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1.
PLoS One ; 7(1): e31120, 2012.
Article in English | MEDLINE | ID: mdl-22295124

ABSTRACT

BACKGROUND: Hypoxia-inducible factor (HIF) is an attractive therapeutic target for renal cell carcinoma (RCC) as its high expression due to the loss of von Hippel-Lindau (VHL) promotes RCC progression. Considering this, we hypothesized that ELR510444, a novel orally available small molecule inhibitor of HIF activity, would reduce angiogenesis and possess significant activity in RCC. The mechanism of action and therapeutic efficacy of ELR510444 were investigated in in vitro and in vivo models of RCC. PRINCIPAL FINDINGS: ELR510444 decreased HIF-1α and HIF-2α levels, reduced RCC cell viability and clonogenic survival, and induced apoptosis. VHL-deficient RCC cells were more sensitive to ELR510444-mediated apoptosis and restoration of VHL promoted drug resistance. Higher concentrations of ELR51044 promoted apoptosis independently of VHL status, possibly due to the microtubule destabilizing properties of this agent. ELR510444 significantly reduced tumor burden in the 786-O and A498 RCC xenograft models. These effects were associated with increased necrosis and apoptosis and inhibition of angiogenesis. CONCLUSIONS: ELR510444 is a promising new HIF inhibitor that reduced RCC cell viability, induced apoptosis, and diminished tumor burden in RCC xenograft models. ELR510444 also destabilized microtubules suggesting that it possesses vascular disrupting and anti-angiogenic properties. Further investigation of ELR510444 for the therapy of RCC is warranted.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/antagonists & inhibitors , Carcinoma, Renal Cell/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Kidney Neoplasms/pathology , Microtubules/drug effects , Neovascularization, Pathologic/drug therapy , Sulfonamides/pharmacology , Thiophenes/pharmacology , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Basic Helix-Loop-Helix Transcription Factors/genetics , Carcinoma, Renal Cell/blood supply , Carcinoma, Renal Cell/metabolism , Cell Hypoxia/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Kidney Neoplasms/blood supply , Kidney Neoplasms/metabolism , Mice , Microtubules/metabolism , Mitosis/drug effects , Polymerization/drug effects , Sulfonamides/therapeutic use , Thiophenes/therapeutic use , Tumor Burden/drug effects , Vascular Endothelial Growth Factor A/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Xenograft Model Antitumor Assays
2.
Biochem Pharmacol ; 74(5): 679-89, 2007 Sep 01.
Article in English | MEDLINE | ID: mdl-17631279

ABSTRACT

The trisubstituted acridine compound BRACO-19 has been developed as a ligand for stabilising G-quadruplex structures. It is shown here that BRACO-19 produces short- and long-term growth arrest in cancer cell lines, and is significantly less potent in a normal cell line. BRACO-19 reduces telomerase activity and long-term telomere length attrition is observed. It is also shown that BRACO-19 binds to telomeric single-stranded overhang DNA, consistent with quadruplex formation, and the single-stranded protein hPOT1 has been shown to be displaced from the overhang in vitro and in cellular experiments. It is concluded that the cellular activity of BRACO-19 can be ascribed both to the uncapping of 3' telomere ends and to telomere shortening that may preferentially affect cells with short telomeres.


Subject(s)
Acridines/pharmacology , Antineoplastic Agents/pharmacology , Telomerase/antagonists & inhibitors , Telomere/metabolism , Acridines/chemistry , Antineoplastic Agents/chemistry , Cell Line , Dose-Response Relationship, Drug , Humans , Molecular Structure
3.
J Med Chem ; 49(2): 582-99, 2006 Jan 26.
Article in English | MEDLINE | ID: mdl-16420044

ABSTRACT

The synthesis is reported of a group of 3,6,9-trisubstituted acridine compounds as telomeric quadruplex-stabilizing ligands with systematic variations at the 3-, 6-, and 9-positions. A new microwave-assisted methodology has been developed for trisubstituted acridine synthesis. Structure-activity relationships are reported using surface plasmon resonance and a fluorescence melting assay to examine quadruplex binding, together with a telomerase inhibition assay. These reveal relationships between G-quadruplex stabilization and telomerase inhibition and optimal 3,6- and 9-substituent side-chain lengths for maximal activity. Qualitative molecular modeling using molecular dynamics simulations has been undertaken on four quadruplex-DNA complexes. Long-term exposure of MCF7 cancer cells to a subset of the most active compounds, at doses lower than the IC(50) values, showed that one compound produced a marked decrease in population growth, accompanied by senescence, which is consistent with telomere targeting by this agent.


Subject(s)
Acridines/chemical synthesis , Antineoplastic Agents/chemical synthesis , DNA/metabolism , Telomerase/antagonists & inhibitors , Telomere/drug effects , Acridines/chemistry , Acridines/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Fluorescence Resonance Energy Transfer , G-Quadruplexes , Humans , Ligands , Models, Molecular , Quantitative Structure-Activity Relationship , Quantum Theory , Surface Plasmon Resonance , Telomerase/metabolism , Telomere/enzymology
4.
Cancer Res ; 65(4): 1489-96, 2005 Feb 15.
Article in English | MEDLINE | ID: mdl-15735037

ABSTRACT

Interference with telomerase and telomere maintenance is emerging as an attractive target for anticancer therapies. Ligand-induced stabilization of G-quadruplex formation by the telomeric DNA single-stranded 3' overhang inhibits telomerase from catalyzing telomeric DNA synthesis and from capping telomeric ends. We report here the effects of a 3,6,9-trisubstituted acridine compound, BRACO-19, on telomerase function in vitro and in vivo. The biological activity of BRACO-19 was evaluated in the human uterus carcinoma cell line UXF1138L, which has very short telomeres (2.7 kb). In vitro, nuclear human telomerase reverse transcriptase (hTERT) expression was drastically decreased after 24 hours, induction of cellular senescence and complete cessation of growth was seen after 15 days, paralleled by telomere shortening of ca. 0.4 kb. In vivo, BRACO-19 was highly active as a single agent against early-stage (68 mm(3)) tumors in a s.c. growing xenograft model established from UXF1138L cells, if given chronically at 2 mg per kg per day i.p. BRACO-19 produced growth inhibition of 96% compared with controls accompanied by partial regressions (P < 0.018). Immunostaining of xenograft tissues showed that this response was paralleled by loss of nuclear hTERT protein expression and an increase in atypical mitoses indicative of telomere dysfunction. Cytoplasmic hTERT expression and its colocalization with ubiquitin was observed suggesting that hTERT is bound to ubiquitin and targeted for enhanced degradation upon BRACO-19 treatment. This is in accord with a model of induced displacement of telomerase from the telomere. The in vitro and in vivo data presented here is consistent with the G-quadruplex binding ligand BRACO-19 producing an anticancer effect by inhibiting the capping and catalytic functions of telomerase.


Subject(s)
Acridines/pharmacology , DNA/metabolism , Telomerase/antagonists & inhibitors , Telomere/drug effects , Uterine Neoplasms/drug therapy , Animals , Cell Line, Tumor , Cell Nucleus/metabolism , Cytoplasm/metabolism , DNA/drug effects , DNA/genetics , DNA, Single-Stranded/drug effects , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , DNA-Binding Proteins , Female , G-Quadruplexes , Guanine/metabolism , Humans , Mice , Mice, Nude , Telomerase/biosynthesis , Telomerase/metabolism , Telomere/genetics , Telomere/metabolism , Ubiquitin/metabolism , Uterine Neoplasms/enzymology , Uterine Neoplasms/genetics , Xenograft Model Antitumor Assays
5.
Mol Cancer Ther ; 3(10): 1201-6, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15486186

ABSTRACT

The trisubstituted acridine derivative BRACO-19 has been designed to interact with and stabilize the quadruplex DNA structures that can be formed by folding of the single-stranded repeats at the 3' end of human telomeres. We suggest that the BRACO-19 complex inhibits the catalytic function of telomerase in human cancer cells and also destabilizes the telomerase-telomere capping complex so that cells enter senescence. Here, we present evidence showing that the inhibition of cell growth caused by BRACO-19 in DU145 prostate cancer cells occurs more rapidly than would be expected solely by the inhibition of the catalytic function of telomerase, and that senescence is accompanied by an initial up-regulation of the cyclin-dependent kinase inhibitor p21, with subsequent increases in p16(INK4a) expression. We also show that treatment with BRACO-19 causes extensive end-to-end chromosomal fusions, consistent with telomere uncapping.


Subject(s)
Cellular Senescence , Chromosomes/ultrastructure , Cyclin-Dependent Kinase Inhibitor p16/genetics , DNA , Prostatic Neoplasms/genetics , Telomere/ultrastructure , 3T3 Cells , Acridines/pharmacology , Animals , Blotting, Western , Catalysis , Cell Line, Tumor , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p16/metabolism , G-Quadruplexes , Humans , Ligands , Male , Metaphase , Mice , Models, Chemical , Nucleic Acid Conformation , Prostatic Neoplasms/metabolism , Rhodamines/pharmacology , Telomerase/metabolism , Time Factors , Up-Regulation
6.
Bioorg Med Chem Lett ; 14(16): 4347-51, 2004 Aug 16.
Article in English | MEDLINE | ID: mdl-15261300

ABSTRACT

Telomerase and telomere maintenance are emerging targets for the treatment of human cancers. We report here on the targeting of the telomere-telomerase complex with a series of small molecules based on an acridine platform. A series of 3,6-bisamidoacridines with extended 9-anilino sidechains were designed and synthesised as potential telomeric G-quadruplex DNA (G4) interacting compounds. G4-stabilisation was assessed using a high-throughput FRET (fluorescence resonance energy transfer) assay and telomerase inhibition quantified by a modified TRAP (telomerase repeat amplification protocol) method. Within the series, the compounds showed significant G4-stabilising ability (Delta T(m) values of 25-36 degrees C at 1 microM concentration) and telomerase inhibition in the nanomolar region ((tel)EC(50) values of 80-318 nM). Furthermore, a direct correlation between the FRET and TRAP assays was observed, supporting the use of the rapid screening FRET assay for early assessment of potential G4-stabilising telomerase inhibitors.


Subject(s)
Acridines/chemical synthesis , Acridines/pharmacology , Aniline Compounds/chemistry , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Telomerase/antagonists & inhibitors , Acridines/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Fluorescence Resonance Energy Transfer , Protein Binding , Telomerase/metabolism
7.
Org Biomol Chem ; 2(7): 981-8, 2004 Apr 07.
Article in English | MEDLINE | ID: mdl-15034620

ABSTRACT

We report here the synthesis and evaluation for telomerase-inhibitory and quadruplex DNA binding properties of several rationally-designed quindoline analogues, substituted at the 2- and 7- positions. The ability of these compounds to interact with and stabilise an intramolecular G-quadruplex DNA against increases in temperature was evaluated by a fluorescence-based (FRET) melting assay. The resulting T(m) values were found to correlate with their potency for telomerase inhibition, as measured in an in vitro telomerase TRAP assay. The interactions of a number of compounds with a quadruplex DNA molecular structure were simulated by molecular modelling methods. It is concluded that this class of compound represents a new chemical type suitable for further development as telomerase inhibitors.


Subject(s)
Alkaloids/chemical synthesis , Alkaloids/pharmacology , DNA/chemistry , Indoles/chemical synthesis , Indoles/pharmacology , Quinolines/chemical synthesis , Quinolines/pharmacology , Telomerase/antagonists & inhibitors , Alkaloids/chemistry , Cell Line, Tumor , Female , G-Quadruplexes , Humans , Indole Alkaloids , Indoles/chemistry , Ligands , Models, Molecular , Molecular Structure , Quinolines/chemistry , Spectrometry, Fluorescence/methods
8.
Mol Pharmacol ; 64(5): 1101-8, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14573759

ABSTRACT

Flavopiridol is a broad-spectrum inhibitor of cyclin-dependent kinases and of global transcription via the inhibition of positive transcription elongation factor b (P-TEFb). Although flavopiridol is currently undergoing phase II clinical trials, acquired cellular resistance to the compound during treatment is a potential problem, as it is with almost all current anticancer agents. A HCT116 human colon carcinoma cell line with an acquired 8-fold resistance to flavopiridol has been established. We report here that there are changes in these resistant cells in terms of telomere length and telomerase activity, whereas no change in the expression of the P-TEFb subunits CDK9, cyclin T1, cyclin T2a, or cyclin T2b was observed. The level of mRNA expression for the telomerase catalytic subunit hTERT was increased over 2-fold in the resistant cells, and mean telomere length was found to be 2 kb longer than the parental length, although telomerase activity was unchanged. The level of mRNA expression for the telomeric binding protein Pot1 was also increased. We also report that treatment of HCT116 cells with a combination of the G-quadruplex interacting telomerase inhibitor BRACO-19 and flavopiridol results in a 3-fold decrease in population doubling and prevents recovery from treatment with either compound alone. Treatment of flavopiridol-resistant cells with BRACO-19 alone also led to rapid inhibition of cell growth, which is not observed in the parental line. The finding that only the resistant line, with up-regulated telomerase, responds to this G-quadruplex inhibitor is consistent with the hypothesis that the mechanism of BRACO-19 down-regulation of cell growth directly involves the targeting of telomeres and telomerase.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/physiology , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Piperidines/pharmacology , Acridines/pharmacology , Catalytic Domain , Colonic Neoplasms/pathology , Cyclin D1/biosynthesis , Cyclin T , Cyclin-Dependent Kinase 9/biosynthesis , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/biosynthesis , DNA-Binding Proteins , Drug Combinations , Humans , Telomerase/antagonists & inhibitors , Telomerase/biosynthesis , Telomerase/metabolism , Telomere/metabolism , Tumor Cells, Cultured , Tumor Suppressor Protein p53/biosynthesis , Up-Regulation/drug effects
9.
Curr Opin Investig Drugs ; 4(6): 675-85, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12901225

ABSTRACT

The ends of chromosomes (telomeres) are subject to progressive shortening in normal somatic cells, leading ultimately to irreversible growth arrest. In contrast, telomeres in all cancer cells are stabilized in length and effectively immortalized by the enzyme telomerase, which catalyzes the synthesis of telomeric DNA repeats. Several strategies have been devised for the inhibition of telomerase in the hope that this will result in anticancer effects. The principal approaches of catalytic inhibitors, antisense to the template, and folding of the DNA substrate, are reviewed and critically evaluated for their potential in anticancer therapy.


Subject(s)
Enzyme Inhibitors/therapeutic use , Neoplasms/drug therapy , Technology, Pharmaceutical/trends , Telomerase/antagonists & inhibitors , Animals , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Forecasting , Humans , Neoplasms/enzymology , Technology, Pharmaceutical/methods , Telomerase/metabolism , Telomere/drug effects , Telomere/metabolism
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