The human genome is replete with repetitive DNA sequences that can fold into thermodynamically stable secondary structures such as hairpins and quadruplexes. Cellular enzymes exist to cope with these structures whose stable accumulation would result in DNA damage through interference with DNA transactions such as transcription and replication. Therefore, chemical stabilization of secondary DNA structures offers an attractive way to foster DNA transaction-associated damages to trigger cell death in proliferating cancer cells. While much emphasis has been recently given to DNA quadruplexes, we focused here on three-way DNA junctions (TWJ) and report on a strategy to identify TWJ-targeting agents through a combination of in vitro techniques (TWJ-Screen, PAGE, FRET-melting, ESI-MS, dialysis equilibrium and SRB assays). We designed a complete workflow and screened 1200 compounds to identify promising TWJ-ligands selected on stringent criteria in terms of TWJ folding ability, affinity and selectivity.

Translocation of DNA and RNA polymerases along their duplex substrates results in DNA supercoiling. This torsional stress promotes the formation of plectonemic structures, including three-way DNA junction (TWJ), which can block DNA transactions and lead to DNA damage. While cells have evolved multiple mechanisms to prevent the accumulation of such structures, stabilizing TWJ through ad hoc ligands offer an opportunity to trigger DNA damage in cells with high level of transcription and replication, such as cancer cells. Here, we develop a series of azacryptand-based TWJ ligands, we thoroughly characterize their TWJ-interacting properties in vitro and demonstrate their capacity to trigger DNA damage in rapidly dividing human cancer cells. We also demonstrate that TWJ ligands are amenable to chemically induced synthetic lethality strategies upon association with inhibitors of DNA repair, thus paving the way towards innovative drug combinations to fight cancers.