Identification of Effective Anticancer G-Quadruplex-Targeting Chemotypes through the Exploration of a High Diversity Library of Natural Compounds

Abstract

In the quest for selective G-quadruplex (G4)-targeting chemotypes, natural compounds have been thus far poorly explored, though representing appealing candidates due to the high structural diversity of their scaffolds. In this regard, a unique high diversity in-house library composed of ca. one thousand individual natural products was investigated. The combination of molecular docking-based virtual screening and the G4-CPG experimental screening assay proved to be useful to quickly and effectively identify—out of many natural compounds—five hit binders of telomeric and oncogenic G4s, i.e., Bulbocapnine, Chelidonine, Ibogaine, Rotenone and Vomicine. Biophysical studies unambiguously demonstrated the selective interaction of these compounds with G4s compared to duplex DNA. The rationale behind the G4 selective recognition was suggested by molecular dynamics simulations. Indeed, the selected ligands proved to specifically interact with G4 structures due to peculiar interaction patterns, while they were unable to firmly bind to a DNA duplex. From biological assays, Chelidonine and Rotenone emerged as the most active compounds of the series against cancer cells, also showing good selectivity over normal cells. Notably, the anticancer activity correlated well with the ability of the two compounds to target telomeric G4s.

Affinity Chromatography‐Based Assays for the Screening of Potential Ligands Selective for G‐Quadruplex Structures

“…Finally, biological assays indicated Chelidonine and Rotenone as the most active anticancer compounds, with IC 50 values of 0.64 and 0.15 μ m , respectively, on BJ‐EHLT cancer cells, and also showing a good selectivity over normal BJ‐hTERT cells. [32] …”

“…[ 25 , 26 ] Specific G4 binding modes include stacking on the outer G‐quartets and/or on nucleobases in loops or in the flanking segments, and/or binding at the grooves or loops mediated by hydrogen bonds or electrostatic interactions. [ 28 , 29 , 30 , 31 , 32 ]…”

“…In the last two decades intensive research efforts have been devoted to the design and synthesis of potential ligands able to selectively target G4 structures and discriminate them over the other and more abundant form of DNA, that is, the B‐DNA duplex. [ 27 , 29 , 32 , 33 ] Currently, four molecules able to interfere with the in vitro and/or in vivo functions of G4 structures reached advanced clinical trials for anticancer treatments, that is, Quarfloxin (ClinicalTrials.gov Identifier: NCT00780663), CX‐5461 (ClinicalTrials.gov Identifier: NCT02719977), APTO‐253 (ClinicalTrials.gov Identifier: NCT02267863) and pyrvinium pamoate (ClinicalTrials.gov Identifier: NCT05055323), providing proof‐of‐principle for the high anticancer therapeutic potential of targeting G4 structures. [ 26 , 34 , 35 , 36 ] Only for Quarfloxin, Phase II studies have been concluded, while Phase I studies are ongoing for the other three G4 ligands.…”

“…Several studies have fully demonstrated the relationship between the anticancer activity of various ligands and their ability to recognize G4 structures [25,26] . Specific G4 binding modes include stacking on the outer G‐quartets and/or on nucleobases in loops or in the flanking segments, and/or binding at the grooves or loops mediated by hydrogen bonds or electrostatic interactions [28–32] …”

“…[25,26] Specific G4 binding modes include stacking on the outer G-quartets and/or on nucleobases in loops or in the flanking segments, and/or binding at the grooves or loops mediated by hydrogen bonds or electrostatic interactions. [28][29][30][31][32] In the last two decades intensive research efforts have been devoted to the design and synthesis of potential ligands able to selectively target G4 structures and discriminate them over the other and more abundant form of DNA, that is, the B-DNA duplex. [27,29,32,33] Currently, four molecules able to interfere with the in vitro and/or in vivo functions of G4 structures reached advanced clinical trials for anticancer treatments, that is, Quarfloxin (ClinicalTrials.…”