Critical for tumour cell growth is the ability of cancer cells to proliferate indefinitely. In most cancers the target discovered to be responsible for this immortal lifespan is the enzyme telomerase, which is illegitimately activated in malignant cells. Telomerase is a reverse transcriptase that elongates telomeres, a process essential for long term proliferation. This enzyme is minimally composed of a catalytic (hTERT) and RNA (hTR) subunit. Telomerase has recently been validated as a target for anti-cancer therapy, as expression of a dominant-negative version of hTERT in a number of human cell lines inhibited enzyme activity and led to widespread apoptosis, while having no measurable effect on telomerase-negative cells. In ovarian and, as we now demonstrate, highly malignant prostate cancer cells, inhibition of telomerase activity by expression of the dominant-negative hTERT protein also abolished the ability of the cells to form tumours in nude mice. We propose to now convert this target into a screen for efictors of telomerase function. Telomerase represent a unique challenge in this regard, as the enzyme is exceedingly large and can not be produced recombinantly in sufficient quantities for high throughput screens. To overcome this limitation we have mapped two RNA-binding regions in hTERT absolutely essential for enzyme activity, which could be targetted by small molecules to inhibit telomerase activity by blocking hTR-hTERT interactions. We therefore propose to produce recombinant versions of these domains from bacteria and confirm their function by assaying hTR binding in vitro. A series of highly complex peptide libraries displayed on the surface of phage will then be screened for interactions with the recombinant RNA-binding proteins. Candidate peptides identified from these phage display screens will be assayed for their ability to inhibit telomerase protein-RNA interactions and telomerase activity. Identification of inhibitory peptides would not only validate these domains as targets for high throughput screens, but also provide first generation inhibitors that could be built upon.
