Allosteric inhibitors of telomerase: oligonucleotide N3'->P5' phosphoramidates

Abstract

Telomerase is a ribonucleoprotein responsible for maintaining telomeres in nearly all eukaryotic cells. The enzyme is able to utilize a short segment of its RNA subunit as the template for the reverse transcription of d(TTAGGG) repeats onto the ends of human chromosomes. Transfection with telomerase was shown to confer immortality on several types of human cells. Moreover, telomerase activation appears to be one of the key events required for malignant transformation of normal cells. Inhibition of telomerase activity in transformed cells results in the cessation of cell proliferation in cultures and provides the rationale for the selection of telomerase as a target for anticancer therapy. Using oligonucleotide N3'-->P5' phosphoramidates (NPs) we have identified a region of the human telomerase RNA subunit (hTR) approximately 100 nt downstream from the template region whose structural integrity appears crucial for telomerase enzymatic activity. The oligonucleotides targeted to this segment of hTR are potent and specific inhibitors of telomerase activity in biochemical assays. Mutant telomerase, in which 3 nt of hTR were not complementary to a 15 nt NP, was found to be refractory to inhibition by that oligonucleotide. We also demonstrated that the binding of NP, oligonucleotides to this hTR allosteric site results in a marked decrease in the affinity of a telomerase substrate (single-stranded DNA primer) for the enzyme.

Mapping Targetable Sites on Human Telomerase RNA Pseudoknot/Template Domain Using 2′-OMe RNA-interacting Polynucleotide (RIPtide) Microarrays

“…Interestingly, we found that several sequenceextended versions of cluster IV RIPtides showed nanomolar IC 50 values in TRAP assays (supplemental Table 1, entries [35][36][37][38][39][40]. These observations are consistent with previous observations that longer DNA oligonucleotides targeting an overlapping region in hTR exhibit inhibitory activity against telomerase in vitro (45).…”

Telomerase inhibition with an oligonucleotide telomerase template antagonist: in vitro and in vivo studies in multiple myeloma and lymphoma

“…38 The hTR contains a crucial 11-nucleotide-long template region, part of which serves as the basis for telomere elongation by TTAGGG repeats. GRN163 forms a high-affinity sequence-specific duplex with the complementary RNA strand that is further stabilized through interactions between the oligonucleotide backbone and hTERT.…”

“…GRN163 is a prototype of a new class of 13-mer 2Ј-deoxy N3Ј3P5Ј thiophosphoramidate oligonucleotides designed to complement the template domain of the RNA component (hTR) in the active site of telomerase. 38 The GRN163 compound forms a high-affinity, sequence-specific duplex with the complementary RNA strand that is further stabilized through interactions between the oligonucleotide backbone and hTERT, is resistant to hydrolysis by cellular nucleases, and exhibits high thermodynamic stability. 30 The effects of GRN163 were evaluated on the growth of human MM and NHL cell lines, primary MM cells, and tumor xenografts.…”

Telomerase as a new target for the treatment of hormone-refractory prostate cancer

“…NP oligonucleotides were targeted at a segment of hTR downstream from the hTR template region, but (Pruzan et al 2002). The same group then tested the anti-telomerase effect of N3 0 !P5 0 thio-phosphoramidate oligonucleotides (NPS).…”