Krisztina Pongracz

Lipid modification of GRN163, an N3' ¿ P5' thio-phosphoramidate oligonucleotide, enhances the potency of telomerase inhibition

The vast majority of human cancers express telomerase activity, while most human somatic cells do not have detectable telomerase activity. Since telomerase plays a critical role in cell immortality, it is an attractive target for a selective cancer therapy. Oligonucleotides complementary to the RNA template region of human telomerase (hTR) have been shown to be effective inhibitors of telomerase and, subsequently, cancer cell growth in vitro. We show here that a lipid-modified N3′ → P5′ thio-phosphoramidate oligonucleotide (GRN163L) inhibits telomerase more potently than its parental nonconjugated thio-phosphoramidate sequence (GRN163). Cells were treated with both the first- (GRN163) and second-generation (GRN163L) oligonucleotides, including a mismatch control, with or without a transfection enhancer reagent. GRN163L inhibited telomerase activity effectively in a dose-dependent manner, even without the use of a transfection reagent. The IC50 values for GRN163 in various cell lines were on average sevenfold higher than for GRN163L. GRN163L inhibition of telomerase activity resulted in a more rapid loss of telomeres and cell growth than GRN163. This report is the first to show that lipid modification enhanced the potency of the novel GRN163 telomerase inhibitor. These results suggest that the lipid-conjugated thio-phosphoramidates could be important for improved pharmacodynamics of telomerase inhibitors in cancer therapy.

Oligonucleotide N3′→P5′ phosphoramidates as efficient telomerase inhibitors

Human telomerase is a unique reverse transcriptase that is expressed in multiple cancers, but not in the vast majority of normal cells. The enzyme is responsible for telomere protection and maintenance, and supports the proliferative immortality of cancer cells. Thus, it has been proposed that the specific inhibition of telomerase activity in tumors might have significant and beneficial therapeutic effects. To this goal we have designed, synthesized, and evaluated several oligonucleotide N3′→P5′ phosphoramidates as telomerase inhibitors. These oligonucleotides are complementary to the template region of the RNA domain of telomerase (hTR). The prepared compounds were evaluated in HME50-5E breast epithelial cells, where their effects on telomerase activity were determined using a cell-based telomerase (TRAP) assay at 24 as well as 72 h after exposure to compounds. The oligo-N3′→P5′ phosphoramidate inhibited telomerase activity in cells in the presence of the cellular up-take enhancer (FuGENE6TM) in a dose- and sequence-dependent manner, with IC50 values of approximately 1 nM. Inhibition of telomerase activity by this compound without the lipid carrier was not efficient. However, the isosequential oligonucleotide N3′→P5′ thio-phosphoramidate was able to inhibit telomerase activity with or without lipid carriers at nM, or low-μM concentrations, respectively. This inhibition of telomerase activity in HME50-5E cells by the oligonucleotide thio-phosphoramidates was also sequence specific. Long-term treatment of the cells with 0.5 μM of FuGENE6 formulated 13-mer thio-phosphoramidates, fully complementary to hTR, resulted in gradual telomere shortening, followed by cellular senescence and apoptosis, as would be predicted for a telomerase inhibitor. The mismatched control compound had no effect on cell proliferation. The results suggest that the oligonucleotide N3′→P5′ phosphoramidates, and particularly thio-phosphoramidates, might be further developed as selective anti-telomerase reagents.

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′ thiophosphoramidates: synthesis and properties

Abstract Uniformly modified oligonucleotides with a novel sugar-phosphate backbone containing internucleoside 3′-NHP(O)(S−)O-5′ linkages were synthesized. These oligonucleotides were found to retain the high RNA binding affinity of the parent oligonucleotide N3′→P5′ phosphoramidates and to exhibit a much higher acid stability.

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.

A Remarkable Stabilization of Complexes Formed by 2,6-Diaminopurine Oligonucleotide N3′→P5′ Phosphoramidates

Abstract 2′-Deoxyribo- and ribo-oligonucleotide N3′→P5′phosphoramidates containing 2,6-diaminopurine nucleosides were synthesized. Thermal denaturation experiments demonstrated a significant stabilization of the complexes formed by these compounds with DNA and RNA complementary strands, relative to adenosine-containing phosphoramidate counterparts. The increase in melting temperature of the complexes reached up to 6.9 °C per substitution. The observed stabilization was attributed to the apparent synergistic effects of N-type sugar puckering of the oligonucleotide N3′→5′ phosphoramidate backbone, and the ability of 2,6-diaminopurine bases to form three hydrogen bonds.

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.

DNA Adducts Formed By Peroxidase Activation of Benzene Metabolites

Abstract DNA adduct formation was examined in HL-60 cells treated with the benzene metabolites p-benzoquinone (p-BQ), hydroquinone (HQ), catechol (CAT), and 1,2,4-benzenetriol (BT). p-BQ was 13-fold more effective at forming DNA adducts than HQ, which was 7-9-fold more effective than CAT and BT. The DNA adduct formed in human bone marrow (HBM) treated with HQ was the same as that in HL-60 cells. Combination treatment of HL-60 cells with HQ and either CAT or BT increased DNA adduct formation 2.2–6.4-fold. In vitro activation of HQ by myeloperoxidase produced the same DNA adduct in purified DNA as in HL-60 cells and HBM treated with HQ. Reaction of calf thymus DNA with p-BQ produced three adducts. The DNA adduct formed in HL-60 cells treated with p-BQ did not correspond to any of the principal adducts formed in DNA reacted with p-BQ. These results suggest that peroxidase enzymes activate benzene metabolites to form DNA adducts in HBM.

A NEW APPROACH TO OLIGONUCLEOTIDE N3′⇉P5′ PHOSPHORAMIDATE BUILDING BLOCKS

A new synthetic approach to 5′-phosphoramidites of 3′-aminonucleosides was developed. The methodology relies upon the use of 3′-amino-2′,3′-dideoxy nucleosides as the key starting materials. The final phosphoramidite products were obtained with high yields via 2–3-step efficient chemical transformations using selective introduction of orthogonal protective groups to the 3′-aminonucleoside sugar and base moieties.

Erratum: Oligonucleotide N3′ → P5′ phosphoramidates as efficient telomerase inhibitors (Oncogene (2002) 21 (638-642))

Correction to: Oncogene (2002) 21, 638–642 DOI: 10.1038/sj.onc.1205064