Tailoring a lead-like compound targeting multiple G-quadruplex structures.

A focused library of analogs of a lead-like G-quadruplex (G4) targeting compound (4), sharing a furobenzoxazine naphthoquinone core and differing for the pendant groups on the N-atom of the oxazine ring, has been here analyzed with the aim of developing more potent and selective ligands. These molecules have been tested vs. topologically different G4s by the G4-CPG assay, an affinity chromatography-based method for screening putative G4 ligands. The obtained results showed that all these compounds were able to bind several G4 structures, both telomeric and extra-telomeric, thus behaving as multi-target ligands, and two of them fully discriminated G4 vs. duplex DNA. Biological assays proved that almost all the compounds produced effective DNA damage, showing marked antiproliferative effects on tumor cells in the low µM range. Combined analysis of the G4-CPG binding assays and biological data led us to focus on compound S4-5, proved to be less cytotoxic than the parent compound 4 on normal cells. An in-depth biophysical characterization of the binding of S4-5 to different G4s showed that the here identified ligand has higher affinity for the G4s and higher ability to discriminate G4 vs. duplex DNA than 4. Molecular docking studies, in agreement with the NMR data, suggest that S4-5 interacts with the accessible grooves of the target G4 structures, giving clues for its increased G4 vs. duplex selectivity.

Common G-Quadruplex Binding Agents Found to Interact With i-Motif-Forming DNA: Unexpected Multi-Target-Directed Compounds.

G-quadruplex (G4) and i-motif (iM) are four-stranded non-canonical nucleic acid structural arrangements. Recent evidences suggest that these DNA structures exist in living cells and could be involved in several cancer-related processes, thus representing an attractive target for anticancer drug discovery. Efforts toward the development of G4 targeting compounds have led to a number of effective bioactive ligands. Herein, employing several biophysical methodologies, we studied the ability of some well-known G4 ligands to interact with iM-forming DNA. The data showed that the investigated compounds are actually able to interact with both DNA in vitro, thus acting de facto as multi-target-directed agents. Interestingly, while all the compounds stabilize the G4, some of them significantly reduce the stability of the iM. The present study highlights the importance, when studying G4-targeting compounds, of evaluating also their behavior toward the i-motif counterpart.

Targeting the BCL2 Gene Promoter G-Quadruplex with a New Class of Furopyridazinone-Based Molecules.

Targeting of G-quadruplex-forming DNA in the BCL2 gene promoter to inhibit the expression of anti-apoptotic Bcl-2 protein is an attractive approach to cancer treatment. So far, efforts made in the discovery of molecules that are able to target the BCL2 G-quadruplex have succeeded mainly in the identification of ligands with poor drug-like properties. Here, a small series of furo[2,3-d]pyridazin-4(5H)-one derivatives were evaluated as a new class of drug-like G-quadruplex-targeting compounds. Biophysical studies showed that two derivatives could effectively bind to BCL2 G-quadruplex with good selectivity. Moreover, one such ligand was found to appreciably inhibit BCL2 gene transcription, with a substantial decrease in protein expression levels, and also showed significant cytotoxicity toward the Jurkat human T-lymphoblastoid cell line.

Discovery of the first dual G-triplex/G-quadruplex stabilizing compound: a new opportunity in the targeting of G-rich DNA structures?

BACKGROUND: Guanine-rich DNA motifs can form non-canonical structures known as G-quadruplexes, whose role in tumorigenic processes makes them attractive drug-target candidates for cancer therapy. Recent studies revealed that the folding and unfolding pathways of G-quadruplexes proceed through a quite stable intermediate named G-triplex. METHODS: Virtual screening was employed to identify a small set of putative G-triplex ligands. The G-triplex stabilizing properties of these compounds were analyzed by CD melting assay. DSC, non-denaturing gel electrophoresis, NMR and molecular modeling studies were performed to investigate the interaction between the selected compound 1 and G-rich DNA structures. Cytotoxic activity of 1 was evaluated by MTT cell proliferation assay. RESULTS: The experiments led to the identification of a promising hit that was shown to bind preferentially to G-triplex and parallel-stranded G-quadruplexes over duplex and antiparallel G-quadruplexes. Molecular modeling results suggested a partial end-stacking of 1 to the external G-triad/G-tetrads as a binding mode. Biological assays showed that 1 is endowed with cytotoxic effect on human osteosarcoma cells. CONCLUSIONS: A tandem application of virtual screening along with the experimental investigation was employed to discover a G-triplex-targeting ligand. Experiments revealed that the selected compound actually acts as a dual G-triplex/G-quadruplex stabilizer, thus stimulating further studies aimed at its optimization. GENERAL SIGNIFICANCE: The discovery of molecules able to bind and stabilize G-triplex structures is highly appealing, but their transient state makes challenging their recognition. These findings suggest that the identification of ligands with dual G-triplex/G-quadruplex stabilizing properties may represent a new route for the design of anticancer agents targeting the G-rich DNA structures. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Toward the Development of Specific G-Quadruplex Binders: Synthesis, Biophysical, and Biological Studies of New Hydrazone Derivatives.

G-Quadruplex-binding compounds are currently perceived as possible anticancer therapeutics. Here, starting from a promising lead, a small series of novel hydrazone-based compounds were synthesized and evaluated as G-quadruplex binders. The in vitro G-quadruplex-binding properties of the synthesized compounds were investigated employing both human telomeric and oncogene promoter G-quadruplexes with different folding topologies as targets. The present investigation led to the identification of potent G-quadruplex stabilizers with high selectivity over duplex DNA and preference for one G-quadruplex topology over others. Among them, selected derivatives have been shown to trap G-quadruplex structures in the nucleus of cancer cells. Interestingly, this behavior correlates with efficient cytotoxic activity in human osteosarcoma and colon carcinoma cells.