Phen‐DC₃ Induces Refolding of Human Telomeric DNA into a Chair‐Type Antiparallel G‐Quadruplex through Ligand Intercalation

Abstract

Human telomeric G-quadruplex DNA structures are attractive anticancer drug targets, but the target's polymorphism complicates the drug design: different ligands prefer different folds, and very few complexes have been solved at high resolution. Here we report that Phen-DC 3 , one of the most prominent G-quadruplex ligands in terms of high binding affinity and selectivity, causes dTAGGG(TTAGGG) 3 to completely change its fold in KCl solution from a hybrid-1 to an antiparallel chair-type structure, wherein the ligand intercalates between a two-quartet unit and a pseudoquartet, thereby ejecting one potassium ion. This unprecedented high-resolution NMR structure shows for the first time a true ligand intercalation into an intramolecular G-quadruplex.

Optimization of G‐Quadruplex Ligands through a SAR Study Combining Parallel Synthesis and Screening of Cationic Bis(acylhydrazones)

“…The results of the FID assay, performed with six Gquadruplexes of different topologies, also suggested the selectivity of 3Ei for the antiparallel structure adopted by 22CTA. The results of CD spectroscopy point to a conformation change of the hybrid-2 structure (25TAG) into an antiparallel structure induced by compounds 3Ab, 3Cb, 3Ei, and, to a lesser extent, 3Ef, most likely due to insertion of the ligands between the two G-quartets, as was recently demonstrated for PhenDC3 [64] and corroborated by molecular modeling. In the case of hybrid-1 structure (24TTG), only compound 3Ei was able to induce a partial conformational change, presumably due to the higher intrinsic stability of this hybrid fold.…”

“…The best‐known example of this transformation is the shift from hybrid conformations adopted by the human telomeric sequence in K + ‐rich conditions to an antiparallel form, induced by bis(quinolinium) carboxamide ligands such as PDC (360A), 3AQN, PhenDC3, and their derivatives, [38,44,56,62,65,66] whereas a shift toward a parallel form was observed with several other ligands, including phenanthroline derivatives devoid of carboxamide groups [67,68] . During the revision of this manuscript, structural details of the PhenDC3‐induced conversion of a hybrid‐1 structure (similar to the one formed by 24TTG ) into an antiparallel chair‐like fold, namely ligand insertion between two G‐quartets and ejection of one K + ion, became available and finally shed light on this intriguing phenomenon [64] . To evaluate the effect of bis(acylhydrazone) ligands on the conformation of G4‐DNA, we recorded CD spectra of unlabeled oligonucleotides in the absence and the presence of 0.5, 1, and 2 molar equivalents of selected ligands in K + ‐rich conditions (K100 buffer, 100 mM K + ).…”

“…In order to get insight into the high affinity of 3Ei to most G4 structures as well as the inconsistencies observed in fluorimetric titrations described above, we performed molecular docking of selected ligands into two G4 receptors whose structures, in the complex with PhenDC3, were recently determined experimentally. Among these, the telomeric sequence 23TAG (for the sequence, see Table S2) adopts a hybrid‐1 conformation in the absence of ligand, whereas the binding of PhenDC3 induces a conformational switch into a chair‐like antiparallel structure with the insertion of the ligand between two G‐quartets (PDB ID: 7Z9L) [64] . Conversely, the snap‐back structure adopted by Pu24T maintains its parallel conformation upon binding of PhenDC3 (PBD ID: 2MGN) [77] .…”

“…(anti-parallel structure adopted by the telomeric sequence 23TAG in the presence of PhenDC3; the top tetrad formed by G3•G11•G15•G23 is not shown for clarity). [4] b) 2MGN (snap-back parallel structure adopted by Pu24T). [10] In both cases, the first frame of the PDB file was used.…”

“…The cytotoxic effects of four bis(acylhydrazones) (3Ab, 3Cb, 3Ef, and 3Ei) were tested in two human cancer cell lines, namely multidrug-resistant glioblastoma line T98G [78] and cisplatin-resistant non-small-cell lung carcinoma line A549, as well as noncancerous lung fibroblast cells MRC-5, and compared with that of PhenDC3. The results (Table 3; for growth inhibition curves, see Figure S16) show that c) 3Ei bound to (left) the antiparallel G4 structure adopted by telomeric sequence 23TAG in the presence of PhenDC3 (from PDB ID: 7Z9L), [64] and (right) the snap-back parallel structure adopted by Pu24T (from PDB ID: 2MGN). [77] Chemistry-A European Journal bis(acylhydrazone) derivatives were not toxic in T98G cells at concentrations of up to ~10 μM, whereas PhenDC3 showed significant cytotoxicity (GI 50 = 2.9 μM upon 96 h of incubation).…”