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The HRAS promoter contains immediately upstream of the transcription start site two neighboring G-elements, each capable of folding into a G-quadruplex structure. We have previously found that these G-quadruplexes bind to the zinc-finger transcription factors MAZ and Sp1. In the present study we have examined the interaction between the HRAS promoter and MAZ, demonstrating for the first time that the protein unfolds the G-quadruplex structures. We also demonstrate that MAZ-GST, in the presence of the complementary strands, promotes a rapid transformation of the two HRAS quadruplexes into duplexes. By a mutational analysis of the HRAS G-elements, we dissected the MAZ-binding sites from the quadruplex-forming motifs, finding that the two neighboring G-quadruplexes bring about a dramatic repression of transcription, in a synergistic manner. We also discovered that the two G-quadruplexes are strong targets for small anticancer molecules. We found that a cell-penetrating anthratiophenedione (ATPD-1), which binds tightly to the G-quadruplexes (ΔT > 15°C), promotes the total extinction of HRAS transcription. In contrast, when one of the two G-quadruplexes was abrogated by point mutations, ATPD-1 repressed transcription by only 50%. Our study provides relevant information for the rationale design of targeted therapy drugs specific for the HRAS oncogene.

We prepared a series of anthrathiophenediones (ATPDs) with guanidino-alkyl side chains of different length (compounds 1, 10-13). The aim was to investigate their interaction with DNA and RNA G-quadruplexes, their uptake in malignant and nonmalignant cells, and their capacity to modulate gene expression and inhibit cell growth. Flow cytometry showed that the ATPDs enter more efficiently in malignant T24 bladder cells than in nonmalignant embryonic kidney 293 or fibroblast NIH 3T3 cells. In T24 malignant cells, compound 1, with two ethyl side chains, is taken up by endocytosis, while 12 and 13, with respectively propyl and butyl side chains, are transported by passive diffusion. The designed ATPDs localize in the cytoplasm and nucleus and tightly bind to DNA and RNA G-quadruplexes. They also decrease HRAS expression, increase the cell population in G0/G1, and strongly inhibit proliferation in malignant T24 bladder cells, but not in nonmalignant 293 or NIH 3T3 cells.

The human KRAS transcript contains a G-rich 5'-UTR sequence (77% GC) harboring several G4 motifs capable to form stable RNA G-quadruplex (RG4) structures that can serve as targets for small molecules. A biotin-streptavidin pull-down assay showed that 4,11-bis(2-aminoethylamino)anthra[2,3-b]furan-5,10-dione (2a) binds to RG4s in the KRAS transcript under low-abundance cellular conditions. Dual-luciferase assays demonstrated that 2a and its analogue 4,11-bis(2-aminoethylamino)anthra[2,3-b]thiophene-5,10-dione (2b) repress translation in a dose-dependent manner. The effect of the G4-ligands on Panc-1 cancer cells has also been examined. Both 2a and 2b efficiently penetrate the cells, suppressing protein p21KRAS to <10% of the control. The KRAS down-regulation induces apoptosis together with a dramatic reduction of cell growth and colony formation. In summary, we report a strategy to suppress the KRAS oncogene in pancreatic cancer cells by means of small molecules binding to RG4s in the 5'-UTR of mRNA.

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A number of newly discovered natural products, the perspective directions for chemical modifications to optimize the anticancer properties, and novel intracellular targets demonstrate that anthracene- 9,10-diones deserve further in-depth investigation as an important source of drug candidates.

Anthraquinones and their analogues, in particular heteroarene-fused anthracendiones, are prospective scaffolds for new compounds with improved antitumor characteristics. We herein report the use of a 'scaffold hopping' approach for the replacement of the core structure in the previously discovered hit compound naphtho[2,3-f]indole-5,10-dione 2 with an alternative anthra[2,3-b]furan-5,10-dione scaffold. Among 13 newly synthesized derivatives the majority of 4,11-dihydroxy-2-methyl-5,10-dioxoanthra[2,3-b]furan-3-carboxamides demonstrated a high antiproliferative potency against a panel of wild type and drug resistant tumor cell lines, a property superior over the reference drug doxorubicin or lead naphtho[2,3-f]indole-5,10-dione 2. At low micromolar concentrations the selected derivative of (R)-3-aminopyrrolidine 3c and its stereoisomer (S)-3-aminopyrrolidine 3d caused an apoptotic cell death preceded by an arrest in the G2/M phase. Studies of intracellular targets showed that 3c and 3d formed stable intercalative complexes with the duplex DNA as determined by spectral analysis and molecular docking. Both 3c and 3d attenuated topoisomerase 1 and 2 mediated unwinding of the supercoiled DNA via a mechanism different from conventional DNA-enzyme tertiary complex formation. Furthermore, 3d decreased the activity of selected human protein kinases in vitro, indicating multiple targeting by the new chemotype. Finally, 3d demonstrated an antitumor activity in a model of murine intraperitoneally transplanted P388 leukemia, achieving the increase of animal life span up to 262% at tolerable doses. Altogether, the 'scaffold hopping' demonstrated its productivity for obtaining new perspective antitumor drug candidates.

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