Intrinsic Thermodynamics and Structure Correlation of Benzenesulfonamides with a Pyrimidine Moiety Binding to Carbonic Anhydrases I,II, VII,XII, and XIII

The early stage of drug discovery is often based on selecting the highest affinity lead compound. To this end the structural and energetic characterization of the binding reaction is important. The binding energetics can be resolved into enthalpic and entropic contributions to the binding Gibbs free energy. Most compound binding reactions are coupled to the absorption or release of protons by the protein or the compound. A distinction between the observed and intrinsic parameters of the binding energetics requires the dissection of the protonation/deprotonation processes. Since only the intrinsic parameters can be correlated with molecular structural perturbations associated with complex formation, it is these parameters that are required for rational drug design. Carbonic anhydrase (CA) isoforms are important therapeutic targets to treat a range of disorders including glaucoma, obesity, epilepsy, and cancer. For effective treatment isoform-specific inhibitors are needed. In this work we investigated the binding and protonation energetics of sixteen [(2-pyrimidinylthio)acetyl]benzenesulfonamide CA inhibitors using isothermal titration calorimetry and fluorescent thermal shift assay. The compounds were built by combining four sulfonamide headgroups with four tailgroups yielding 16 compounds. Their intrinsic binding thermodynamics showed the limitations of the functional group energetic additivity approach used in fragment-based drug design, especially at the level of enthalpies and entropies of binding. Combined with high resolution crystal structural data correlations were drawn between the chemical functional groups on selected inhibitors and intrinsic thermodynamic parameters of CA-inhibitor complex formation.     

Effects of litter on seedling establishment: an indoor experiment with short-lived Brassicaceae species

Recruitment by seeds is essential both in vegetation dynamics and in supporting biodiversity in grasslands. The recruitment by seeds is feasible in suitable vegetation gaps from the seed rain and/or by establishment from persistent soil seed banks. Cessation of grassland management results in litter accumulation, which leads to the decline of species diversity by the decreased availability of open patches. Low amounts of litter is often beneficial, while high amounts of litter is detrimental for seed germination and seedling establishment of short-lived species. In a designed indoor experiment, we explored the effect of litter on seedling establishment by germinating six short-lived Brassicaceae species with both increasing seed mass and litter cover. We found that both seed mass and litter had significant effect on germination and establishment of the sown species. Small-seeded species were significantly negatively affected by the 300 and/or 600 g/m² litter layers. No negative litter effect was detected for species with high seed masses (Lepidium spp.). No overall significant positive litter effect was found, although for most of the species cumulative seedling numbers were not the highest at the bare soil pots. Our results suggest that the negative effects of litter are less feasible on the large-seeded short-lived species than on that of small-seeded ones.     

New evidence on the southeast Baltic Late Bronze Age agrarian intensification and the earliest AMS dates of Lens culinaris and Vicia faba

Vegetation History and Archaeobotany - This article explores the Late Bronze Age agrarian intensification in the south-east Baltic. In recent years several studies have illustrated that to date...     

The influence of sodium dodecyl sulfate from different sources on the separation of virus proteins in polyacrylamide gel electrophoresis

Sodium dodecyl sulfate (SDS) preparations from different sources were investigated with regard to their effect on the separation of the proteins of foot and mouth disease virus (FMDV) types 0₁K, A₂S and C-Obb as well as of Sindbis virus during gel electrophoresis in continuous and discontinuous buffer systems in the presence of 8 m urea. In the continuous system, the different SDS preparations did not markedly alter the separations of any of the virus proteins, but the distances between the FMDV protein bands were small. In the discontinuous system, the source of SDS and the kind of virus protein strongly affected the separations obtained. In this system, adequate resolutions were obtained only with A₂S and C-Obb proteins in the presence of MCB or Pierce SDS preparations. An appreciable percentage of the carbon chains in these SDS preparations were found to be longer than C₁₂. The separations of Sindbis virus proteins which occurred in the continuous buffer PAGE system using any of the SDS preparations were preferable to those which were obtained in the discontinuous system.     

Sustaining recovered grasslands is not likely without proper management: vegetation changes after cessation of mowing

Grasslands recovered by sowing low diversity seed mixtures of local provenance are usually managed by mowing. Besides restoration success only a few studies have focused on the direct effects of post-restoration mowing on recovered grassland vegetation. In this study we followed vegetation changes in 13 successfully recovered grasslands in 5 × 5-m-sized sites with continuous and ceased mowing at Hortobágy National Park, East-Hungary. We asked the following questions: (i) What are the effects of cessation of mowing on the vegetation structure and diversity of recovered grasslands? (ii) What are the effects of cessation of mowing on the abundance of sown grasses, target and undesirable species? (iii) Is yearly mowing an appropriate management tool for the maintenance of recovered grasslands? Our results showed that the cessation of mowing caused litter accumulation, while diversity, total vegetation cover and the cover of sown grasses decreased compared to the mown sites. The cover of undesirable perennial species was significantly higher in unmown sites than in mown ones. The species composition of mown sites remained more similar to near-natural grasslands than the unmown ones. Our results suggest that without regular post-restoration mowing the favourable status of recovered grasslands can rapidly decline due to litter accumulation and by the expansion of undesirable species, even in the short-run. We also stress that while yearly mowing is enough to maintain grasslands recovered by low-diversity seed sowing, it cannot be considered to be enough to recover target vegetation composition.     

Tetrapeptide-amide analogues of enkephalin: the role of C-terminus in determining the character of opioid activity

The opioid activities of tetrapeptide-amide analogues of enkephalin /H-Tyr-D-Met-Gly-Phe-NH₂; H-Tyr-D-Nle-Gly-Phe-NH₂/ were studied in isolated, electrically stimulated longitudinal muscle strip of guinea-pig ileum and mouse vas deferens preparations in vitro and in vivo in the rat tail-flick test. Their effects were compared to those of the parent L-Pro⁵-NH₂ containing analogues, and to other enkephalin derivatives substituted with D-Met in position 2 and L-amino/imino acids of different character in position 5. It was found that whilst the opioid receptor in mouse vas deferens preferred aliphatic residues of acidic character at the C-terminus of pentapeptides and was highly sensitive to the removal of C-terminal amino acid, the other systems were either much less responsive to these changes, or the effects were opposite to those found in mouse vas deferens.     

Attenuation of morphine tolerance and dependence by alpha-melanocyte stimulating hormone(alpha-MSH).

Repeated administration of morphine resulted in significant reduction of its analgesic potency. If 0.1 mg/kg α-MSH was coadministered, the tolerance development was attenuated, 1 mg/kg MIF (MSH release inhibiting factor), given simultaneously with morphine, did not affect tolerance. Injecting, however, MIF 1 hour prior to the daily opiate treatment resulted in accelerated development of tolerance supposedly by lowering the plasma α-MSH level at the time of morphine administration. Of the morphine abstinence symptoms the naloxone-induced jumping in morphine pretreated mice could not be modified either by α-MSH coadministration or by MIF pretreament, but the withdrawal body weight loss was found to be diminished by the former and increased by the latter peptide. The possible role of α-MSH in preventing the development of tolerance to the analgesic effect of endogenous opioid peptides is discussed.     

Benzenesulfonamides with pyrimidine moiety as inhibitors of human carbonic anhydrases I,II, VI,VII, XII,and XIII

Two groups of benzenesulfonamide derivatives, bearing pyrimidine moieties, were designed and synthesized as inhibitors of carbonic anhydrases (CA). Their binding affinities to six recombinant human CA isoforms I, II, VI, VII, XII, and XIII were determined by the thermal shift assay (TSA). The binding of several inhibitors was measured by isothermal titration calorimetry (ITC). Direct demonstration of compound inhibition was achieved by determining the inhibition constant by stopped-flow CO₂ hydration assay. The most potent compounds demonstrated selectivity towards isoform I and affinities of 0.5 nM. The crystal structures of selected compounds in complex with CA II, XII, and XIII were determined to atomic resolution. Compounds described here were compared with previously published pyrimidinebenzenesulfonamides.¹ Systematic structure–activity analysis of 40 compound interactions with six isoforms yields clues for the design of compounds with greater affinities and selectivities towards target CA isoforms.