Inhibition Of Human Telomerase Research Articles

Imetelstat, a Telomerase Inhibitor, Induces Morphologic and Molecular Remissions In Myelofibrosis and Reversal Of Bone Marrow Fibrosis

BackgroundJAK inhibitors, including ruxolitinib, are to date incapable of inducing complete (CR) or partial (PR) remissions, reversal of bone marrow (BM) fibrosis or molecular responses in myelofibrosis (MF). This is consistent with the fact that JAK2 mutations are neither specific nor pathogenetically essential for the disease. Other currently available drugs in MF are equally ineffective in terms of disease-modifying activity. MethodsIn an investigator-sponsored single-center study (ClinicalTrials.gov Identifier: NCT01731951), imetelstat, a lipid-conjugated oligonucleotide inhibitor of human telomerase, was administered to patients with high or intermediate-2 risk MF (JCO 2011). Adverse events were monitored by common terminology criteria (Version 4.03) and responses by the International Working Group criteria (Blood 2013). Eligibility criteria included platelets ≥50 x 10(9)/L. Study drug and funding were provided by Geron Corporation (Menlo Park, CA, USA).Imetelstat was administered by a 2-hour intravenous infusion (9.4 mg/kg) every three weeks (cohort A) or weekly x 3 followed by every three weeks (cohort B). Mutations with prognostic (ASXL1 and SRSF2) or phenotypic (SF3B1 and U2AF1) relevance were screened by DNA sequencing. Quantitative PCR was used to measure JAK2V617F burden (assay sensitivity 0.01%). Laboratory correlative studies included analyses of granulocyte telomere length, mononuclear cell telomerase activity and human telomerase reverse transcriptase (hTERT) isoforms. ResultsThirty-three patients were accrued; the first 18 patients enrolled and followed for a minimum of 3 months or discontinued are presented in this abstract: 11 cohort A and 7 cohort B; 44% PMF, 33% post-PV MF and 22% post-ET MF. Median age was 68 years and baseline risk was high in 56% and intermediate-2 in 44%. Seven patients were transfusion-dependent. Median spleen size was 13 cm and 11 patients had constitutional symptoms. Karyotype was abnormal in 7 patients and 89% were JAK2-mutated. Fifteen (83%) patients were previously treated including 7 with a JAK inhibitor and 3 with pomalidomide.i) ToxicityAt a median f/u of 3.2 months, 16 (89%) patients remain on treatment; the two discontinuations were from unrelated death and disease progression. In cohort A, there were no grade-4 treatment-related adverse events; grade-3 events were limited to thrombocytopenia in 27% and anemia in 9%. In cohort B, two (29%) patients experienced grade-4 thrombocytopenia; grade-3 events were limited to thrombocytopenia, neutropenia and anemia in one patient each. Dose reduction was necessary in only two (11%) patients because of grade 3 or 4 myelosuppression.ii) EfficacyOverall response rate was 44%. This included five (28%) patients who met the BM and peripheral blood morphologic criteria for CR (n=4) or PR (n=1) and 3 patients with clinical improvement, pending validation of response duration and resolution of drug-induced grade-1 thrombocytopenia. The four (22%) CR patients experienced reversal of BM fibrosis and recovery of normal megakaryocyte morphology. Two CR patients were transfusion-dependent at baseline and became transfusion-independent. Complete molecular responses were documented in 2 CR patients: one had U2AF1Q157P and 10% JAK2V617F and the other SF3B1K666E and 50% JAK2V617F. A third CR patient had a >50% reduction in U2AF1 469_insAGTATG mutation. Among 13 patients with leukocytosis, 10 (77%) normalized their count or had >50% reduction. Eleven (61%) patients had complete or partial resolution of leukoerythroblastosis.iii) Laboratory correlative studiesThree (50%) of 6 spliceosome-mutated vs. 1 (8%) of 12 unmutated (p=0.045) achieved CR. Spliceosome-mutated patients were also more likely to experience grade-3/4 myelosuppression (67% vs. 25% ; p=0.09). Treatment was associated with suppression of telomerase activity, shortening of telomere length and alteration of hTERT isoform pattern. ConclusionsThe current study signifies the potential value of telomerase-based treatment strategies in MF and identifies imetelstat as an active drug in that regard. The observed morphologic and molecular remissions confirm selective anti-clonal activity, which has thus far eluded other drugs in MF, including JAK inhibitors. The association between response and spliceosome mutations suggests a broader application for the drug in myeloid malignancies.</abstract> Disclosures:Stuart:Geron: Consultancy.

A three-state model for the regulation of telomerase by TERRA and hnRNPA1

Telomeres, the physical ends of eukaryotic chromosomes, are transcribed into telomeric repeat-containing RNA (TERRA), a large non-coding RNA, which forms an integral part of telomeric heterochromatin. In vitro, naked TERRA molecules are efficient inhibitors of human telomerase, base-pairing via their 5′-UUAGGG-3′ repeats with the template sequence of telomerase RNA, in addition to contacting the telomerase reverse transcriptase protein subunit. In vivo, however, TERRA-mediated inhibition of telomerase can be prevented by unknown mechanisms. Also, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) has been implicated in telomere length control. In vivo, TERRA is partially associated with hnRNPA1, and hnRNPA1 is also detected at telomeres. We demonstrate that on binding of TERRA, hnRNPA1 can alleviate the TERRA-mediated inhibition of telomerase. However, when in excess over TERRA, hnRNPA1 becomes itself an inhibitor of telomere extension, on binding of the telomeric DNA substrate. Yet, hnRNPA1 has no notable direct effects on the telomerase catalysis. Our in vitro results suggest that TERRA-mediated telomerase inhibition may be prevented by hnRNPA1 in vivo. Telomere extension by telomerase may require balanced levels of TERRA and hnRNPA1 at telomeres. Thus, TERRA and hnRNPA1 can function as a bimolecular regulator to turn telomerase and the telomere on and off.

Yeast chemical genetic screen for inhibitors of human telomerase

Yeast chemical genetic screen for inhibitors of human telomerase

A Yeast Chemical Genetic Screen Identifies Inhibitors of Human Telomerase

SummaryTelomerase comprises a reverse transcriptase and an internal RNA template that maintains telomeres in many eukaryotes, and it is a well-validated cancer target. However, there is a dearth of small molecules with efficacy against human telomerase in vivo. We developed a surrogate yeast high-throughput assay to identify human telomerase inhibitors. The reversibility of growth arrest induced by active human telomerase was assessed against a library of 678 compounds preselected for bioactivity in S. cerevisiae. Four of eight compounds identified reproducibly restored growth to strains expressing active human telomerase, and three of these four compounds also specifically inhibited purified human telomerase in vitro. These compounds represent probes for human telomerase function, and potential entry points for development of lead compounds against telomerase-positive cancers.

In Vitro and Ex Vivo Inhibition of Human Telomerase by Anti-HIV Nucleoside Reverse Transcriptase Inhibitors (NRTIs) but Not by Non-NRTIs

Telomerase is a specialized reverse transcriptase responsible for the de novo synthesis of telomeric DNA repeats. In addition to its established reverse transcriptase and terminal transferase activities, recent reports have revealed unexpected cellular activities of telomerase, including RNA-dependent RNA polymerization. This telomerase characteristic, distinct from other reverse transcriptases, indicates that clinically relevant reverse transcriptase inhibitors might have unexpected telomerase inhibition profiles. This is particularly important for the newer generation of RT inhibitors designed for anti-HIV therapy, which have reported higher safety margins than older agents. Using an in vitro primer extension assay, we tested the effects of clinically relevant HIV reverse transcriptase inhibitors on cellular telomerase activity. We observed that all commonly used nucleoside reverse transcriptase inhibitors (NRTIs), including zidovudine, stavudine, tenofovir, didanosine and abacavir, inhibit telomerase effectively in vitro. Truncated telomere synthesis was consistent with the expected mode of inhibition by all tested NRTIs. Through dose-response experiments, we established relative inhibitory potencies of NRTIs on in vitro telomerase activity as compared to the inhibitory potencies of the corresponding dideoxynucleotide triphosphates. In contrast to NRTIs, the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine and efavirenz did not inhibit the primer extension activity of telomerase, even at millimolar concentrations. Long-term, continuous treatment of human HT29 cells with select NRTIs resulted in an accelerated loss of telomere repeats. All tested NRTIs exhibited the same rank order of inhibitory potencies on telomerase and HIV RT, which, according to published data, were orders-of-magnitude more sensitive than other DNA polymerases, including the susceptible mitochondria-specific DNA polymerase gamma. We concluded that telomerase activity could be inhibited by common NRTIs, including currently recommended RTI agents tenofovir and abacavir, which warrants large-scale clinical and epidemiological investigation of the off-target effects of long-term highly active antiretroviral therapy (HAART) with these agents.

Anti-Cancer Targeting Telomerase Inhibitors: &amp;beta;-Rubromycin and Oleic Acid

Telomerase is a ribonucleoprotein complex that elongates telomeric DNA and appears to play an important part in the cellular immortalization of cancers. In the screening of potent inhibitors of human telomerase, several inhibitors have been discovered from natural and chemical sources. Some compounds potently inhibit the activity of human telomerase. Rubromycins and fatty acids such as β-rubromycin and oleic acid, respectively, were found to be inhibitors of human telomerase. The IC(50) values of β-rubromycin and oleic acid were 8.60 and 8.78 μM, respectively. A kinetic study revealed that these compounds competitively inhibited the activity of telomerase with respect to the substrate of the primer and dNTP. The energy-minimized three-dimensional structure of β-rubromycin and oleic acid was calculated and designed. The V-shaped curve and molecule length of 18.7-20.3 Å in these compound structures were suggested to be important for telomerase inhibition. The three-dimensional structure of the active site of telomerase (i.e., the binding site of the primer and dNTP substrate) might have a "pocket" that could "join" these compounds. These results appear to suggest a potential structure for the development of more potent inhibitors of human telomerase.

Pt(ii) squares as selective and effective human telomeric G-quadruplex binders and potential cancer therapeutics

A series of four self-assembled Pt(II) molecular squares with 4,4'-dipyridyl or pyrazine bridges, including the previously reported Pt(II) squares [Pt(en)(4,4'-dipyridyl)](4)(NO(3))(8) (1), were investigated for their abilities to act as selective and effective human telomeric (htelo) G-quadruplex binders. FRET and SPR studies demonstrated that Pt(II) squares could discriminate against duplex DNA, and show promising selectivity between intramolecular G-quadruplexes. PCR-stop assays and CD studies showed that Pt(II) squares strongly induced the formation of parallel G-quadruplexes. ITC experiments indicated that Pt(II) squares could bind to the G-quadruplex with high binding constants (K(b) values ranging from 10(4)-10(8) M(-1)). All four Pt(II) squares were effective inhibitors of human telomerase, and showed anticancer efficacy. This was particularly the case for [Pt(NH(3))(2)(4,4'-dipyridyl)](4)(NO(3))(8) (2), which exhibited a 15-fold higher antiproliferative effect on A549/cisR cells than cisplatin.

Different roles of p53 in the regulation of DNA damage caused by 1,2-heteroannelated anthraquinones and doxorubicin

The anthracyclin antibiotic agent doxorubicin (DXR) has been widely used as a chemotherapeutic drug for more than 40 years, but its clinical use has been limited by its cardiotoxicity. The mechanism of action of DXR remains uncertain and controversial. A series of 1,2-heteroannelated anthraquinones and anthra[1,2-d]imidazole-6,11-dione compounds were synthesized and their cytotoxicity profiles were analyzed using the National Cancer Institute 60 (NCI 60) platform and human telomerase inhibition assays. In the current study, three of the 1,2-heteroannelated anthraquinones, NSC745795, NSC745885 and NSC745887, were found to differ from each other with respect to their effects on cell cycle regulation, apoptosis, autophagy, senescence and their abilities to induce DNA damage. The differences depended on the presence or absence of a heterocyclic moiety, which suggested that the differences were due, at least in part, to differential effects on specific cellular targets, such as p53. In contrast to DXR, which induced p53 expression, treatment with NSC745885 resulted in the degradation of several proteins, including p53, via proteasome-dependent and -independent pathways in HeLa cells. These results provide insights into the molecular mechanisms governing cell inhibition by 1,2-heteroannelated anthraquinone derivatives and suggest that these mechanisms could serve as the basis for new structure-based drug designs.

The non-coding RNA TERRA is a natural ligand and direct inhibitor of human telomerase

Telomeres, the physical ends of eukaryotes chromosomes are transcribed into telomeric repeat containing RNA (TERRA), a large non-coding RNA of unknown function, which forms an integral part of telomeric heterochromatin. TERRA molecules resemble in sequence the telomeric DNA substrate as they contain 5′-UUAGGG-3′ repeats near their 3′-end which are complementary to the template sequence of telomerase RNA. Here we demonstrate that endogenous TERRA is bound to human telomerase in cell extracts. Using in vitro reconstituted telomerase and synthetic TERRA molecules we demonstrate that the 5′-UUAGGG-3′ repeats of TERRA base pair with the RNA template of the telomerase RNA moiety (TR). In addition TERRA contacts the telomerase reverse transcriptase (TERT) protein subunit independently of hTR. In vitro studies further demonstrate that TERRA is not used as a telomerase substrate. Instead, TERRA acts as a potent competitive inhibitor for telomeric DNA in addition to exerting an uncompetitive mode of inhibition. Our data identify TERRA as a telomerase ligand and natural direct inhibitor of human telomerase. Telomerase regulation by the telomere substrate may be mediated via its transcription.

Oligonucleotide N3′→P5′ Phosphoramidates and Thio‐Phoshoramidates as Potential Therapeutic Agents

Nucleic acids analogues, i.e., oligonucleotide N3'-->P5' phosphoramidates and N3'-->P5' thio-phosphoramidates, containing 3'-amino-3'-deoxy nucleosides with various 2'-substituents were synthesized and extensively studied. These compounds resist nuclease hydrolysis and form stable duplexes with complementary native phosphodiester DNA and, particularly, RNA strands. An increase in duplexes' melting temperature, DeltaT(m), relative to their phosphodiester counterparts, reaches 2.2-4.0 degrees per modified nucleoside. 2'-OH- (RNA-like), 2'-O-Me-, and 2'-ribo-F-nucleoside substitutions result in the highest degree of duplex stabilization. Moreover, under close to physiological salt and pH conditions, the 2'-deoxy- and 2'-fluoro-phosphoramidate compounds form extremely stable triple-stranded complexes with either single- or double-stranded phosphodiester DNA oligonucleotides. Melting temperature, T(m), of these triplexes exceeds T(m) values for the isosequential phosphodiester counterparts by up to 35 degrees . 2'-Deoxy-N3'-->P5' phosphoramidates adopt RNA-like C3'-endo or N-type nucleoside sugar-ring conformations and hence can be used as stable RNA mimetics. Duplexes formed by 2'-deoxy phosphoramidates with complementary RNA strands are not substrates for RNase H-mediated cleavage in vitro. Oligonucleotide phosphoramidates and especially thio-phosphoramidates conjugated with lipid groups are cell-permeable and demonstrate high biological target specific activity in vitro. In vivo, these compounds show good bioavailability and efficient biodistribution to all major organs, while exerting acceptable toxicity at therapeutically relevant doses. Short oligonucleotide N3'-->P5' thio-phosphoramidate conjugated to 5'-palmitoyl group, designated as GRN163L (Imetelstat), was recently introduced as a potent human telomerase inhibitor. GRN163L is not an antisense agent; it is a direct competitive inhibitor of human telomerase, which directly binds to the active site of the enzyme and thus inhibits its activity. This compound is currently in multiple Phase-I and Phase-I/II clinical trials as potential broad-spectrum anticancer agent.

Synthesis, cytotoxicity and human telomerase inhibition activities of a series of 1,2-heteroannelated anthraquinones and anthra[1,2- d]imidazole-6,11-dione homologues

A series of 1,2-heteroannelated anthraquinones and anthra[1,2- d]imidazole-6,11-dione tetracyclic analogues with different side chain were prepared using an various synthetic route via acylation, cyclization, condensation, and intramolecular heterocyclization. Tetracyclic system containing alkyl and aryl, aromatic and heterocyclic, linear and cyclic, polar and apolar, and basic and acids residues were incorporated. They were evaluated for their effects on telomerase activity, hTERT expression, cell proliferations, and in vitro cytotoxicity against NCI’s 60 cell line human tumor screen. Compounds 4, 11, 12, 14, 15, 16, 17, 19, 20, 23, 25, and 26 were selected by the NCI for one dose screening program and further studies on 4, 23 and 25 where the curves cross these lines represent the interpolated values to cause 50% growth inhibition (GI 50), total growth inhibition (TGI) and 50% cell killing (LC 50), respectively. Further studies did not reveal any compound that showed potent and significant on telomerase inhibitory activity and hTERT repressing ability. Comparative testing of these compounds in the NCI’s screen revealed varying levels of potency and differential cytotoxicity, apparently related to the unsaturation levels in and substitution patterns on the core ring system. It appeared that addition of a fourth planar aromatic system to a tricyclic chromophore might enhances potent cytotoxic agents, at a level equivalent to a second side chain in one of the tricyclic series. Although the exact mechanism of how this pharmacophore contributes to its activity is still unclear, however, the group in the extended arm of the tetracyclic system might contribute to proper binding to the residues within the grove of G-quadruplex structure.

BRACO19 analog dimers with improved inhibition of telomerase and hPot 1

Human chromosomes terminate with telomeres, which contain double-stranded G-rich, repetitive DNA followed by a single-stranded overhang of the G-rich sequence. Single-stranded oligonucleotides containing G-rich telomeric repeats have been observed in vitro to fold into a variety of G-quadruplex topologies depending on the solution conditions. G-quadruplex structures are notable in part because G-quadruplex ligands inhibit both the enzyme telomerase and other telomere-binding proteins. Because telomerase is required for growth by the majority of cancers, G-quadruplex-stabilizing ligands have become an attractive platform for anticancer drug discovery. Here, we present the preparation and biochemical activities of a novel series of 3,6-disubstituted acridine dimers modeled after the known G-quadruplex ligand BRACO19. These BRACO19 Analog Dimer (BAD) ligands were shown to bind to human telomeric DNA and promote the formation of intramolecular G-quadruplexes in the absence of monovalent cations. As expected, the BAD ligands bound to telomeric DNA with a 1:1 stoichiometry, whereas the parent compound BRACO19, a monomer, bound with a 2:1 stoichiometry. The BAD ligands exhibited potent inhibition of human telomerase with IC 50 values similar to or lower than those of BRACO19. Furthermore, the BAD ligands displayed greater potency in the inhibition of hPot1 and increased selectivity for G-quadruplex DNA when compared to BRACO19. Collectively, these experiments support the hypothesis that there is an increased potency and selectivity to be gained in the design of G-quadruplex-stabilizing agents that incorporate multiple interactions.

Platinum(II) Complexes with Dipyridophenazine Ligands as Human Telomerase Inhibitors and Luminescent Probes for G-Quadruplex DNA

A series of platinum(II) complexes containing dipyridophenazine (dppz) and C-deprotonated 2-phenylpyridine (N-CH) ligands were prepared and assayed for G-quadruplex DNA binding activities. [PtII(dppz-COOH)(N-C)]CF3SO3 (1; dppz-COOH = 11-carboxydipyrido[3,2-a:2',3'-c]phenazine) binds G-quadruplex DNA through an external end-stacking mode with a binding affinity of approximately 10(7) dm3 mol-1. G-quadruplex DNA binding is accompanied by up to a 293-fold increase in the intensity of photoluminescence at lambdamax = 512 nm. Using a biotinylated-primer extension telomerase assay, 1 was shown to be an effective inhibitor of human telomerase in vitro, with a telIC50 value of 760 nM.

Antisense Inhibition of Human Telomerase by Phosphorothioate Oligonucleotide-Peptide Conjugates

Oligonucleotides can be covalently linked to peptides composed of any sequence of amino acids by SPFC. The peptides incorporated into the conjugates include nuclear localizing signals (NLS), nuclear export signals (NES), membrane fusion domain of some viral proteins and some designed peptides with amphipathic character. Evaluation of biological properties of DNA-peptide conjugates indicated that (a) the conjugates could bind to target RNA and dsDNA with increased affinity, (b) the conjugates were more resistant to cellular nuclease degradation, (c) the conjugates-RNA hybrids could activate RNase H as effective as native oligonucleotides, (d) the conjugates with fusion peptides showed largely enhanced cellular uptake, (e) the conjugates with NLS could be predominantly delivered into cell nucleus, (f) the conjugates with NES could be localized in cytoplasm. As a result, antisense oligonucleotides conjugated with NLS could inhibit human telomerase in human leukemia cells much more strongly than phosphorothioate oligonucleotides.

Tumor therapy targeting human telomerase reverse transcriptase

Human telomerase reverse transcriptase (hTERT) is one of the focus researches of cancer therapy in recent years,including the studies of human telomerase inhibitor, RNA interfere targeting hTERT and gene therapy research, as well as hTERT-mediated immunology research. All these studies have gotten some a-chievements. Key words: Telomerase: Neoplasms; RNA interference

Synthesis, human telomerase inhibition and anti-proliferative studies of a series of 2,7-bis-substituted amido-anthraquinone derivatives

Telomerase is important in tumor initiation and cellular immortalization. Given the striking correlations between telomerase activity and proliferation capacity in tumor cells, telomerase had been considered as a potentially important molecular target in cancer therapeutics. A series of 2,7-diamidoanthraquinone were designed and synthesized. They were evaluated for their effects on telomerase activity, hTERT expression, cell proliferations, and cytotoxicity. In the series, compounds ( 6, 10, 13, 16, 18, 19, 20– 22, and 24) showed potent telomerase inhibitory activity, while compounds 19, 21, and 22 activated hTERT expression in normal human fibroblasts. The results indicated that 2,7-diamidoanthraquinones represent an important class of compounds for telomerase-related drug developments. Compounds 8, 16, 18, 26, and 32 were also selected by the NCI for Screening Program and demonstrated high anti-proliferative activity against 60 human cancer cell lines. Structure–activity relationships (SAR) study revealed that the test compounds with side chains two carbon spacer between amido and amine are important structural moiety for telomerase inhibition. Although the exact mechanism of how this amine group contributes to its activity is still unclear, however, the amine group in the extended arm of the bis-substituted anthraquinone might contribute to proper binding to the residues within the grove of G-quadruplex structure. Our results indicated that the 2,7-disubstituted amido-anthraquinones are potent telomerase inhibitors that have the potential to be further developed into novel anticancer chemotherapeutic agents.

Total Synthesis of (±)‐γ‐Rubromycin on the Basis of Two Aromatic Pummerer‐Type Reactions

Double or nothing: In a convergent synthesis of the title compound, a potent inhibitor of human telomerase, two different aromatic Pummerer-type reactions were employed to construct the pivotal bisbenzannelated spiroketal skeleton from the two fragments shown in the scheme. This methodology offers convenient access to a wide range of substituted bisbenzannelated spiroketals from naphthol derivatives. MOM=methoxymethyl.

Synthesis and Human Telomerase Inhibition of a Series of Regioisomeric Disubstituted Amidoanthraquinones

Telomerase is the enzymatic activity that maintains the ends of eukaryotic chromosomes. Telomerase activity is detected in most tumor cells whereas it is low or undetectable in most normal somatic cells. Expression of the telomerase catalytic component, the human telomerase reverse transcriptase (hTERT), is believed to be controlled primarily at the level of transcription. Because of this selective expression property of telomerase, it has been touted as a specific target for antitumor chemotherapeutics. However, a concern for the applicability of telomerase inhibitors is that they require a long lag time for telomeres to be shortened to critical length before cancer cells stop proliferating. Here we investigate telomerase inhibitory, cytotoxicity and the hTERT repressing effects on a number of synthesized 2,6-diamidoanthraquinones and 1,5-diamidoanthraquinones as compared to their disubstituted homologues. We found that several of the 1,5-diamidoanthraquinones and 2,6-diamidoanthraquinones inhibited telomerase activity effectively with IC50 at the sub-micro to micro molar range and caused acute cytotoxicity to cancer cells with EC50 similar or better than that of mitoxantrone. Particularly, 2,6-diamidoanthraquinone with 2-ethylaminoacetamido side chains 33, even though not affecting cell proliferation, showed to be endowed with a strong telomerase effect, probably related to a marked stabilization of the G-quadruplex-binding structure. The results suggested that these compounds caused multiple effects to cancer cells. More significantly, they overcome the long lag period problem of classical telomerase inhibitors that they are also potent cytotoxic agents. These results greatly expand the potential of tricyclic anthraquinone pharmacophore in preventive and/or curative therapy.

Potent inhibition of human telomerase by U-73122

Telomerase activity is repressed in normal human somatic cells, but is activated in most cancers, suggesting that telomerase may be an important target for cancer therapy. In this study, we report that U-73122, an amphiphilic alkylating agent that is commonly used as an inhibitor for phospholipase C, is also a potent and selective inhibitor of human telomerase. The inhibition of telomerase by U-73122 was attributed primarily to the pyrrole-2,5-dione group, since its structural analog U-73343 did not inhibit telomerase. In confirmation, we observed that telomerase was inhibited by N-ethylmaleimide, but not N-ethylsuccinimide. The IC(50) value of U-73122 for the in vitro inhibition of telomerase activity is 0.2 microM, which is comparable to or slightly more sensitive than that for phospholipase C. The inhibitory action of U-73122 on telomerase appears to be rather selective since the presence of externally added proteins did not protect the inhibition and the IC(50) values for the other enzymes tested in this study were at least an order of magnitude higher than that for telomerase. Furthermore, we demonstrate that U-73122 can inhibit telomerase in hematopoietic cancer cells. The potent and selective inhibition of telomerase by U-73122 raises the potential exploitation of this drug and other alkylating agents as telomerase inhibitor.

Potent Inhibitory Activity of Chimeric Oligonucleotides Targeting Two Different Sites of Human Telomerase

Suppression of telomerase activity in tumor cells has been considered as a new anticancer strategy. Here, we present chimeric oligonucleotides (chimeric ODNs) as a new type of telomerase inhibitor that contains differently modified oligomers to address two different sites of telomerase: the RNA template and a suggested protein motif. We have shown previously that phosphorothioate-modified oligonucleotides (PS ODNs) interact in a length-dependent rather than in a sequence-dependent manner, presumably with the protein part of the primer-binding site of telomerase, causing strong inhibition of telomerase. In the present study, we demonstrate that extensions of these PS ODNs at their 3'-ends with an antisense oligomer partial sequence covering 11 bases of the RNA template cause significantly increased inhibitory activity, with IC(50) values between 0.60 and 0.95 nM in a Telomeric Repeat Amplification Protocol (TRAP) assay based on U-87 cell lysates. The enhanced inhibitory activity is observed regardless of whether the antisense part is modified (phosphodiester, PO; 2'-O-methylribosyl, 2'-OMe/PO; phosphoramidate, PAM). However, inside intact U-87 cells, these modifications of the antisense part proved to be essential for efficient telomerase inhibition 20 hours after transfection. In particular, the chimeric ODNs containing PAM or 2'-OMe/PO modifications, when complexed with lipofectin, were most efficient telomerase inhibitors (ID(50) = 0.04 and 0.06 microM, respectively). In conclusion, ODNs of this new type emerged as powerful inhibitors of human telomerase and are, therefore, promising candidates for further investigations of the anticancer strategy of telomerase inhibition.