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.

DNA G-quadruplex (G4) structures, either within gene promoter sequences or at telomeres, have been extensively investigated as potential small-molecule therapeutic targets. However, although G4s forming at the telomeric DNA have been extensively investigated as anticancer targets, few studies focus on the telomeric repeat-containing RNA (TERRA), transcribed from telomeres, as potential pharmacological targets. Here, a virtual screening approach to identify a library of drug-like putative TERRA G4 binders, in tandem with circular dichroism melting assay to study their TERRA G4-stabilizing properties, led to the identification of a new hit compound. The affinity of this compound for TERRA RNA and some DNA G4s was analyzed through several biophysical techniques and its biological activity investigated in terms of antiproliferative effect, DNA damage response (DDR) activation, and TERRA RNA expression in high vs. low TERRA-expressing human cancer cells. The selected hit showed good affinity for TERRA G4 and no binding to double-stranded DNA. In addition, biological assays showed that this compound is endowed with a preferential cytotoxic effect on high TERRA-expressing cells, where it induces a DDR at telomeres, probably by displacing TERRA from telomeres. Our studies demonstrate that the identification of TERRA G4-targeting drugs with potential pharmacological effects is achievable, shedding light on new perspectives aimed at discovering new anticancer agents targeting these G4 structures.

G-quadruplexes (G4s) are noncanonical DNA or RNA secondary structures involved in numerous biological processes. Their recognition by G4-related proteins (G4RPs) is essential for modulating biological pathways, particularly those associated with transcription and cancer progression. Identifying G4RPs is crucial for understanding their role in diseases like cancer, as these proteins may represent promising therapeutic targets. In this study, a proteomic-based fishing-for-partners approach was employed to identify putative interactors of G4-forming DNA sequences from the promoter regions of cancer-related genes DAP, HIF-1α, JAZF-1, and PDGF-A. A total of eighty-six G4RPs were identified, including nineteen known RNA and/or DNA G4 interactors. Notably, fourteen proteins were identified as potential interactors of all four investigated G4-forming DNA, seven of which were novel G4RPs. Direct interactions with G4s were validated for five of these proteins (AHNAK, GAPDH, HNRNP M, LMNA, and PPIA) using surface plasmon resonance experiments, which showed nanomolar binding affinities. This study not only validated known G4RPs but also led to the discovery of new G4/protein interactions, providing the basis for further investigation into their biological significance and potential implications in disease-associated pathways.

Under slightly acidic conditions, cytosine-rich DNA sequences can form non-canonical secondary structures called i-motifs, which occur as four stretches of cytosine repeats form hemi-protonated C·C⁺ base pairs. The growing interest in the i-motif structures as important components in functional DNA-based nanotechnology or as potential targets of anticancer drugs, increases the need for a deep understanding of the energetics of their structural transitions. Here, a combination of spectroscopic and calorimetric techniques is used to unravel the thermodynamics of folding of an i-motif DNA under favorable conditions. The results give new insights into the energetic aspects of i-motifs and show that thermodynamic and thermal stability are related but not identical properties of such DNA structures.

Besides the well-known double-helical conformation, DNA is capable of folding into various noncanonical arrangements, such as G-quadruplexes (G4s) and i-motifs (iMs), whose occurrence in gene promoters, replication origins, and telomeres highlights the breadth of biological processes that they might regulate. Particularly, previous studies have reported that G4 and iM structures may play different roles in controlling gene transcription. Anyway, molecular tools able to simultaneously stabilize/destabilize those structures are still needed to shed light on what happens at the biological level. Herein, a multicomponent reaction and a click chemistry functionalization were combined to generate a set of 31 bis-triazolyl-pyridine derivatives which were initially screened by circular dichroism for their ability to interact with different G4 and/or iM DNAs and to affect the thermal stability of these structures. All the compounds were then clustered through multivariate data analysis, based on such capability. The most promising compounds were subjected to a further biophysical and biological characterization, leading to the identification of two molecules simultaneously able to stabilize G4s and destabilize iMs, both in vitro and in living cells.

The synthesis and characterization of a small series of cyclic triimidazole derivatives functionalized with methylated pyridine or guanidine moieties is reported. These compounds were tested for their ability to bind to G‐quadruplexes from both telomeric and oncogene promoter sequences. Some of them exhibited high affinity and effective G‐quadruplex‐stabilizing properties. Overall, these compounds may represent useful leads for developing more potent and selective analogues.