Towards DNA-targeting magic bullets: searching for potential conformation-selective G-quadruplex ligands

In the search for effective and minimally toxic anticancer drugs, G-quadruplex (G4) structures emerged as appealing targets for their crucial roles in human telomeres and oncogene promoters. G4s are non-canonical nucleic acid secondary structures exhibiting marked structural polymorphism. In the last decade, increasing efforts in the G4 research field have been devoted to the search of selective G4-binders, able not only to fully discriminate G4 vs. duplex DNA, but also to specifically recognize different G4 topologies, thus resulting into effective ligands, and expectedly into useful anticancer drugs with limited side effects. To reach this ambitious goal, an integrated approach is required, involving the massive production of large libraries of new putative G4 ligands, coupled with High Throughput Screening methodologies for the fast and reliable analysis of the selected compounds. In this context, we here describe: i) a method for the on-line synthesis of support-bound, fully deprotected secondary structure-forming oligonucleotides, and ii) an affinity chromatography-based assay, named G-quadruplex on Controlled Pore Glass (G4-CPG), for the screening of libraries of putative conformation-selective G4 ligands. It consists in flowing solutions of the potential ligands through a glass support functionalized with a G4-forming DNA sequence and quantifying the bound ligand by spectrophotometric measurements. After full optimization of the G4-CPG assay, two different libraries of putative G4 selective ligands, based either on furobenzoxazine naphthoquinone or naphthalene diimide scaffold, were evaluated. Overall, the novel developed G4-CPG method based on our newly designed CPG support allowed us selecting two promising candidate drugs for in vivo studies, showing binding preferences for a specific G-quadruplex conformation, in addition to their high G4s vs. duplex DNA selectivity.