Elucidation of DNA-Eltrombopag Binding: Electrochemical, Spectroscopic and Molecular Docking Techniques

Abstract

Eltrombopag is a powerful adjuvant anticancer drug used in treating MS (myelodysplastic syndrome) and AML (acute myeloid leukemia) diseases. In this study, the interaction mechanism between eltrombopag and DNA was studied by voltammetry, spectroscopic techniques, and viscosity measurements. We developed a DNA-based biosensor and nano-biosensor using reduced graphene oxide-modified glassy carbon electrode to detect DNA-eltrombopag binding. The reduction of desoxyguanosine (dGuo) and desoxyadenosine (dAdo) oxidation signals in the presence of the drug demonstrated that a strong interaction could be established between the eltrombopag and dsDNA. The eltrombopag-DNA interaction was further investigated by UV absorption and fluorescence emission spectroscopy to gain more quantitative insight on binding. Viscosity measurements were utilized to characterize the binding mode of the drug. To shed light on the noncovalent interactions and binding mechanism of eltrombopag molecular docking and molecular dynamics (MD), simulations were performed. Through simultaneously carried out experimental and in silico studies, it was established that the eltrombopag binds onto the DNA via intercalation.

Keywords: DNA; biosensor; eltrombopag; molecular docking; voltammetry.

Scheme 1

The fabrication procedure of DNA/rGO/GCE.

Figure 1

DPVs of (a) DNA/GCE and (b) DNA/rGO/GCE in pH 4.7 acetate buffer.

Figure 2

DPVs of DNA/rGO/GCE in the absence (a) and the presence of 3 mg L⁻¹ ELT (b).

Figure 3

DPVs of DNA/rGO/GCE in acetate buffer solution (0.5 M, pH 4.8) containing 2 mg L⁻¹ ELT under different incubation time: 15 s (a), 30 s (b), 1 min (c), 3 min (d), and 5 min (e). The arrow shows the increasing peak current of ELT with the interaction time.

Figure 4

The plot of the guanine (blue dots) and adenine (red dots) peaks current vs. different interaction times with the ELT.

Figure 5

DPVs of DNA/rGO/GCE in the absence (black) and in the presence of (a) 0.5, (b) 1, (c) 2, (d) 3, and (e) 4 mg L^—1 ELT. Insert: The plot of the peak currents of guanine (blue dots) and adenine (red dots) vs. ELT concentration.

Figure 6

UV-Vis spectra of ELT (8 × 10⁻⁶M) upon the titration of dsDNA (20–140 × 10⁻⁶M) in buffer media. The increase in absorbance for an increase in dsDNA concentration is indicated by an “arrow”. The maximum wavelength of ELT (λ_max = 249 nm) shifted to 256 nm. K_b was calculated from this linearity, showing the linear agreement between “[dsDNA]/(ε_a − ε_f)” and [dsDNA] in the Appendix (R² = 0.9930).

Figure 7

Thermal denaturation curves of dsDNA (120 µM) in the absence and in the presence of ELT and some intercalator/groove agents (10 µM) in Tris/HCl buffer solution.

Figure 8

Fluorescence titration of (a) EtBr–dsDNA, (b) Hoechst-33258–dsDNA mixtures with ELT (r = [ELT]/[dsDNA]).

Figure 9

Stern–Volmer plots show the fluorescence quenching of different dye-dsDNA solutions by the increasing addition of ELT. (R² = 0.9879 for EtBr–dsDNA, R² = 0.9932 for Hoechst-33258–dsDNA).

Figure 10

The effect of increasing amount of ELT on the relative viscosity of dsDNA. (r = [ELT]/[dsDNA] = 0.25–3.0).

Figure 11

3D structure of ELT molecule.

Figure 12

3-D structure of ELT bound to 1BNA via intercalation mechanism at pH 7.4.

Figure 13

RMSD plots of ELT-BNA complex (in green) and non-ligand bound 1-BNA (in purple), respectively.