Molecular Recognition of Parallel G-quadruplex [d-(TTGGGGT)]4 Containing Tetrahymena Telomeric DNA Sequence by Anticancer Drug Daunomycin: NMR-Based Structure and Thermal Stability

Abstract

The anticancer drug daunomycin exerts its influence by multiple strategies of action to interfere with gene functioning. Besides inhibiting DNA/RNA synthesis and topoisomerase-II, it affects the functional pathway of telomere maintenance by the telomerase enzyme. We present evidence of the binding of daunomycin to parallel-stranded tetramolecular [d-(TTGGGGT)]4 guanine (G)-quadruplex DNA comprising telomeric DNA from Tetrahymena thermophilia by surface plasmon resonance and Diffusion Ordered SpectroscopY (DOSY). Circular Dichroism (CD) spectra show the disruption of daunomycin dimers, suggesting the end-stacking and groove-binding of the daunomycin monomer. Proton and phosphorus-31 Nuclear Magnetic Resonance (NMR) spectroscopy show a sequence-specific interaction and a clear proof of absence of intercalation of the daunomycin chromophore between base quartets or stacking between G-quadruplexes. Restrained molecular dynamics simulations using observed short interproton distance contacts depict interaction at the molecular level. The interactions involving ring A and daunosamine protons, the stacking of an aromatic ring of daunomycin with a terminal G6 quartet by displacing the T7 base, and external groove-binding close to the T1–T2 bases lead to the thermal stabilization of 15 °C, which is likely to inhibit the association of telomerase with telomeres. The findings have implications in the structure-based designing of anthracycline drugs as potent telomerase inhibitors.

Binding of anticancer drug adriamycin to parallel G‐quadruplex DNA [d‐(TTAGGGT)]4 comprising human telomeric DNA leads to thermal stabilization: A multiple spectroscopy study

“…Thermal transitions monitored independently by differential scanning calorimetry give more accurate values within ±0.4°C and show intermediates during transition from ordered state to disordered state in G4 DNA. The DSC thermogram of 100 μM [d‐(TTAGGGT)] 4 showed two “two state” transition with apparent T m 1 = 58.8°C and T m 2 = 64.3°C .…”

“…The NMR spectra of free [d‐(TTAGGGT)] 4 (Figure 8a) is well characterized and shows presence of Hoogstein hydrogen bond in G‐quartets (NH resonating at 10.7–11.7 ppm) in tetramolecular parallel quadruplex having right handed B‐DNA conformation and anti glycosidic bond rotation . The two dimensional 1 H‐ 1 H NOESY spectra of free [d‐(TTAGGGT)] 4 (Figure 8b) and 2:1 adriamycin‐[d‐(TTAGGGT)] 4 complex (Figure 8c) shows several short inter proton distance contacts which refer to intra molecular connectivities within DNA (Figure 8b) and within adriamycin (referred to as D1 and D2 in Figure 8c) as well as inter molecular distance correlations between adriamycin and DNA (marked with asterisk and numbered 1–4 in Figure 8c), which are absent in free DNA (Figure 8b). The presence of intra nucleotide and sequential inter nucleotide Nuclear Overhauser Enhancement (NOE) correlations shows that overall conformation of G‐quadruplex with anti glycosidic bond rotation is unchanged on interaction with adriamycin and no opening of base pair occurs at any step for intercalation of ligand chromophore and thus adriamycin binds externally to DNA quadruplex .…”

“…The two dimensional 1 H‐ 1 H NOESY spectra of free [d‐(TTAGGGT)] 4 (Figure 8b) and 2:1 adriamycin‐[d‐(TTAGGGT)] 4 complex (Figure 8c) shows several short inter proton distance contacts which refer to intra molecular connectivities within DNA (Figure 8b) and within adriamycin (referred to as D1 and D2 in Figure 8c) as well as inter molecular distance correlations between adriamycin and DNA (marked with asterisk and numbered 1–4 in Figure 8c), which are absent in free DNA (Figure 8b). The presence of intra nucleotide and sequential inter nucleotide Nuclear Overhauser Enhancement (NOE) correlations shows that overall conformation of G‐quadruplex with anti glycosidic bond rotation is unchanged on interaction with adriamycin and no opening of base pair occurs at any step for intercalation of ligand chromophore and thus adriamycin binds externally to DNA quadruplex . Inter molecular NOE connectivities of 3H of adriamycin with T1CH 3 , T7H2″, G6H2″ of DNA and 1H/2H with G6H2″, T2H2′ (marked with asterik, numbered 1–5 in Figure 8c) show close proximity of ring D proton of adriamycin to T1‐T2‐A3 and G6‐T7 bases.…”

“…Melting profiles were obtained in temperature range 25–100°C at the rate of 1°C/min and measured at 263 nm at an interval of 5°C. Thermal transition profiles were also obtained excess heat capacity as a function of temperature using VP DSC Microcalorimeter (Microcal Inc. Northampton, MA) and analyzing thermograms using inbuilt VP Viewer software with origin 7.0 . NMR spectroscopy experiments were carried out on 1.16 mM of [d‐(TTAGGGT)] 4 dissolved in 10 mM KBPES buffer prepared in 90% H 2 O and 10% D 2 O with 0.1 μl of 0.1 M Trimethyl Silyl Propionic acid (TSP) and EDTA added to it.…”

“…Besides, long telomeric sequences existing in several folded forms with different topologies form inter molecular aggregates making interpretation of results on ligand‐intra molecular G‐quadruplex complex very difficult. Inter molecular quadruplexes have therefore been used in past as motifs to get high resolution data on drug‐DNA interactions for various ligands using X‐ray/NMR techniques, which is necessary to understand their molecular basis of action. X‐ray crystallographic structure of daunomycin co‐crystallized with inter molecular hexameric G‐quadruplex DNA [d‐(TGGGGT)] 4 , containing telomeric DNA sequence from Tetrahymena thermophila , showed two layers of daunomycin, each containing three molecules, sandwiched between two G4 DNA molecules stacked end to end .…”