Síntesis y estudios de interacción de compuestos con capacidad de unión al ADN cuádruple-G

Abstract

One of the main goals of this doctoral thesis has been the synthesis and structural characterization of compounds capable of recognizing and stabilizing G-quadruplex DNA secondary structures. In particular, we have focused our work on the human telomeric DNA sequence, with the purpose of setting a basis for the discovery of novel anticancer compounds. Moreover, other of the main objectives has been the realization of a series of experiments aimed at studying the nature of G-quadruplex DNA/ligand interactions and at preliminary assessing the biological in vitro activity of the synthesized compounds, as well as of other compounds previously synthesized by other members of our research group. First, different series of ligands based on the 2,2'-bipyridine, 1,10- phenanthroline and diquaternized 1,10-phenanthroline cores (figure 1) were synthesized. The 2,2'-bipyridine ligands were disubstituted with various side chains at the 4,4' positions (figure 1, a), while in the case of the 1,10- phenanthroline system, the heterocycle was disubstituted at 4 and 7 positions (figure 1, a). All side chains (R) contained as a terminal group either a guanidinium group or an aliphatic heterocycle, such as a morpholine, piperidine, piperazine or N-methylpiperazine. The length of the linker between the aromatic scaffold and the terminal group varied from 3 to 8 atoms. Afterwards, some metal complexes of Cu(II) or Ni(II) with 2,2'-bipyridine or 1,10-phenanthroline ligands (figure 1, b) were prepared and characterized by IR, UV-visible and high resolution mass spectrometry. After that, DNA interaction and biological activity studies were held on the synthesized compounds as well as on other metal complexes previously synthesized in the laboratory. In all experiments, the interactions between the tested compounds and telomeric G-quadruplex DNA, with its general sequence (TTAGGG)n, were studied, both under potassium or sodium-rich conditions. Different biophysical techniques were employed, such as a DNA melting assay monitored by fluorescence resonance energy transfer (FRET), circular dichroism, and fluorescence DNA probe displacement assay (FID assay). Competitive experiments were also held to determine the selectivity of the ligands in their binding to G- quadruplex structures versus duplex DNA. For this kind of experiments, techniques such as competitive equilibrium dialysis or the FRET melting assay in the presence of a competitor oligonucleotide were employed. In addition, a complementary technique was utilized to establish the binding modes between several ligands and duplex DNA, as an attempt to gain an insight into the general recognition mode of nucleic acids by these compounds. The viscometric measurements were used to differentiate whether the interaction between the ligand and DNA occurred by intercalation, by groove binding to the double helix or by mixed binding mode (intercalation/groove binding). Finally, during a three-month stay in the research group of Prof. Stephen Neidle (Biomedical Structure Group, School of Pharmacy, University College London) some biological activity assays were performed to preliminary assess the potential as anticancer agents of a small selection of ligands. On the one hand, the TRAP-LIG assay (Telomere Repeat Amplification Protocol without ligand) was used to test for in vitro inhibition of human telomerase. Simultaneously, a cytotoxicity test on several cancer cell lines was carried out by using the Sulforhodamine B short term cytotoxicity test. Both techniques were used to determine IC50 values, the concentrations required for the 50% inhibition of telomerase activity (TRAP-LIG) or 50% inhibition of cell growth, in comparison with a control sample.