Asymmetric triplex metallohelices stabilise DNA G-quadruplexes in promoter oncogene sequences and efficiently reduce their expression in cancer cells

Jaroslav Malinaa Czech Academy of Sciences, Institute of Biophysics, Brno, Czech RepublicView further author information

Hana Kostrhunovaa Czech Academy of Sciences, Institute of Biophysics, Brno, Czech RepublicView further author information

Hualong Songb Department of Chemistry, University of Warwick, Coventry, UKView further author information

Peter Scottb Department of Chemistry, University of Warwick, Coventry, UKView further author information

Viktor Brabeca Czech Academy of Sciences, Institute of Biophysics, Brno, Czech RepublicCorrespondence[email protected]
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Figure 1. Triplex metallohelices used in this study. Each assembly comprises three ligand strands L, colourised separately and shown inset in black for clarity. The R_c,Δ_Fe,HHT-[Fe₂L₃]⁴⁺ enantiomers shown are denoted Δ-1 and Δ-2. The mirror images S_c,Λ_Fe,HHT-[Fe₂L₃]⁴⁺ are denoted Λ-1 and Λ-2 in the text.

Figure 2. DC₅₀ values [μM] (upper panels) for DNA G4s and DNA duplex determined by FID upon the addition of metallohelices in 10 mM potassium phosphate buffer (pH 7) and increasing concentrations of KCl (total concentrations of K⁺ are indicated in the ). The results are expressed as mean ± SD from 2 independent experiments. Selectivity indexes (lower panels) of metallohelices towards DNA G4s in 10 mM potassium phosphate buffer (pH 7) and increasing concentrations of KCl. The values of the binding selectivity for each metallohelix were calculated as a ratio between the DC₅₀ values obtained for the duplex and G4 DNA.

Figure 2. DC50 values [μM] (upper panels) for DNA G4s and DNA duplex determined by FID upon the addition of metallohelices in 10 mM potassium phosphate buffer (pH 7) and increasing concentrations of KCl (total concentrations of K+ are indicated in the Figure 2). The results are expressed as mean ± SD from 2 independent experiments. Selectivity indexes (lower panels) of metallohelices towards DNA G4s in 10 mM potassium phosphate buffer (pH 7) and increasing concentrations of KCl. The values of the binding selectivity for each metallohelix were calculated as a ratio between the DC50 values obtained for the duplex and G4 DNA.

Figure 3. ΔT_m values for the fluorescently labelled hTelo, c-kit1, c-kit2, c-myc, and k-ras DNA G4s (0.4 μM) determined by FRET upon addition of 0.8 μM enantiomers of 1 and 2 in the presence of increasing concentrations of dsDNA (indicated in ). The measurements were carried out in 10 mM potassium phosphate (pH 7) (upper panels) and 10 mM potassium phosphate and 30 mM KCl (pH 7) (lower panels). The results are expressed as mean ± SD from three independent experiments.

Table 1. Loss of the stabilising ability of metallohelices (0.8 µM) towards DNA G4s (0.4 µM) in the presence of 240 µM dsDNA in 10 and 40 mM K⁺.

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Figure 4. Inhibition of Taq polymerase DNA synthesis on templates (30 nM) with c-myc, c-kit2, c-kit1, hTelo, and k-ras G4-forming sequences in the presence of increasing concentrations of metallohelices. A. Autoradiograms of 12% PAA sequencing gels with products of DNA synthesis on the template containing c-myc G4-forming sequence in the presence of increasing concentrations of the Λ- and Δ-enantiomers of 1 and 2. fp, ps, and p correspond to full-length product, pausing site by G4, and primer, respectively. B. Plots showing the ratio of the radiation corresponding to pausing sites to total radiation of the lane vs. the concentration of 1 and 2 enantiomers. The results are expressed as mean ± SD from two independent experiments.

Figure 5. A. Relative expression of c-MYC and k-RAS mRNA in HCT116 cells treated with Λ- and Δ-enantiomers of 1 and 2 at concentrations corresponding to 1×, 2×, and 3 × IC₅₀, respectively, normalised to GAPDH. B. Western blot images of c-myc, k-ras, and GAPDH levels in HCT116 cells treated with increasing concentrations (indicated above gels) of 1 and 2. C. Relative expression of c-myc and k-ras proteins normalised to GAPDH. Data represent the mean ± SD of two to four independent experiments. The stars indicate a statistically significant difference determined by the Student t-test (*p ≤ .05; **p ≤ .01; $p ≤ .001; #p ≤ .0001).

Table 2. IC₅₀ values determined with MTT assay in HCT116 cells after a 72-h treatment.

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Asymmetric triplex metallohelices stabilise DNA G-quadruplexes in promoter oncogene sequences and efficiently reduce their expression in cancer cells

Table 1. Loss of the stabilising ability of metallohelices (0.8 µM) towards DNA G4s (0.4 µM) in the presence of 240 µM dsDNA in 10 and 40 mM K⁺.

Table 2. IC₅₀ values determined with MTT assay in HCT116 cells after a 72-h treatment.

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Asymmetric triplex metallohelices stabilise DNA G-quadruplexes in promoter oncogene sequences and efficiently reduce their expression in cancer cells

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Table 1. Loss of the stabilising ability of metallohelices (0.8 µM) towards DNA G4s (0.4 µM) in the presence of 240 µM dsDNA in 10 and 40 mM K+.

Table 2. IC50 values determined with MTT assay in HCT116 cells after a 72-h treatment.

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Table 1. Loss of the stabilising ability of metallohelices (0.8 µM) towards DNA G4s (0.4 µM) in the presence of 240 µM dsDNA in 10 and 40 mM K⁺.

Table 2. IC₅₀ values determined with MTT assay in HCT116 cells after a 72-h treatment.