Irene Bessi

Spectroscopic, molecular modeling, and NMR-spectroscopic investigation of the binding mode of the natural alkaloids berberine and sanguinarine to human telomeric G-quadruplex DNA.

G-quadruplex structures can be formed at the single-stranded overhang of telomeric DNA, and ligands able to stabilize this structure have recently been identified as potential anticancer drugs. Among the potential G-quadruplex binders, we have studied the binding ability of berberine and sanguinarine, two members of the alkaloid family, an important class of natural products long known for medicinal purpose. Our spectroscopic (CD, NMR, and fluorescence) studies and molecular modeling approaches revealed binding modes at ligand-complex stoichiometries >1:1 and ligand self-association induced by DNA for the interactions of the natural alkaloids berberine and sanguinarine with the human telomeric G-quadruplex DNA.

A nucleus-imaging probe that selectively stabilizes a minor conformation of c-MYC G-quadruplex and down-regulates c-MYC transcription in human cancer cells

The c-MYC proto-oncogene is a regulator of fundamental cellular processes such as cell cycle progression and apoptosis. The development of novel c-MYC inhibitors that can act by targeting the c-MYC DNA G-quadruplex at the level of transcription would provide potential insight into structure-based design of small molecules and lead to a promising arena for cancer therapy. Herein we report our finding that two simple bis-triazolylcarbazole derivatives can inhibit c-MYC transcription, possibly by stabilizing the c-MYC G-quadruplex. These compounds are prepared using a facile and modular approach based on Cu(I) catalysed azide and alkyne cycloaddition. A carbazole ligand with carboxamide side chains is found to be microenvironment-sensitive and highly selective for “turn-on” detection of c-MYC quadruplex over duplex DNA. This fluorescent probe is applicable to visualize the cellular nucleus in living cells. Interestingly, the ligand binds to c-MYC in an asymmetric fashion and selects the minor-populated conformer via conformational selection.

Photoresponsive Formation of an Intermolecular Minimal G-Quadruplex Motif.

The ability of three different bifunctional azobenzene linkers to enable the photoreversible formation of a defined intermolecular two-tetrad G-quadruplex upon UV/Vis irradiation was investigated. Circular dichroism and NMR spectroscopic data showed the formation of G-quadruplexes with K(+)  ions at room temperature in all three cases with the corresponding azobenzene linker in an E conformation. However, only the para-para-substituted azobenzene derivative enables photoswitching between a nonpolymorphic, stacked, tetramolecular G-quadruplex and an unstructured state after E-Z isomerization.

A Fluorescent Guanosine Dinucleoside as a Selective Switch‐On Sensor for c‐myc G‐Quadruplex DNA with Potent Anticancer Activities

Like likes like! A novel fluorescent C2 -symmetric guanosine-based dinucleoside has been engineered by chemical ligation of two guanosine units with a biocompatible dansyl tag. The nucleoside exhibits high selectivity for c-myc G-quadruplex DNA through fluorescence enhancement over duplex DNA and other promoter G-quadruplexes (see scheme). It stains the nucleus preferentially, arrests the cell cycle at the G2/M phase, inhibits cell growth, and induces apoptosis in A375 cancer cells.

Fluorescent Dansyl-Guanosine Conjugates that Bind c-MYC Promoter G-Quadruplex and Downregulate c-MYC Expression.

The four‐stranded G‐quadruplex present in the c‐MYC P1 promoter has been shown to play a pivotal role in the regulation of c‐MYC transcription. Small‐molecule compounds capable of inhibiting the c‐MYC promoter activity by stabilising the c‐MYC G‐quadruplex could potentially be used as anticancer agents. In this context, here we report the synthesis of dansyl‐guanosine conjugates through one‐pot modular click reactions. The dansyl‐guanosine conjugates can selectively detect c‐MYC G‐quadruplex over other biologically relevant quadruplexes and duplex DNA and can be useful as staining reagents for selective visualisation of c‐MYC G‐quadruplex over duplex DNA by gel electrophoresis. NMR spectroscopic titrations revealed the preferential binding sites of these dansyl ligands to the c‐MYC G‐quadruplex. A dual luciferase assay and qRT‐PCR revealed that a dansyl‐bisguanosine ligand represses the c‐MYC expression, possibly by stabilising the c‐MYC G‐quadruplex.

Cell penetrating thiazole peptides inhibit c-MYC expression via site-specific targeting of c-MYC G-quadruplex

Abstract The structural differences among different G-quadruplexes provide an opportunity for site-specific targeting of a particular G-quadruplex structure. However, majority of G-quadruplex ligands described thus far show little selectivity among different G-quadruplexes. In this work, we delineate the design and synthesis of a crescent-shaped thiazole peptide that preferentially stabilizes c-MYC quadruplex over other promoter G-quadruplexes and inhibits c-MYC oncogene expression. Biophysical analysis such as Förster resonance energy transfer (FRET) melting and fluorescence spectroscopy show that the thiazole peptide TH3 can selectively interact with the c-MYC G-quadruplex over other investigated G-quadruplexes and duplex DNA. NMR spectroscopy reveals that peptide TH3 binds to the terminal G-quartets and capping regions present in the 5′- and 3′-ends of c-MYC G-quadruplex with a 2:1 stoichiometry; whereas structurally related distamycin A is reported to interact with quadruplex structures via groove binding and end stacking modes with 4:1 stoichiometry. Importantly, qRT-PCR, western blot and dual luciferase reporter assay show that TH3 downregulates c-MYC expression by stabilizing the c-MYC G-quadruplex in cancer cells. Moreover, TH3 localizes within the nucleus of cancer cells and exhibits antiproliferative activities by inducing S phase cell cycle arrest and apoptosis.

Human Telomeric G-Quadruplex Selective Fluoro-Isoquinolines Induce Apoptosis in Cancer Cells

Small molecules that stabilize G-quadruplex structures in telomeres can prevent telomerase enzyme mediated telomere lengthening and subsequently lead to cell death. We herein report two fluoro-isoquinoline derivatives IQ1 and IQ2 as selective ligands for human telomeric G-quadruplex DNA. IQ1 and IQ2 containing different triazolyl side chains have been synthesized by Cu (I) catalyzed azide-alkyne cycloaddition. Fluorescence Resonance Energy Transfer (FRET) melting assay and fluorescence binding titrations indicate that both these ligands exhibit binding preference for telomeric G-quadruplex DNA ( h-TELO) over other promoter DNA quadruplexes and duplex DNA. However, ligand IQ1, containing pyrrolidine side chains, is capable of discriminating among quadruplexes by showing higher affinity toward h-TELO quadruplex DNA. On the contrary, IQ2, containing benzamide side chains, interacts with all the investigated quadruplexes. NMR analysis suggests that IQ1 interacts strongly with the external G-quartets of h-TELO. Biological studies reveal that IQ1 is more potent than IQ2 in inhibiting telomerase activity by selectively interacting with telomeric DNA G-quadruplex. Moreover, a dual luciferase reporter assay indicates that IQ1 is unable to reduce the cellular expression of c-MYC and BCL2 at transcriptional level. Significantly, IQ1 mostly stains the nucleus, induces cell cycle arrest in G0/G1 phase, triggers apoptotic response in cancer cells, and activates caspases 3/7.