PhenQE8, a Novel Ligand of the Human Telomeric Quadruplex

Abstract

A novel quadruplex ligand based on 1,10-phenanthroline and incorporating two guanyl hydrazone functionalities, PhenQE8, is reported herein. Synthetic access was gained in a two-step procedure with an overall yield of 61%. X-ray diffraction studies revealed that PhenQE8 can adopt an extended conformation that may be optimal to favor recognition of quadruplex DNA. DNA interactions with polymorphic G-quadruplex telomeric structures were studied by different techniques, such as Fluorescence resonance energy transfer (FRET) DNA melting assays, circular dichroism and equilibrium dialysis. Our results reveal that the novel ligand PhenQE8 can efficiently recognize the hybrid quadruplex structures of the human telomeric DNA, with high binding affinity and quadruplex/duplex selectivity. Moreover, the compound shows significant cytotoxic activity against a selected panel of cultured tumor cells (PC-3, HeLa and MCF-7), whereas its cytotoxicity is considerably lower in healthy human cells (HFF-1 and RPWE-1).

Keywords: 1,10-phenanthroline; DNA interactions; antitumor agents; guanyl hydrazones; human telomeric quadruplex.

Figure 1

(A) Chemical structure of novel G4 ligand PhenQE8; (B) Examples of other quadruplex ligands containing the guanyl hydrazone group [47,48].

Scheme 1

Synthetic route for compound PhenQE8.

Figure 2

Ortep diagram for one of the two crystallographically independent molecules of PhenQE8. Thermal ellipsoids are drawn at the 50% probability level.

Figure 3

DNA FRET melting experiments for PhenQE8 at a concentration range between 1–10 μM, in the buffer system 10 mM lithium cacodylate, 90 mM LiCl, 10 mM KCl with (A) telomeric oligonucleotide F21T; (B) duplex DNA F10T.

Figure 4

Competition DNA FRET melting experiments for PhenQE8 at a 5 μM concentration with 0.2 μM G4 sequence Tel22 (10 mM lithium cacodylate, 90 mM LiCl, 10 mM KCl) in the absence (grey) and presence (blue and dark pink) of competitor dsDNA sequence ds26: 3 μM, 15-fold DNA excess (blue) and 10 μM, 50-fold DNA excess (dark pink).

Figure 5

(A) Changes in circular dichroism (CD) spectra of quadruplex Tel22 (4 μM strand molarity), folded in the presence of potassium ions (10 mM lithium cacodylate, 90 mM LiCl, 10 mM KCl, pH 7.3, 25 °C) upon addition of PhenQE8 in a molar ratio quadruplex DNA/PhenQE8 ranging from 1:1 to 1:5. (B) CD changes at 1:2 Tel22/ligand stoichiometry for PhenQE8 and reference compound 360A.

Figure 6

Viscometric titration experiment of calf thymus (CT) DNA and ligand PhenQE8 at 25 °C (10 mM sodium phosphate buffer, pH 7.2). Averaged slope value over replicate experiments is 0.24 ± 0.12.