Synthesis and evaluation of 9-O-substituted berberine derivatives containing aza-aromatic terminal group as highly selective telomeric G-quadruplex stabilizing ligands

A series of new 9-O-substituted berberine derivatives (4a–j) as telomeric quadruplex ligands was synthesized and evaluated. The results from biophysical and biochemical assay indicated that introducing of positive charged aza-aromatic terminal group into the side chain of 9-position of berberine significantly improved the binding ability with G-quadruplex, and exhibited the inhibitory effect on the hybridization and on telomerase activity. These derivatives showed excellent selectivity for telomeric G-quadruplex DNA over duplex.     

Benzofuroquinoline Derivatives Had Remarkable Improvement of their Selectivity for Telomeric G-Quadruplex DNA over Duplex DNA upon Introduction of Peptidyl Group

In order to improve the selectivity of 5-N-methyl quindoline (cryptolepine) derivatives as telomeric quadruplex binding ligands versus duplex DNA, a series of peptidyl-benzofuroquinoline (P-BFQ) conjugates (2a-2n) were designed and synthesized. Their interactions with telomeric quadruplex and duplex DNA were examined by using the fluorescence resonance energy transfer (FRET) melting assay, surface plasmon resonance (SPR), circular dichroism spectroscopy (CD), and molecular modeling studies. Introduction of a peptidyl group at 11-position of the aromatic benzofuroquinoline scaffold not only effectively increased its binding affinity, but also significantly improved its selectivity toward telomeric quadruplex versus duplex DNA. Combined with the data for their inhibitory effects on telomerase activity, their structure-activity relationships (SARs) studies showed that the types of amino acid residues and the length of the peptidyl side chains were important for the improvement of their interactions with the telomeric G-quadruplex. Long-term exposure of human cancer cells to 2c showed a remarkable cessation in population growth and cellular senescence phenotype, and accompanied by a shortening of the telomere length.     

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 microM). 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.     

Design, synthesis, biophysical and biological studies of trisubstituted naphthalimides as G-quadruplex ligands

A series of trisubstituted naphthalimides have been synthesized and evaluated as telomeric G-quadruplex ligands by biophysical methods. Affinity for telomeric G-quadruplex AGGG(TTAGGG)(3) binding was first screened by fluorescence titrations. Subsequently, the interaction of the telomeric G-quadruplex with compounds showing the best affinity has been studied by isothermal titration calorimetry and UV-melting experiments. The two best compounds of the series tightly bind the telomeric quadruplex with a 2:1 drug/DNA stoichiometry. These derivatives have been further evaluated for their ability to inhibit telomerase by a TRAP assay and their pharmacological properties by treating melanoma (M14) and human lung cancer (A549) cell lines with increasing drug concentrations. A dose-dependent inhibition of cell proliferation was observed for all cellular lines during short-term treatment.     

Specific binding of telomeric G-quadruplexes by hydrosoluble perylene derivatives inhibits repeat addition processivity of human telomerase

Telomerase is responsible for the immortal phenotype of cancer cells and telomerase inhibition may specifically target cancer cell proliferation. Ligands able to selectively bind to G-quadruplex telomeric DNA have been considered as telomerase inhibitors but their mechanisms of action have often been deduced from a non-quantitative telomerase activity assay (TRAP assay) that involves a PCR step and that does not provide insight on the mechanism of inhibition. Furthermore, quadruplex ligands have also been shown to exert their effects by affecting association of telomere binding proteins with telomeres. Here, we use quantitative direct telomerase activity assays to evaluate the strength and mechanism of action of hydrosoluble perylene diimides (HPDIs). HPDIs contain a perylene moiety and different numbers of positively charged side chains. Side chain features vary with regard to number and distances of the charges. IC₅₀ values of HPDIs were in the low micromolar (0.5–5 μM) range depending on the number and features of the side chains. HPDIs having four side chains emerged as the best compounds of this series. Analysis of primer elongation products demonstrated that at low HPDI concentrations, telomerase inhibition involved formation of telomeric G-quadruplex structures, which inhibited further elongation by telomerase. At high HPDI concentrations, telomerase inhibition occurred independently of G-quadruplex formation of the substrate. The mechanism of action of HPDIs and their specific binding to G-quadruplex DNA was supported by PAGE analysis, CD spectroscopy and ESI-MS. Finally, competition Telospot experiments with duplex DNA indicated specific binding of HPDIs to the single-stranded telomeric substrates over double stranded DNA, a result supported by competitive ESI-MS. Altogether, our results indicate that HPDIs act by stabilizing G-quadruplex structures in single-stranded telomeric DNA, which in turn prevents repeat addition processivity of telomerase.     

Selective interaction of ethidium derivatives with quadruplexes: an equilibrium dialysis and electrospray ionization mass spectrometry analysis

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analyzed the selectivity of four ethidium derivatives and ethidium itself toward different G-quadruplex species, with electrospray mass spectrometry and competitive equilibrium dialysis and evaluated their inhibitory properties against telomerase. A selectivity profile may be obtained through electrospray ionization mass spectrometry (ESI-MS), which is in fair agreement with competitive equilibrium dialysis data. It also provides unambiguous data on the number of binding sites per nucleic acid (maximal number of two ethidium derivatives per quadruplex, in agreement with external stacking). Our experiments also demonstrate that one compound (4) is the most active and selective G-quadruplex ligand within this series and the most selective telomerase inhibitor in a modified TRAP-G4 assay.     

Spectroscopic study and G-quadruplex DNA binding affinity of two bioactive papaverine-derived ligands

The interactions of G-quadruplex DNA with two oxidation products of papaverine, 6a,12a-diazadibenzo-[a,g]fluorenylium derivative (1) and 2,3,9,10-tetramethoxy-12-oxo-12H-indolo[2,1-a]isoquinolinium cation (2) were investigated. Their activity against telomerase was assessed using the conventional telomeric repeat amplification protocol (TRAP) assay. Effect of TRAP buffer and oligonucleotide length on the DNA-binding affinity of 1 and 2 were also studied. Three quadruplex-forming oligonucleotides with human telomeric sequence: dG₃(T₂AG₃)₃ (htel21), dAG₃(T₂AG₃)₃ (htel22), and d(T₂AG₃)₄ (htel24) were used in these investigations. Both ligands were capable of interacting with G4 DNA with binding stoichiometry indicating that two ligand molecules bind to G-quadruplex, which agrees with the binding model of end-stacking on terminal G-tetrads. Circular dichroism spectra revealed that preferences of quadruplex-forming oligonucleotide to adopt a particular topological structure may be also affected by the external ligand that binds to quadruplex. Telomerase activity was suppressed at very low ligand 1 and ligand 2 concentrations with an appreciable selectivity comparing with inhibition of Taq polymerase.     

Telomerase recognizes G-quadruplex and linear DNA as distinct substrates

Telomeric DNA can assemble into a nonlinear, higher-order conformation known as a G-quadruplex. Here, we demonstrate by electrospray ionization mass spectrometry that the two repeat telomeric sequence d(TGGGGTTGGGGT) from Tetrahymena thermophila gives rise to a novel parallel four-stranded G-quadruplex in the presence of sodium. The G-quadruplex directly interacts with the catalytic subunit of Tetrahymena telomerase (TERT) with micromolar affinity, and the presence of telomerase RNA is not obligatory for this interaction. Both N- and C-terminal halves of TERT bind the G-quadruplex independently. This G-quadruplex is a robust substrate for both recombinant and cell extract-derived telomerase in vitro. Furthermore, the G-quadruplex weakens the affinity of wild-type telomerase for the incoming nucleotide (dTTP) and likely perturbs the nucleotide binding pocket of the enzyme. In agreement with this, a lysine to alanine substitution at amino acid 538 (K538A) within motif 1 of TERT dramatically reduces the ability of telomerase to extend G-quadruplex but not linear DNA. The K538A mutant retains binding affinity for the quadruplex. This suggests that telomerase undergoes changes in conformation in its active site to specifically accommodate binding and subsequent extension of G-quadruplex DNA. We propose that telomerase recognizes G-quadruplex DNA as a substrate that is distinct from linear DNA.     

Tetrasubstituted naphthalene diimide ligands with selectivity for telomeric G-quadruplexes and cancer cells

A series of tetrasubstituted naphthalene diimide compounds with N-methylpiperazine end groups has been synthesized and evaluated as G-quadruplex ligands. They have high affinity and selectivity for telomeric G-quadruplex DNA over duplex DNA. CD studies show that they induce formation of a parallel G-quadruplex topology. They inhibit the binding of hPOT1 and topoisomerase IIIα to telomeric DNA and inhibit telomerase activity in MCF7 cells. The compounds have potent activity in a panel of cancer cell lines, with typical IC(50) values of ～0.1 μM, and up to 100-fold lower toxicity in a normal human fibroblast cell line.     

Interaction of an acridine dimer with DNA quadruplex structures.

The reactivation of telomerase activity in most cancer cells supports the concept that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. The telomeric G-rich single-stranded DNA can adopt an intramolecular G-quadruplex structure in vitro, which has been shown to inhibit telomerase activity. The C-rich sequence can also adopt a quadruplex (intercalated) structure (i-DNA). Two acridine derivatives were shown to increase the melting temperature of the G- quadruplex and the C-quadruplex at 1 microM dye concentration. The increase in Tm value of the G-quadruplex was associated with telomerase inhibition in vitro. The most active compound, "BisA", showed an IC(50) value of 0.75 microM in a standard TRAP assay.     

Disubstituted 1,8-dipyrazolcarbazole derivatives as a new type of c-myc G-quadruplex binding ligands

A series of 1,8-dipyrazolcarbazole (DPC) derivatives (6a-6d, 7a-7d) designed as G-quadruplex ligands have been synthesized and characterized. The FRET-melting and SPR results showed that the DPC derivatives could well recognize G-quadruplex with strong discrimination against the duplex DNA. In addition, the DPC derivatives showed much stronger stabilization activities and binding affinities for c-myc G-quadruplex rather than telomeric G-quadruplex. Therefore, their interactions with c-myc G-quadruplex were further explored by means of CD spectroscopy, PCR-stop assay, and molecular modeling. In cellular studies, all compounds showed strong cytotoxicity against cancer cells, while weak cytotoxicity towards normal cells. RT-PCR assay showed that compound 7b could down-regulate c-myc gene expression in Ramos cell line, while had no effect on c-myc expression in CA46 cell line with NHE III(1) element removed, indicating its effective binding with G-quadruplex on c-myc oncogene in vivo.     

Real-time determination of telomerase activity in cell extracts using an optical biosensor

A biosensoric approach has been developed to determine the activity of telomerase in tumor cell lysates. An optical sensor, the grating coupler, was used to monitor the association and dissociation of unlabeled compounds on the sensor surface in real time, by virtue of an evanescent field. An oligonucleotide was immobilized on the surface of the optical biosensor and linked with two other oligonucleotides by complementary sequences in an overlapping manner. The 3'-end of the last one carried the sequence of the telomeric substrate (TS) primer used for elongation by telomerase in the telomeric repeat amplification protocol (TRAP) assay. This primer sequence was phosphorothioate (PS)-modified, which is known to strongly increase the affinity to the primer binding site of telomerase protein and consequently the velocity of the telomerase reaction. We show that the PS primer binds to the modified biosensor and is elongated effectively by the telomerase from HL-60 cell lysates. A synthesis rate of 1 nucleotide/min was determined. The inhibitory effect of peptide nucleic acid (PNA) was shown by using immobilized TS. The velocity of the telomerase reaction was slowed down and the signal intensity was below the signal-to-noise ratio. Most nucleic acid detection systems use amplification steps such as polymerase chain reaction (PCR) to increase the amount of the probe. Since telomerase is a polymerase itself amplification of DNA by PCR is not required. Furthermore, no purification steps were required since all measurements were performed with crude cell extract.     

A DNA polymerase stop assay for G-quadruplex-interactive compounds.

We have developed and characterized an assay for G-quadruplex-interactive compounds that makes use of the fact that G-rich DNA templates present obstacles to DNA synthesis by DNA polymerases. Using Taq DNA polymerase and the G-quadruplex binding 2, 6-diamidoanthraquinone BSU-1051, we find that BSU-1051 leads to enhanced arrest of DNA synthesis in the presence of K+by stabilizing an intramolecular G-quadruplex structure formed by four repeats of either TTGGGG or TTAGGG in the template strand. The data provide additional evidence that BSU-1051 modulates telomerase activity by stabilization of telomeric G-quadruplex DNA and point to a polymerase arrest assay as a sensitive method for screening for G-quadruplex-interactive agents with potential clinical utility.     

Spectroscopic, molecular modeling, and NMR-spectroscopic investigation of the binding mode of the natural alkaloids berberine and sanguinarine to human telomeric G-quadruplex DNA

G-quadruplex structures can be formed at the single-stranded overhang of telomeric DNA, and ligands able to stabilize this structure have recently been identified as potential anticancer drugs. Among the potential G-quadruplex binders, we have studied the binding ability of berberine and sanguinarine, two members of the alkaloid family, an important class of natural products long known for medicinal purpose. Our spectroscopic (CD, NMR, and fluorescence) studies and molecular modeling approaches revealed binding modes at ligand-complex stoichiometries >1:1 and ligand self-association induced by DNA for the interactions of the natural alkaloids berberine and sanguinarine with the human telomeric G-quadruplex DNA.     

Design,synthesis and evaluation of 4,5-di-substituted acridone ligands with high G-quadruplex affinity and selectivity,together with low toxicity to normal cells

A series of 4,5-di-substituted acridones have been designed and synthesized. Several compounds show high affinity for telomeric G-quadruplex DNA in classical and competition FRET assays, together with low duplex DNA affinity, although they do not show activity in a telomerase assay or evidence of telomere shortening. They have low toxicity against a panel of cancer cell lines and a normal human fibroblast line, and produce potent senescence-based long-term growth arrest in the MCF7 and A549 cancer cell lines.     

Targeting pancreatic cancer with a G-quadruplex ligand

The integrity of telomeres in most cancer cells is maintained by the action of the telomerase enzyme complex, which catalyzes the synthesis of telomeric DNA repeats in order to replace those lost during replication. Telomerase is especially up-regulated in metastatic cancer and is thus emerging as a major therapeutic target. One approach to telomerase inhibition involves the sequestration of the single-stranded 3' ends of telomeric DNA into higher-order quadruplex structures. We have recently shown that tetra-substituted naphthalene diimide compounds are potent quadruplex-stabilizing molecules with telomerase inhibitory activity in cells. We show here that one such compound, BMSG-SH-3, which has been optimized by computer modeling, has significant in vivo antitumor activity against a model for pancreatic cancer, a cancer that is especially resistant to current therapies. A large reduction in telomerase activity in treated tumors was observed and the naphthalene diimide compound was found to be selectively localized in the treated tumors. We find that the expression of the therapeutically important chaperone protein HSP90, a regulator of telomerase is also reduced in vivo by BMSG-SH-3 treatment. The compound is a potent stabilizer of two G-quadruplex sequences found in the promoter region of the HSP90 gene, as well as a G-quadruplex from human telomeric DNA. It is proposed that the simultaneous targeting of these quadruplexes may be an effective anti-tumor strategy.