Elie Lescot

Self-association and Domains of Interactions of an Amphipathic Helix Peptide Inhibitor of HIV-1 Integrase Assessed by Analytical Ultracentrifugation and NMR Experiments in Trifluoroethanol/H2O Mixtures

EAA26 (VESMNEELKKIIAQVRAQAEHLKTAY) is a better inhibitor of human immunodeficiency virus, type 1, integrase than its parent Lys-159, reproducing the enzyme segment 147–175 with a nonpolar-polar/charged residue periodicity defined by four helical heptads (abcdefg) prone to collapse into a coiled-coil. Circular dichroism, nuclear magnetic resonance, sedimentation equilibrium, and chemical cross-linking were used to analyze EAA26 in various trifluoroethanol/H2O mixtures. In pure water the helix content is weak but increases regularly up to 50–60% trifluoroethanol. In contrast the multimerization follows a bell-shaped curve with monomers in pure water, tetramers at 10% trifluoroethanol, and dimers at 40% trifluoroethanol. All suggest that interhelical interactions between apolar side chains are required for the coiled-coil formation of EAA26 and subsist at medium trifluoroethanol concentration. The NH temperature coefficients measured by nuclear magnetic resonance show that at low trifluoroethanol concentration the amide groups buried in the hydrophobic interior of four α-helix bundles are weakly accessible to trifluoroethanol and are only weakly subject to its hydrogen bond strengthening effect. The increased accessibility of trifluoroethanol to buried amide groups at higher trifluoroethanol concentration entails the reduction of the hydrophobic interactions and the conversion of helix tetramers into helix dimers, the latter displaying a smaller hydrophobic interface. The better inhibitory activity of EAA26 compared with Lys-159 could arise from its better propensity to form a helix bundle structure with the biologically important helical part of the 147–175 segment in integrase.

SEQUENCE DEPENDENT EFFECTS OF CPG CYTOSINE METHYLATION : A JOINT 1H-NMR AND 31P-NMR STUDY

The impact of cytosine methylation in the central CpG step of two closely related octanucleotide duplexes d(CATCGATG)2 and d(CTTCGAAG)2 was examined by 1H-NMR and 31P-NMR experiments, and a quantitative structural analysis was performed using the NOE-derived distances, the sugar puckers and the epsilon torsion angles. The two starting oligonucleotides displayed a B-DNA conformation with, however, significant local structure differences. Although the methylated oligonucleotides retained their B-DNA conformation, different structural and thermal stability effects were observed. The magnitude of the methylation effects was to depend on the initial conformation of the CpG site, which is governed by the nature of the dinucleotide AT or TT located on the CpG flanks. As an example of sequence dependence, the methylation of CpG entailed larger conformational variation in d(CATCGATG)2 than in d(CTTCGAAG)2. In this study, the 1H and 31P chemical-shift parameters averred as extremely sensitive probes for detecting subtle conformational changes. Finally, our comparative results may aid our understanding of the structural and related biological effects produced by cytosine methylation in DNA.

A CD study of interactions of ellipticine derivatives with DNA : relations with the in vitro cytotoxicity

UV‐absorption and circular dichroism (CD) experiments showed that ellipticine derivatives may interact with DNA according to 3 possible binding modes depending on their structure and concentration. The first mode concerned intercalation of 1‐methyl‐9‐hydroxyellipticine (1‐Me‐HE) with its long axis perpendicular to the long axis of base pairs. The same drug was able to bind to external sites (second mode) once the intercalation sites were saturated at high concentration. The third mode illustrated by 1,2‐dimethyl‐9‐hydroxyisoellipticinium (1‐Me‐isoNMHE), concerned self‐stacked molecules interacting at the surface of DNA. Biological significance of these different binding modes was then discussed in connection with in vitro cytotoxic activity of compounds.

DNA binding, cytotoxicity and inhibitory effect on RNA synthesis of two new 1-nitro-9-aminoacridine dimers.

Two 1-nitro-9-aminoacridine dimers were prepared: one bearing a spermine flexible linking chain, compound 4, the other a rigid dipiperidine-type linker, compound 7. Both dimers elicited a higher affinity constant for DNA than the parent monomeric drug nitracrine 2. This affinity was several orders lower than what was found for other dimeric compounds having the same linkers and no nitro group on the acridine ring (3, 5, 6 and 8). Bisintercalation was evidenced for compound 4 by viscosimetric measurements. In the absence of dithiothreitol, an inhibitory effect of RNA synthesis in vitro was observed for all the tested compounds except 2 and 7. In the presence of dithiothreitol, 4 and 7 formed irreversible complexes with DNA of decreased template properties. The level of the dimers binding was lower than that of the parent compound 2. Cross-links were detected by means of hydroxylapatite chromatography in a complex of the dimer bearing a flexible linking chain, compound 4 with DNA, while the compound 7-DNA complex eluted in the single-stranded DNA region. The extent of cytotoxicity of the two 1-nitro-9-aminoacridine dimers against L1210 cultured cells was different.

31p NMR of a hairpin structure within a 19-Mer DNA

The 31P resonances corresponding to the eighteen phosphate groups of a 19-mer DNA hairpin structure, belonging to a major cleavable site for topoisomerase II were unequivocally assigned and unsual 31P chemical shifts corresponding to those phosphate groups located in the loop were noted.