Exploring the Interaction of G-quadruplex Binders with a (3 + 1) Hybrid G-quadruplex Forming Sequence within the PARP1 Gene Promoter Region

Abstract

The enzyme PARP1 is an attractive target for cancer therapy, as it is involved in DNA repair processes. Several PARP1 inhibitors have been approved for clinical treatments. However, the rapid outbreak of resistance is seriously threatening the efficacy of these compounds, and alternative strategies are required to selectively regulate PARP1 activity. A noncanonical G-quadruplex-forming sequence within the PARP1 promoter was recently identified. In this study, we explore the interaction of known G-quadruplex binders with the G-quadruplex structure found in the PARP gene promoter region. The results obtained by NMR, CD, and fluorescence titration, also confirmed by molecular modeling studies, demonstrate a variety of different binding modes with small stabilization of the G-quadruplex sequence located at the PARP1 promoter. Surprisingly, only pyridostatin produces a strong stabilization of the G-quadruplex-forming sequence. This evidence makes the identification of a proper (3+1) stabilizing ligand a challenging goal for further investigation.

Keywords: G-quadruplex; NMR; PARP1 promoter; circular dichroism; fluorescence; molecular modeling; stabilizing ligand.

Figure 1

Structures of selected G-quadruplex binders.

Figure 2

Schematic representation of TP3-T6 oligomer G-quadruplex. Adenine in red, Cytosine in yellow, Guanine in green and Thymine in blue.

Figure 3

Imino proton region of the 1D NMR titration spectra of TP3-T6 with curaxin (Left), recorded at 25 °C and different R = (drug)/(DNA) ratios. The side (Center) and 3′-end (Right) views of the TP3-T6/curaxin complex obtained by Molecular Docking were created by using the Chimera-X software. In the side (Center) view, nucleotides are shown in sticks to reveal atoms and bonds, while in the 3’-end (Right) view the nucleotides are shown as slabs and filled sugars: Adenine in red, Cytosine in yellow, Guanine in green and Thymine in blue. The curaxin molecule is rendered in sticks and colored according to the atoms.

Figure 4

(a) Fluorescence spectra recorded along the titration of curaxin with TP3-T6; (b) Experimental (red symbols) and calculated (green line) fluorescence intensity at 450 nm for the titration of curaxin at different concentrations of TP3-T6. Conditions: 20 mM phosphate buffer (pH 7.1), 70 mM KCl of 3 μM curaxin and increasing amounts of 69 μM of TP3-T6. Excitation wavelength 334 nm.

Figure 5

(a) Selected CD spectra recorded along the melting of the TP3-T6/curaxin 1:3 mixture. The whole data set is given in Supplementary Materials; (b) Fraction of folded DNA vs. temperature calculated from the melting of TP3-T6 and of the TP3-T6/curaxin 1:3 mixture at 265 nm. DNA and ligand concentration were 2 and 6 μM, respectively, 20 mM phosphate buffer (pH 7.1), 70 mM KCl.

Figure 6

Imino proton region of 1D NMR titration spectra of TP3-T6 with CX-5461 at 25 °C at different R = (drug)/(DNA) ratios (Left). The complex between TP3-T6 and CX-5461 was obtained by molecular docking and the side (Center) and 3′-end (Right) views were created by using the ChimeraX software., nucleotides are shown in sticks to reveal atoms and bonds, the nucleotides in the side (Center) view are rendered in stick while in the 3’-end (Right) view they are shown as slabs and filled sugars. Adenine residues are colored in red, Cytosine in yellow, Guanine in green and Thymine in blue. In both views the ligand is represented as sticks and colored according to the atoms.

Figure 7

Imino proton region of 1D NMR titration spectra of TP3-T6 with BMH21 at 25 °C at different R = (drug)/(DNA) ratios.

Figure 8

Imino proton region of 1D NMR titration spectra of TP3-T6 with BA41 at 25 °C at different R = (drug)/(DNA) ratios.

Figure 9

Side (Left) and 3′-end (Right) views of the complexes predicted by molecular docking of the TP3-T6 target with BMH21 and BA41. In the graphical representations, nucleotides in the side views are rendered in stick while those in the 3′-end views are rendered as slabs and filled sugars. Adenine residues are colored in red, Cytosine in yellow, Guanine in green and Thymine in blue. The ligands are always represented as sticks and colored according to the atoms. Both graphical representations were created with the ChimeraX software.

Figure 10

Imino proton region of 1D NMR titration spectra of TP3-T6 with RHPS4 at 25 °C at different R = (drug)/(DNA) ratios.

Figure 11

Imino proton region of 1D NMR titration spectra of TP3-T6 with pyridostatin at 25 °C at different R = (drug)/(DNA) ratios.