High throughput screening of a new fluorescent G-quadruplex ligand having telomerase inhibitory activity in human A549 cells

Genome-wide analysis showed that putative G-quadruplex DNA structures are prevalent in the human genome. The presence of G-quadruplex structure in the telomere and promoter region of certain oncogenes inspired people to use G-quadruplex ligand as anti-cancer agents. G-quadruplex structures, stabilized by ligand at telomere are resolved by telomerase making the cancer cells resistant to G-quadruplex ligand. So, identification of a new G-quadruplex ligand having anti-telomerase activity would be a promising strategy for cancer therapy as about 85% of human cancers are telomerase positive. A set of the drug-like compounds were screened from the ZINC database randomly and 2284 ligands were chosen following Lipinski rule of five that were docked with five different G-quadruplex DNA sequences in idock. We screened 43 potential G-quadruplex binders using Z-score as a normalization scoring function. The compound (ZINC ID- 05220992) gave the best score (average idock = -10.17 kcal/mol, average normalized idock = -3.42). We performed G4 FID assay, CD analysis to understand its binding with three different G-quadruplex DNA sequences, and checked its anti-telomerase activity in A549 cells using TRAP assay. We observed that this compound had an intrinsic fluorescence, capability to stain live cells with a blue fluorescence, and a specific affinity to only 22AG out of three different G-quadruplex DNA sequences under study. It showed cytotoxicity, good permeability to live cells, and a significant reduction of telomerase activity in human A549 cells at a very low dose. So, this compound has strong potential to be an anti-cancer drug.

QSAR, pharmacophore modeling and molecular docking studies to identify structural alerts for some nitrogen heterocycles as dual inhibitor of telomerase reverse transcriptase and human telomeric G-quadruplex DNA

Background Telomerase reverse transcriptase (TERT) and human telomeric G-quadruplex DNA are amongst the favorable target for researchers to discover novel and more effective anticancer agents. To understand and elucidate structure activity relationship and mechanism of inhibition of telomerase reverse transcriptase (TERT) and human telomeric G-quadruplex DNA, a QSAR modeling and molecular docking were conducted. Results Two robust QSAR model were obtained which consist of full set QSAR model (R2: 0.8174, CCCtr: 0.8995, Q2loo: 0.7881, Q2LMO: 0.7814) and divided set QSAR model (R2: 0.8217, CCCtr: 0.9021, Q2loo: 0.7886, Q2LMO: 0.7783, Q2-F1: 0.7078, Q2-F2: 0.6865, Q2-F3: 0.7346) for envisaging the inhibitory activity of telomerase reverse transcriptase (TERT) and human telomeric G-quadruplex DNA. The analysis reveals that carbon atom exactly at 3 bonds from aromatic carbon atom, nitrogen atom exactly at six bonds from planer nitrogen atom, aromatic carbon atom within 2 A0 from the center of mass of molecule and occurrence of element hydrogen within 2 A0 from donar atom are the key pharmacophoric features important for dual inhibition of TERT and human telomeric G-quadruplex DNA. To validate this analysis, pharmacophore modeling and the molecular docking is performed. Molecular docking analysis support QSAR analysis and revealed that, dual inhibition of TERT and human telomeric DNA is mainly contributed from hydrophobic and hydrogen bonding interactions. Conclusion The findings of molecular docking, pharmacophore modelling, and QSAR are all consistent and in strong agreement. The validated QSAR analyses can detect structural alerts, pharmacophore modelling can classify a molecule's consensus pharmacophore involving hydrophobic and acceptor regions, whereas docking analysis can reveal the mechanism of dual inhibition of telomerase reverse transcriptase (TERT) and human telomeric G-quadruplex DNA. The combination of QSAR, pharmacophore modeling and molecular docking may be useful for the future drug design of dual inhibitors to combat the devastating issue of resistance. Graphical abstract

Studies on the Interactions of 3,11-Difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium and Insulin with the Quadruplex-Forming Oligonucleotide Sequence a2 from the Insulin-Linked Polymorphic Region

Recently, several quadruplex-DNA-forming sequences have been identified in the insulin-linked polymorphic region (ILPR), which is a guanine-rich oligonucleotide sequence in the promoter region of insulin. The formation of this non-canonical quadruplex DNA (G4-DNA) has been shown to be involved in the biological activity of the ILPR, specifically with regard to its interplay with insulin. In this context, this contribution reports on the investigation of the association of the quadruplex-forming ILPR sequence a2 with insulin as well as with the well-known G4-DNA ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium (1), also named RHPS4, by optical and NMR spectroscopy. CD- and NMR-spectroscopic measurements confirmed the preferential formation of an antiparallel quadruplex structure of a2 with four stacked guanine quartets. Furthermore, ligand 1 has high affinity toward a2 and binds by terminal π stacking to the G1–G11–G15–G25 quartet. In addition, the spectroscopic studies pointed to an association of insulin to the deoxyribose backbone of the loops of a2.

A quadruplex-DNA binder functionalised with a SPECT probe for in vivo imaging

Guanine-rich sequences of DNA can fold into intramolecular tetra-helical assemblies known as G-quadruplexes (G4). Their formation in vivo has been associated to a range of biological functions and therefore they have been identified as potential drug targets. Consequently, a broad range of small molecules have been developed to target quadruplexes. However, to date, there is very limited information of the biodistribution of quadruplex binders in whole organisms. Here, we report the functionalisation of a well-established G4 DNA binder (based on a square planar platinum(II)-salphen complex) with two different radiolabelled complexes. An 111In-conjugate was successfully used to assess its in vivo distribution in a mouse tumour model using single-photon emission computed tomography (SPECT) imaging. These studies have shown the overall biodistribution of the G4 DNA binder highlighting its accumulation in the tumour.

Interaction of 9-Methoxyluminarine with Different G-Quadruplex Topologies: Fluorescence and Circular Dichroism Studies

The interactions of G–quadruplexes of different topologies with highly fluorescent 9-methoxyluminarine ligand 9-MeLM were investigated by fluorescence and circular dichroism spectroscopy. The results showed that 9-methoxyluminarine was able to interact and did not destabilize any investigated molecular targets. The studied compound was selectively quenched by parallel c-MYC G-quadruplex DNA, whereas hybrid and antiparallel G4 topology caused only a negligible decrease in the fluorescence of the ligand. A high decrease of fluorescence of the ligand after binding with c-MYC G-quadruplex suggests that this molecule can be used as a selective probe for parallel G-quadruplexes.