Targeting C-Myc Promoter: Helquats As Novel G-Quadruplex Stabilizing Ligands

Influencing stability of G-quadruplexes that can be formed in specific guanine-rich sequences has emerged as a promising route to anticancer drug design in recent years. These sequences are highly prevalent in human genome and occur in the telomeres as well as in promoters of certain genes upregulated in cancer (e.g. c-Myc, K-ras, VEGF, bcl-2, c-kit, or HIF-1α). Functional role of these G-quadruplex structures has been tested using G-quadruplex-stabilizing ligands in cell-based assays, resulting in suppression of transcription of oncogenes. As c-Myc deregulation is found in many types of human leukemia and lymphoma and its overexpression is often associated with disease progression, we chose it as our primary target.

We designed a library of helical structures with cationic aromatic moieties called helquats to find a novel class of G-quadruplex specific ligands. Their non-planar structure minimizes potential of intercalation into DNA duplex. To screen a library of about 1500 compounds synthesized in-house and find ligands that stabilize c-Myc G-quadruplex at low micromolar concentrations, we used G-quadruplex forming sequence NHE III₁ of c-Myc promoter in various biophysical and biochemical experiments such as FRET (Förster resonance energy transfer) melting experiments, CD spectroscopy, and DNA polymerase stop assay. We tested promising candidates for reducing c-Myc mRNA and protein expression in several leukemic cell lines and in reporter assay using an experimental vector with firefly luciferase under control of c-Myc promoter region containing either wild-type or mutated G-quadruplex NHE III₁ sequence. The most potent helquats showed more than 50 % inhibition of c-Myc mRNA and protein expression (at 25 μM, after 24 hours) in HL-60, Ramos, and U937 cell lines overexpressing c-Myc. On the other hand, no such an effect of helquats was observed in CA46 Burkitt lymphoma cells in which NHE III₁ element has been lost during the translocation. Similar results as with cell lines were obtained in primary cells with or without c-Myc translocation.

Our results provide the foundation for the development of new helquat derivatives as effective inhibitors of c-Myc expression and as potential anticancer agents in hematooncology.

This study was supported by IOCB, AS CR, v.v.i. (RVO: 61388963), Project InterBioMed LO1302 from Ministry of Education of the Czech Republic and PRVOUK-27/LF1/1