# Screening of Scaffolds for the Design of G-Quadruplex Ligands
###### Abstract  
In the last decade, progress has been made in G-quadruplex (G4) ligands development, but for most compounds, the ligand binding mode is speculative or based on low resolution methods, with its discovery based on structure-based approaches. Herein, we report the synthesis of small (MW \(<\) 400 Da) heterocyclic compounds, containing different aromatic scaffolds, such as phenyl, quinoline, naphthalene, phenanthroline and arcidine moieties, in order to explore their stabilization effect towards different DNA G4s, such as those found in c-MYC, KRAS21 and VEGF promoters, 21G human telomeric motif and pre-MIR150. The fluorescence resonance energy transfer (FRET) melting assay indicates that the arcidine moiety is the most active scaffold, followed by phenanthroline. The different scaffolds are promising in terms of drug-like properties and, in general, the IC50 values of the respective heterocycle compounds are lower in a cancer cell line, when compared with a normal cell line. The arcidine derivative C\({}_{5}\)NH\({}_{2}\) has the most favorable cytotoxic profile in terms of cell selectivity.  
G-quadruplex; heterocycle compounds; scaffolds; drug-design +
Footnote †: journal: Accepted: 16 February 2022  
compounds, such as PhenDC3, have been used to broadly promote G4 formation in high-throughput sequencing of G4 structures [9]. Some of these G4 ligands have entered clinical trials, namely the fluoroquinolones CX-3543 and CX-5461, that selectively bind and stabilize a broad spectrum of G4 structures, including those harboured in c-MYC, c-KIT, and telomeres [10]. These fluoroquinolones interact with a G4 found in ribosomal DNA and disrupt the binding between these G4s and protein complexes, thereby inhibiting ribosome biogenesis [11]. Other novel ligands were reported to be selective for a sub-class of quadruplex structures, such as the diquinolinly-pyridine ligands, that show a preference for the parallel conformation of telomeric, c-MYC and c-KIT G4s [12], or ToxaP, which prefers an antiparallel fold [13].  
However, there is still a long way to go for the development of G4 ligands. Indeed, the multiplicity of potential targets, the variety of ligand binding sites and the differences in the ligand effects, in vitro and in vivo, make it difficult to unravel if the stabilization or destabilization of G4s promotes or inhibits gene expression. A major limitation for the clinical application of G4 ligands is related to their selectivity and potential side effects on normal cells. In this regard, it is important to screen different scaffolds and analyze the binding to G4s and the cellular effects on normal cells and cancer cells. In this context, we synthetized and tested some heterocyclic compounds that will be able to act as a scaffold for G4 ligand/drug design. Different scaffolds, such as quinoline, phenyl, acridine and phenanthroline (Figure 1), were investigated to their capacity to stabilize different DNA and RNA G4 structures by the FRET melting assay [14]. Additionally, the in silico molecular properties were predicted and the cytotoxicity effects were also evaluated in a lung cancer cell line (A549) vs. normal cell (NHDF).  
Figure 1: Chemical structures of the different scaffolds used in the study.