Date Published: December 27, 2013
Publisher: Public Library of Science
Author(s): Shuo Shi, Shane Gao, Tongcheng Cao, Jie Liu, Xing Gao, Jian Hao, Chunyan Lv, Hailiang Huang, Jun Xu, Tianming Yao, Andrea Cavalli.
Inhibition of telomerase by inducing/stabilizing G-quadruplex formation is a promising strategy to design new anticancer drugs. We synthesized and characterized a new dinuclear complex [(dmb)2Ru(obip)Ru(dmb)2]4+ (dmb = 4,4’-dimethyl-2,2’-bipyridine, obip = (2-(2-pyridyl)imidazo[4,5-f][1,10]phenanthroline) with high affinity for both antiparallel and mixed parallel / antiparallel G-quadruplex DNA. This complex can promote the formation and stabilize G-quadruplex DNA. Dialysis and TRAP experiments indicated that [(dmb)2Ru(obip)Ru(dmb)2]4+ acted as an excellent telomerase inhibitor due to its obvious selectivity for G-quadruplex DNA rather than double stranded DNA. In vitro co-culture experiments implied that [(dmb)2Ru(obip)Ru(dmb)2]4+ inhibited telomerase activity and hindered cancer cell proliferation without side effects to normal fibroblast cells. TUNEL assay indicated that inhibition of telomerase activity induced DNA cleavage further apoptosis in cancer cells. Therefore, RuII complex represents an exciting opportunity for anticancer drug design by specifically targeting cancer cell G-quadruplexes DNA.
Telomeres are believed to play a vital role in genome integrity by protecting the genomic DNA from degradation and deleterious recombination events such as end-to-end fusion, rearrangements, chromosomal translocations, and chromosomal loss [1,2]. Human telomeric DNA consists of tandem repeats of double-stranded DNA sequence (5′-TTAGGG): (5′-CCCTAA) with the 3′-end capped with a 100 to 200 nucleotides single-stranded overhang [3,4]. In suitable conditions, the overhang can readily fold into a four-stranded structure known as G-quadruplex through Hoogsteen hydrogen bonds (Figure 1A). Significantly, G-quadruplex has been suggested to act as a negative regulator of telomere elongation by inhibiting telomerase activity in vivo thus considered as a potential cancer therapy target [5-10]. Meanwhile, G-quadruplex structures in gene promoter regions were also investigated as a potential new class of therapeutic targets, since the promoter regions with their diverse sequences may provide unique scaffolds ideally for designing selective ligands [11,12]. Successful forming and stabilizing in vivo G-quadruplex may be an ideal strategy to inhibit telomere elongation and telomerase activity. Besides, some important G-quadruplexes formed by human telomeric RNA sequences were also proved to be attractive therapeutic targets [13-15]. In contrast to duplex structures, G-quadruplexes show a high degree of polymorphism in terms of topological features including individual strand orientation and loop connectivity. For example, the NMR structure of 5′-AG3[T2AG3]3-3′ (denoted 22AG) in the presence of Na+ was an antiparallel basket quadruplex (Figure 1B) , but the X-ray structure for the same sequence in the presence of K+ revealed a parallel propeller quadruplex . Furthermore, circular dichroism studies indicated that it favored a mixed parallel / antiparallel structure in the presence of K+ solution (Figure 1C) [18,19].
DNA polymorphism exerts a fascination on a large scientific community. It is worth mentioning that G-quadruplexes are often induced and further stabilized by the coordination of interstitial cations (e.g., Na+ and K+). This study finds that [(dmb)2Ru(obip)Ru(dmb)2]4+ can promote the human telomeric repeat 5′-AG3[T2AG3]3-3′ to fold into intramolecular antiparallel G-quadruplexes in the absence of Na+ or K+ cations. Clarification of the binding modes between G-quadruplex and ligand (complex) is a challenging job without crystallographic structural data. Herein, both fluorescence quenching and molecular modelling studies figure out their binding modes, through which [(dmb)2Ru(obip)Ru(dmb)2]4+ can strongly stabilize both the antiparallel and the mixed parallel/antiparallel G-quadruplex DNA. Moreover, [(dmb)2Ru(obip)Ru(dmb)2]4+ shows an obvious selectivity for G-quadruplex DNA rather than double stranded DNA and exhibits as a nanomolar potency inhibitor for telomerase. The telIC50 value is much lower than that of the most potent G-quadruplex interacting compounds reported in the literature. Biological function analysis demonstrated that [(dmb)2Ru(obip)Ru(dmb)2]4+ can significantly (P<0.05) inhibit cancer cell proliferation with minor/no side-effects to normal cells and decrease the telomerase activity of human cancer cells, implying its possibly cancer cell targeting when used as an anti-cancer drug. TUNEL assay indicated that telomerase inhibition caused genome deprotection by telomere and further DNA instability, which led to cell apoptosis. All these cellular results point to a promising candidate of [(dmb)2Ru(obip)Ru(dmb)2]4+ as a specific telomerase targeting anti-cancer drug even though further experiments on the underlying signaling pathways and animals models need to be performed. Source: http://doi.org/10.1371/journal.pone.0084419