Multicharged Phthalocyanines as Selective Ligands for G-Quadruplex DNA Structures

Abstract

The stabilization of G-Quadruplex DNA structures by ligands is a promising strategy for telomerase inhibition in cancer therapy since this enzyme is responsible for the unlimited proliferation of cancer cells. To assess the potential of a compound as a telomerase inhibitor, selectivity for quadruplex over duplex DNA is a fundamental attribute, as the drug must be able to recognize quadruplex DNA in the presence of a large amount of duplex DNA, in the cellular nucleus. By using different spectroscopic techniques, such as ultraviolet-visible, fluorescence and circular dichroism, this work evaluates the potential of a series of multicharged phthalocyanines, bearing four or eight positive charges, as G-Quadruplex stabilizing ligands. This work led us to conclude that the existence of a balance between the number and position of the positive charges in the phthalocyanine structure is a fundamental attribute for its selectivity for G-Quadruplex structures over duplex DNA structures. Two of the studied phthalocyanines, one with four peripheral positive charges (ZnPc1) and the other with less exposed eight positive charges (ZnPc4) showed high selectivity and affinity for G-Quadruplex over duplex DNA structures and were able to accumulate in the nucleus of UM-UC-3 bladder cancer cells.

Keywords: G-Quadruplexes; G4-FID; UV-Vis; circular dichroism; hyperchromism; multicharged phthalocyanines; salmon sperm DNA; selectivity; telomerase inhibition.

Figure 1

Schematic demonstration of telomerase inhibition by formation and stabilization of G-Quadruplex structures.

Figure 2

Structures of the studied TMPyP and thiopyridinium (ZnPc1, ZnPc2) and methoxypyridinium (ZnPc3, ZnPc4) phthalocyanines.

Figure 3

UV-Vis absorption spectra (550–800 nm) for the titrated solutions of ZnPc1 (2.0 μM) with (A) tetramolecular GQ, (B) bimolecular GQ, (C) unimolecular GQ, (D) salmon sperm, (E) 5GC, and (F) 20 mM PBS buffer with 100 mM KCl.

Figure 4

UV-Vis absorption spectra (550–800 nm) for the titrated solutions of ZnPc2 (2.0 μM) with (A) tetramolecular GQ, (B) bimolecular GQ, (C) unimolecular GQ, (D) salmon sperm, (E) 5GC, and (F) 20 mM PBS buffer with 100 mM KCl.

Figure 5

UV-Vis absorption spectra (550–800 nm) for the titrated solutions of ZnPc3 (2.0 μM) with (A) tetramolecular GQ, (B) unimolecular GQ, (C) salmon sperm, (D) 5GC, and (E) 20 mM PBS buffer with 100 mM KCl.

Figure 6

UV-Vis absorption spectra (550–800 nm) for the titrated solutions of ZnPc4 (2.0 μM) with (A) tetramolecular GQ, (B) bimolecular GQ, (C) unimolecular GQ, (D) salmon sperm, (E) 5GC, and (F) 20 mM PBS buffer with 100 mM KCl.

Figure 7

UV-Vis spectra of Pcs (2.0 μM) in DMSO (---), in PBS (- . -), and PBS + 200 μL PBS (.....), PBS + 200 μL GQ (T₂G₅T)₄ (—); (A) ZnPc1, (B) ZnPc2, (C) ZnPc3, (D) ZnPc4.

Figure 8

G4-FID assay performed in PBS at 25 °C with (T₂G₅T)₄ (green squares), (AG₃(T₂AG₃)₃) (blue circles), duplex (5GC) (red triangles) and (A) ZnPc1, (B) ZnPc2, (C) ZnPc4, (D) TMPyP.

Figure 9

Fluorescence spectra obtained in PBS at 25 °C for ZnPc1 in the concentration range of 0–4.0 uM with (A) tetramolecular GQ (T₂G₅T)₄, (B) unimolecular GQ AG₃(T₂AG₃)₃, and (C) duplex sequence 5GC.

Figure 10

Fluorescence emission spectra of (A) ZnPc1, (B) ZnPc2, (C) ZnPc4 and (D) TMPyP with increasing concentration of unimolecular GQ AG₃(T₂AG₃)₃.

Figure 11

CD melting profiles obtained for GQ unimolecular AG₃(T₂AG₃)₃ in the absence or in the presence of ZnPc1, ZnPc4 and TMPyP.

Figure 12

Representative fluorescence images of UM-UC-3 bladder cancer cells incubated with the ligands (red) TMPyP (30 μM), ZnPc1 (20 μM) or ZnPc4 (45 μM) for 48 h of incubation. DAPI is staining the nucleus. Scale bars 20 μM.

Figure 13

Red shifts obtained in the UV-Vis titration with the selected ligands and DNA sequences.