Exploring Guanidinium Group Involvement in Hordatine Interactions with the G-Quadruplex Motif Within the c-MYC Promoter Region

Abstract

G-quadruplexes (G4s) are four-stranded DNA or RNA structures formed by guanine-rich sequences. They occur in functional regions of the genomic material, including the promoter part of genes, regulatory region, and telomeric threads. G4s play a key role in various biological processes, including transcription, replication, and telomere maintenance. Guanidine-containing derivatives can bind to G-quadruplexes, either by intercalating into the structure or by interacting with the grooves or loops. The binding can stabilize the G-quadruplex, potentially affecting its biological function. In this paper, the ability of guanidinium-containing hordatines to interact with G4 was evaluated. Analogues lacking the guanidinium group or showing the benzofuran system instead of the dihydrobenzofuran core were prepared and tested as well. NMR titration and docking calculations were used to probe the binding of the compounds to G4 of c-MYC oncogene. Spectroscopic analyses were consistent with a significant interaction of benzofurans 3 and 4 at the 5'-end and 3'-end tetrads and with the formation of ligand/G-quadruplex complexes with a 2:1 stoichiometry. The resulting data were supported by docking simulations. Cytotoxic activity was evaluated on a model of U2OS osteosarcoma (ATCC HTB-96) and breast cancer (MDA-MB-231) cell lines, further highlighting the key role of the guanidinium fragment and the benzofuran core in the G-quadruplex stabilization.

Keywords: G-quadruplex; NMR spectroscopy; cytotoxic activity; docking; guanidinium; hordatine.

Figure 1

Structure of compounds 1–4.

Scheme 1

Synthesis of acids 7 and 9.

Scheme 2

Synthesis of compounds 1–4.

Figure 2

Imino proton region of the 1D NMR titration spectra of Pu22T14T23 with 4 (top) and with 3 (bottom) at 25 °C in H₂O/D₂O (9:1), 25 mM KH₂PO₄, 70 mM KCl, pH 6.9, at different R = [ligand]/[DNA] ratios.

Figure 3

Imino and aromatic protons region of the 2D-NOESY spectrum at 25 °C in H₂O/D₂O (9:1), 25 mM KH₂PO₄, 70 mM KCl, pH 6.9, at R = [3]/[DNA] = 2.0. The boxes and the blue dashed lines show the NOE interactions between imino protons of Pu22T14T23 and aromatic protons and OCH₃ of compound 3. The red line indicates the intramolecular NOE interactions between aromatic protons of 3.

Figure 4

Lateral view (left) and docking site at 3′-end of 3 complexed with the Pu22T14T23. The nucleotides are shown as slabs and filled sugars: adenine in red, cytosine in yellow, guanine in green, and thymine in blue. The Pu22T14T23 is represented in CPK, the binding site is contoured by the solid solvent accessible surface (SAS), and the ligand is in CPK (right).

Figure 5

Lateral view (left) and docking site at 5′-end of 3 complexed with the Pu22T14T23 (right). The nucleotides are shown as slabs and filled sugars: adenine in red, cytosine in yellow, guanine in green, and thymine in blue. The Pu22T14T23 is represented in CPK, the binding site is contoured by the solid SAS (solvent accessible surface), and the ligand is in CPK.

Figure 6

Two-dimensional representation of the molecular interactions between 3 and the Pu22T14T23. On the left 3′-end and on the right 5′-end binging site. Here, the colors used refer to the different interactions, as indicated in the legend.

Figure 7

Side views of the 4 complex with the c-MYC G-quadruplex sequence at the 5′-end (a) and 3′-end (b). The aromatic moiety of the ligand is located near the center of the quadruplex, while the side chain fits into the groove between G17 and G20. The nucleotides are shown as follows: adenine in red, guanine in green, and thymine in blue.

Figure 8

Docking sites of 4 on the left 5′-end and on the right 3′-end: the Pu22T14T23 is represented in CPK, the binding site is contoured by the solid SAS (solvent accessible surface), and the ligand is in CPK. The nucleotides are shown as follows: adenine in red, guanine in green, and thymine in blue.

Figure 9

Two-dimensional representation of the molecular interactions between 4 and the Pu22T14T23, on the left 5′-end and on the right 3′-end binging site. Here, the colors used refer to the different interactions, as indicated in the legend.