Stereoselectivity in β-cyclodextrin complexation of 1,4-dihydropyridine derivatives

Structure–interaction relationships, stereoselectivity, and solubility enhancement in inclusion compexation of β-cyclodextrins (CDs) with some racemic and enantiomerically pure 1,4-dihydropyridine derivatives (DHPs) were investigated. 1:1 and 1:2 (mole ratio) complexes were prepared and characterized by X-ray powder diffraction, differential scanning calorimetry (DSC), MS-FAB spectrometry, ¹H-NMR spectroscopy, water and phase solubility. The solubility studies have revealed different complexation equilibria for optically pure DHP enantiomers, and corresponding racemic mixtures in water solutions. By means of ¹H-NMR chemical shift measurements, the inclusion of aromatic fragments of racemic and enantiomerically pure DHP molecules within the cavities of different CDs was elucidated. Considerable stereoselectivity in complexation interactions was observed. The results indicate the potential use of cyclodextrins as chiral selectors for enantiomeric resolution of 1,4-DHP calcium antagonists. © 1993 Wiley-Liss, Inc.     

Interactions of Archaeal Chromatin Proteins Alba1 and Alba2 with Nucleic Acids

Background

Architectural proteins have important roles in compacting and organising chromosomal DNA. There are two potential histone counterpart peptide sequences (Alba1 and Alba2) in the Aeropyrum pernix genome (APE1832.1 and APE1823).

Methodology/Principal Findings

These two peptides were expressed and their interactions with various DNAs were studied using a combination of various experimental techniques: surface plasmon resonance, UV spectrophotometry, circular dichroism–spectropolarimetry, gel-shift assays, and isothermal titration calorimetry.

Conclusions/Significance

Our data indicate that there are significant differences in the properties of the Alba1 and Alba2 proteins. Both of these Alba proteins can thermally stabilise DNA polynucleotides, as seen from UV melting curves. Alba2 and equimolar mixtures of Alba1/Alba2 have greater effects on the thermal stability of poly(dA-dT).poly(dA-dT). Surface plasmon resonance sensorgrams for binding of Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 to DNA oligonucleotides show different binding patterns. Circular dichroism indicates that Alba2 has a less-ordered secondary structure than Alba1. The secondary structures of the Alba proteins are not significantly influenced by DNA binding, even at high temperatures. Based on these data, we conclude that Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 show different properties in their binding to various DNAs.     

NMR structure of 2′-O-(2-methoxyethyl) modified and C5-methylated RNA dodecamer duplex

The solution-state structure of 2′-O-(2-methoxyethly) substituted dodecamer r(*CG*CGAA*U*U*CG*C)d(G), 2′-MOE RNA, with all cytosines and uracils methylated at the C5-position has been determined by NMR spectroscopy. The chemical modifications were used to improve the oligonucleotide's drug-like properties. The 2′-MOE group drives pseudorotational equilibrium of the ribofuranose moiety to the N-type conformation and supposedly results in structural preorganization leading to high affinity of a modified oligonucleotide towards its complementary biological target, improved pharmacokinetic and toxicological properties. The high melting temperature of the antiparallel duplex structure adopted by 2′-MOE RNA was explained through the formation of a stable A-form RNA consistent with effective base-pairing and stacking interactions. The comparison of the solution-state structure with the crystal structure of a non-methylated analogue shows an increase in the stacking at the base pair steps for the C5-methylated 2′-MOE RNA duplex. The MOE substituents adopt a well-defined structure in the minor groove with the predominant gauche conformations around the ethylene bond.     

Cell-mediated immune response to high-passage Borrelia spirochetes in C57bl/6 mice is strictly dependent on antigen specificity

Inbred C57bl/6 mice were challenged with high-passage Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto and tested for antigen specific T-cell response in vitro. Sonicated preparations of washed spirochetes were potent cell activators, capable of stimulating polyclonal proliferation after 72h of culture while increasing the incubation time up to 120h provoked specific cell-mediated response. Isolated murine spleocytes previously sensitized to B. burgdorferi sensu lato but not those from control mice could be induced for antigen-specific proliferation in vitro, as revealed by [3H]thymidine incorporation assay, Moreover, in mice presensitized to B. burgdorferi sensu lato, detectable cell-mediated response could be induced only with antigen preparations derived from a corresponding strain but not with those obtained from other Borrelia genospecies. The current study emphasises that the B. burgdorferi antigen-specific response may also be expected in different genospecies infections in men.     

Structural and functional characterization of enantiomeric glutamic acid derivatives as potential transition state analogue inhibitors of MurD ligase

Mur ligases play an essential role in the intracellular biosynthesis of bacterial peptidoglycan, the main component of the bacterial cell wall, and represent attractive targets for the design of novel antibacterials. UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) catalyses the addition of D-glutamic acid to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA) and is the second in the series of Mur ligases. MurD ligase is highly stereospecific for its substrate, D-glutamic acid (D-Glu). Here, we report the high resolution crystal structures of MurD in complexes with two novel inhibitors designed to mimic the transition state of the reaction, which contain either the D-Glu or the L-Glu moiety. The binding modes of N-sulfonyl-D-Glu and N-sulfonyl-L-Glu derivatives were also characterised kinetically. The results of this study represent an excellent starting point for further development of novel inhibitors of this enzyme.     

In silico fragment-based discovery of indolin-2-one analogues as potent DNA gyrase inhibitors

We describe here the fragment-based design of potent DNA gyrase inhibitors. Using the tools of virtual screening and NMR spectroscopy we identified the binding of two low-molecular weight fragments (2-aminobenzimidazole and indolin-2-one) to the 24kDa N-terminal fragment of DNA gyrase B. Further in silico optimization of indolin-2-one led to the discovery of potent DNA gyrase inhibitors.     

Unlike Diablo/smac, Grim promotes global ubiquitination and specific degradation of X chromosome-linked inhibitor of apoptosis (XIAP) and neither cause apoptosis

Grim is a Drosophila inhibitor of apoptosis (IAP) antagonist that directly interferes with inhibition of caspases by IAPs. Expression of Grim, or removal of DIAP1, is sufficient to activate apoptosis in fly cells. Transient expression of Grim in mammalian cells induces apoptosis, arguing for the conservation of apoptotic pathways, but cytoplasmic expression of the mammalian IAP antagonist Diablo/smac does not. To understand why, we compared Grim and Diablo. Although they have the same IAP binding specificity, only Grim promoted XIAP ubiquitination and degradation. Grim also synergized with XIAP to promote an increase in total cellular ubiquitination, whereas Diablo antagonized this activity. Surprisingly, Grim-induced ubiquitination of XIAP did not require the IAP RING finger. Analysis of a Grim mutant that promoted XIAP degradation, but was not cytotoxic, suggests that Grim killing in transient assays is due to a combination of IAP depletion, blocking of IAP-mediated caspase inhibition, and at least one other unidentified function. Unlike transiently transfected cells, inducible mammalian cell lines can sustain continuous expression of Grim and selective degradation of XIAP without undergoing apoptosis, demonstrating that down-regulation and antagonism of IAPs is not sufficient to cause apoptosis of mammalian cells.