The solution structures of d(GAAAACGTTTTC) 2 and of its methylated derivative d(GAAAAMe 5 CGTTTTC) 2 have been determined by NMR and molecular modelling in order to examine the impact of cytosine methylation on the central CpG conformation. Detailed 1 H NMR and 31 P NMR investigation of the two oligomers includes quantitative NOESY, 2D homonuclear Hartmann±Hahn spectroscopy, double-quantum-filtered COSY and heteronuclear 1 H-31 P correlation. Back-calculations of NOESY spectra and simulations of double-quantumfiltered COSY patterns were performed to gain accurate information on interproton distances and sugar phase angles. Molecular models under experimental constraints were generated by energy minimization by means of the molecular mechanics program jumna. The morass software was used to iteratively refine the structures obtained. After methylation, the oligomer still has a B-DNA conformation. However, there are differences in the structural parameters and the thermal stability as compared to the unmethylated molecule. Careful structural analysis shows that after methylation CpG departs from the usual conformation observed in other ACGT tetramers with different surroundings. Subtle displacements of bases, sugars and backbone imposed by the steric interaction of the two methyl groups inside the major groove are accompanied by severe pinching of the minor groove at the C-G residues.Keywords: DNA; 2D NMR; modelling; cytosine methylation; CpG; adenine tract.DNA methylation at cytosines in CpG steps is recognized as an important event in biological processes that control gene expression. Methylation turns off the transcription of genes involved in mammal development [1] and genomic imprinting [2], and abolishes the immune response induced by oligonucleotide motifs containing a CpG step flanked by two purines and two pyrimidines in the 5 H and 3 H directions, respectively [3]. The hypothesis that gene silencing stems from blocking the binding of transcription factors at methylated CpG has led to several investigations designed to unveil possible structural singularities of the methylated sequence as compared to the unmodified one. Crystallographic data show that cytosine methylation in the CMe 5 CTG oligomer only induces a slight structural alteration [4]. NMR analysis has underlined the marked conformational difference between the octamers d(CATCGATG) 2 and d(CTTCGAAG) 2 , despite the same central four-base sequence, and their specific structural response to the methylation of the central cytosine [5]. The 1 H NMR results show that cytosine methylation induces more significant structural effects in the first oligomer than in the other. The conclusions reached by 1 H NMR have been corroborated by 1 H-31 P coupling constant and 31 P chemical shift investigation. These studies have shown that the CpG backbone is more sensitive to methylation in d(CATCGATG) 2 than in the second octamer. The observation that the conformation of the central tetranucleotide is strongly affected by its 5 H and 3 H environment is in line wit...
Comparative molecular field analysis (CoM-FA) and comparative molecular similarity indices analysis (CoMSIA) based on three-dimensional quantitative structure-activity relationship (3D-QSAR) studies were conducted on a series (39 molecules) of peptidyl vinyl sulfone derivatives as potential Plasmodium Falciparum cysteine proteases inhibitors. Two different methods of alignment were employed: (i) a receptor-docked alignment derived from the structure-based docking algorithm GOLD and (ii) a ligand-based alignment using the structure of one of the ligands derived from a crystal structure from the PDB databank. The best predictions were obtained for the receptor-docked alignment with a CoMFA standard model (q 2 = 0.696 and r 2 = 0.980) and with CoMSIA combined electrostatic, and hydrophobic fields (q 2 = 0.711 and r 2 = 0.992). Both models were validated by a test set of nine compounds and gave satisfactory predictive r 2 pred values of 0.76 and 0.74, respectively. CoMFA and CoM-SIA contour maps were used to identify critical regions where any change in the steric, electrostatic, and hydrophobic fields may affect the inhibitory activity, and to highlight the key structural features required for biological activity. Moreover, the results obtained from 3D-QSAR analyses were superimposed on the Plasmodium Falciparum cysteine proteases active site and the main interactions were studied. The present work provides extremely useful guidelines for future structural modifications of this class of compounds towards the development of superior antimalarials.
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