Structure-based design of selective and potent G quadruplex-mediated telomerase inhibitors

Abstract

The telomerase enzyme is a potential therapeutic target in many human cancers. A series of potent inhibitors has been designed by computer modeling, which exploit the unique structural features of quadruplex DNA. These 3,6,9-trisubstituted acridine inhibitors are predicted to interact selectively with the human DNA quadruplex structure, as a means of specifically inhibiting the action of human telomerase in extending the length of single-stranded telomeric DNA. The anilino substituent at the 9-position of the acridine chromophore is predicted to lie in a third groove of the quadruplex. Calculated relative binding energies predict enhanced selectivity compared with earlier 3,6-disubstituted compounds, as a result of this substituent. The ranking order of energies is in accord with equilibrium binding constants for quadruplex measured by surface plasmon resonance techniques, which also show reduced duplex binding compared with the disubstituted compounds. The 3,6,9-trisubstututed acridines have potent in vitro inhibitory activity against human telomerase, with EC50 values of up to 60 nM. T he telomeric ends of chromosomes consist of tandem repeats of simple guanine-rich DNA protein-associated motifs whose function is to protect the ends from unwanted DNA damage-repair, recombination, and end-fusions. In eukaryotics the repeat is TTAGGG, with telomere length varying between ca. 5 and 15 kb (1, 2). Cancer cells typically have short telomeres, whereas stem cell telomere length tends to be at the high end of this range. The terminal 150-200 bases at the 3Ј end of human telomeres form a single-stranded overhang, whose exact structure is not fully established, although loop-type arrangements have been suggested from electron microscope studies (3). Telomeres shorten in somatic cells on each round of replication, by 50-200 bases, as a consequence of the inability of DNA polymerase to fully replicate the ends (4). Once telomeres reach a critically short length, cells enter a senescent state and do not replicate further (5). By contrast, the short telomeres in tumor cells are stable in length, maintained by the action of a specialized DNA polymerase, the telomerase enzyme complex, which catalyses the synthesis of further telomere repeats (6). Telomerase is activated in 80-90% of human tumors and is undetectable in most normal somatic cells (7). This activation has been shown to be a key step in the immortalization process in human cells, leading to tumorigenesis (8). A small proportion of tumor cells have an alternative telomere maintenance pathway (ALT) which appears to be independent of telomerase and involves recombination events. Inhibition of telomerase by a dominant negative mutant (9, 10), or by synthetic oligonucleotides targeted to the RNA template (11), leads to telomere shortening, growth arrest and apoptosis for tumor cells in culture. Telomerase is thus a highly attractive target for selective anti-cancer therapy (12).We have focused on the rational discovery of small-molecule telomerase inhibitors with pharma...

Interactions of cryptolepine and neocryptolepine with unusual DNA structures

“…Cryptolepine stabilizes the F21MB quadruplex by 3 °C at 3 µM, whereas the best G4 ligands stabilize by 20 °C or more at 1 µM compound [44]. It is not a potent telomerase inhibitor (IC 50 = 9.4 µM); the best inhibitors described so far have an IC 50 of 50 nM or lower [37,44,[48][49][50].…”

Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration

“…BRACO-19 was designed to bind specifically to G-quadruplexes with the assumption that each of its chains would occupy a groove of its ligand, as it stacks between two G-quadruplexes: its aromatic planar core stacks between two G-quadruplexes, and each of the two positively charged chains occupies the groove of each bound G-quadruplex (35,36,43). As a positive control, the BG4 antibody was used.…”

“…To achieve this, we used BRACO-19, a 3,6,9-trisubstituted acridine derivative designed by molecular modeling to interact with and stabilize the G-quadruplex DNA structures formed in human telomeres while displaying very low affinity toward duplex DNA (35). BRACO-19 binds and stabilizes G-quadruplexes present in the single-stranded region of telomeres.…”

Structure-based design of selective high-affinity telomeric quadruplex-binding ligands

“…1) has high affinity for human telomeric quadruplex DNAs and is a potent inhibitor of the telomerase enzyme. 10 It has selective cytotoxic activity against a range of human tumour cell lines and shows antitumour activity against xenograft models. 11 The BRACO-19 molecule was designed using qualitative molecular modeling, with the crystal structure of the native parallel human telomeric quadruplex as a template.…”

“…It was rationalized that each of the three substituents emanating from the acridine core of BRACO-19 would be able to interact with a quadruplex groove. 10 This feature would, it was suggested, provide binding selectivity over duplex DNA, which has just two grooves. A more recent crystal structure of a BRACO-19 complex with a bimolecular quadruplex has confirmed the essential correctness of this hypothesis and has also provided a more detailed view of the interactions involved.…”