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...

The synthesis and evaluation for telomerase-inhibitory and quadruplex DNA binding properties of three related series of rationally designed trisubstituted acridine derivatives are described. These are substituted on the acridine ring at the 2,6,9; 2,7,9; and 3,6,9 positions. The ability of several of the most potent compounds to interact with and stabilize an intramolecular G-quadruplex DNA was evaluated by surface plasmon resonance methods, and affinities were found to correlate with potency in a telomerase assay. The interactions of a number of compounds with a parallel quadruplex DNA structure were simulated by molecular modeling methods. The calculated interaction energies were compared with telomerase activity and showed generally consistent correlations between quadruplex affinity and telomerase inhibition. These data support a model for the action of these compounds that involves the stabilization of intermediate quadruplex structures that inhibit the elongation of telomeric DNA by telomerase in tumor cells.

No abstract

G-Quadruplex DNAs are folded, non-Watson-Crick structures that can form within guanine-rich DNA sequences such as telomeric repeats. Previous studies have identified a series of trisubstituted acridine derivatives that are potent and selective ligands for G-quadruplex DNA. These ligands have been shown previously to inhibit the activity of telomerase, the specialized reverse transcriptase that regulates telomere length. The RecQ family of DNA helicases, which includes the Bloom's (BLM) and Werner's (WRN) syndrome gene products, are apparently unique among cellular helicases in their ability to efficiently disrupt G-quadruplex DNA. This property may be relevant to telomere maintenance, since it is known that the sole budding yeast RecQ helicase, Sgs1p, is required for a telomerase-independent telomere lengthening pathway reminiscent of the "ALT" pathway in human cells. Here, we show that trisubstituted acridine ligands are potent inhibitors of the helicase activity of the BLM and WRN proteins on both G-quadruplex and B-form DNA substrates. Inhibition of helicase activity is associated with both a reduction in the level of binding of the helicase to G-quadruplex DNA and a reduction in the degree to which the G-quadruplex DNA can support DNA-dependent ATPase activity. We discuss these results in the context of the possible utility of trisubstituted acridines as antitumor agents for the disruption of both telomerase-dependent and telomerase-independent telomere maintenance.