Phen-DC3

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

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-DC3 , 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 pseudo-quartet, 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.

Ligand Binding to Dynamically Populated G-Quadruplex DNA

Several small-molecule ligands specifically bind and stabilize G-quadruplex (G4) nucleic acid structures, which are considered to be promising therapeutic targets. G4s are polymorphic structures of varying stability, and their formation is dynamic. Here, we investigate the mechanisms of ligand binding to dynamically populated human telomere G4 DNA by using the bisquinolinium based ligand Phen-DC3 and a combination of single-molecule FRET microscopy, ensemble FRET and CD spectroscopies. Different cations are used to tune G4 polymorphism and folding dynamics. We find that ligand binding occurs to pre-folded G4 structures and that Phen-DC3 also induces G4 formation in unfolded single strands. Following ligand binding to dynamically populated G4s, the DNA undergoes pronounced conformational redistributions that do not involve direct ligand-induced G4 conformational interconversion. On the contrary, the redistribution is driven by ligand-induced G4 folding and trapping of dynamically populated short-lived conformation states. Thus, ligand-induced stabilization does not necessarily require the initial presence of stably folded G4s.

A complementary chemical probe approach towards customized studies of G-quadruplex DNA structures in live cells

G-quadruplex (G4) DNA structures are implicated in central biological processes and are considered promising therapeutic targets because of their links to human diseases such as cancer. However, functional details of how, when, and why G4 DNA structures form in vivo are largely missing leaving a knowledge gap that requires tailored chemical biology studies in relevant live-cell model systems. Towards this end, we developed a synthetic platform to generate complementary chemical probes centered around one of the most effective and selective G4 stabilizing compounds, Phen-DC3. We used a structure-based design and substantial synthetic devlopments to equip Phen-DC3 with an amine in a position that does not interfere with G4 interactions. We next used this reactive handle to conjugate a BODIPY fluorophore to Phen-DC3. This generated a fluorescent derivative with retained G4 selectivity, G4 stabilization, and cellular effect that revealed the localization and function of Phen-DC3 in human cells. To increase cellular uptake, a second chemical probe with a conjugated cell-penetrating peptide was prepared using the same amine-substituted Phen-DC3 derivative. The cell-penetrating peptide conjugation, while retaining G4 selectivity and stabilization, increased nuclear localization and cellular effects, showcasing the potential of this method to modulate and direct cellular uptake e.g. as delivery vehicles. The applied approach to generate multiple tailored biochemical tools based on the same core structure can thus be used to advance the studies of G4 biology to uncover molecular details and therapeutic approaches.

Solution structure of a G-quadruplex bound to the bisquinolinium compound Phen-DC(3)

Phen-DC3 is a highly promising compound that specifically targets G-quadruplexes, with potent biological effects observed in vivo. We used NMR spectroscopy to solve the structure of the complex formed between Phen-DC3 and an intramolecular G-quadruplex derived from the c-myc promoter. Structural information revealed that Phen-DC3 interacts with the quadruplex through extensive π-stacking with guanine bases of the top G-tetrad. On the basis of our structure, modifications are proposed for the development of this compound for selective targeting of a specific G-quadruplex conformation.

Screening of a chemical library by HT-G4-FID for discovery of selective G-quadruplex binders

Due to the lack of structural guidelines about G-quadruplex ligands, rational design cannot be the only approach to discover potent G4-ligands. As a complementary approach, screening of chemical library may provide interesting scaffolds known as hits provided that specific tools are available. In this work, the Institut Curie-CNRS chemical library was firstly screened by chemoinformatics methods. Similarity estimations by comparison with reference compounds (Phen-DC3, 360A, MMQ12) provided a set of molecules, which were then evaluated by high-throughput G4-FID (HT-G4-FID) against various G-quadruplex DNA. A full investigation of the most interesting molecules, using the HT-G4-FID assay and molecular modeling, supplied an interesting structure-activity relationship confirming the efficiency of this general approach. Overall, we demonstrated that HT-G4-FID coupled with screening of chemical libraries is a powerful tool to identify new G4-DNA binding scaffolds.