Potent Telomerase Inhibitor Research Articles

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.

Telomerase and cancer therapeutics

Telomerase is an attractive cancer target as it appears to be required in essentially all tumours for immortalization of a subset of cells, including cancer stem cells. Moreover, differences in telomerase expression, telomere length and cell kinetics between normal and tumour tissues suggest that targeting telomerase would be relatively safe. Clinical trials are ongoing with a potent and specific telomerase inhibitor, GRN163L, and with several versions of telomerase therapeutic vaccines. The prospect of adding telomerase-based therapies to the growing list of new anticancer products is promising, but what are the advantages and limitations of different approaches, and which patients are the most likely to respond?

Synthesis of RHPS4 via an anionic ring closing cascade

A new and convergent synthesis of the potent telomerase inhibitor RHPS4 is presented. The key step is the construction of the pentacyclic framework via an anionic ring closing cascade in which two new rings are formed.

Synthesis and evaluation of cationic phthalocyanine derivatives as potential inhibitors of telomerase

A series of water-soluble cationic phthalocyanine derivatives ( 1– 10) were designed and synthesized to develop novel and potent telomerase inhibitors. These phthalocyanine derivatives as inhibitors of telomerase were investigated via modified telomerase repeat amplification protocol (TRAP) assay. The TRAP assay indicates that these cationic compounds had strong telomerase inhibitory activity (IC 50 < 1.65 μM). To determine whether the phthalocyanine derivatives binding to G-quadruplex enhance the block to DNA synthesis, primer extension reactions were carried out in the presence of phthalocyanines. The interaction of the G-quadruplex of telomerase DNA with these molecules was examined by CD melting and PCR stop assay. These cationic phthalocyanine derivatives can stabilize G-quadruplex, which is demonstrated by the increased T m values. All these results indicate that the phthalocyanine derivatives might be potential lead compounds for the development of new telomerase inhibitor.

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.

Quaternary Ammonium Zinc Phthalocyanine: Inhibiting Telomerase by Stabilizing G quadruplexes and Inducing G‐Quadruplex Structure Transition and Formation

Water-soluble, octacationic zinc phthalocyanine (ZnPc) was found to be a very good G-quadruplex DNA stabilizer by using UV-melting studies and DNA polymerase stop assays, and a potent telomerase inhibitor by using the telomeric repeat amplification protocol (TRAP) assay. The compound's DNA-binding properties were also studied by surface plasmon resonance (SPR). Furthermore, CD experiments demonstrated that ZnPc could induce intramolecular G-quadruplex structure transition from the antiparallel to parallel form. More importantly, ZnPc was found to induce parallel structure formation in cation-deficient conditions. The stability of the induced structure was determined with CD melting assays.

Cancer Stem Cell Targeting in Multiple Myeloma by GRN163L, a Novel and Potent Telomerase Inhibitor.

Telomerase activity (TA) is required within normal cells capable of long-term replication, including stem cells and is upregulated in many cancers. In the absence of TA or presence of TA inhibitors, the progressive shortening of telomeres ultimately results in cellular senescence and/or apoptosis. These observations support that TA inhibitors represent a novel class of anti-tumor agents. Much evidence suggests that human cancers display a hierarchical cellular organization that mirrors normal tissues. Cancer stem cells (CSC) are derived from the malignant transformation of normal stem cells and progenitors and retain the capacity to self-renew. Moreover, CSC give rise to differentiated tumor cells that form the bulk of the tumor mass, but have little or no capacity for long-term proliferation. We recently demonstrated that the malignant CD138+ plasma cells in multiple myeloma (MM) have limited replicative potential; instead they arise from the differentiation of clonogenic CSC that resemble normal memory B cells (CD138negCD19+CD27+). In addition, several groups have demonstrated that telomerase inhibitors are active against human MM cell lines in vitro and in vivo. We examined TA in CD138+ plasma cells and CD138neg precursors, and studied the effects of telomerase inhibition against both cell populations. We isolated CD138+ and CD138neg cells by FACS from three human MM cell lines (RPMI 8225, NCI-H929, and U266) and measured TA using a PCR-based assay of activity. For each cell line, TA was detectable within both the CD138neg and CD138+ cell populations. GRN163L is a lipid conjugated 13 nucleotide thio-phosphoramidate oligonucleotide that acts as a potent and specific active site inhibitor of telomerase. We found that treatment with GRN163L (0.1–5μM) markedly reduced TA within 48 hours. To examine the effects of telomerase inhibition on clonogenic growth, we continuously cultured CD138+ and CD138neg RPMI 8226 cells with GRN163L (1μM). Cells were collected weekly, washed to remove GRN163L, and then plated in methylcellulose to assess colony formation. We found that GRN163L was active against both CD138+ and CD138neg cells and eliminated the colony forming potential of both by 5 weeks. Similarly, we found that GRN163L inhibited the in vitro clonogenic growth of CD138neg MM CSC isolated from the bone marrow aspirates of patients with MM. These data demonstrate that TA is detectable within both immature MM CSC and mature MM plasma cells, and that CSC from both cell lines and primary clinical samples are targeted by the telomerase inhibitor GRN163L. Therefore, this agent may offer a novel therapeutic approach to myeloma as well as other diseases in which CSC have been identified.

GRN163L, a Potent and Specific Inhibitor of Telomerase: Integrated Pharmacokinetic, Pharmacodynamic, and Safety Data Guides Design of Practical Treatment Regimens for Targeting Inhibitory Levels in Patients.

GRN163L is a lipidated thiophosphoramidate oligonucleotide that binds with nanomolar affinity to the template region of human telomerase RNA, causing direct enzyme inhibition. Cellular IC50 values range from 0.8 to 6.5 μg/mL among 13 solid and hematologic tumor lines, and GRN163L inhibits tumor growth in multiple human tumor xenograft studies. Due to the high affinity and slow off-rate of binding, telomerase inhibition is long-lasting following exposure to the drug (Oncogene , 2005, 24, 5262). Previously reported studies demonstrated that exposure of human myeloma cell lines INA6 and ARP to 1 μM GRN163L resulted in ≥ 90% inhibition of telomerase activity and was followed by reduced cell growth and survival (Masood et al, ASH 2005). GRN163L uptake and telomerase inhibition have also been demonstrated in primary human CLL cells tested at 2 μM (Lin et al., ASH, 2005). Many polyanionic oligonucleotides can cause reversible inhibition of the intrinsic coagulation pathway and complement activation at high concentrations. The studies reported here were designed to determine safe and well-tolerated dose schedules and dose ranges for GRN163L in primates in order to attain plasma levels effective for telomerase inhibition and anti-tumor effects.Methods: GRN163L was administered i.v. to cynomolgus monkeys at varying doses and infusion durations. Plasma levels were determined using a validated hybridization-ELISA method.Results: In multiple studies, the T½α from two compartment modeling was 2.9 to 5.3 hr for doses of 5 to 15 mg/kg. Actual (from separate but representative studies) and modeled peak plasma levels (Cmax) as well as duration of plasma levels above the 1 μM (~4.8 μg/mL) cellular telomerase inhibitory level are shown in the table below.5 mg/kg doseObserved CmaxFrom ModelInf DurMean ±SDCmaxLevel ≥ 1 μg/mlhrsμg/mlhrsBolus185±8153150.5200±16144162128±312216663±98319At 10 mg/kg over 6 h, Cmax was 90–115 μg/mL, with < 2-fold increases in APTT and no significant complement activation. Repeated doses up to 15 mg/kg were well tolerated at all infusion durations. A Phase I/II clinical trial of GRN163L infusions in CLL patients is in progress and the observed T½α values from initial cohorts are consistent with the model presented here.Conclusions: Due to the relatively long T½α of GRN163L, telomerase inhibitory concentrations should be attainable with clinically practical infusions of 1 to 6 hr duration without dose-limiting peak plasma concentrations. Given the high target affinity, such infusions repeated intermittently could maintain near continual target inhibition. GRN163L is the first telomerase targeted agent to enter clinical trials. Initial results from these clinical trials support the hypothesis that telomerase inhibitory levels can be achieved at well tolerated doses.

Telomerase inhibition by an siRNA directed against hTERT leads to telomere attrition in HT29 cells

Human telomerase is a ribonucleoprotein complex composed of at least the reverse catalytic transcriptase hTERT and RNA component hTR. The enzyme stabilizes telomere length and thereby contributes to unlimited cell proliferation, i.e. immortality. Reactivation of telomerase activity during carcinogenesis is a common hallmark in most human tumor types. Consequently, telomerase is an attractive molecular target toward which to direct cancer therapeutic agents. RNA interference (RNAi) has been shown to be an effective method for inhibiting the expression of a given gene in human cells by targeting with short duplex RNA (short-interfering RNA or siRNA). Thus, we evaluated the ability of siRNAs to inhibit telomerase activity in the HT29 immortal human colorectal adenocarcinoma cell line as a model for colorectal carcinogenesis. By transient expression of a specific siRNA directed against hTERT, we reduced telomerase activity in the transfected cells. Moreover, telomere lengths were reduced in cells stably expressing this particular RNA sequence, cloned as an shRNA into an eukaryotic expression vector. The cell clone that displayed a telomerase-negative phenotype showed dramatically reduced telomere lengths and stopped proliferation. We observed that the vector was integrated into the cell genome and, despite telomere shortening, cells retained their MSI phenotype. We conclude that we have developed a potent telomerase inhibitor leading to cell death.

A Phenol Quaternary Ammonium Porphyrin as a Potent Telomerase Inhibitor by Selective Interaction with Quadruplex DNA

A Phenol Quaternary Ammonium Porphyrin as a Potent Telomerase Inhibitor by Selective Interaction with Quadruplex DNA

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.

Neomycin-capped aromatic platforms: quadruplex DNA recognition and telomerase inhibition

A series of aminoglycoside-capped macrocyclic structures has been prepared using intramolecular bis-tethering of neomycin on three aromatic platforms (phenanthroline, acridine, quinacridine). Based on NMR and calculations studies, it was found that the cyclic compounds adopt a highly flexible structure without conformational restriction of the aminoglycoside moiety. FRET-melting stabilization measurements showed that the series displays moderate to high affinity for the G4-conformation of human telomeric repeats, this effect being correlated with the size of the aromatic moiety. In addition, a FRET competition assay evidenced the poor binding ability of all macrocycles for duplex DNA and a clear binding preference for loop-containing intramolecular G4 structures compared to tetramolecular parallel G4 DNA. Finally, TRAP experiments demonstrated that the best G4-binder (quinacridine ) is also a potent and selective telomerase inhibitor with an IC(50) in the submicromolar range (200 nM).

Synthesis and Evaluation of Quindoline Derivatives as G-Quadruplex Inducing and Stabilizing Ligands and Potential Inhibitors of Telomerase

A new series of quindoline derivatives (4a-j) were designed and synthesized to develop novel and potent telomerase inhibitors. The interaction of the G-quadruplex of human telomere DNA with these newly designed molecules was examined via circular dichroism spectroscopy and electrophoretic mobility shift assay (EMSA). The selectivity between the quindoline derivative (4a) and G-quadruplex or duplex DNA was investigated by competition dialysis. These new compounds as inhibitors of telomerase were also investigated through the utilization of modified telomerase repeat amplification protocol (TRAP) assay. The results revealed that the introduction of electron-donating groups such as substituted amino groups at the C-11 position of quindoline significantly improved the inhibitory effect on telomerase activity ((Tel)IC50 > 138 microM for quindoline, 0.44-12.3 microM for quindoline derivatives 4a-j). The quindoline derivatives not only stabilized the G-quadruplex structure but also induced the G-rich telomeric repeated DNA sequence to fold into quadruplex.

Senescence and telomere shortening induced by novel potent G-quadruplex interactive agents, quindoline derivatives, in human cancer cell lines

Agents stabilizing G-quadruplexes have the potential to interfere with telomere replication by blocking the elongation step catalysed by telomerase or telomerase-independent mechanism and could therefore act as antitumor agents. In this study, we found that quindoline derivatives interacted preferentially with intramolecular G-quadruplex structures and were novel potent telomerase inhibitors. Treatment with quindoline derivatives reproducibly inhibited telomerase activity in human leukemia K562 cells and colon cancer SW620 cells. N'-(10H-Indolo [3,2-b] quinolin-11-yl)-N, N-dimethyl-propane-1,3-diamine (SYUIQ-5), (one of quindoline derivatives), when added to K562 and SW620 cell culture at nonacute cytotoxic concentrations, increased time of population doublings of K562 and SW620 cells, induced a marked cessation in cell growth and cellular senescence phenotype after 35 and 18 days, respectively. Growth cessation was accompanied by a shortening of telomere length, and induction of p16, p21 and p27 protein expression. However, another compound SYUIQ-7 with greater IC(50) for telomerase had no obvious cellular effect in nonacute cytotoxic concentrations. These results indicate that quindoline derivatives as novel potent G-quadruplex interactive agents induce senescence and telomere shortening in cancer cells and therefore are promising agents for cancer treatment.

Preclinical efficacy, safety, and ADME of GRN163L, a novel telomerase inhibitor developed for the treatment of cancer

3168 Background: Telomerase maintains telomeric DNA and plays a critical role in tumor cell immortality. Human telomerase is repressed or transiently active in normal somatic cells and telomeres gradually shorten over decades. In most cancers, telomeres are short, but maintained by telomerase. A correlation between tumor progression and telomerase activation makes the inhibition of telomerase a promising target for the treatment of cancer. GRN163L is a lipid-conjugated thio-phosphoramidate (N3’-P5’) oligonucleotide, developed as a potent and specific telomerase inhibitor for the treatment of cancer. Methods: GRN163L was administered systemically as a single agent or in combination with other chemotherapeutics in tumor models to assess activity of the molecule. IND-supporting preclinical toxicology, pharmacodynamic and ADME studies were carried out in multiple species to characterize the safety, pharmacokinetics and activity of GRN163L, and to determine the desired dose and schedule to be used in initial clinical trials for the treatment of cancer. Results: GRN163L administration in vivo inhibited telomerase activity and tumor growth in tumor xenografts. Models studied included melanoma, myeloma, hepatocellular, ovarian, and lung cancer. Combination studies of GRN63L with melphalan, Velcade, or taxol in various tumor models demonstrated increased efficacy of the combination regimens versus single agent treatment. ADME, pharmacodynamic and preclinical safety studies showed wide biodistribution and support an intermittent schedule and dose that should achieve efficacious levels of GRN163L in patients with an acceptable safety profile. Conclusions: Sufficient GRN163L has been manufactured under GLP and GMP conditions to support IND-enabling preclinical and Phase I clinical studies. The efficacy, PD, and ADME data have identified a target dose range and schedule for GRN163L, and preclinical safety studies have shown a robust safety margin that should allow attainment of this dose and schedule in the clinic. Additional studies to support the clinical development strategy are ongoing. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Geron Geron

Telomerase Inhibition, Telomere Shortening and Apoptotic Cell Death in Multiple Myeloma Cells Following Exposure to a Novel and Potent Telomerase Inhibitor (GRN163L), Targeting RNA component of Telomerase.

Telomeres, the specialized nucleoprotein structures at the ends of chromo-somes, shorten at each DNA replication, and if unopposed leads to chromosomal erosion and cell death. Telomere shortening below a critical length is prevented by telomerase. We have previously observed elevated telomerase activity and shortened telomeres in multiple myeloma (MM), making the telomere maintenance mechanism an important target for therapy. Based on success with other non-specific telomerase inhibitors, in this study, we evaluated the effects of a thio-phosphoramidate oligonucleotide specifically targeting the RNA component of telomerase (GRN163L), with modifications to facilitate its delivery into human cells. Nuclear uptake of GRN163L without need for transfection enhancer at 24h was confirmed in >99% MM cells using fluorescein isothiocyanate-tagged GRN163L and confocal microscopy. Next we evaluated the effects of different concentrations and length of exposure of GRN163L on telomerase activity in diff MM cell lines (ARP and INA6). Whereas control oligonucleotide did not significantly affect telomerase activity, ≥ 80% loss of telomerase activity was observed in MM cell lines at day 3 at submicromolar concentrations of GRN163L. This inhibition of telomerase activity was associated with inhibition of myeloma cell growth and survival. Treatment of INA6 cells with GRN163L for three weeks induced 96±4% and 100% cell death at 0.5 and 1 μM concentrations, respectively, while ARP cells with higher telomerase activity and longer telomeres showed 67 ± 4% cell death at 5 weeks with 0.5 μM inhibitor and 82 ± 3% and 100% cell death at 4 and 5 weeks respectively with 2 μM concentration. The cell death was predominantly apoptotic, as determined by 51% annexin V-positive INA6 cells at two weeks and >80 % annexin V positive ARP cells at four weeks. The apoptotic cell death was associated with reduction in telomere length as analyzed using Telomere-FISH. While the control oligo treated ARP cells showed mean Telomere Fluorescence Intensity (TFI) on interphase chromosomes of 17.2 ± 1.5 (range 1.3–146.4), GRN163L treated cells showed reduction of mean TFI to 13.6 ± 0.46 (range 0.74 – 65.7). Although values above 100 were observed on 2 chromosomes in control oligo treated cells, GRN163L-treated cells had no chromosome with a TFI of more than 70.0. A similar reduction in telomere length was observed for INA6 cells. Subsequently, we were also able to increase cytotoxicity of DNA damaging agents in MM cells treated with GRN163L and shortened telomeres; providing a rationale for evaluation of combination therapies. These data demonstrate GRN163L as a potent and specific telomerase inhibitor able to disrupt telomere integrity and inducing apoptotic death of multiple myeloma cells. Evaluation of this agent in a SCIDhu model of myeloma is underway prior to its clinical evaluation.

A novel pyrazolone, 4,4-dichloro-1-(2,4-dichlorophenyl)-3-methyl-5-pyrazolone, as a potent catalytic inhibitor of human telomerase

A new derivative of 1-phenyl-3-methyl-5-pyrazolone, 4,4-dichloro-1-(2,4-dichlorophenyl)-3-methyl-5-pyrazolone, named TELIN, was chemically synthesized and identified as a potent inhibitor of human telomerase in the cell-free telomeric repeat amplification protocol. TELIN inhibited telomerase activity at submicromolar level with IC50 of ∼0.3μM. Kinetic studies revealed that TELIN does not bind to DNA but to telomerase protein, and the mode of inhibition by this substance was competitive–noncompetitive mixed-type with respect to the TS primer, whereas it was uncompetitive or noncompetitive–uncompetitive mixed-type with respect to the three deoxyribonucleosides. These results demonstrate that TELIN is a specific potent catalytic blocker of telomerase, and is considered to be a valuable substance for medical treatment of cancer and related diseases.

Telomerase inhibition by sulfoquinovosyldiacylglycerol from edible purple laver ( Porphyra yezoensis)

As high telomerase activity is detected in most cancer cells, telomerase represents a promising cancer therapeutic target. We investigated the inhibitory effect of sulfoquinovosyldiacylglycerol (SQDG), distributed in plants and seaweeds, on human telomerase in a cell-free system. SQDG inhibited telomerase activity dose-dependently with 50% inhibition at 22 μM, whereas monogalactosyldiacylglycerol and digalactosyldiacylglycerol did not, even at concentrations of 100 μM. Moreover, we confirmed that eicosapentaenoic acid, one of the fatty acid components of SQDG, is a potent telomerase inhibitor with 50% inhibition at 19 μM. We speculate that the structure of the sulfate group and fatty acid of SQDG is important for the potent telomerase-inhibitory effect. Our findings suggest that SQDG has potential use as a therapeutic dietary compound for telomerase inhibition.

Human PinX1, a potent telomerase inhibitor, is not involved in human gastrointestinal tract carcinoma

PinX1 was isolated as a Pin2/TRF1 binding protein that also binds to the telomerase catalytic subunit hTERT. The gene is a potent telomerase inhibitor and a putative tumor suppressor since it inhibits telomerase activity and affects tumorigenicity in nude mice. This study investigated aberrations of PinX1 gene in gastrointestinal tract carcinomas (GITCs). We examined mutations, mRNA expression and promoter methylation of PinX1 gene in 15 GITC cell lines, and 20 patients with primary GITC. We found a missense mutation at codon 254 (AGC/TGC) in a colon and an esophageal carcinoma cell line, and in cancerous and matching normal tissues of 2 patients with primary GITC (10%). It might be a benign polymorphism. No hyper-methylation was found in the promoter region and the treatment by 5-Aza-2'-deoxycytidine did not affect PinX1 mRNA expression level in any of the cell lines. It was concluded that the human PinX1 does not affect tumorigenesis of human GITC.

Effects of triethylene tetraamine on telomerase activity and proliferation in HeLa cells

Telomerase, which is required to maintain telomeres, has attracted considerable attention as a target for anticancer therapy. In this study, we investigated the inhibition of HeLa cell telomerase activity and cell cycle progression by triethylene tetraamine (TETA), using a modified telomeric repeat amplification protocol (TRAP) assay, and flow cytometry. TETA inhibited telomerase activity in HeLa cell extracts, with an IC50 of about 7.8 microM. Coupled with this inhibition, TETA also increased the proportion of cells in the G1 phase of the cell cycle in a dose-dependent manner. These findings demonstrate that TETA is a potent inhibitor of telomerase in micromolar concentrations, and inhibits the proliferation of HeLa cells by arresting them in G1.