Gyula Batta

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.

Preparation of acetylated C-(1-bromo-d-glycosyl) heterocycles and 1-bromo-d-glycosyl cyanides☆

Abstract The reaction of acetylated C -( d -glycosyl) heterocycles and d -glycosyl cyanides with either N -bromosuccinimide in hot carbon tetrachloride or bromine under irradiation resulted in bromination at the anomeric carbon atom. The location of the bromine substituent and the conformations of these products were determined by n.m.r. spectroscopy. Absolute configurations of the bromo compounds were established.

Anti-influenza virus activity and structure-activity relationship of aglycoristocetin derivatives with cyclobutenedione carrying hydrophobic chains

Abstract Previous studies have demonstrated that glycopeptide compounds carrying hydrophobic substituents can have favorable pharmacological (i.e. antibacterial and antiviral) properties. We here report on the in vitro anti-influenza virus activity of aglycoristocetin derivatives containing hydrophobic side chain-substituted cyclobutenedione. The lead compound 8e displayed an antivirally effective concentration of 0.4μM, which was consistent amongst influenza A/H1N1, A/H3N2 and B viruses, and a selectivity index ≥50. Structural analogues derived from aglycovancomycin were found to be inactive. The hydrophobic side chain was shown to be an important determinant of activity. The narrow structure–activity relationship and broad activity against several human influenza viruses suggest a highly conserved interaction site, which is presumably related to the influenza virus entry process. Compound 8e proved to be inactive against several unrelated RNA and DNA viruses, except for varicella-zoster virus, against which a favorable activity was noted.

NMR Studies on Structure and Dynamics of the Monomeric Derivative of BS-RNase: New Insights for 3D Domain Swapping

Three-dimensional domain swapping is a common phenomenon in pancreatic-like ribonucleases. In the aggregated state, these proteins acquire new biological functions, including selective cytotoxicity against tumour cells. RNase A is able to dislocate both N- and C-termini, but usually this process requires denaturing conditions. In contrast, bovine seminal ribonuclease (BS-RNase), which is a homo-dimeric protein sharing 80% of sequence identity with RNase A, occurs natively as a mixture of swapped and unswapped isoforms. The presence of two disulfides bridging the subunits, indeed, ensures a dimeric structure also to the unswapped molecule. In vitro, the two BS-RNase isoforms interconvert under physiological conditions. Since the tendency to swap is often related to the instability of the monomeric proteins, in these paper we have analysed in detail the stability in solution of the monomeric derivative of BS-RNase (mBS) by a combination of NMR studies and Molecular Dynamics Simulations. The refinement of NMR structure and relaxation data indicate a close similarity with RNase A, without any evidence of aggregation or partial opening. The high compactness of mBS structure is confirmed also by H/D exchange, urea denaturation, and TEMPOL mapping of the protein surface. The present extensive structural and dynamic investigation of (monomeric) mBS did not show any experimental evidence that could explain the known differences in swapping between BS-RNase and RNase A. Hence, we conclude that the swapping in BS-RNase must be influenced by the distinct features of the dimers, suggesting a prominent role for the interchain disulfide bridges.

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.

Glucose-based spiro-heterocycles as potent inhibitors of glycogen phosphorylase.

Glucopyranosylidene-spiro-1,4,2-oxathiazoles were prepared in high yields by NBS-mediated spiro-cyclization of the corresponding glucosyl-hydroximothioates. In an effort to synthesize analogous glucopyranosylidene-spiro-1,2,4-oxadiazolines, with a nitrogen atom instead of the sulphur, attempted cyclizations resulted in aromatization of the heterocycle with opening of the pyranosyl ring. Enzymatic measurements showed that some of the glucose-based inhibitors were active in the micromolar range. The 2-naphthyl-substituted 1,4,2-oxathiazole displayed the best inhibition against RMGPb (K(i)=160 nM), among glucose-based inhibitors known to date.

Calretinin and calbindin D28k have different domain organizations.

The domain organization of calretinin (CR) was predicted to involve all six EF‐hand motifs (labeled I to VI) condensed into a single domain, as characterized for calbindin D28k (Calb), the closest homolog of calretinin. Unperturbed 1H,15N HSQC NMR spectra of a 15N‐labeled calretinin fragment (CR III–VI, residues 100–271) in the presence of the unlabeled complimentary fragment (CR I–II, residues 1–100) show that these fragments do not interact. Size exclusion chromatography and affinity chromatography data support this conclusion. The HSQC spectrum of 15N‐labeled CR is similar to the overlaid spectra of individual 15N‐labeled CR fragments (CR I–II and CR III–VI), also suggesting that these regions do not interact within intact CR. In contrast to these observations, but in accordance with the Calb studies, we observed interactions between other CR fragments: CR I (1–60) with CR II–VI (61–271), and CR I–III (1–142) with CR IV–VI (145–271). We conclude that CR is formed from at least two independent domains consisting of CR I–II and CR III–VI. The differences in domain organization of Calb and CR may explain the specific target interaction of Calb with caspase‐3. Most importantly, the comparison of CR and Calb domain organizations questions the value of homologous modeling of EF‐hand proteins, and perhaps of other protein families.

Thiazole C-nucleosides. III, Synthesis of pyranose analogues of tiazofurin

Abstract A large-scale synthesis of 3,4,5-tri-0-acetyl-2,6-anhydro-L-mannono-and-D-gulonothioamides (5, 6) has been achieved from the corresponding nitriles. The Hantzsch reaction of (5) or (6) with ethyl bromopyruvate afforded the expected thiazoles (7.8) only in a low yield along with furan derivatives (9-11), the formation of which is rationalized by an acid-catalysed rearrangement-elimination process. The some Hantzsch reaction in the presence of barium carbonate yielded hydroxythiazolines (16,17). Attempted dehydration of (16) or (17) with trifluoroacetic anhydride or trifluoroacetic anhydride/pyridine resulted in the formation of pent-1′-enopyranosylthiazoles (18-20). Deprotected thioamides (24,25) furnished with ethyl bromopyruvate thiazoles (27,28). The obtained thiazole esters (7,8, 18-20. 27,28) were transformed into new tiazofurin analogues (12, 13, 21- 23).

3-Substituted xanthines as promising candidates for quadruplex formation: computational, synthetic and analytical studies

Our computational studies suggest that 3-substituted xanthines are good candidates for tetrad and quadruplex structures. 3-Methylxanthine (3MX) has been synthesized from 7-benzylxanthine, and the existence of tetrameric and octameric aggregates of 3MX with NH4+, Na+ and K+ ions in the gas phase (MS) and in DMSO-d6 solution (NMR) has been observed. The “internal” H-bonds (N1H⋯O6) are stronger than the “external” ones (N7H⋯O2) in these clusters (NMR).

The small molecular mass antifungal protein of Penicillium chrysogenum – a mechanism of action oriented review

The β‐lactam producing filamentous fungus Penicillium chrysogenum secretes a 6.25 kDa small molecular mass antifungal protein, PAF, which has a highly stable, compact 3D structure and is effective against a wide spectrum of plant and zoo pathogenic fungi. Its precise physiological functions and mode of action need to be elucidated before considering possible biomedical, agricultural or food technological applications. According to some more recent experimental data, PAF plays an important role in the fine‐tuning of conidiogenesis in Penicillium chrysogenum. PAF triggers apoptotic cell death in sensitive fungi, and cell death signaling may be transmitted through two‐component systems, heterotrimeric G protein coupled signal transduction and regulatory networks as well as via alteration of the Ca2+‐homeostasis of the cells. Possible biotechnological applications of PAF are also outlined in the review. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)