greater grain number and grain weight at the end of the growing period. The chlorophyll content of the flag leaf in phenophases Z77 and Z83 was significantly correlated with the grain number and grain weight of the main spike. The grain yield was greatly influenced by the treatment, while the genotype had a significant effect on the thousand-kernel weight and on the grain number and grain weight of the main spike. When the lines were compared in the non-irrigated treatment, significantly more grains and significantly higher grain weight were observed in the main spike in NIL++ lines, confirming the theory that the higher yields of the QYld.idw-3B++ lines when sown in spring and exposed to drought stress could be attributed to the positive effect of the “Kofa” QTL on chromosome 3B.     

Electrophysiological Effects of the Transient Receptor Potential Melastatin 4 Channel Inhibitor (4-Chloro-2-(2-chlorophenoxy)acetamido) Benzoic Acid (CBA) in Canine Left Ventricular Cardiomyocytes

Transient receptor potential melastatin 4 (TRPM4) plays an important role in many tissues, including pacemaker and conductive tissues of the heart, but much less is known about its electrophysiological role in ventricular myocytes. Our earlier results showed the lack of selectivity of 9-phenanthrol, so CBA ((4-chloro-2-(2-chlorophenoxy)acetamido) benzoic acid) was chosen as a new, potentially selective inhibitor. Goal: Our aim was to elucidate the effect and selectivity of CBA in canine left ventricular cardiomyocytes and to study the expression of TRPM4 in the canine heart. Experiments were carried out in enzymatically isolated canine left ventricular cardiomyocytes. Ionic currents were recorded with an action potential (AP) voltage-clamp technique in whole-cell configuration at 37 °C. An amount of 10 mM BAPTA was used in the pipette solution to exclude the potential activation of TRPM4 channels. AP was recorded with conventional sharp microelectrodes. CBA was used in 10 µM concentrations. Expression of TRPM4 protein in the heart was studied by Western blot. TRPM4 protein was expressed in the wall of all four chambers of the canine heart as well as in samples prepared from isolated left ventricular cells. CBA induced an approximately 9% reduction in AP duration measured at 75% and 90% of repolarization and decreased the short-term variability of APD₉₀. Moreover, AP amplitude was increased and the maximal rates of phase 0 and 1 were reduced by the drug. In AP clamp measurements, CBA-sensitive current contained a short, early outward and mainly a long, inward current. Transient outward potassium current (I_to) and late sodium current (I_Na,L) were reduced by approximately 20% and 47%, respectively, in the presence of CBA, while L-type calcium and inward rectifier potassium currents were not affected. These effects of CBA were largely reversible upon washout. Based on our results, the CBA induced reduction of phase-1 slope and the slight increase of AP amplitude could have been due to the inhibition of I_to. The tendency for AP shortening can be explained by the inhibition of inward currents seen in AP-clamp recordings during the plateau phase. This inward current reduced by CBA is possibly I_Na,L, therefore, CBA is not entirely selective for TRPM4 channels. As a consequence, similarly to 9-phenanthrol, it cannot be used to test the contribution of TRPM4 channels to cardiac electrophysiology in ventricular cells, or at least caution must be applied.