Katalin E. Kövér

Functional aspects of the solution structure and dynamics of PAF – a highly‐stable antifungal protein from Penicillium chrysogenum

Penicillium antifungal protein (PAF) is a promising antimycotic without toxic effects on mammalian cells and therefore may represent a drug candidate against the often lethal Aspergillus infections that occur in humans. The pathogenesis of PAF on sensitive fungi involves G‐protein coupled signalling followed by apoptosis. In the present study, the solution structure of this small, cationic, antifungal protein from Penicillium chrysogenum is determined by NMR. We demonstrate that PAF belongs to the structural classification of proteins fold class of its closest homologue antifungal protein from Aspergillus giganteus. PAF comprises five β‐strands forming two orthogonally packed β‐sheets that share a common interface. The ambiguity in the assignment of two disulfide bonds out of three was investigated by NMR dynamics, together with restrained molecular dynamics calculations. The clue could not be resolved: the two ensembles with different disulfide patterns and the one with no S–S bond exhibit essentially the same fold. 15N relaxation dispersion and interference experiments did not reveal disulfide bond rearrangements via slow exchange. The measured order parameters and the 3.0 ns correlation time are appropriate for a compact monomeric protein of this size. Using site‐directed mutagenesis, we demonstrate that the highly‐conserved and positively‐charged lysine‐rich surface region enhances the toxicity of PAF. However, the binding capability of the oligosaccharide/oligonucleotide binding fold is reduced in PAF compared to antifungal protein as a result of less solvent‐exposed aromatic regions, thus explaining the absence of chitobiose binding. The present study lends further support to the understanding of the documented substantial differences between the mode of action of two highly homologous antifungal proteins.

Gradient enhanced selective experiments in the 1H NMR chemical shift assignment of the skeleton and side-chain resonances of stigmasterol, a phytosterol derivative.

The applicability of homonuclear gradient enhanced NMR experiments is demonstrated in the structure determination of steroid derivatives using stigmasterol as a model compound. High resolution 1H NMR spectra of steroids very often display well resolved multiplets usually in the low-field region, and these signals can be used as starting points in several selective NMR experiments to study scalar (J coupling), and dipolar (NOE) interactions. Selective excitation was carried out using a double pulsed-field gradient spin-echo sequence (DPFGSE) in which 180 degrees Gaussian pulses are sandwiched between sine shaped z-gradients. Scalar interactions were studied by homonuclear DPFGSE-COSY, DPFGSE-relay-COSY and DPFGSE-TOCSY experiments, while DPFGSE-NOESY was used to monitor spatial environment of the selectively excited proton. These methods provided unambiguous assignments for signals of the main skeleton and the side-chain of the steroid molecule. In addition, they allowed determination of the conformationally important homonuclear proton-proton coupling constants (J).

Accurate determination of small one-bond heteronuclear residual dipolar couplings by F1 coupled HSQC modified with a G-BIRD(r) module.

We report a G-BIRD(r) modified coupled HSQC experiment for the accurate determination of one-bond heteronuclear residual dipolar couplings. The G-BIRD(r) module has been employed to refocus the long-range coupling evolution of the heteronucleus during the t1 frequency labeling period. As a result, the crosspeaks obtained are split by only the direct one-bond coupling that can be extracted by measuring simple frequency differences between singlet maxima. Additionally the decoupling of long-range multiple bond splittings leads to considerable sensitivity enhancement. The modification also has been applied in a TROSY sequence resulting in a significant sensitivity and resolution improvement.

Accurate determination of one-bond heteronuclear coupling constants with "pure shift" broadband proton-decoupled CLIP/CLAP-HSQC experiments.

We report broadband proton-decoupled CLIP/CLAP-HSQC experiments for the accurate determination of one-bond heteronuclear couplings and, by extension, for the reliable measurement of small residual dipolar coupling constants. The combination of an isotope-selective BIRD((d)) filter module with a non-selective (1)H inversion pulse is employed to refocus proton-proton coupling evolution prior to the acquisition of brief chunks of free induction decay that are subsequently assembled to reconstruct the fully-decoupled signal evolution. As a result, the cross-peaks obtained are split only by the heteronuclear one-bond coupling along the F2 dimension, allowing coupling constants to be extracted by measuring simple frequency differences between singlet maxima. The proton decoupling scheme presented has also been utilized in standard HSQC experiments, resulting in a fully-decoupled pure shift correlation map with significantly improved resolution.

New endomorphin analogues containing alicyclic β-amino acids: Influence on bioactive conformation and pharmacological profile

Endomorphins were subjected to a number of structural modifications in a search for their bioactive conformations. The alicyclic beta-amino acids cis-(1 S,2 R)ACPC/ACHC, cis-(1 R,2 S)ACPC/ACHC, trans-(1 S,2 S)ACPC/ACHC, and trans-(1 R,2 R)ACPC/ACHC were introduced into endomorphins to examine the conformational effects on the bioactivity. Use of a combination of receptor binding techniques, (1)H NMR, and molecular modeling allowed the conclusion that Pro (2) substitution by these residues causes changes in structure, proteolytic stability, and pharmacological activity. It seems that the size of the alicyclic beta-amino acids does not have marked influence on the receptor binding affinities and/or selectivities. Among the new analogues, the cis-(1 S,2 R)ACPC (2) and cis-(1 S,2 R)ACHC (2)-containing derivatives displayed the highest binding potencies and efficacies in receptor binding and ligand-stimulated [ (35)S]GTPgammaS functional experiments. Molecular dynamic simulations and (1)H NMR studies of the cis-ACPC/ACHC-containing analogues revealed that many conformations are accessible, though it is most likely that these peptides bind to the mu-opioid receptor in a compact, folded structure rather than extended.

Comparative analysis of binary and ternary cyclodextrin complexes with econazole nitrate in solution and in solid state

The aim of this work was to investigate in-depth interactions of econazole nitrate (ECN), a very poorly water-soluble antifungal agent, with different β-cyclodextrin (βCD) derivatives, and to evaluate the potential synergistic effect of suitable third compounds (l-amino acids, citric acid, hydrophilic polymers). Phase-solubility studies showed the formation of equimolar complexes with all tested CDs, and indicated sulfobutyl-βCD (SBEβCD) as the best complexing and solubilizing agent for ECN, followed by hydroxypropyl-βCD (HPβCD). 1D and 2D (1)H NMR studies demonstrated the actual formation of inclusion complexes of 1:1mol:mol stoichiometry, and gave insight about different inclusion modes of ECN molecule into the CD cavity, simultaneously existing in solution. Among the different tested ternary systems, only those with citric acid (CA) enabled a significant increase in complexing and solubilizing ability towards the drug with respect to the binary ones, indicating a synergistic effect between SBEβCD and CA and the formation of highly soluble ternary complexes, which was further supported by NMR studies. Solid equimolar binary and ternary systems of ECN, CDs and CA were prepared by co-grinding in high energy vibrational micro-mills and characterized by differential scanning calorimetry, X-ray powder diffractometry and in vitro dissolution studies. In the case of binary systems, total sample amorphization, indicative of strong solid state interactions and possible inclusion complex formation, was obtained only for co-ground products with HPβCD and SBEβCD, but they both presented a dissolution profile typical of a supersaturated system, with a limited improvement of drug dissolution efficiency (8.3 and 22.13 times, respectively). On the contrary, the ternary ECN/SBEβCD/CA co-ground product presented superior dissolution properties, increasing the ECN dissolution efficiency of 66.62 times, clearly having the best potential for further development of a novel ECN delivery system for efficient delivery of the drug to the oral cavity, thus improving the therapy of oral candidosis.

Heteronuclear coupling constants of hydroxyl protons in a water solution of oligosaccharides: trehalose and sucrose

Abstract Relatively few details are known about the conformational preferences of hydroxyl groups in carbohydrates in water solution, though these would be informative about solvation and H-bonding. We show that highly concentrated solutions of sucrose and trehalose exhibit surprisingly well-resolved 1H NMR spectra in a deuterium oxide–water solvent mixture at subzero temperatures. Measurement conditions are suitable to extract nearly all homonuclear and, for the first time, heteronuclear coupling constants of OH groups of carbohydrates in their natural abundance. For 2,3JHO,C coupling constants new, powerful variants of HETLOC and HECADE techniques were applied. The present data do not support the presence of persistent H-bonds in these two cryogenic disaccharides.

Experimental and computational studies of nJ(77Se,1H) selenium–proton couplings in selenoglycosides

Selenoglycosides are important starting materials in synthetic carbohydrate chemistry and play a role in biological interactions as well. Both aspects are influenced by the conformation around the glycosidic bond. Here, we present a combined experimental and computational approach to measure and evaluate nJ(77Se,1H) coupling constants for their use in conformational analysis. The measurements were carried out using a modified CPMG‐HSQMBC pulse scheme which yields pure absorption antiphase multiplets to allow accurate determination of the nJXH values regardless of the size of the proton‐proton couplings. Theoretical calculations were performed at the Second‐Order Polarization Propagator Approach (SOPPA) level. Population‐averaged values calculated for geminal and vicinal couplings are in a good agreement with experiment indicating an adequate theoretical level of the calculations. Experimental observations and computations alike have indicated that two‐bond 77Se‐1H couplings, 2J(77Se,1H), in a H1‐C1‐Se‐X moiety are very sensitive to the torsion angle around the C1‐Se‐bond and will, therefore, be useful for conformational studies. Copyright

Chiroptical properties and solution conformations of protected endothiodipeptide esters

Abstract The syntheses and CD spectra in different solvents are described for four series of N-benzyloxycarbonyl-endothiodipeptide esters of types a - d with Ala, Val, Phe and Pro residues. Difference 1 H NOE and IR measurements were also performed on selected models. The spectroscopic evidence pointed to the existence of two main conformers in solution. Conformation A, predominating in nonpolar solvents, can be characterized as a folded C 7 C 5 form with a bifurcated system of intramolecular H-bonds formed by the thioamide N-H and the CO groups of the N-terminal urethane and the C-terminal ester moieties. Conformation B, the major conformation in H-bond-accepting solvents such as DMSO, is more extended and flexible in the N-terminal region and assumes a “proline-like” rotamer state (φ 2 ~ -60°) at the C-terminus. The sign and magnitude of the Cotton effect due to the n →π * transition (near 350 nm) can be rationalized with an octant-like sector rule. The optical activity of the π→π * transition is determined in part by exciton interaction of the thioamide with the urethane and/or ester chromophores. Through application of the sector rule, finer details of the correlation between the chiroptical properties and the conformation can be explained.

Epigallocatechin‐3‐gallate and penta‐O‐galloyl‐β‐d‐glucose inhibit protein phosphatase‐1

Protein phosphatase‐1 (PP1) and protein phosphatase‐2A (PP2A) are responsible for the dephosphorylation of the majority of phosphoserine/threonine residues in cells. In this study, we show that (–)‐epigallocatechin‐3‐gallate (EGCG) and 1,2,3,4,6‐penta‐O‐galloyl‐β‐d‐glucose (PGG), polyphenolic constituents of green tea and tannins, inhibit the activity of the PP1 recombinant δ‐isoform of the PP1 catalytic subunit and the native PP1 catalytic subunit (PP1c) with IC50 values of 0.47–1.35 μm and 0.26–0.4 μm, respectively. EGCG and PGG inhibit PP2Ac less potently, with IC50 values of 15 and 6.6 μm, respectively. The structure–inhibitory potency relationships of catechin derivatives suggests that the galloyl group may play a major role in phosphatase inhibition. The interaction of EGCG and PGG with PP1c was characterized by NMR and surface plasmon resonance‐based binding techniques. Competitive binding assays and molecular modeling suggest that EGCG docks at the hydrophobic groove close to the catalytic center of PP1c, partially overlapping with the binding surface of microcystin‐LR or okadaic acid. This hydrophobic interaction is further stabilized by hydrogen bonding via hydroxyl/oxo groups of EGCG to PP1c residues. Comparative docking shows that EGCG binds to PP2Ac in a similar manner, but in a distinct pose. Long‐term treatment (24 h) with these compounds and other catechins suppresses the viability of HeLa cells with a relative effectiveness reminiscent of their in vitro PP1c‐inhibitory potencies. The above data imply that the phosphatase‐inhibitory features of these polyphenols may be implicated in the wide spectrum of their physiological influence.