G4 Ligands

G-quadruplexes are four-stranded nucleic acids structures that can form in guanine-rich sequences. These four-stranded structures are widespread throughout the genome and play key roles in fundamental biological processes such as replication, transcription, and genome stability. Notably, G-quadruplexes are enriched in regulatory regions, including telomeres, gene promoters, and untranslated regions, where they influence gene expression and cellular homeostasis. Their presence has been linked to various human diseases, ranging from cancer to neurodegenerative and viral infections. Due to their biological relevance, G-quadruplexes have emerged as attractive targets for therapeutic intervention, leading to the development of small molecules capable of modulating their structure and function.

Functional ligands

Selective Cleavage

NDI-Cu-DETA is a novel oligonucleotide cleaving agent targeting G-quadruplex nucleic acids (G4), which has been synthesised and characterised by us. The presence of a NDI unit, acting simultaneously as excellent Cu(II) and G4 ligands, promotes an efficient cleavage by a catalytic reactive oxygen species (ROS)generation. This irreversible cleavage depends on the G4 structure and conformation occuring at the available loop phosphate bonds of the exposed bases. The results obtained confirm the formation and the involvement of hydroxyl radicals. Close proximity of Cu(II) coordination sphere to G4s, opens the opportunity to achieve G4 selective cleavage, dependent on structure and topology of the target.

J. Am. Chem. Soc., 2018, 140(44), 14528-14532

Chem. Eur. J., 2022, 28(35), e202200734

Selective Alkylation

In the recent past we have developed a class of G4 ligands acting through a dual covalent/non-covalent binding mode. Selective alkylation of G4 has been achieved by tethering modified naphthalene diimides (NDIs) to different alkylating moiety i.e. quinone methides (QMs) as activatable electrophiles. The resulting ligand/alkylating hybrid NDIs have been synthesized and tested with G-rich oligomers.

Reversible ligands

Biomolecules, 2025, 15(2), 311

Cancer

Targeting G-quadruplexes (G4s) has emerged as a promising strategy in cancer therapy due to their crucial role in genome regulation. These four-stranded nucleic acid structures are highly enriched in genomic regions associated with cancer, including telomeres, oncogene promoters, and replication origins. In tumor cells, G4s can influence key processes such as gene expression, genome stability, and DNA damage response, making them attractive targets for therapeutic intervention. Small molecules capable of stabilizing G4 structures have been shown to selectively modulate oncogene transcription, disrupt telomere maintenance, and induce DNA damage, ultimately leading to cancer cell death. However, most studies have focused on monomeric G4s, while growing evidence suggests that multimeric G4 assemblies, which better mimic physiological conditions, may play an even more significant role in genome regulation. In recent years, our research has been dedicated to exploring multimeric G4 structures as more relevant targets for drug discovery, aiming to develop ligands with enhanced selectivity and biological efficacy.

Bacteria

The information regarding the role and use of G4-ligands as therapeutic drug targets in bacteria is still scarce. Recently we tested the biological activity of a G4-DNA ligand library, based on the naphthalene diimide (NDI) pharmacophore, against both Gram-positive and Gram-negative bacteria. For the best compound identified, NDI-Pham, a different action mechanism was described for Gram-positive or negative bacteria. This asymmetric activity profile could be related to the different prevalence of putative G4-DNA structures in each group, the influence that they can exert on gene expression, and the different roles of the G4 structures in these bacteria, which seem to promote transcription in Gram-positive bacteria and repress transcription in Gram-negatives.

J. Med. Chem., 2021, 65(6), 4752-4766

ACS Inf. Dis., 2024, 10(2), 489-499

Viruses

G4s also play crucial roles in viral genomes, where they regulate key steps of the viral life cycle, including replication, transcription, and immune evasion. Several viruses, including Epstein-Barr virus (EBV), human papillomavirus (HPV), and herpes simplex virus (HSV), harbor G4-forming sequences that influence viral gene expression and pathogenicity. Among RNA viruses, G4s have been identified in flaviviruses, coronaviruses, and retroviruses, where they serve as potential regulatory elements and therapeutic targets. In the case of Human Immunodeficiency Virus (HIV), G4s have been mapped in critical genomic regions, such as the long terminal repeat (LTR), a key regulatory sequence controlling viral transcription. Notably, the LTR-III G-quadruplex plays a pivotal role in modulating HIV gene expression by influencing the recruitment of transcription factors and the activity of the viral promoter. The stabilization of LTR-III by small molecules has been explored as a potential antiviral strategy, highlighting the therapeutic potential of targeting G4 structures in HIV.

Parasites

Bioinformatic and Biophysical experiments showed that EBR1, a 29 nucleotide long highly repeated PQS in T. brucei, forms a stable G4 structure. G4 ligands based on carbohydrate conjugated naphthalene diimides (carb-NDIs) which bind G4’s including hTel, could bind EBR1 with selectivity versus dsDNA. These ligands showed important antiparasitic activity. IC50 values were in the nanomolar range against T.brucei with high selectivity against MRC-5 human cells. Starting from this family we develop binders in an attempt to understand the rules of activity and selectivity towards these particular G4

J. Med. Chem, 2024, 67(13), 10643-10654

Int. J. Biol. Macromol., 2024, 277(4), 134417

NEURODEGENERATIVE DISEASES

Novel G-quadruplex sequences, identified on the SNCA gene's transcription starting site and 5′-UTR of SNCA mRNAs, were experimentally confirmed for their stability through biophysical assays and in vitro experiments on human genomic DNA. Biological validation in differentiated SH-SY5Y cells revealed that well-known G-quadruplex ligands remarkably stabilized these structures, inducing the modulation of SNCA mRNAs expression, and the effective decrease in alpha-synuclein amount.

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