Ronald Pruzan

Reconstitution of human telomerase activity and identification of a minimal functional region of the human telomerase RNA

Telomerase is a ribonucleoprotein that catalyzes telomere elongation through the addition of TTAGGG repeats in humans. Activation of telomerase is often associated with immortalization of human cells and cancer. To dissect the human telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay. After removal of the essential 445 nucleotide human telomerase RNA (hTR) by micrococcal nuclease digestion of partially purified human telomerase, the addition of in vitro transcribed hTR reconstituted telomerase activity. The activity was dependent upon and specific to hTR. Using this assay, truncations at the 5′ and 3′ ends of hTR identified a functional region of hTR, similar in size to the full‐length telomerase RNAs from ciliates. This region is located between positions 1‐203. Furthermore, we found that residues 1‐44, 5′ to the template region (residues 46–56) are not essential for activity, indicating a minimal functional region is located between residues 44–203. Mutagenesis of full‐length hTR between residues 170–179, 180–189 or 190–199 almost completely abolished the ability of the hTR to function in the reconstitution of telomerase activity, suggesting that sequences or structures within this 30 nucleotide region are required for activity, perhaps by binding telomerase protein components.

TELOMERASE INHIBITORS – OLIGONUCLEOTIDE PHOSPHORAMIDATES AS POTENTIAL THERAPEUTIC AGENTS

We have designed, synthesized, and evaluated using physical, chemical and biochemical assays various oligonucleotide N3′ → P5′ phosphoramidates, as potential telomerase inhibitors. Among the prepared compounds were 2′-deoxy, 2′-hydroxy, 2′-methoxy, 2′-ribo-fluoro, and 2′-arabino-fluoro oligonucleotide phosphoramidates, as well as novel N3′ → P5′ thio-phosphoramidates. The compounds demonstrated sequence specific and dose dependent activity with IC50 values in the sub-nM to pM concentration range.

Oligonucleotide N3′ → P5′ Thio-phosphoramidate Telomerase Template Antagonists as Potential Anticancer Agents

Abstract Human telomerase is a reverse transcriptase that is expressed in essentially all cancer cells, but not in the vast majority of normal somatic cells. Therefore, the specific inhibition of telomerase activity in tumors might have significant beneficial therapeutic effects. We have designed and evaluated oligonucleotide N3′ → P5′ thio-phosphoramidates as telomerase template antagonists. In biochemical cell-free assays 11-13-mer thio-phosphoramidate oligonucleotides demonstrated sequence specific and dose dependent inhibition of telomerase with pico-molar IC50 values. Optimization of the oligonucleotide sequence and length resulted in the identification of a 13-mer-oligonucleotide thio-phosphoramidate GRN163 as a drug development candidate. In cell cultures GRN163 was able to inhibit telomerase activity in the absence of cationic lipid with ∼1 µM IC50 values. Telomerase inhibition by GRN163 produced gradual telomere shortening, followed by cellular senescence and/or apoptosis of cancer derived cell lines.

Novel short oligonucleotide conjugates as inhibitors of human telomerase.

Abstract A series of oligonucleotide conjugates were designed and synthesized as novel inhibitors of human telomerase. These compounds contain a relatively short (6–7-mer) oligonucleotide domain, with an N3′ → P5′ phosphoramidate (np) or thio-phosphoramidate (nps) backbone, targeted to the template region of the RNA component of the enzyme and various pendant groups attached to either their 5′- or preferably to the 3′- termini. The most potent compounds in the series inhibited telomerase with low nM IC50 values in biochemical assays whereas the cognate oligonucleotides without the pendant groups were significantly less active having IC50 values 100-1000-fold higher.

Stereopure oligonucleotide phosphorothioates as human telomerase substrates

To address the role of sulfur in the telomerase–primer recognition process, we investigated the effects of substitution of natural phosphodiester linked DNA telomerase substrates with phosphorothioate group-containing counterparts. Model dodecanucleotides with either stereorandom or stereopure all-Rp/Sp internucleoside phosphorothioate linkages were evaluated and the stereorandom models found to have slightly reduced affinity for the enzyme.