BackgroundTelomeric 3′ overhangs can fold into a four-stranded DNA structure termed G-quadruplex (G4), a formation which inhibits telomerase. As telomerase activation is crucial for telomere maintenance in most cancer cells, several classes of G4 ligands have been designed to directly disrupt telomeric structure.MethodsWe exposed brain tumor cells to the G4 ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) and investigated proliferation, cell cycle dynamics, telomere length, telomerase activity and activated c-Myc levels.ResultsAlthough all cell lines tested were sensitive to RHPS4, PFSK-1 central nervous system primitive neuroectodermal cells, DAOY medulloblastoma cells and U87 glioblastoma cells exhibited up to 30-fold increased sensitivity compared to KNS42 glioblastoma, C6 glioma and Res196 ependymoma cells. An increased proportion of S-phase cells were observed in medulloblastoma and high grade glioma cells whilst CNS PNET cells showed an increased proportion of G1-phase cells. RHPS4-induced phenotypes were concomitant with telomerase inhibition, manifested in a telomere length-independent manner and not associated with activated c-Myc levels. However, anti-proliferative effects were also observed in normal neural/endothelial cells in vitro and ex vivo.ConclusionThis study warrants in vivo validation of RHPS4 and alternative G4 ligands as potential anti-cancer agents for brain tumors but highlights the consideration of dose-limiting tissue toxicities.

Guanine-rich nucleic acids can fold into four-stranded G-quadruplex structures which are found in telomeric DNA repeats as well as in sequences in the promoter and other regulatory regions of genes. Small molecules that can selectively bind and stabilize quadruplex structure are of increasing significance as potential anticancer agents. We recently reported a series of novel biaryl polyamides with significant selectively towards G-quadruplex compared to duplex DNA, and modest selectivity between different quadruplex types such as c-kit1, c-kit2 and HT4 (Chemical Commun., 4097, 2009). These molecules were based on a distamycin scaffold but included biaryl building blocks in place of pyrroles to switch preference from duplex to quadruplex DNA. This alteration in shape ensured that the molecules had low affinity for duplex DNA while increasing their interaction with G-quadruplex structure, since the ligands had similar dimensions. We have now synthesized a 34-member second-generation biaryl polyamide library based on the previously obtained structural information. Initial assessment of the G-quadruplex interaction of library members was carried out using a FRET-based melting assay. Two compounds (KN-88 and KN-119) containing hybrid benzofused and biaryl building blocks provided significant selective stabilization of human Telomeric G-quadruplex. At 1 µM they stabilize human Telomeric G-quadruplex by 21 and 18 °C, respectively, while showing insignificant affinity for duplex DNA. FRET competition assays with C-kit quadruplexes and CT DNA further confirmed selective stabilization of telomeric quadruplexes. CD titrations of KN-88 and KN-119 with h-telo, c-kit1 and c-kit2 quadruplex-forming DNA sequences showed concentration-dependent enhancement of major CD signals, and in some cases the ligands induced the folding of a particular quadruplex structure. Kd values for KN-88 and KN-119 for telomeric quadruplex sequences were determined by CD titration and were found to be 2.8 × 106 and 7.0 × 106, respectively. Short-term growth inhibitory experiments against a panel of tumour cell lines (MiaPaCa2, A549, MCF7, HeLa, U87MG and A431) gave low micromolar IC50 values for these two compounds, and significant selectivity for tumour cell lines compared to the non-tumour fibroblast WI38 cell line. Given their low molecular weight, good water solubility and cellular penetration properties, molecules of this type have potential as therapeutic agents and reagents that can selectively probe quadruplex structure and/or selectively down-regulate signaling pathways in cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2517. doi:10.1158/1538-7445.AM2011-2517