Novel Synthesis of IMC-48 and Affinity Evaluation with Different i-Motif DNA Sequences

Abstract

During the last decade, the evidence for the biological relevance of i-motif DNA (i-DNA) has been accumulated. However, relatively few molecules were reported to interact with i-DNA, and a controversy concerning their binding mode, affinity, and selectivity persists in the literature. In this context, the cholestane derivative IMC-48 has been reported to modulate bcl-2 gene expression by stabilizing an i-motif structure in its promoter. In the present contribution, we report on a novel, more straightforward, synthesis of IMC-48 requiring fewer steps compared to the previous approach. Furthermore, the interaction of IMC-48 with four different i-motif DNA sequences was thoroughly investigated by bio-layer interferometry (BLI) and circular dichroism (CD) spectroscopy. Surprisingly, our results show that IMC-48 is a very weak ligand of i-DNA as no quantifiable interaction or significant stabilization of i-motif structures could be observed, stimulating a quest for an alternative mechanism of its biological activity.

Keywords: DNA ligands; IMC-48; affinity; bio-layer interferometry; circular dichroism; i-motif DNA; steroid derivatives.

Figure 1

(A) Hemi-protonated CC base pair. (B) Schematic representation of the structure of the telomeric i-DNA.

Scheme 1

Synthesis of IMC-48: (A) synthesis from reference [25]; (B) new synthesis.

Figure 2

X-ray crystal structure of IMC-48 synthesized by the new route.

Figure 3

Sensorgrams recorded at pH 5.5 for the recognition of IMC-48 with immobilized sequences: (A) HRAS; (B) bcl-2; (C) Htelo-C; and (D) c-myc. Concentration range: 0.1 (blue), 0.5 (brown), 1 (green), 5 (dark cyan), 10 (black), 25 (orange), 50 (violet), and 100 µM (pink). Buffer composition: 50 mM TrisHCl, 50 mM KCl, 2% DMSO and 0.05% v/v surfactant P20.

Figure 4

Sensorgrams recorded at pH 7.5 for the recognition of IMC-48 with immobilized sequences: (A) HRAS; (B) bcl-2; (C) HTelo-C; and (D) c-myc. Concentration range: 0.1 (blue), 0.5 (brown), 1 (green), 5 (dark cyan), 10 (black), 25 (orange), 50 (violet), and 100 µM (pink). Buffer composition: 50 mM TrisHCl, 50 mM KCl, 2% DMSO and 0.05% v/v surfactant P20.

Figure 5

Representative pH-dependent denaturation/renaturation of i-DNA: normalized changes of ellipticity at 285 nm in the course of potentiometric titrations of bcl-2 performed in the absence (black points and curves) and in the presence (red points and curves) of 20 µM IMC-48. For the detailed conditions, cf. Table 2 legend. (Left) alkali (denaturing) titration; (right) acid (renaturing) titration. The arrows indicate the denaturing/renaturing processes.