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Targeting telomeres and telomerase
Abstract
Telomeres and telomerase represent, at least in theory, an extremely attractive target for cancer therapy. The objective of this review is to present the latest view on the mechanism(s) of action of telomerase inhibitors, with an emphasis on a specific class of telomere ligands called G-quadruplex ligands, and to discuss their potential use in oncology.
... In addition, the groove binding pockets and loop arrangement are ideal for a specific ligand-based therapeutic design. The mode of action of the G-quadruplex depends on stability with the ligands and the basis of forming different structures (parallel, antiparallel and hybrid) in the presence of ions such as K + , Na + , Mg + , and the pH [15][16][17][18][19]. Several G-quadruplex ligands (e.g., anthraquinone derivatives, flavonoids, alkaloids, telomastatins, anthracyclines, etc.), bind through external groove binding mode [2][3][4]20,21]. Few others, such as RHPS4, daunomycin (an anthracycline) and epiberberine alkaloid bind through stacking mode at the ends of DNA or intercalate between the G-quartets via favorable π-π interactions between the aromatic surface of the G-quartets and ligands [2][3][4]17]. ...
... The mode of action of the G-quadruplex depends on stability with the ligands and the basis of forming different structures (parallel, antiparallel and hybrid) in the presence of ions such as K + , Na + , Mg + , and the pH [15][16][17][18][19]. Several G-quadruplex ligands (e.g., anthraquinone derivatives, flavonoids, alkaloids, telomastatins, anthracyclines, etc.), bind through external groove binding mode [2][3][4]20,21]. Few others, such as RHPS4, daunomycin (an anthracycline) and epiberberine alkaloid bind through stacking mode at the ends of DNA or intercalate between the G-quartets via favorable π-π interactions between the aromatic surface of the G-quartets and ligands [2][3][4]17]. Aromatic end stackers and groove binding ligands increase the binding affinity by interacting with side chains. To the best of our knowledge, there are three reports of ligands tested against some of the genes comprising G4s in Mtb for their ...
... In humans, the G4 DNA/RNA is associated with many vital cellular processes, such as replication, transcription, etc. In addition, this G-quartet is also found in the telomere, which inhibits telomerase enzyme or the promoters of several proto-oncogenes, such as BCL-2, c-MYC, KRAS, and c-KIT, making it a potential therapeutic target for cancer [2][3][4]. The RNA/DNA G-quadruplex structure selectivity for towards tetraplex structures over duplex [5][6][7]. ...
Occurrence of non-canonical G-quadruplex (G4) DNA structures in the genome have been recognized as key factors in gene regulation and several other cellular processes. The mosR and ndhA genes involved in pathways of oxidation sensing regulation and ATP generation, respectively, make Mycobacterium tuberculosis (Mtb) bacteria responsible for oxidative stress inside host macrophage cells. Circular Dichroism spectra demonstrate stable hybrid G4 DNA conformations of mosR/ndhA DNA sequences. Real-time binding of mitoxantrone to G4 DNA with an affinity constant ~105–107 M−1, leads to hypochromism with a red shift of ~18 nm, followed by hyperchromism in the absorption spectra. The corresponding fluorescence is quenched with a red shift ~15 nm followed by an increase in intensity. A change in conformation of the G4 DNA accompanies the formation of multiple stoichiometric complexes with a dual binding mode. The external binding of mitoxantrone with a partial stacking with G-quartets and/or groove binding induces significant thermal stabilization, ~20–29 °C in ndhA/mosR G4 DNA. The interaction leads to a two/four-fold downregulation of transcriptomes of mosR/ndhA genes apart from the suppression of DNA replication by Taq polymerase enzyme, establishing the role of mitoxantrone in targeting G4 DNA, as an alternate strategy for effective anti-tuberculosis action in view of deadly multi-drug resistant tuberculosis disease causing bacterial strains t that arise from existing therapeutic treatments.
... Based on the similarity principle, some ligands were selected with high affinity and selectivity toward DNA G-quadruplex, drug-like property, chemical diversity, different mechanism of interaction and in vitro activity in tumor cells as query molecules [14]. For example, D. Cian et al. surveyed the G-quadruplex ligands tested for telomerase inhibition [14,24]. ...
... Based on the similarity principle, some ligands were selected with high affinity and selectivity toward DNA G-quadruplex, drug-like property, chemical diversity, different mechanism of interaction and in vitro activity in tumor cells as query molecules [14]. For example, D. Cian et al. surveyed the G-quadruplex ligands tested for telomerase inhibition [14,24]. S. Neidle critically examined the major classes of the currently developed quadruplex-binding small molecules [5,25,26]. ...
In order to develop new anti-cancer drugs more efficiently and reduce side effects based on active drug targets, the virtual drug screening was carried out through the target of G-quadruplexes and 23 hit compounds were, thus, screened out as potential anticancer drugs. Six classical G-quadruplex complexes were introduced as query molecules, and the three-dimensional similarity of molecules was calculated by shape feature similarity (SHAFTS) method so as to reduce the range of potential compounds. Afterwards, the molecular docking technology was utilized to perform the final screening followed by the exploration of the binding between each compound and four different structures of G-quadruplex. In order to verify the anticancer activity of the selected compounds, compounds 1, 6 and 7 were chosen to treat A549 cells in vitro, the lung cancer epithelial cells, for further exploring their anticancer activity. These three compounds were found to be of good characteristics in the treatment of cancer, which revealed the great application prospect of the virtual screening method in developing new drugs.
... Structural studies of human telomeric repeats have potential applications in the development of telomeric quadruplextargeting small molecules for the treatment of telomerasepositive cancer [29,30,[43][44][45]. Therefore, the search for the inhibitory effects of small molecules on the catalytic activity of the telomerase is growing interestingly in the development of specific telomeric quadruplex-targeting drugs for potential anticancer treatment [39,[46][47][48][49][50][51][52]. ...
... For example, the c-MYC-MAX heterodimeric complex promotes cell proliferation by switching on cyclins and cyclin-dependent kinases (CDK4) that are required for cell-cycle progression and also at the same time represses the transcription of cell-cycle checkpoint genes and inhibits the function of cyclin-dependent kinase inhibitors [66]. There are varieties of c-MYC responsive genes involved in cellular functions (cell proliferation, metabolic transformation, and metastatics) [51]. The c-MYC protein's function in tumor cells is upregulated, and therefore, the downregulation of overexpression of c-MYC is effective for cancer therapy in 80% of solid tumors [67][68][69]. ...
The self-association of DNA formed by Hoogsteen hydrogen bonding comprises several layers of four guanine or G-tetrads or G4s. The distinct feature of G4s, such as the G-tetrads and loops, qualify structure-selective recognition by small molecules and various ligands and can act as potential anticancer therapeutic molecules. The G4 selective-ligands, can influence gene expression by targeting a nucleic acid structure rather than sequence. Telomere G4 can be targeted for cancer treatment by small molecules inhibiting the telomerase activity whereas c-MYC is capable of controlling transcription, can be targeted to influence transcription. The k-RAS is one of the most frequently encountered oncogenic driver mutations in pancreatic, colorectal, and lung cancers. The k-RAS oncogene plays important role in acquiring and increasing the drug resistance and can also be directly targeted by small molecules to combat k-RAS mutant tumors. Modular G4 ligands with different functional groups, side chains and rotatable bonds as well as conformation affect the binding affinity/selectivity in cancer chemotherapeutic interventions. These modular G4 ligands act by targeting the diversity of G4 loops and groves and assists to develop more drug-like compounds with selectivity. In this review, we present the recent research on synthetic G4 DNA-interacting ligands as an approach toward the discovery of target specific anticancer chemotherapeutic agents.
... According to the calculations data (Table 2), the reactions of the model system 34a with the investigated nucleophiles proceed within complexes according to reaction (9), which is in complete agreement with the experimental data: only in the case of reaction (9) with the participation of an electroneutral nucleophile and a monohydrated substrate all processes for different substituents R are exothermic, while reactions (7), (8) and (10), (11) predict endothermic processes for some substituents R. ...
... To calculate the minimum energy path (MEP) of reaction (9), the simplest ketone, acetone, was chosen as the nucleophile RH which replaces the hydroxy group by the acetonyl fragment. It is known that the nucleophilicity of acetone is close to the nucleophilicity of haloalkanes and nitriles, and therefore the reactive properties of the latter can be rather reliably modeled with acetone. ...
The possibility of obtaining electroneutral 8-substituted berberines was demonstrated experimentally as well as by DFT/B3LYP and ab initio MP2/HF quantum-chemical calculations with the 6-311+G(d,p) basis set. In alkaline media, it is possible to substitute the C-8 position of berberine with such C-nucleophiles as methyl ketones, acetic acid esters, nitriles, geminal di- and trihaloalkanes. This process proceeds stepwise. First, berberine is attacked by a hydroxide anion to form a covalently bonded 8-hydroxyberberine, then 8-hydroxyberberine reacts with a nucleophile in the nonionized (molecular) form.
... Most human cancer cells, however, express high levels of telomerase, allowing these cells to proliferate and cause malignancies [7,8]. Considering this central role in cell proliferation, telomerase inhibition has become a promising way to combat cancer [9,10]. ...
... Kypr and Vorlíčková previously described that some oligonucleotides with stacked guanine runs, like G-quadruplexes, are characterized by a strong CD band at *260 nm, which originates from intrastrand guanineguanine (G-G) stacking. More specifically, similar CD traces were observed for parallel-stranded DNA tetraplex of d(G 4 ), ordered single strand of d(GA) 10 , and B-and A-forms of the antiparallel duplex of d(C 4 G 4 ). Because Band A-forms have a different backbone geometry, the authors concluded that the backbone does not influence that CD band. ...
Imetelstat (GRN163L) is a potent and specific telomerase inhibitor currently in clinical development for the treatment of hematological malignancies such as myelofibrosis and myelodysplastic syndrome. It is a 13-mer N3'-P5' thio-phosphoramidate oligonucleotide covalently functionalized at the 5'-end with a palmitoyl lipid moiety through an aminoglycerol linker. As a competitive inhibitor of human telomerase, imetelstat directly binds to the telomerase RNA component sequence (hTR) in the catalytic site of the enzyme and acts as a direct competitor of human telomere binding. Administration of imetelstat causes progressive shortening of the telomeres, thereby inhibiting malignant cells' proliferation. We report here the ability of imetelstat to form stable, parallel, intermolecular G-quadruplex structures in vitro. The impact of the ionic environment on the formation and stability of imetelstat higher-order structure was investigated through circular dichroism spectroscopy, thermal denaturation analysis, and size-exclusion chromatography. We demonstrated that different structural elements, such as the 5'-palmitoyl linker and the thio-phosphoramidate backbone, critically contribute to G-quadruplex stability. Experiments further showed that G-quadruplex formation does not hamper binding to the hTR oligonucleotide sequence in vitro.
... 300,000-400,000 guanine-rich sequences that can potentially form quadruplex DNA structures. [17][18][19][20] Formation of G-quadruplex structures has been confirmed in telomere, thrombin binding aptamer (TBA), immunoglobulin switch regions, gene transcriptional regulatory regions such as the insulin gene, and also the promoter of certain oncogenes, such as c-MYC and BCL2. Formation of G-quadruplex in vitro is confirmed by NMR spectroscopy, X-ray crystallography, and CD spectroscopy. ...
G-quadruplex DNA is formed in telomer. It inhibits telomerase enzyme which is found active in more than 85 % of cancer cells. In this work, the effect of Tween 80 on telomeric G-quadruplex DNA, AGGG(TTAGGG)3 was studied using circular dichroism spectroscopic technique. The obtained results showed that using Tween 80 with telomeric quadruplex is safe up to less than 1.0 %.
... The inhibition of telomerase activity by ligands stabilisation of DNA secondary structures, namely of G4 present in telomeric ends, is being pointed out as a promising antitumour strategy [7,8,[14][15][16]. Moye et al. [17] reported that telomerase has the ability to recognize and partially unwind G-quadruplexes during the 3 -end extension process. ...
Since the occurrence of tumours is closely associated with the telomerase function and oncogene expression, the structure of such enzymes and genes are being recognized as targets for new anticancer drugs. The efficacy of several ligands in telomerase inhibition and in the regulation of genes expression, by an effective stabilisation of G-quadruplexes (G4) DNA structures, is being considered as a promising strategy in cancer therapies. When evaluating the potential of a ligand for telomerase inhibition, the selectivity towards quadruplex versus duplex DNA is a fundamental attribute due to the large amount of double-stranded DNA in the cellular nucleus. This study reports the evaluated efficacy of three tetracationic opp-dibenzoporphyrins, a free base, and the corresponding zinc(II) and nickel(II) complexes, to stabilise G4 structures, namely the telomeric DNA sequence (AG3(T2AG3)3). In order to evaluate the selectivity of these ligands towards G4 structures, their interaction towards DNA calf thymus, as a double-strand DNA sequence, were also studied. The data obtained by using different spectroscopic techniques, such as ultraviolet-visible, fluorescence, and circular dichroism, suggested good affinity of the free-base porphyrin and of its zinc(II) complex for the considered DNA structures, both showing a pattern of selectivity for the telomeric G4 structure. A pattern of aggregation in aqueous solution was detected for both Zn(II) and Ni(II) metallo dibenzoporphyrins and the ability of DNA sequences to induce ligand disaggregation was observed.
... In particular, this is well documented for PDC 360A and PhenDC3 which are indeed strong (nanomolar) in vitro inhibitors of telomerase ). Nonetheless, this notion has been challenged when using cellular models, and it soon appeared that the action of bisquinolinium compounds (and more broadly, G4 ligands) in cells might be independent of telomerase inhibition (De Cian et al. 2008). Furthermore, this is consistent with recent findings showing that telomerase has a G4-resolvase activity that is not inhibited even by potent G4-stabilizing ligands such as PhenDC3 (Paudel et al. 2020). ...
... [4][5][6][7] However, recently shelterin proteins have been proposed as new-class targets for anticancer strategy. [8][9][10][11] The studies of these targets are still very new and it is an unexplored area but is promising and relevant. [12][13][14][15] Thus targeting shelterin components by short peptides/modified peptides or small molecules emerge as a new concept for cancer therapy. ...
In this work, we present studies on relatively new and still not well-explored potential anticancer targets which are shelterin proteins, in particular the TRF1 protein can be blocked by in silico designed "peptidomimetic" molecules. TRF1 interacts directly with the TIN2 protein, and this protein-protein interaction is crucial for the proper functioning of telomere, which could be blocked by our novel modified peptide molecules. Our chemotherapeutic approach is based on assumption that modulation of TRF1-TIN2 interaction may be more harmful for cancer cells as cancer telomeres are more fragile than in normal cells. We have shown in vitro within SPR experiments that our modified peptide PEP1 molecule interacts with TRF1, presumably at the site originally occupied by the TIN2 protein. Disturbance of the shelterin complex by studied molecule may not in short term lead to cytotoxic effects, however blocking TRF1-TIN2 resulted in cellular senescence in cellular breast cancer lines used as a cancer model. Thus, our compounds appeared useful as starting model compounds for precise blockage of TRF proteins.
... In particular, this is well documented for PDC 360A and PhenDC3 which are indeed strong (nanomolar) in vitro inhibitors of telomerase ). Nonetheless, this notion has been challenged when using cellular models, and it soon appeared that the action of bisquinolinium compounds (and more broadly, G4 ligands) in cells might be independent of telomerase inhibition (De Cian et al. 2008). Furthermore, this is consistent with recent findings showing that telomerase has a G4-resolvase activity that is not inhibited even by potent G4-stabilizing ligands such as PhenDC3 (Paudel et al. 2020). ...
G-quadruplexes (G4s) are non-canonical secondary structures that can form in single-stranded DNA and RNA sequences containing multiple guanine tracts. G4s can accommodate and be stabilized by small molecules (G4 ligands) that typically interact by π-stacking with their external quartets. Along these lines, numerous G4 ligands acting as probes and drug prototypes have been reported, but only a few meet the criteria of selectivity and affinity necessary to achieve efficient G4 targeting in cells. The present chapter is focused on bisquinolinium compounds comprising two quinolinium units, typically linked to a (hetero)aromatic dicarboxamide core and which represent the “gold standard” of G4 ligands. The seminal works that led to their design, the development of functional derivatives and of new analogues are described. In addition, a brief overview of their applications to imaging and covalent trapping of G4s in cells and of their therapeutic potential in treating cancer and other diseases is presented.
... G4-DNAs have gained attention as prospective cancer therapeutic targets. It has been hypothesized that ligands that stabilize and bind selectively to the telomeric G4-DNA in cancer cells could limit the telomere elongation process, resulting in senescence (growth arrest) or delayed cell death, as well as the elimination of tumorigenicity [9]. While stabilization of the promoter G-quadruplex of the Hsp90 gene by G4 binders could theoretically downregulate the gene expression and its product Hsp90 chaperone protein, resulting in the deactivation and eventual degradation of its clients known to be involved in oncogenesis, such as kinases and transcription factors, this is not the case in practice [10][11][12]. ...
Here, we describe the anticancer activity of our novel bis-triazoles MS47 and MS49, developed previously as G-quadruplex stabilizers, focusing specifically upon the human melanoma MDA-MB-435 cell line. At the National Cancer Institute (NCI), USA, bis-triazole MS47 (NCS 778438) was evaluated against a panel of sixty human cancer cell lines, and showed selective, distinct multi-log differential patterns of activity, with GI50 and LC50 values in the sub-micromolar range against human cancer cells. MS47 showed highly selective cytotoxicity towards human melanoma, ovarian, CNS and colon cancer cell lines; in contrast, the leukemia cell lines interestingly showed resistance to MS47 cytotoxic activity. Further studies revealed the potent cell growth inhibiting properties of MS47 and MS49 against the human melanoma MDA-MB-435 cell line, as verified by MTT assays; both ligands were more potent against cancer cells than MRC-5 fetal lung fibroblasts (SI > 9). Melanoma colony formation was significantly suppressed by MS47 and MS49, and time- and dose-dependent apoptosis induction was also observed. Furthermore, MS47 significantly arrested melanoma cells at the G0/G1 cell cycle phase. While the expression levels of Hsp90 protein in melanoma cells were significantly decreased by MS49, corroborating its binding to the G4-DNA promoter of the Hsp90 gene. Both ligands failed to induce senescence in the human melanoma cells after 72 h of treatment, corroborating their weak stabilization of the telomeric G4-DNA.
... These noncanonical structures have been extensively studied because their importance in biological processes is high and probably still not fully understood. The G-quadruplex is better recognized, and its role in the suppression of telomerase activity has been confirmed [6][7][8][9][10][11]. The biological role of the i-motif is still not fully understood, and there are some remarks that formation of the i-motif may lead to a similar effect as the G-quadruplex in this direction. ...
Cytosine-rich DNA sequences are able to fold into noncanonical structures, in which semi-protonated cytosine pairs develop extra hydrogen bonds, and these bonds are responsible for the overall stability of a structure called the i-motif. The i-motif can be formed in many regions of the genome, but the most representative is the telomeric region in which the CCCTAA sequences are repeated thousands of times. The ability to reverse folding/unfolding in response to pH change makes the above sequence and i-motif very promising components of nanomachines, extended DNA structures, and drug carriers. Molecular dynamics analysis of such structures is highly beneficial due to direct insights into the microscopic structure of the considered systems. We show that Amber force fields for DNA predict the stability of the i-motif over a long timescale; however, these force fields are not able to predict folding of the cytosine-rich sequences into the i-motif. The reason is the kinetic partitioning of the folding process, which makes the transitions between various intermediates too time-consuming in atomistic force field representation. Application of coarse-grained force fields usually highly accelerates complex structural transitions. We, however, found that three of the most popular coarse-grained force fields for DNA (oxDNA, 3SPN, and Martini) were not able to predict the stability of the i-motif structure. Obviously, they were not able to accelerate the folding of unfolded states into an i-motif. This observation must be strongly highlighted, and the need to develop suitable extensions of coarse-grained force fields for DNA is pointed out. However, it will take a great deal of effort to successfully solve these problems.
... 28, 29 The presence of putative G4 and C-rich sequences in plants and mammals may help understand the inhibitory actions of binding proteins and ligands especially at telomeric regions, which facilitate finding novel therapeutic effects. 30 The physical properties like DNA meltability, flexibility of non-B-DNA structures were discussed in eukaryotic core promoters by a recent research. 31 The pathophysiological studies of neurodegenerative diseases affecting the brain in higher primates like humans can be done by using G-quadruplex motifs and aids in designing new diagnostic ways. ...
The evolutionary constraints of a gene consist of cis-gene regulatory regions, such as promoters and enhancers, which contribute to the regulation of gene expression. Several genomic and computational studies stated the roles of G-quadruplex and i-motif structures in vital cellular processes like transcription, translation, gene regulation, etc. The formation of these non-B DNA structures is supported by the occurrence of unique repeated sequences. However, many studies lean toward understanding the role of the G-quadruplex, and only recent studies indicated i-motif significance. In this study, we attempted to dissect the enrichment of G-quadruplex and i-motifs in promoter regions of mammals and plants. To this end, we employed the genomic sequences encompassing -500 to +500 region relative to the gene start positions in mammals and plants retrieved from the UCSC browser and Plant Genome database (PlantGDB). We computed the putative G-quadruplexes and i-motifs with well-recognized regular expression sequence patterns. We observed that G-quadruplex motifs showed preponderance in mammals, algal species, namely, green algae, and Chlamydomonas when compared to plants. Contrastingly i-motifs are enriched in both monocot and dicot plants compared to G-quadruplex motifs. The comparative examinations in this study revamp our understanding of the two quadruplex structures and their emerging functional roles in complex eukaryotes.
... Stabilization of G-quadruplex structures by small molecules was first demonstrated using an anthraquinone derivative 1 (Figure 3, diamidoanth) [68] and other studies have followed [20], mainly reporting the use of planar aromatic compounds, one end of which being positively charged [14,15,68]. Several studies have shown that the planar part of the molecule comes to rest on the external quartet, while intercalation between two guanine quartets has not been less observed so far [69][70][71][72][73][74]. ...
Due to their optical and electrochemical properties, ruthenium(II) polypyridyl complexes have been used in a wide array of applications. Since the discovery of the light-switch ON effect of [Ru(bpy)2dppz]2+ when interacting with DNA, the design of new Ru(II) complexes as light-up probes for specific regions of DNA has been intensively explored. Amongst them, G-quadruplexes (G4s) are of particular interest. These structures formed by guanine-rich parts of DNA and RNA may be associated with a wide range of biological events. However, locating them and understanding their implications in biological pathways has proven challenging. Elegant approaches to tackle this challenge relies on the use of photoprobes capable of marking, reversibly or irreversibly, these G4s. Indeed, Ru(II) complexes containing ancillary π-deficient TAP ligands can create a covalently linked adduct with G4s after a photoinduced electron transfer from a guanine residue to the excited complex. Through careful design of the ligands, high selectivity of interaction with G4 structures can be achieved. This allows the creation of specific Ru(II) light-up probes and photoreactive agents for G4 labelling, which is at the core of this review composed of an introduction dedicated to a brief description of G-quadruplex structures and two main sections. The first one will provide a general picture of ligands and metal complexes interacting with G4s. The second one will focus on an exhaustive and comprehensive overview of the interactions and (photo)reactions of Ru(II) complexes with G4s.
... The possibility of targeting secondary structures, such as the G-quadruplexes (G4) present in most human oncogene promoters, and also in telomeres, using interactive ligands is being considered a promising antineoplastic strategy since can contribute to downregulate transcription or to block telomere elongation in cancer cells, by stalling the DNA replication machinery [3]. More specifically, the presence of G4 at the end of telomeres (whose function is to protect their ends from nuclease attack) enables indirect targeting of telomerase [4][5][6]. G4 are recognized and partially unwound by telomerase for 3 ′ end extension, thus binding of stabilizing compounds to G4 structures will "lock" the telomeres in the quadruplex configuration, preventing telomere lengthening by telomerase ( Fig. 1) [7]. ...
Telomerase, oncogenes and tumor suppressors are closely associated with tumour occurrence, therefore these structures are being recognized as targets for the development of new anticancer drugs.
The efficacy of several molecules in telomerase inhibition and regulation of genes expression, by adduct formation with G-quadruplexes (G4), has been studied by biophysical and biochemical methods with promising results. We report here the synthesis and structural characterization of a small positively charged diketopyrrolo[3,4–c]pyrrole derivative, identified as DPP(PyMe)2, that showed very promising results as G4 stabilizing ligand.
The data obtained from UV–Vis and fluorescence experiments suggest that DPP(PyMe)2 presents high affinity to G4 structures. Docking studies and molecular dynamics simulations unraveled the binding modes of the ligand with four G4 structures. The obtained results also allowed us to conclude that the DPP(PyMe)2 ligand binds into the top G-tetrad or in a mixed binding mode depending on the GQ structure.
A remarkable selectivity of DPP(PyMe)2 for c-MYC and KRAS 32R in the presence of ds26 was observed by circular dichroism (CD) and fluorescence resonance energy transfer (FRET) melting experiments. CD titrations revealed a stabilization higher than 30 °C in the case of c-MYC G4 structure and, for the same sequence, DPP(PyMe)2 showed the ability to block the activity of Taq polymerase in a dose-dependent manner. The subcellular localization obtained with confocal microscopy corroborates the results obtained by the other techniques and the obtained data suggest that DPP(PyMe)2 is an attractive ligand for the development of G4 labelling probes.
... Each four guanines in a plane form a layer called G-tetrads or G-quartets where Watson-Crick edge of a guanine base forms two hydrogen bonds with Hoogsteen edge of a neighbor guanine base. G-quartets are held together by ππ stacking to form the quadruplex structure and stabilized by electrostatic interactions between the guanine carbonyl groups and alkali metal cations as shown in scheme 1. [26][27][28][29][30][31][32][33][34][35][36][37]. ...
GW-2974 is a potent inhibitor for the tyrosine kinase receptor. It inhibits EGFR and ErbB-2 in tumor cells with high selectivity towards malignant over normal cells. SCH-442416 is a potent antagonist for adenosine receptor with high selectivity towards human A2a receptor over other adenosine receptors. The two compounds were reported to possess antitumor properties. In this work, we studied the stabilization of human telomeric G-quadruplex DNA by GW-2974 and SCH-442416, as a probable underlying mechanism for their anticancer effects. Interactions of human telomeric G-quadruplex DNA - sequence AG3(TTAGGG)3 – with both compounds were studied using UV-Vis, fluorescence quenching, melting temperature, circular dichroism and molecular docking techniques.
Results obtained from absorption, fluorescence and CD indicated that GW2974 and SCH-442416 interacted with G-quadruplex through intercalation binding modes on two types of dependent binding sites. Binding affinities of 1.3x108 - 1.72x106 M-1 and of 1.55x107 - 3.74x105 M-1 were respectively obtained for GW-2974 for SCH-442416. An average number of binding sites between 1 and 2 was obtained for both compounds. The melting temperature curves indicated that complexations of both compounds to G-quadruplex DNA have respectively stabilized it by ΔTm = 9.9 oC and 9.6 oC, relative non-complexed G-quadruplex DNA. Increasing the molar ratios of GW-2974 or SCH-442416 relative to G-quadruplex DNA over 1:1 has shown to destabilized G-quadruplex DNA. Our selectivity experiment indicated 4.7- and 4.0-folds better selectivity for GW-2974 and SCH-442516 towards G-quadruplex DNA over ds-DNA, respectively. The two compounds have also proved to selectively bind and stabilize G-quadruplex DNA over calf thymus duplex ct-DNA. In silico molecular docking indicated exothermic intercalation bindings on two sites of the G-quadruplex DNA
These results supported our hypothesis that both GW-2974 and SCH-442416 strongly stabilize human telomeric G-quadruplex DNA in additions to modulating tyrosine-kinase and adenosine receptors. Consequently, stabilizing G-quadruplex DNA could contribute to the mechanism of their anticancer activity.
... To date, this is the first demonstration that ZnPP has direct antitelomerase activity and these findings support consideration of (Table 2) is also close to the micromolar ranges of IC 50 reported for other synthetic experimental porphyrins. 6,46 The major components of the telomerase holoenzyme complex include TERT, dyskerin, p23, Hsp90, TERC and telomerase-associated protein. 53 TERT is absent from telomeres until it is assembled into telomerase holoenzyme and then recruited to selective chromosomal telomeric sites at the start of S phase. ...
Zinc protoporphyrin (ZnPP), a naturally occurring metalloprotoporphyrin (MPP), is currently under development as a chemotherapeutic agent although its mechanism is unclear. When tested against other MPPs, ZnPP was the most effective DNA synthesis and cellular proliferation inhibitor while promoting apoptosis in telomerase positive but not telomerase negative cells. Concurrently, ZnPP down-regulated telomerase expression and was the best overall inhibitor of telomerase activity in intact cells and cellular extracts with IC50 and EC50 values of ca 2.5 and 6 µM, respectively. The natural fluorescence properties of ZnPP enabled direct imaging in cellular fractions using non-denaturing agarose gel electrophoresis, western blots, and confocal fluorescence microscopy. ZnPP localized to large cellular complexes (>600 kD) that contained telomerase and dysskerin as confirmed with immunocomplex mobility shift, immunoprecipitation, and immunoblot analyses. Confocal fluorescence studies showed that ZnPP co-localized with telomerase reverse transcriptase (TERT) and telomeres in the nucleus of synchronized S-phase cells. ZnPP also co-localized with TERT in the perinuclear regions of log phase cells but did not co-localize with telomeres on the ends of metaphase chromosomes, a site known to be devoid of telomerase complexes. Overall, these results suggest that ZnPP does not bind to telomeric sequences per se, but alternatively, interacts with other structural components of the telomerase complex to inhibit telomerase activity. In conclusion, ZnPP actively interferes with telomerase activity in neoplastic cells, thus promoting pro-apoptotic and anti-proliferative properties. These data support further development of natural or synthetic protoporphyrins for use as chemotherapeutic agents to augment current treatment protocols for neoplastic disease.
... This test measures the elongation of a telomeric strand in order to indirectly measure the effect of a ligand on a GQ sequence. 67 Other tests, such as the Taq polymerase stop assay and the PCR stop assay, also allow an analysis of the effects of the binders in nontelomeric GQs. 68,69 ■ ELECTROSPRAY IONIZATION MASS SPECTROMETRY Increased instrument availability and improved flexibility pushed the development of ESI-MS as an always more commonly used tool for the discovery of GQ binders along with the traditional techniques cited above. ...
Guanine-rich sequences forming G-quadruplexes (GQs) are present in several genomes, ranging from viral to human. Given their peculiar localization, the induction of GQ formation or GQ stabilization with small molecules represents a strategy for interfering with crucial biological functions. Investigating the recognition event at the molecular level, with the aim of fully understanding the triggered pharmacological effects, is challenging. Native electrospray ionization mass spectrometry (ESI-MS) is being optimized to study these noncovalent assemblies. Quantitative parameters retrieved from ESI-MS studies, such as binding affinity, the equilibrium binding constant, and sequence selectivity, will be overviewed. Computational experiments supporting the ESI-MS investigation and boosting its efficiency in the search for GQ ligands will also be discussed with practical examples. The combination of ESI-MS and in silico techniques in a hybrid high-throughput-screening workflow represents a valuable tool for the medicinal chemist, providing data on the quantitative and structural aspects of ligand–GQ interactions.
... Systems capable of controlling DNA and RNA G-quadruplex structures would be useful for the modulation of various cellular events and different G-quadruplex-targeting ligands have been described [17,18], including phthalocyanine [19], porphyrin [20], and other derivatives [21,22]. From these, peptides represent a class of highly specific ligands with a greater degree of functionality including binding on-off switching, cellular penetration, and the ability to target organelles [23][24][25][26][27]. ...
G-quadruplex (G4) forming DNA sequences were recently found to play a crucial role in the regulation of genomic processes such as replication, transcription and translation, also related to serious diseases. Therefore, systems capable of controlling DNA and RNA G-quadruplex structures would be useful for the modulation of various cellular events. In particular, peptides represent good candidates for targeting G-quadruplex structures, since they are easily tailored to enhance their functionality. In this work, we analyzed, by circular dichroism and synchrotron radiation circular dichroism spectroscopies, the interaction of a 25-residue peptide deriving from RHAU helicases (Rhau25) with three G-quadruplex-forming oligonucleotide sequences, in both sodium- and potassium-containing buffers, the most relevant monovalent cations in physiological conditions. The peptide displayed greater affinity for the G4 sequences adopting a parallel structure. However, it showed the ability to also interact with antiparallel or hybrid G-quadruplex structures, inducing a conformation conversion to the parallel structure. The stability of the oligonucleotide structure alone or in presence of the Rhau25 peptide was studied by temperature melting and UV denaturation experiments, and the data showed that the interaction with the peptide stabilized the conformation of oligonucleotide sequences when subjected to stress conditions.
... They are concentrated in regions implicated in essential cellular processes such as initiation of DNA replication, transcription and telomere maintenance (1,2). Due to their biological significance, extensive effort has been directed towards eliciting a therapeutic response by manipulating non-B-DNA structures found in telomeric and promoter regions (3,4). The KIT receptor is a transmembrane protein that belongs to a family of growth factor receptors with intrinsic tyrosine kinase activity and participates, upon activation by endogenous ligands, in a variety of physiological processes such as cell growth, proliferation, migration and survival (5). ...
Several sequences forming G-quadruplex are highly conserved in regulatory regions of genomes of different organisms and affect various biological processes like gene expression. Diverse G-quadruplex properties can be modulated via their interaction with small polyaromatic molecules such as pyrene. To investigate how pyrene interacts with G-rich DNAs, we incorporated deoxyuridine nucleotide(s) with a covalently attached pyrene moiety (Upy) into a model system that forms parallel G-quadruplex structures. We individually substituted terminal positions and positions in the pentaloop of the c-kit2 sequence originating from the KIT proto-oncogene with Upy and performed a detailed NMR structural study accompanied with molecular dynamic simulations. Our results showed that incorporation into the pentaloop leads to structural polymorphism and in some cases also thermal destabilization. In contrast, terminal positions were found to cause a substantial thermodynamic stabilization while preserving topology of the parent c-kit2 G-quadruplex. Thermodynamic stabilization results from π–π stacking between the polyaromatic core of the pyrene moiety and guanine nucleotides of outer G-quartets. Thanks to the prevalent overall conformation, our structures mimic the G-quadruplex found in human KIT proto-oncogene and could potentially have antiproliferative effects on cancer cells.
... The incrimination of G-quadruplexes gives rise to several biological dysfunctions leading to selectively alternation of the integrity of cancer cells (Oganesian and Bryan, 2007). Specifically, the arrangement of G-quadruplex-DNA towards the end of telomeres has been accounted for not only to hinder the telomerase interconnection and activity but also severely hampering genomic stability by hindering the recognition ability of telomerase binding proteins to their targets (Kelland, 2007;De Cian et al., 2008). Many cytotoxic anti-cancer drugs such as Gemcitabine are explicitly delivered to cancerous cells using G-quadruplexes as vehicle (Park et al., 2018). ...
Of the multiple areas of applications of DNA nanotechnology, stimuli-responsive nanodevices have emerged as an elite branch of research owing to the advantages of molecular programmability of DNA structures and stimuli-responsiveness of motifs and DNA itself. These classes of devices present multiples areas to explore for basic and applied science using dynamic DNA nanotechnology. Herein, we take the stake in the recent progress of this fast-growing sub-area of DNA nanotechnology. We discuss different stimuli, motifs, scaffolds, and mechanisms of stimuli-responsive behaviours of DNA nanodevices with appropriate examples. Similarly, we present a multitude of biological applications that have been explored using DNA nanodevices, such as biosensing, in vivo pH-mapping, drug delivery, and therapy. We conclude by discussing the challenges and opportunities as well as future prospects of this emerging research area within DNA nanotechnology.
... G-quadruplex DNA is a typical secondary structure of DNA-, formed from one or more chains and by self-assembly of guanine-rich nucleic acid sequences. DNA can fold into G-quadruplex structures under conditions of the physiological range [4] . ...
DNA is an important target for the development of chemotherapeutic drug design due to increased and uncontrolled cell proliferation of cancer cells. In the present study, the affinity of water-soluble gallium(III) phthalocyanine chloride (GaPc) non-peripherally substituted with 2-mercapto-N-methylpyridinium groups to different types of DNA (double-stranded ctDNA and G-quadruplex DNA) was investigated. The interaction mechanisms were evaluated by UV-Vis, fluorescence and circular dichroism spectroscopic techniques, competitive dialysis assay, and capillary gel electrophoresis (CGE) system integrated with a high sensitivity LED-induced fluorescence (LIF) detector. Binding mode studies were carried out by molecular docking and molecular dynamic simulations. The experimental binding constants were determined as1.9 × 10⁶ M⁻¹, 1.1 × 10⁶ M⁻¹, 8.5 × 10⁵ M⁻¹, 2.9 × 10⁴ M⁻¹ for c-MYC, AS1411, Tel21, and ctDNA, respectively. Competitive binding assay confirmed the higher affinity towards G-quadruplex structure rather than ctDNA. The GaPc-G-quadruplex DNA conjugates showed higher Kb constants and a strong absorption band closer to NIR window which is a desired feature for the photodynamic therapy applications. Besides, experimental data show that the formation of GaPc-G-quadruplex DNA conjugates reduced the aggregation of GaPc. Reduction of aggregation of phthalocyanine is important. Because aggregation decreases the photoactivity of most photosensitizers. The experimental results suggested that both intercalative and non‑intercalative binding contributions occurred in the case of double-stranded ctDNA. Confirming experiments, molecular docking studies showed that the GaPc can bind from two different major groove regions in double-stranded DNA while only one binding mode was observed in the G-quadruplex DNA. Both experimental and computational studies suggested that the conformational change and unfolding mechanism in G-quadruplex DNA structure were determined upon interaction with GaPc.
... While in cancer cells, telomerase, a nucleoprotein reverse transcriptase that can produce telomere repeated sequences at the end of chromosome, is aberrantly overexpressed. Hence, telomere shortening in cancer cells is suppressed, and they obtain the potential to divide infinitely [140]. The relationship between G4, telomerase, and cancer therapy will be discussed later. ...
G-quadruplex is a three-dimensional secondary structure of nucleic acids formed by the Hoogsteen hydrogen pairing of four guanines. Diverse topologies of G-quadruplex could be employed in biosensing and bioimaging. By intercalating fluorescence dyes into G-quadruplex or forming a horseradish peroxidase (HRP)-mimicking G-quadruplex/hemin DNAzyme, G-quadruplexes based biosensors realized the sensitive and selective detection of nucleic acids, protein, enzyme activity, ions, small molecules, exosomes, cells, and microorganisms. The vital role that cellular G-quadruplexes played in genome further facilitated the application of G-quadruplex stabilizing on cancer therapy. Combined with G-quadruplex aptamer, which is an efficient therapeutic tool, a current landscape of the application potential of this fascinate nucleic acids structure from clinical diagnosis to cancer therapy is summarized here.
... 196 Telomeres are thus implicated in both cancer and the ageing process. 199,200 It is largely accepted that G4s form in telomere ends, 201,202 and the earliest evidence of biological G4 formation was obtained at telomeres. Even if telomeric G4s can either protect telomeres against exonucleases 203 or jeopardise telomere organisation by preventing telomeric loop (Tloop) formation and telomerase recognition, [204][205][206][207] they have been mostly studied as targets for fostering chromosomal fragility in cancers. ...
Damaging DNA is a current and efficient strategy to fight against cancer cell proliferation. Numerous mechanisms exist to counteract DNA damage, collectively referred to as the DNA damage response (DDR) and which are commonly dysregulated in cancer cells. Precise knowledge of these mechanisms is necessary to optimise chemotherapeutic DNA targeting. New research on DDR has uncovered a series of promising therapeutic targets, proteins and nucleic acids, with application notably via an approach referred to as combination therapy or combinatorial synthetic lethality. In this review, we summarise the cornerstone discoveries which gave way to the DNA being considered as an anticancer target, and the manipulation of DDR pathways as a valuable anticancer strategy. We describe in detail the DDR signalling and repair pathways activated in response to DNA damage. We then summarise the current understanding of non-B DNA folds, such as G-quadruplexes and DNA junctions, when they are formed and why they can offer a more specific therapeutic target compared to that of canonical B-DNA. Finally, we merge these subjects to depict the new and highly promising chemotherapeutic strategy which combines enhanced-specificity DNA damaging and DDR targeting agents. This review thus highlights how chemical biology has given rise to significant scientific advances thanks to resolutely multidisciplinary research efforts combining molecular and cell biology, chemistry and biophysics. We aim to provide the non-specialist reader a gateway into this exciting field and the specialist reader with a new perspective on the latest results achieved and strategies devised.
... G-quadruplex DNA is a noncanonical DNA consists of secondary structures located in specific genome sequences having oncogene promoters. They help to indicate the onset of cancer in the human body [106][107][108]. G-quadruplex ligands exhibit apoptotic effects through telomere dependent and independent mechanisms, however mode of action of these ligands inducing cell death are still unexplored [109]. ...
Owing to cisplatin have extensive side-effects which led the investigators to go for non-platinum metal complexes for anti-cancer drug discovery. Among these, further ruthenium(II) and iridium(III), all others retains high toxicity in normal cells in addition to the cancerous ones. In this review newly developed several Ru(II) arene complexes, cyclometalated Ru(II) complexes, Ir(III)-cp* complexes and cyclometalated Ir(III) complexes have been reported. Most of the cases molecules are selectively targeting to the cancer cells with respect to normal cell. The mechanistic pathway of apoptosis was also highlighted in briefly.
The nucleus is an essential organelle for the function of cells. It holds most of the genetic material and plays a crucial role in the regulation of cell growth and proliferation. Since many antitumoral therapies target nucleic acids to induce cell death, tumor‐specific nuclear drug delivery could potentiate therapeutic effects and prevent potential off‐target side effects on healthy tissue. Due to their great structural variety, good biocompatibility, and unique physico‐chemical properties, organometallic complexes and other metal‐based compounds have sparked great interest as promising anticancer agents. In this review, strategies for specific nuclear delivery of metal complexes are summarized and discussed to highlight crucial parameters to consider for the design of new metal complexes as anticancer drug candidates. Moreover, the existing opportunities and challenges of tumor‐specific, nucleus‐targeting metal complexes are emphasized to outline some new perspectives and help in the design of new cancer treatments.
ABSTRACT: We used circular dichroism spectroscopy, UV spectrophotometry, and
differential scanning calorimetry to investigate pH-dependent structural transitions in
an equimolar mixture of complementary G-rich d[5′-A(GGGTTA)3GGG-3′]
(TelG) and C-rich d[3′-T(CCCAAT)3CCC-5′] (TelC) human telomeric DNA
strands. Our studies were conducted at neutral (pH 7.0) and slightly acidic (pH 5.5
and 6.5) pH. We analyzed the melting thermodynamics of TelG and TelC and their
equimolar mixture. Our analysis revealed that the preferred conformation of an
equimolar mixture of TelG and TelC is the duplex. At pH 5.5, however, in addition
to the duplex state, we observed a significant population of the i-motif state formed
by TelC. Our results are consistent with the picture in which an increase in pH from
5.5 to 7.0 has little effect on the melting enthalpy of an isolated G-quadruplex while
causing a strong reduction in the melting enthalpy of an isolated i-motif (the latter
diminishes to 0 at pH 7.0). These effects summarily lead to a decrease in the
contribution of the i-motif to the melting enthalpy of the mixture and, hence, an increase in the apparent melting enthalpy and
overall stability of the duplex state.
It is one of the challenging works to deal with second major cause of death due to cancer and to reduce the
mortality rate caused by this. Researchers have been finding various approaches to minimise the unwanted effect
of cancer therapy and increase the selectivity of the cancer cells that are cancerous without harming the normal
cells. Accordingly, they have discovered and reported a series of anti-cancer complexes with moderate to high
selective behaviour. Conventional cancer therapies are resistant to some of standard drugs. When we consider the
group of metallotherapeutic anticancer agents, platinum (Pt) based anti-neoplastic complexes have some
drawbacks and lower potency and selectivity than complexes like Ruthenium (Ru), Iridium (Ir), Rhodium (Rh)
and Rhenium (Re) towards various cancer cell lines (in vitro). Many of them have proved the in vivo applications.
This review aims to provide a comprehensive summary of prior literature pertaining to the cytotoxic impact and
cellular uptake of those metallocomplexes (Ru, Ir, Rh and Re), with particular emphasis on recently developed
metal-based complexes. Most of them target the DNA, mitochondria, and induces cancer cell apoptosis lowering
the adverse drug reaction.
Telomeres and their single stranded overhangs gradually shorten with successive cell divisions, as part of the natural aging process, but can be elongated by telomerase, a nucleoprotein complex which is activated in the majority of cancers. This prominent implication in cancer and aging has made the repetitive telomeric sequences (TTAGGG repeats) and the G-quadruplex structures that form in their overhangs the focus of intense research in the past several decades. However, until recently most in vitro efforts to understand the structure, stability, dynamics, and interactions of telomeric overhangs had been focused on short sequences that are not representative of longer sequences encountered in a physiological setting. In this review, we will provide a broad perspective about telomeres and associated factors, and introduce the agents and structural characteristics involved in organizing, maintaining, and protecting telomeric DNA. We will also present a summary of recent research performed on long telomeric sequences, nominally defined as those that can form two or more tandem G-quadruplexes, i.e., which contain eight or more TTAGGG repeats. Results of experimental studies using a broad array of experimental tools, in addition to recent computational efforts will be discussed, particularly in terms of their implications for the stability, folding topology, and compactness of the tandem G-quadruplexes that form in long telomeric overhangs.
DNA replication, the process of copying one double stranded DNA molecule to form two identical copies, is highly conserved at the mechanistic level across evolution. Interesting in its own right as a fascinating feat of biochemical regulation and coordination, DNA replication is at the heart of modern advances in molecular biology. An understanding of the process at both the biological and chemical level is essential to developing new techniques in molecular biology. Insights into the process at the molecular level provide opportunities to modulate and intervene in replication. Rapidly dividing cells need to replicate their DNA prior to division, and targeting components of the replication process is a potentially powerful strategy in cancer treatment. Conversely, ageing may be associated with loss of replication activity and restoring it to cells may moderate some of the diseases associated with old age. Replication is, therefore, fundamental to a huge range of molecular biological and biochemical applications, and provides many potential targets for drug design. The fast pace of replication research, particularly in providing new structural insights, has outdated the majority of available texts. This learned, yet accessible, book contains the latest research written by those conducting it. It examines conserved themes providing a biological background for biochemical, chemical and pharmaceutical studies of this huge and exciting field. Rather than simply "itemising" the replication steps and the proteins involved, replication is tackled from a novel perspective. The book provides logical groupings of processes based upon biochemical similarities. The emphasis on mechanisms and the relationship between structure and function targets the chapters towards biochemists and biological chemists as well as molecular and cell biologists. The book highlights new insights into the replication process, from the assembly of pre-replication complexes, through polymerisation mechanisms, to considering replication in the context of chromatin and chromosomes. It also covers mitochondrial DNA replication, and includes archaeal paradigms, which are proving increasingly relevant to the study of replication in higher eukaryotes. Exciting potential drug targets in DNA replication are discussed, particularly in the context of treating malaria and cancer.
Here, we describe further cytotoxic studies and reverse pharmacophore mapping (pharmacophore profiling) for bis-triazoles MS44-53, which were designed and synthesized previously to stabilize the G-quadruplex nucleic acids capable of being formed at the telomeric region and promoter sequences of genes involved in cellular proliferation and oncogenes. Pharmacophore-based activity profiling screen demonstrated some biological targets that MS44-53 may modulate their biological response, and thus can be considered as potential drugs to treat different kinds of diseases, such as carcinoma, diabetes type II, bacterial infection and cardiovascular diseases. Potent cell growth inhibitory properties were shown by ligands MS47 and MS49 against human melanoma MDA-MB-435, colon cancer HCT-116 and COLO 205, and pancreatic cancer MIA PaCa-2 cell lines, as evidenced by MTT assay. Both ligands were more potent against cancer cells than in skin normal CCD-1064Sk fibroblasts.
Aim
The aim of this study is to identify the molecular target and mechanism of action of our promising anticancer bis-triazoles MS44-53, focusing specifically on the G-quadruplex stabilizers MS47 and MS49.
Background
In molecular biology, G-quadruplexes (also known as G4-DNA), one of the higher-order structures of polynucleotides, are four stranded structures formed by nucleic acid sequences which are rich in guanine. They are formed mainly at the single-stranded G-overhang of telomeric DNA and within promoter sequences of genes involved in cellular proliferation and oncogenes such as c - myc , c-kit, and Hsp90. Stabilization of DNA G-quadruplexes is one of the anticancer strategies that has the potential to treat all cancers regardless of the type. A new series of bis-triazoles MS44-53 were developed to stabilize G-quadruplex structures selectively, as G4 ligands and experimental antitumour agents. FRET assay showed that MS47 and MS49 were only the best binders towards the Hsp90 promoter G-guadruplexes. While all bis-triazoles MS44-53 exhibited potent cell growth inhibitory activity against human carcinoma cell lines, suggesting that the ligands perturb molecular targets and mechanisms of action, other than stabilizing G-quadruplexes, contributing to antitumor activity. Therefore, the molecular targets and mechanisms of action of bis-triazoles MS44-53 in different types of human cancer cell lines should be determined by performing further computational studies to MS44-53 and in vitro evaluations for the G-quadruplex stabilizers MS47 and MS49.
Objectives
1- Determining the exact IC 50 for bis-triazoles MS47 & MS49 against four different types of human cancer cell lines; melanoma MDA-MB-435, pancreatic cancer MIA PaCa-2, and colon cancer HCT-116 and COLO 205 cell lines.
2- Predicting the biological targets that bis-triazoles MS44-53 may interact with to trigger or block their biological response.
Methods
1- MTT assay was used for in vitro evaluation of the antiproliferative activities of MS47 and MS49, and determination of IC 50 values.
2- Reverse pharmacophore mapping (pharmacophore profiling) was used for predicting the biological targets of bis-triazoles MS44-53, and determining the % binding probabilities.
Results
MS49 exhibited more potent proliferation inhibitory activity than MS47 and higher IC 50 value against skin normal fibroblasts. Pharmacophore profiling demonstrated FGFR1, PDGFR2, FLT3, mTOR, PPAR-gamma, MUR-F and CETP as biological targets for bis-triazoles MS44-53.
Conclusion
Bis-triazoles MS47 and MS49 are promising selective innovative compounds with wide spectrum cytotoxic activities against distinct cancer types. Bis-triazoles MS44-53 can be considered as potential drugs to treat different types of carcinoma, in addition to diabetes type II, bacterial infection and cardiovascular diseases.
Other
Further in vitro evaluations will be performed for bis-triazoles MS44-53 in order to identify their molecular targets and mechanisms of action in different types of human cancer cell lines.
tMetallacrowns (MCs) due to the enormous structural diversity have found great interest in many areas of fundamental studies and applications. One such fundamental study is research concerning the interaction of MCs with DNA in order to elucidate their potential biological activity. In the earlier reports, most studies have focused on the binding of various MCs (e.g., inverse-9-MC-3, 15-MC-5, or 36-MC-6) with double-stranded DNA. In recent years, new reports appeared about the interaction of MCs (e.g., 12-MC-4, 15-MC-5) with different structural forms of tetraplex DNA known as G-quadruplexes (G4 DNA). Published results have shown that the MC/G4 interactions are dependent on the structure and the net charge of the metallacrown as well as on the particular G4 topology. The research carried out on the group of diverse G-quadruplex structures can demonstrate whether the tested MC compounds are expected to serve in the future as agents in anticancer or gene therapy or as fluorescent probes for structural investigation of G4 DNA or for detection of trace amounts of DNA. The chapter presents the results of the investigation concerning the binding affinity of MCs to various DNA structures using several analytical techniques e.g., UV–Vis spectrophotometry, spectrofluorescence, circular dichroism spectroscopy, and gel electrophoresis.
Guanine‐rich sequences are known to fold into G‐quadruplex (G4) arrangements, which are present in oncogenes and in the telomeric regions of chromosomes. In particular, G4s represent an obstacle to functioning of telomerase, an enzyme overexpressed in cancer cells causing their immortalization. Therefore, G4 stabilization using small molecules represents an appealing strategy for the medicinal chemist. Ligands based on an anthraquinone scaffold, to which peptidic side chains were attached by an amide bond, were previously reported. We envisioned improving this ligand concept leveraging the click chemistry approach, which, besides representing a flexible, high yielding synthetic strategy, allows an elongation of the side chains and an increase of pi‐pi stacking and H‐bond interactions with the nucleobases through the triazole ring. Compounds were tested for their ability to interact with G4 DNA with a multiple analytical approach, demonstrating an elevated aptitude to stabilize the G4 and high selectivity over double stranded DNA.
The evidence that telomerase is overexpressed in almost 90% of human cancers justifies the proposal of this enzyme as a potential target for anticancer drug design. The inhibition of telomerase by quadruplex stabilizing ligands is being considered a useful approach in anticancer drug design proposals. Several aromatic ligands, including porphyrins, were exploited for telomerase inhibition by adduct formation with G-Quadruplex (GQ). 5,10,15,20-Tetrakis(N-methyl-4-pyridinium)porphyrin (H2TMPyP) is one of the most studied porphyrins in this field, and although reported as presenting high affinity to GQ, its poor selectivity for GQ over duplex structures is recognized. To increase the desired selectivity, porphyrin modifications either at the peripheral positions or at the inner core through the coordination with different metals have been handled. Herein, studies involving the interactions of TMPyP and analogs with different DNA sequences able to form GQ and duplex structures using different experimental conditions and approaches are reviewed. Some considerations concerning the structural diversity and recognition modes of G-Quadruplexes will be presented first to facilitate the comprehension of the studies reviewed. Additionally, considering the diversity of experimental conditions reported, we decided to complement this review with a screening where the behavior of H2TMPyP and of some of the reviewed metal complexes were evaluated under the same experimental conditions and using the same DNA sequences. In this comparison under unified conditions, we also evaluated, for the first time, the behavior of the AgII complex of H2TMPyP. In general, all derivatives showed good affinity for GQ DNA structures with binding constants in the range of 106–107 M−1 and ligand-GQ stoichiometric ratios of 3:1 and 4:1. A promising pattern of selectivity was also identified for the new AgII derivative.
A novel strategy to design “turn-on” fluorescent receptors for G-quadruplexes of DNA is presented, which relies on the connection of phosphate binding macrocycles (PBM) with naphthalimide dyes. A new PBM-dye...
G-quadruplexes have become attractive drug targets in cancer therapy. However, due to the polymorphism of G-quadruplex structures, it is difficult to experimentally verify the relevant structures of multiple intermediates and transition states in dynamic equilibrium. Hence, understanding the mechanism by which structural conversions of G-quadruplexes occur is still challenging. We conducted targeted molecular dynamics simulation with umbrella sampling to investigate how salt affects the conformational conversion of human telomeric G-quadruplex. Our results explore a unique view into the structures and energy barrier of the intermediates and transition states in the interconversion process. The pathway of G-quadruplex conformational interconversion was mapped out by a free energy landscape, consisting of branched parallel pathways with multiple energy basins. We propose a salt-controlled mechanism that as the salt concentration increases, the conformational conversion mechanism switches from multi-pathway folding to sequential folding pathways. The hybrid-I and hybrid-II structures are intermediates in the basket-propeller transformation. In high-salt solutions, the conformational conversion upon K+ binding is more feasible than upon Na+ binding. The free energy barrier for conformational conversions ranges from 1.6 to 4.6 kcal/mol. Our work will be beneficial in developing anticancer agents.
Background: Zinc protoporphyrin (ZnPP) is a naturally occurring metalloprotoporphyrin (MPP) that is currently under development as a chemotherapeutic agent although its mechanism is unclear. Similar to natural and synthetic porphyrins, MPPs are thought to bind DNA and stabilize secondary structures such as guanine quadruplexes (G-4) and thus potentially impact telomerase activity and DNA synthesis which are important targets for chemotherapy. Interactions of MPPs with telomerase have not been previously reported.
Methods: We wished to evaluate the effects of common MPPs, i.e., ZnPP, tin protoporphyrin (SnPP), and iron protoporphyrin (FePP), on cellular proliferation, apoptosis, and telomerase activity in hepatoma cells. The cytotoxicities of porphyrins were determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Native agarose gel electrophoresis was used to identify ZnPP binding of telomerase complexes. Inhibition of telomerase activity by ZnPP was assessed by conventional telomeric repeat amplification protocol (TRAP) and direct telomerase activity assays. Colocalization of ZnPP with telomerase was analyzed with immunofluorescence staining and confocal microscopic analysis.
Results: ZnPP was the most effective MPP for decreasing DNA synthesis and cellular proliferation, while promoting apoptosis in cultured hepatocytes. Concurrently, ZnPP down-regulated telomerase expression and was the best overall inhibitor of telomerase activity in intact cells and in vitro assays, with IC50 and EC50 values of ca 2.5 and 6 µM respectively. The natural fluorescence properties of ZnPP enabled direct imaging in cellular fractions using non-denaturing agarose gel electrophoresis, western blots, and confocal fluorescence microscopy. ZnPP localized to large cellular complexes (> 600kD) that contained telomerase and dyskerin as confirmed with immunocomplex mobility shift, immunoprecipitation, and immunoblot analyses. Confocal fluorescence studies showed that ZnPP co-localized with telomerase reverse transcriptase (TERT) and telomeres in the nucleus of synchronized S-phase cells. ZnPP also co-localized with TERT in the perinuclear regions of log phase cells but did not co-localize with telomeres on the ends of metaphase chromosomes, a site known to be devoid of telomerase complexes. Taken together, these results suggest that ZnPP does not bind to telomeric sequences per se, but alternatively, interacts with other structural components of the telomerase complex to inhibit telomerase enzymatic activity.
Conclusions. ZnPP can actively interfere with telomerase activity in neoplastic cells, thus eliciting pro-apoptotic and anti-proliferative properties. These data support further development of natural or synthetic protoporphyrins for use as chemotherapeutic agents to augment current treatment protocols for a number of neoplasms.
Human c‐KIT oncogene is known to regulate cell growth and proliferation, and thus, acts as a probable target in the treatment of gastrointestinal tumors (GIST). To identify small molecule ligands which can specifically bind with the G‐quadruplex (G4) in the c‐KIT promoter region as potential antitumor agents, we propose the combination of electrospray ionization mass spectrometry (ESI‐MS), capillary electrophoresis frontal analysis (CE‐FA), and Taylor dispersion analysis (TDA) to accurately investigate the G4/ligands binding properties. First, ESI‐MS was used for initial screening of natural products (NPs). CE‐FA was then used to calculate specific binding constants and the stoichiometry of the native state binding pair in solution. Next, TDA, a micro‐capillary flow technique was used to examine the effect of the ligand binding on the diffusivity and particle size of the c‐KIT G4. Two of the screened NPs, scopolamine butylbromide (L1) and isorhamnetin‐3‐O‐neohesperidoside (L3), were found to specifically bind to the c‐KIT G4 with binding constants of around 104 M–1 and 1:1 stoichiometry in a free solution. TDA data showed that ligand binding (both L1 and L3) induced the c‐KIT strands to fold into a tightly structured G4 with a decreased hydrodynamic radius. These ligands have the potential to be drug candidates for the regulation of c‐KIT gene transcription by stabilizing the G4 structure. This methodology not only increased the speed of analysis but also improved its accuracy and specificity compared with the conventional binding approaches. This article is protected by copyright. All rights reserved
Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in catecholamine (CA) biosynthesis pathway making TH a molecular target for controlling CA production, specifically dopamine. Dysregulation of dopamine is correlated with neurological diseases such as Parkinson's disease (PD) and post-traumatic stress disorder (PTSD) among others. Previously, we showed that a 49-nucleotide guanine (G)-rich sequence within the human TH promoter adopts two different sets of G-quadruplex (GQ) structures (5ʹGQ and 3ʹGQ) where the 5ʹGQ uses G-stretches I, II, IV and VI in TH49 which enhances TH transcription, while the 3ʹGQ utilizes G-stretches II, IV, VI and VII which represses transcription. Herein, we demonstrated targeted switching of these GQs to their active state using rationally designed DNA GQ Clips (5ʹGQ and 3ʹGQ Clips) to modulate endogenous TH gene expression and dopamine production. As a translational approach, we synthesized a targeted nanoparticle delivery system to effectively deliver the 5ʹGQ Clip in vivo. We believe this strategy could potentially be an improved approach for controlling dopamine production in a multitude of neurological disorders including PD.
Recently, non-canonical DNA structures, such as G-quadruplexes (GQs), were found to be highly pressure sensitive, suggesting that pressure modulation studies can provide additional mechanistic details of such biomolecular systems. Using FRET and CD spectroscopy as well as binding equilibrium measurements, we investigated the effect of pressure on the binding reaction of the ligand ThT to the quadruplex 22AG in solutions containing different ionic species and a crowding agent mimicking the intracellular milieu. Pressure modulation helped us to identify the different conformational substates adopted by the quadruplex at the different solution conditions and to determine the volumetric changes during complex formation and the conformational transitions involved. The magnitudes of the binding volumes are a hallmark of packing defects and hydrational changes upon ligand binding. The conformational substates of the GQ as well as the binding strength and the stoichiometry of complex formation depend strongly on the solution conditions as well as on pressure. High hydrostatic pressure can also impact GQs inside living cells and thus affect expression of genetic information in deep sea organisms. We show that sub-kbar pressures do not only affect the conformational dynamics and structures of GQs, but also their ligand binding reactions.
A static external magnetic field is included in molecular dynamics simulations of aqueous solutions, and the effects on static and dynamic properties are determined. It is pointed out that diamagnetic properties are generally negligible if small molecules or ions are considered. Therefore, particular attention is paid to the effects arising from the Lorentz force acting on the (partial) charges on water, atomic ions, and larger molecules in solution. Careful analysis of many macroscopic and microscopic properties is performed for systems in magnetic fields of up to 1 T. None of the properties is found to be significantly affected by the Lorentz force. It is shown that any apparent variations with magnetic-field strength are simply statistical fluctuations. It is emphasized that the magnitude of the Lorentz force is up to eight orders of magnitude smaller than the average force coming from nonmagnetic forces and thermal fluctuations in the liquid phase at normal temperatures. The results are complemented by a theoretical argument which precludes Lorentz-force effects. A discussion of earlier reports of significant magnetic-field effects is presented.
We compare K⁺‐induced folding of an RNA G‐quadruplex (G4) with a sequence homologous DNA G4 using CD spectroscopy and real‐time NMR spectroscopy. The folding kinetics of RNA and DNA G4 are markedly different: while DNA folding is biphasic, shows kinetic partitioning and involves kinetically favoured off‐pathway intermediates, RNA folding is faster and monophasic. We attribute the differences to different stabilities of the torsion angle χ.
Abstract
We investigated the folding kinetics of G‐quadruplex (G4) structures by comparing the K⁺‐induced folding of an RNA G4 derived from the human telomeric repeat‐containing RNA (TERRA25) with a sequence homologous DNA G4 (wtTel25) using CD spectroscopy and real‐time NMR spectroscopy. While DNA G4 folding is biphasic, reveals kinetic partitioning and involves kinetically favoured off‐pathway intermediates, RNA G4 folding is faster and monophasic. The differences in kinetics are correlated to the differences in the folded conformations of RNA vs. DNA G4s, in particular with regard to the conformation around the glycosidic torsion angle χ that uniformly adopts anti conformations for RNA G4s and both, syn and anti conformation for DNA G4s. Modified DNA G4s with ¹⁹F bound to C2′ in arabino configuration adopt exclusively anti conformations for χ. These fluoro‐modified DNA (antiTel25) reveal faster folding kinetics and monomorphic conformations similar to RNA G4s, suggesting the correlation between folding kinetics and pathways with differences in χ angle preferences in DNA and RNA, respectively.
We investigate here the folding kinetics of G-quadruplex (G4) structures. In particular, we compare K + -induced folding of an RNA G4 derived from the human telomeric repeat-containing RNA (TERRA25) with a sequence homologous DNA G4 (wtTel25) using CD spectroscopy and real-time NMR spectroscopy. The folding kinetics of RNA and DNA G4 are markedly different: while DNA G4 folding is biphasic, reveals kinetic partitioning and involves kinetically favoured off-pathway intermediates, RNA G4 folding is faster and monophasic. The differences in kinetics are correlated to the differences in the folded conformations of RNA vs DNA G4s, in particular with regard to the conformation around the glycosidic torsion angle χ that uniformly adopts anti conformations for RNA G4s and both, syn and anti conformation for DNA G4s. Modified DNA G4s with 19 F bound to C2' in arabino configuration adopt exclusively anti conformations for χ. These fluoro-modified DNA (antiTel25) reveal faster folding kinetics and monomorphic conformations similar to RNA G4s, suggesting the correlation between folding kinetics and pathways with differences in χ angle preferences in DNA and RNA, respectively.
Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is active in most human cancers and in germline cells but, with few exceptions, not in normal human somatic tissues. Telomere maintenance is essential to the replicative potential of malignant cells and the inhibition of telomerase can lead to telomere shortening and cessation of unrestrained proliferation. We describe novel chemical compounds which selectively inhibit telomerase in vitro and in vivo. Treatment of cancer cells with these inhibitors leads to progressive telomere shortening, with no acute cytotoxicity, but a proliferation arrest after a characteristic lag period with hallmarks of senescence, including morphological, mitotic and chromosomal aberrations and altered patterns of gene expression. Telomerase inhibition and telomere shortening also result in a marked reduction of the tumorigenic potential of drug-treated tumour cells in a mouse xenograft model. This model was also used to demonstrate in vivo efficacy with no adverse side effects and uncomplicated oral administration of the inhibitor. These findings indicate that potent and selective, non-nucleosidic telomerase inhibitors can be designed as novel cancer treatment modalities.
The extremities of eukaryotic chromosomes are called telomeres. They have a structure unlike the bulk of the chromosome, which
allows the cell DNA repair machinery to distinguish them from ‘broken’ DNA ends. But these specialised structures present
a problem when it comes to replicating the DNA. Indeed, telomeric DNA progressively erodes with each round of cell division
in cells that do not express telomerase, a specialised reverse transcriptase necessary to fully duplicate the telomeric DNA.
Telomerase is expressed in tumour cells but not in most somatic cells and thus telomeres and telomerase may be proposed as
attractive targets for the discovery of new anticancer agents.
Although broken chromosomes can induce apoptosis, natural chromosome ends (telomeres) do not trigger this response. It is
shown that this suppression of apoptosis involves the telomeric-repeat binding factor 2 (TRF2). Inhibition of TRF2 resulted
in apoptosis in a subset of mammalian cell types. The response was mediated by p53 and the ATM (ataxia telangiectasia mutated)
kinase, consistent with activation of a DNA damage checkpoint. Apoptosis was not due to rupture of dicentric chromosomes formed
by end-to-end fusion, indicating that telomeres lacking TRF2 directly signal apoptosis, possibly because they resemble damaged
DNA. Thus, in some cells, telomere shortening may signal cell death rather than senescence.
A recent approach in anticancer chemotherapy envisages telomerase as a potentially useful target. An attractive strategy deals with the development of compounds able to stabilize telomeric DNA in the G-quadruplex folded structure and, among them, a prominent position is found in the perylenes. With the aim to further investigate the role of drug structure, in view of possible pharmaceutical applications, we synthesized a series of compounds related to PIPER, a well-known perylene-based telomerase inhibitor. We modified the number of condensed aromatic rings and introduced different side chains to modulate drug protonation state and extent of self-aggregation. Effective telomerase inhibition was induced by heptacyclic analogues only, some showing a remarkably wide selectivity index with reference to inhibition of Taq polymerase. G-quadruplex stabilization was monitored by circular dichroism and melting experiments. Cell cytotoxicity measurements indicated a poor short-term cell killing ability for the best G-quartet binders. Besides the presence of a planar seven-condensed ring system, the introduction of a cyclic amine in the side chains critically affects the selectivity window.
Repair of single strand breaks in telomeric DNA is less efficient than in other genomic regions. This leads to an increased vulnerability of telomeric DNA towards damage induced by reactive oxygen species (ROS) and to accelerated telomere shortening under oxidative stress. The causes for the diminished repair efficacy in telomeres are unknown. We show here that overexpression of the telomere-binding protein TRF2 further reduces telomeric, but not genomic, single strand break repair. This suggests the possibility of strand break repair in telomeres being sterically hindered by the three-dimensional structure of the telomere DNA-protein complex and explains the effect of TRF2 on telomere shortening rates in telomerase-negative cells.
Most studies on the structure of DNA in telomeres have been dedicated to the double-stranded region or the guanosine-rich strand and consequently little is known about the factors that may bind to the telomere cytosine-rich (C-rich) strand. This led us to investigate whether proteins exist that can recognise C-rich sequences. We have isolated several nuclear factors from human cell extracts that specifically bind the C-rich strand of vertebrate telomeres [namely a d(CCCTAA)n repeat] with high affinity and bind double-stranded telomeric DNA with a 100× reduced affinity. A biochemical assay allowed us to characterise four proteins of apparent molecular weights 66–64, 45 and 35 kDa, respectively. To identify these polypeptides we screened a λgt11-based cDNA expression library, obtained from human HeLa cells using a radiolabelled telomeric oligonucleotide as a probe. Two clones were purified and sequenced: the first corresponded to the hnRNP K protein and the second to the ASF/SF2 splicing factor. Confirmation of the screening results was obtained with recombinant proteins, both of which bind to the human telomeric C-rich strand in vitro.
Most eukaryotic telomeres contain a repeating motif with stretches of guanine residues that form a 3′-terminal overhang extending beyond the telomeric duplex region. The telomeric repeat of hypotrichous ciliates, d(T4G4), forms a 16-nucleotide 3′-overhang. Such sequences can adopt parallel-stranded as well as antiparallel-stranded quadruplex conformations in vitro. Although it has been proposed that guanine-quadruplex conformations may have important cellular roles including telomere function, recombination, and transcription, evidence for the existence of this DNA structure in vivo has been elusive to date. We have generated high-affinity single-chain antibody fragment (scFv) probes for the guanine-quadruplex formed by the Stylonychia telomeric repeat, by ribosome display from the Human Combinatorial Antibody Library. Of the scFvs selected, one (Sty3) had an affinity of Kd = 125 pM for the parallel-stranded guanine-quadruplex and could discriminate with at least 1,000-fold specificity between parallel or antiparallel quadruplex conformations formed by the same sequence motif. A second scFv (Sty49) bound both the parallel and antiparallel quadruplex with similar (Kd = 3–5 nM) affinity. Indirect immunofluorescence studies show that Sty49 reacts specifically with the macronucleus but not the micronucleus of Stylonychia lemnae. The replication band, the region where replication and telomere elongation take place, was also not stained, suggesting that the guanine-quadruplex is resolved during replication. Our results provide experimental evidence that the telomeres of Stylonychia macronuclei adopt in vivo a guanine-quadruplex structure, indicating that this structure may have an important role for telomere functioning.
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 in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. We used a fluorescence assay to identify molecules that stabilize G-quadruplexes. Intramolecular folding of an oligonucleotide with four repeats of the human telomeric sequence into a G-quadruplex structure led to fluorescence excitation energy transfer between a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalently attached to the 5′ and 3′ ends of the oligonucleotide, respectively. The melting of the G-quadruplex was monitored in the presence of putative G-quadruplex-binding molecules by measuring the fluorescence emission of the donor. A series of compounds (pentacyclic crescent-shaped dibenzophenanthroline derivatives) was shown to increase the melting temperature of the G-quadruplex by 2–20°C at 1 μM dye concentration. This increase in Tm value was well correlated with an increase in the efficiency of telomerase inhibition in vitro. The best telomerase inhibitor showed an IC50 value of 28 nM in a standard telomerase repeat amplification protocol assay. Fluorescence energy transfer can thus be used to reveal the formation of four-stranded DNA structures, and its stabilization by quadruplex-binding agents, in an effort to discover new potent telomerase inhibitors.
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 describe ethidium derivatives that stabilize
G-quadruplexes. These molecules were shown to increase the melting temperature of an intramolecular quadruplex structure,
as shown by fluorescence and absorbance measurements, and to facilitate the formation of intermolecular quadruplex structures.
In addition, these molecules may be used to reveal the formation of multi-stranded DNA structures by standard fluorescence
imaging, and therefore become fluorescent probes of quadruplex structures. This recognition was associated with telomerase
inhibition in vitro: these derivatives showed a potent anti-telomerase activity, with IC50 values of 18–100 nM in a standard TRAP assay.
The enzyme telomerase is involved in the replication of telomeres, specialized structures that cap and protect the ends of chromosomes. Its activity is required for maintenance of telomeres and for unlimited lifespan, a hallmark of cancer cells. Telomerase is overexpressed in the vast majority of human cancer cells and therefore represents an attractive target for therapy. Several approaches have been developed to inhibit this enzyme through the targeting of its RNA or catalytic components as well as its DNA substrate, the single-stranded 3'-telomeric overhang. Telomerase inhibitors are chemically diverse and include modified oligonucleotides as well as small diffusable molecules, both natural and synthetic. This review presents an update of recent investigations pertaining to these agents and discusses their biological properties in the context of the initial paradigm that the exposure of cancer cells to these agents should lead to progressive telomere shortening followed by a delayed growth arrest response.
The terminus of a DNA helix has been called its Achilles' heel. Thus to prevent possible incomplete replication and instability of the termini of linear DNA, eukaryotic chromosomes end in characteristic repetitive DNA sequences within specialized structures called telomeres. In immortal cells, loss of telomeric DNA due to degradation or incomplete replication is apparently balanced by telomere elongation, which may involve de novo synthesis of additional repeats by novel DNA polymerase called telomerase. Such a polymerase has been recently detected in HeLa cells. It has been proposed that the finite doubling capacity of normal mammalian cells is due to a loss of telomeric DNA and eventual deletion of essential sequences. In yeast, the est1 mutation causes gradual loss of telomeric DNA and eventual cell death mimicking senescence in higher eukaryotic cells. Here, we show that the amount and length of telomeric DNA in human fibroblasts does in fact decrease as a function of serial passage during ageing in vitro and possibly in vivo. It is not known whether this loss of DNA has a causal role in senescence.
Immortalization of human cells is often associated with reactivation of telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats onto telomeres and compensates for their shortening. We examined whether telomerase activation is necessary for immortalization. All normal human fibroblasts tested were negative for telomerase activity. Thirteen out of 13 DNA tumor virus-transformed cell cultures were also negative in the pre-crisis (i.e. non-immortalized) stage. Of 35 immortalized cell lines, 20 had telomerase activity as expected, but 15 had no detectable telomerase. The 15 telomerase-negative immortalized cell lines all had very long and heterogeneous telomeres of up to 50 kb. Hybrids between telomerase-negative and telomerase-positive cells senesced. Two senescent hybrids demonstrated telomerase activity, indicating that activation of telomerase is not sufficient for immortalization. Some hybrid clones subsequently recommenced proliferation and became immortalized either with or without telomerase activity. Those without telomerase activity also had very long and heterogeneous telomeres. Taken together, these data suggest that the presence of lengthened or stabilized telomeres is necessary for immortalization, and that this may be achieved either by the reactivation of telomerase or by a novel and as yet unidentified mechanism.
Synthesis of DNA at chromosome ends by telomerase may be necessary for indefinite proliferation of human cells. A highly sensitive
assay for measuring telomerase activity was developed. In cultured cells representing 18 different human tissues, 98 of 100
immortal and none of 22 mortal populations were positive for telomerase. Similarly, 90 of 101 biopsies representing 12 human
tumor types and none of 50 normal somatic tissues were positive. Normal ovaries and testes were positive, but benign tumors
such as fibroids were negative. Thus, telomerase appears to be stringently repressed in normal human somatic tissues but reactivated
in cancer, where immortal cells are likely required to maintain tumor growth.
In virtually all eukaryotic organisms, telomeric DNA is composed of a variable number of short direct repeats. While the primary sequence of telomeric repeats has been determined for a great variety of species, the actual physical DNA structure at the ends of a bona fide metazoan chromosome with a centromere is unknown. It is shown here that an overhang of the strand forming the 3' ends of the chromosomes, the G-rich strand, is found at mammalian chromosome ends. Moreover, on at least some telomeres, the overhangs are > or = 45 bases long. Such surprisingly long overhangs were present on chromosomes derived from fully transformed tissue culture cells and normal G0-arrested peripheral leukocytes. Thus, irrespective of whether the cells were actively dividing or arrested, a very similar terminal DNA arrangement was found. These data suggest that the ends of mammalian and possibly all vertebrate chromosomes consist of an overhang of the G-rich strand and that these overhangs may be considerably larger than previously anticipated.
Telomeres protect the ends of linear chromosomes from degradation and abnormal recombination events, and in vertebrates may be important in cellular senescence and cancer. However, very little is known about the structure of human telomeres. In this report we purify telomeres and analyze their termini. We show that following replication the daughter telomeres have different terminal overhangs in normal diploid telomerase-negative human fibroblasts. Electron microscopy of those telomeres that have long overhangs yields 200 +/- 75 nucleotides of single-stranded DNA. This overhang is four times greater than the amount of telomere shortening per division found in these cells. These results are consistent with models of telomere replication in which leading-strand synthesis generates a blunt end while lagging-strand synthesis produces a long G-rich 3' overhang, and suggest that variations in lagging-strand synthesis may regulate the rate of telomere shortening in normal diploid human cells. Our results do not exclude the possibility that nuclease processing events following leading strand synthesis result in short overhangs on one end.
Telomerase is the RNA-protein complex which elongates telomeric DNA (TTAGGG)n and appears to play an important role in cellular immortalization. The almost exclusive expression of telomerase in tumor cells, and not in most normal cells, offers an exciting opportunity for therapy by inhibiting its function. Here, we have investigated the effect of inhibition of telomerase on the growth and survival of human malignant glioma cells in vitro and in vivo by using a 19-mer antisense oligonucleotide against human telomerase RNA linked to a 2',5'-oligoadenylate (2-5A). 2-5A antisense functions by activating the endoribonuclease, RNase L, resulting in the degradation of single stranded, targeted RNA. We have shown that the 2-5A antisense treatment effectively suppressed tumor cell growth and survival in vitro. Furthermore, treatment of tumors grown in nude mice with the antisense oligonucleotide inhibited survival of the tumor cells. TUNEL assays suggest that this effect is mediated through the induction of apoptosis. Targeting telomerase RNA with 2-5A antisense, therefore, may represent an effective and novel approach for treatment of a broad range of cancers.
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 p16INK4a expression. We also show that treatment with BRACO-19 causes extensive end-to-end chromosomal fusions, consistent with telomere uncapping.
Immortalization of human cells is often associated with reactivation of telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats onto telomeres and compensates for their shortening. We examined whether telomerase activation is necessary for immortalization. All normal human fibroblasts tested were negative for telomerase activity. Thirteen out of 13 DNA tumor virus‐transformed cell cultures were also negative in the pre‐crisis (i.e. non‐immortalized) stage. Of 35 immortalized cell lines, 20 had telomerase activity as expected, but 15 had no detectable telomerase. The 15 telomerase‐negative immortalized cell lines all had very long and heterogeneous telomeres of up to 50 kb. Hybrids between telomerase‐negative and telomerase‐positive cells senesced. Two senescent hybrids demonstrated telomerase activity, indicating that activation of telomerase is not sufficient for immortalization. Some hybrid clones subsequently recommenced proliferation and became immortalized either with or without telomerase activity. Those without telomerase activity also had very long and heterogeneous telomeres. Taken together, these data suggest that the presence of lengthened or stabilized telomeres is necessary for immortalization, and that this may be achieved either by the reactivation of telomerase or by a novel and as yet unidentified mechanism.
The requirement for the maintenance of telomeres by telomerase by most cancer cells for continued proliferation is a target in anticancer strategies. Tankyrases are poly(ADP-ribose) polymerases that enhance telomerase access to telomeres. Tankyrase I modulates telomerase inhibition in human cancer cells and is reviewed in this report as a potential telomere-directed anticancer target.
The relationship between the variation of friction coefficient of steel GCr15 against the hard alloy YG8 and the normal load as well as the sliding speed under dry sliding condition was studied. The results showed that the friction coefficient decreases with the increase of load and speed. On the basis of surface temperature calculation and of the change on worn surface, it was considered that the flash temperature increases with the increase in load and/or speed. This results in the partial melting of the frictional surface, i.e. the boundary lubrication is formed, as a consequence, the friction coefficient decreases. Besides, there is a decrease in the difference of friction coefficients among different microstructures under the condition of high load and/or speed.
Telomerase is a ribonucleoprotein responsible for maintaining telomeres in nearly all eukaryotic cells. The enzyme is able
to utilize a short segment of its RNA subunit as the template for the reverse transcription of d(TTAGGG) repeats onto the
ends of human chromosomes. Transfection with telomerase was shown to confer immortality on several types of human cells. Moreover,
telomerase activation appears to be one of the key events required for malignant transformation of normal cells. Inhibition
of telomerase activity in transformed cells results in the cessation of cell proliferation in cultures and provides the rationale
for the selection of telomerase as a target for anticancer therapy. Using oligonucleotide N3′→P5′ phosphoramidates (NPs) we
have identified a region of the human telomerase RNA subunit (hTR) ∼100 nt downstream from the template region whose structural
integrity appears crucial for telomerase enzymatic activity. The oligonucleotides targeted to this segment of hTR are potent
and specific inhibitors of telomerase activity in biochemical assays. Mutant telomerase, in which 3 nt of hTR were not complementary
to a 15 nt NP, was found to be refractory to inhibition by that oligonucleotide. We also demonstrated that the binding of
NP, oligonucleotides to this hTR allosteric site results in a marked decrease in the affinity of a telomerase substrate (single-stranded
DNA primer) for the enzyme.
Most studies on the structure of DNA in telomeres have been dedicated to the double-stranded region or the guanosine-rich strand and consequently little is known about the factors that may bind to the telomere cytosine-rich (C-rich) strand. This led us to investigate whether proteins exist that can recognise C-rich sequences. We have isolated several nuclear factors from human cell extracts that specifically bind the C-rich strand of vertebrate telomeres [namely a d(CCCTAA)n repeat] with high affinity and bind double-stranded telomeric DNA with a 100× reduced affinity. A biochemical assay allowed us to characterise four proteins of apparent molecular weights 66–64, 45 and 35 kDa, respectively. To identify these polypeptides we screened a λgt11-based cDNA expression library, obtained from human HeLa cells using a radiolabelled telomeric oligonucleotide as a probe. Two clones were purified and sequenced: the first corresponded to the hnRNP K protein and the second to the ASF/SF2 splicing factor. Confirmation of the screening results was obtained with recombinant proteins, both of which bind to the human telomeric C-rich strand in vitro.
Telomerase activity is expressed in most types of cancer cells but not in normal somatic cells, suggesting that telomerase may be an important target for cancer chemotherapy. Inhibition of telomerase results in telomere erosion, leading to the subsequent growth arrest of cancer cells followed by senescence or cell death. In this study, we screened a chemical library for the inhibition of human telomerase, identifying three inhibitors. All compounds contained a common nitrostyrene moiety conjugated to different side chains. One of these compounds, 3-(3,5-dichlorophenoxy)-nitrostyrene (DPNS), showed the most potent inhibitory effect, with 50% inhibition at approximately 0.4 microM and did not inhibit DNA and RNA polymerases, including retroviral reverse transcriptase. A series of enzyme kinetic experiments suggests that DPNS is a mixed-type noncompetitive inhibitor, with an inhibitor-binding site distinct from the binding sites for the telomeric substrate primer and the deoxynucleoside-5'-triphosphates. Extensive propagation of cancer cell line in the presence of DPNS resulted in progressive telomere erosion followed by the induction of senescence phenotype. The results presented here demonstrate that DPNS is a highly selective, small-molecule telomerase inhibitor in vitro and could be useful as a lead molecule for the further development of inhibitors with an improved potential for efficacy in vivo.
Telomere maintenance is essential for the continued proliferation of dividing cells, and is implicated in chromosome stability and cell immortalization. Telomerase activity allows cells to maintain their telomeric DNA and contributes to the indefinite replicative capacity of cancer cells. Telomerase is expressed in most cancer cells, but not in normal somatic cells, suggesting that telomerase is an attractive target for cancer chemotherapy. Here we screened a chemical library for inhibition of human telomerase, and identified 2,3,7-trichloro-5-nitroquinoxaline (TNQX) as a potent inhibitor. TNQX showed a potent inhibitory effect, with 50% inhibition at approximately 1.4 microM, and did not inhibit DNA and RNA polymerases, including retroviral reverse trancriptase. A series of enzyme kinetic experiments suggested that TNQX is a mixed-type non-competitive inhibitor, with an inhibitor-binding site distinct from the binding sites for the telomeric substrate (TS) primer and the dNTPs. Long-term cultivation of the MCF7 cell line with a drug concentration that did not cause acute cytotoxicity resulted in progressive telomere erosion followed by an increased incidence of chromosome abnormalities and induction of the senescence phenotype. The results presented here indicate that TNQX is a highly potent and selective anti-telomerase agent with good potential for further development as a promising anti-cancer agent.
Telomerase is an attractive target for the design of new anticancer drugs. We have previously described a series of 1,4- and 2,6-difunctionalized amidoanthracene-9,10-diones that inhibit human telomerase via stabilization of telomeric G-quadruplex structures. The present study details the preparation of three further, distinct series of regioisomeric difunctionalized amidoanthracene-9,10-diones substituted at the 1,5-, 1,8-, and 2,7-positions, respectively. Their in vitro cytotoxicity and Taq DNA polymerase and human telomerase inhibition properties are reported and compared with those of their 1,4- and 2,6-isomers. Potent telomerase inhibition (telIC50 values 1.3−17.3 μM) is exhibited within each isomeric series. In addition, biophysical and molecular modeling studies have been conducted to examine binding to the target G-quadruplex structure formed by the folding of telomeric DNA. These studies indicate that the isomeric diamidoanthracene-9,10-diones bind to the human telomeric G-quadruplex structure with a stoichiometry of 1:1. Plausible G-quadruplex−ligand complexes have been identified for each isomeric family, with three distinct modes of intercalative binding being proposed. The exact mode of intercalative binding is dictated by the positional placement of substituent side chains. Furthermore, in contrast to previous studies directed toward triplex DNA, it is evident that stringent control over positional attachment of substituents is not a necessity for effective telomerase inhibition.
G-Quadruplex DNAs are folded, non-Watson−Crick structures that can form within guanine-rich DNA sequences such as telomeric repeats. Previous studies have identified a series of trisubstituted acridine derivatives that are potent and selective ligands for G-quadruplex DNA. These ligands have been shown previously to inhibit the activity of telomerase, the specialized reverse transcriptase that regulates telomere length. The RecQ family of DNA helicases, which includes the Bloom's (BLM) and Werner's (WRN) syndrome gene products, are apparently unique among cellular helicases in their ability to efficiently disrupt G-quadruplex DNA. This property may be relevant to telomere maintenance, since it is known that the sole budding yeast RecQ helicase, Sgs1p, is required for a telomerase-independent telomere lengthening pathway reminiscent of the “ALT” pathway in human cells. Here, we show that trisubstituted acridine ligands are potent inhibitors of the helicase activity of the BLM and WRN proteins on both G-quadruplex and B-form DNA substrates. Inhibition of helicase activity is associated with both a reduction in the level of binding of the helicase to G-quadruplex DNA and a reduction in the degree to which the G-quadruplex DNA can support DNA-dependent ATPase activity. We discuss these results in the context of the possible utility of trisubstituted acridines as antitumor agents for the disruption of both telomerase-dependent and telomerase-independent telomere maintenance.
The telomeric sequence (T2G4)4 was platinated in aqueous solutions containing 50 mM LiClO4, NaClO4, or KClO4. The identification of the guanines which reacted with [Pt(NH3)3(H2O)]2+ revealed that the same type of folding exists in the presence of the three cations and that the latter determine the relative stabilities of the G-quadruplex structures in the order K+ > Na+ Li+. The tri-ammine complex yielded ca. 40−90% of adducts, mono- and poly-platinated, bound to 4 guanines out of the 16 guanines in the sequence, in the decreasing amounts G9 > G15 G3 > G21. The formation of these adducts was interpreted with a G-quadruplex structure obtained by restrained molecular dynamics (rMD) simulations which confirms the schematic model proposed by Williamson et al. [(1989) Cell 59, 871−880]. The bifunctional complexes cis- and trans-[Pt(NH3)2(H2O)2]2+ also first reacted with G9 and G15 and gave cross-linked adducts between two guanines, which did not exceed 5% each of the products formed. Both the cis and trans isomers formed a G3-G15 platinum chelate, and the second also formed bis-chelates at both ends of the G-quadruplex structure: G3-G15/G9-G21 and G3-G15/G9-G24. The rMD simulations showed that the cross-linking reactions by the trans complex can occur without disturbing the stacking of the three G-quartets.
Telomeres, guanine-rich tandem DNA repeats of chromosomal ends, provide chromosomal stability, and cellular replication causes
their loss. In somatic cells, the activity of telomerase, a reverse transcriptase that can elongate telomeric repeats, is
usually diminished after birth so that the telomere length is gradually shortened with cell divisions and triggers cellular
senescence. In embryonic stem cells, telomerase is activated and maintains telomere length and cellular immortality. On the
other hand, in adult stem cells, the level of telomerase activity is low and insufficient to maintain telomere length. Thus,
even in stem cells, except for embryonic stem cells and cancer stem cells, telomere shortening occurs during replicative aging,
possibly at a slower rate than that in normal somatic cells. In the past few years, the importance of telomere maintenance
in human stem cells has been highlighted by the studies on dyskeratosis congenita, aplastic anemia, and idiopathic pulmonary
fibrosis, a part of which are genetic disorders in the human telomerase component and are characterized by premature loss
of tissue regeneration with stem cell dysfunction. The regulation of telomere length and telomerase activity is a complex
and dynamic process that is tightly linked to cell cycle regulation in human stem cells. Here we review the role of telomeres
and telomerase in human stem cells.
KeywordsTelomere–Telomerase–Stem cell–Cancer stem cell–Dyskeratosis congenita–Aplastic anemia–Idiopathic pulmonary fibrosis
Telomerase is a ribonucleoprotein enzyme complex that adds 5′-TTAGGG-3′ repeats onto the ends of human chromosomes, providing
a telomere maintenance mechanism for ∼90% of human cancers. We have purified human telomerase ∼108-fold, with the final elution dependent on the enzyme's ability to catalyze nucleotide addition onto a DNA oligonucleotide
of telomeric sequence, thereby providing specificity for catalytically active telomerase. Mass spectrometric sequencing of
the protein components and molecular size determination indicated an enzyme composition of two molecules each of telomerase
reverse transcriptase, telomerase RNA, and dyskerin.
(Figure Presented) The single-stranded region of the d-(TTAGGG) repeat is known to form a G-quadruplex structure in vitro. We report here a novel naphthyridine tetramer (NT) ligand that induces a nonquadruplex structure in the single-stranded region of the human telomeric sequence.
In most human cancers, the telomere erosion problem has been bypassed through the activation of a telomere maintenance system (usually activation of telomerase). Therefore, telomere and telomerase are attractive targets for anti-cancer therapeutic interventions. Here, we review a large panel of strategies that have been explored to date, from small inhibitors of the catalytic sub-unit of telomerase to anti-telomerase immunotherapy and gene therapy. The many positive results that are reported from anti-telomere/telomerase assays suggest a prudent optimism for a possible clinical application in a close future. However, we discuss some of the main limits for these approaches of antitumour drug development and why significant work remains before a clinically useful drug can be proposed to patients.
The ribonucleoprotein telomerase, a specialized cellular reverse transcriptase, synthesizes one strand of the telomeric DNA of eukaryotes. We analyzed telomere maintenance in two immortalized human cell lines: the B-cell line JY616 and the T-cell line Jurkat E6-1, and determined whether known inhibitors of retroviral reverse transcriptases could perturb telomere lengths and growth rates of these cells in culture. Dideoxyguanosine (ddG) caused reproducible, progressive telomere shortening over several weeks of passaging, after which the telomeres stabilized and remained short. However, the prolonged passaging in ddG caused no observable effects on cell population doubling rates or morphology. Azidothymidine (AZT) caused progressive telomere shortening in some but not all T- and B-cell cultures. Telomerase activity was present in both cell lines and was inhibited in vitro by ddGTP and AZT triphosphate. Prolonged passaging in arabinofuranyl-guanosine, dideoxyinosine (ddI), dideoxyadenosine (ddA), didehydrothymidine (d4T), or phosphonoformic acid (foscarnet) did not cause reproducible telomere shortening or decreased cell growth rates or viabilities. Combining AZT, foscarnet, and/or arabinofuranyl-guanosine with ddG did not significantly augment the effects of ddG alone. Strikingly, with or without inhibitors, telomere lengths were often highly unstable in both cell lines and varied between parallel cell cultures. We propose that telomere lengths in these T- and B-cell lines are determined by both telomerase and telomerase-independent mechanisms.
The ends or telomeres of the linear chromosomes of eukaryotes are composed of tandem repeats of short DNA sequences, one strand being rich in guanine (G strand) and the complementary strand in cytosine. Telomere synthesis involves the addition of telomeric repeats to the G strand by telomere terminal transferase (telomerase). Telomeric G-strand DNAs from a variety of organisms adopt compact structures, the most stable of which is explained by the formation of G-quartets. Here we investigate the capacity of the different folded forms of telomeric DNA to serve as primers for the Oxytricha nova telomerase in vitro. Formation of the K(+)-stabilized G-quartet structure in a primer inhibits its use by telomerase. Furthermore, the octanucleotide T4G4, which does not fold, is a better primer than (T4G4)2, which can form a foldback structure. We conclude that telomerase does not require any folding of its DNA primer. Folding of telomeric DNA into G-quartet structures seems to influence the extent of telomere elongation in vitro and might therefore act as a negative regulator of elongation in vivo.
I have identified an activity in crude HeLa cell extracts that satisfies the requirements for a human telomere terminal transferase or telomerase. It catalyzes the addition of a 6 nucleotide repeating pattern to oligonucleotide primers containing human or nonhuman telomeric repeat sequences. Direct sequence analyses of reaction products reveal the added sequence to be TTAGGG in all cases. Under optimal conditions 65-70 repeats can be synthesized. The enzyme has the properties of a ribonucleoprotein. Telomerase has previously been observed only in ciliated protozoans, which possess 10(4) - 10(7) macronuclear telomeres. The identification of telomerase in HeLa cells with only approximately 100 telomeres indicates that telomerase-mediated telomere maintenance is conserved throughout eukaryotes.
A highly conserved repetitive DNA sequence, (TTAGGG)n, has been isolated from a human recombinant repetitive DNA library. Quantitative hybridization to chromosomes sorted by flow cytometry indicates that comparable amounts of this sequence are present on each human chromosome. Both fluorescent in situ hybridization and BAL-31 nuclease digestion experiments reveal major clusters of this sequence at the telomeres of all human chromosomes. The evolutionary conservation of this DNA sequence, its terminal chromosomal location in a variety of higher eukaryotes (regardless of chromosome number or chromosome length), and its similarity to functional telomeres isolated from lower eukaryotes suggest that this sequence is a functional human telomere.
We have found a novel activity in Tetrahymena cell free extracts that adds tandem TTGGGG repeats onto synthetic telomere primers. The single-stranded DNA oligonucleotides (TTGGGG)4 and TGTGTGGGTGTGTGGGTGTGTGGG, consisting of the Tetrahymena and yeast telomeric sequences respectively, each functioned as primers for elongation, while (CCCCAA)4 and two nontelomeric sequence DNA oligomers did not. Efficient synthesis of the TTGGGG repeats depended only on addition of micromolar concentrations of oligomer primer, dGTP, and dTTP to the extract. The activity was sensitive to heat and proteinase K treatment. The repeat addition was independent of both endogenous Tetrahymena DNA and the endogenous alpha-type DNA polymerase; and a greater elongation activity was present during macronuclear development, when a large number of telomeres are formed and replicated, than during vegetative cell growth. We propose that the novel telomere terminal transferase is involved in the addition of telomeric repeats necessary for the replication of chromosome ends in eukaryotes.
Repeats of Gn sequences are detected as single strand overhangs at the ends of eukaryotic chromosomes together with associated binding proteins. Such telomere sequences have been implicated in the replication and maintenance of chromosomal termini. They may also mediate chromosomal organization and association during meiosis and mitosis.
We have determined the three-dimensional solution structure of the human telomere sequence, d[AG3(T2AG3)3] in Na(+)-containing solution using a combined NMR, distance geometry and molecular dynamics approach (including relaxation matrix refinement). The sequence, which contains four AG3 repeats, folds intramolecularly into a G-tetraplex stabilized by three stacked G-tetrads which are connected by two lateral loops and a central diagonal loop. Of the four grooves that are formed, one is wide, two are of medium width and one is narrow. The alignment of adjacent G-G-G segments in parallel generates the two grooves of medium width whilst the antiparallel arrangement results in one wide and one narrow groove. Three of the four adenines stack on top of adjacent G-tetrads while the majority of the thymines sample multiple conformations.
The availability of the d[AG3(T2AG3)3] solution structure containing four AG3 human telomeric repeats should permit the rational design of ligands that recognize and bind with specificity and affinity to the individual grooves of the G-tetraplex, as well as to either end containing the diagonal and lateral loops. Such ligands could modulate the equilibrium between folded G-tetraplex structures and their unfolded extended counterparts.
The ribonucleoprotein enzyme telomerase is a specialized type of cellular reverse transcriptase which synthesizes one strand of telomeric DNA, using as the template a sequence in the RNA moiety of telomerase. We analyzed the effects of various nucleoside analogs, known to be chain-terminating inhibitors of retroviral reverse transcriptases, on Tetrahymena thermophila telomerase activity in vitro. We also analyzed the effects of such analogs on telomere length and maintenance in vivo, and on vegetative growth and mating of Tetrahymena cells. Arabinofuranyl-guanosine triphosphate (Ara-GTP) and ddGTP both efficiently inhibited telomerase activity in vitro, while azidothymidine triphosphate (AZT-TP), dideoxyinosine triphosphate (ddITP) or ddTTP were less efficient inhibitors. All of these nucleoside triphosphate analogs, however, produced analog-specific alterations of the normal banding patterns seen upon gel electrophoresis of the synthesis products of telomerase, suggesting that their chain terminating and/or competitive actions differ at different positions along the RNA template. The analogs AZT, 3'-deoxy-2',3'-didehydrothymidine (d4T) and Ara-G in nucleoside form caused consistent and rapid telomere shortening in vegetatively growing Tetrahymena. In contrast, ddG or ddI had no effect on telomere length or cell growth rates. AZT caused growth rates and viability to decrease in a fraction of cells, while Ara-G had no such effects even after several weeks in culture. Neither AZT, Ara-G, acycloguanosine (Acyclo-G), ddG nor ddI had any detectable effect on cell mating, as assayed by quantitation of the efficiency of formation of progeny from mated cells. However, AZT decreased the efficiency of programmed de novo telomere addition during macronuclear development in mating cells.
The study of eukaryotic telomeres at the molecular level began with the discovery of short, tandem repeats at Tetrahymena chromosome ends. In the following two decades, major insights about telomere structure and function have come from investigations of telomerase, the DNA polymerase that synthesizes these repeats. In the past year, three areas of telomerase research have been particularly intense: assays of telomerase activity, isolation of telomerase components, and studies of the regulation of telomerase and telomere length in vivo.
The chromosomes of lower eukaryotes have short telomeric 3' extensions. Using a primer-extension/nick-translation technique and nondenaturing hybridization, we find long 3' G-rich tails at human chromosome ends in mortal primary fibroblasts, umbilical vein endothelial cells, and leukocytes, as well as in immortalized fibroblasts. For all cells tested, >80% of the telomeres have long G-rich overhangs, averaging 130-210 bases in length, in disagreement with the conventional model for incomplete lagging-strand replication, which predicts overhangs on 50% of the chromosome ends. The observed G tails must exist during most of the cell cycle and probably result from degradation of both chromosome ends. The average lengths of the G tails are quantitatively consistent with the observed rates of human chromosome shortening.
Telomerase is a ribonucleoprotein that participates in the maintenance of telomere length. Its activity is up-regulated in many tumor types, suggesting that it may be a novel target for chemotherapy. The RNA component of telomerase contains an active site that plays at least two roles&sbd;binding telomere ends and templating their replication [Greider, C. W., & Blackburn, E. H. (1989) Nature 337, 331-337]. The accessibility of RNA nucleotides for inhibitor binding cannot be assumed because of the potential for RNA secondary structure and RNA-protein interactions. Here we use high-affinity recognition by overlapping peptide nucleic acids (PNAs) [Nielsen, P. E., et al. (1991) Science 254, 1497-1500] to identify nucleotides within the RNA active site of telomerase that are determinants for inhibitor recognition. The IC50 for inhibition decreases from 30 microM to 10 nM as cytidines 50-52 (C50-52) at the boundary between the alignment and elongation domains are recognized by PNAs overlapping from the 5' direction. As C50-52 are uncovered in the 3' direction, IC50 increases from 10 nM to 300 nM. As cytidine 56 at the extreme 3' end of the active site is uncovered, IC50 values increase from 0.5 microM to 10 microM. This analysis demonstrates that C50-C52 and C56 are important for PNA recognition and are physically accessible for inhibitor binding. We use identification of these key determinants to minimize the size of PNA inhibitors, and knowledge of these determinants should facilitate design of other small molecules capable of targeting telomerase. The striking differences in IC50 values for inhibition of telomerase activity by related PNAs emphasize the potential of PNAs to be sensitive probes for mapping complex nucleic acids. We also find that PNA hybridization is sensitive to nearest-neighbor interactions, and that consecutive guanine bases within a PNA strand increase binding to complementary DNA and RNA sequences.
We report the inhibition of human telomerase activity by peptide nucleic acids (PNAs). PNAs recognize the RNA component of human telomerase (hTR) and inhibit activity of the enzyme with IC50 values in the picomolar to nanomolar range. Inhibition depends on targeting exact functional boundaries of the hTR template and is 10- to 50-fold more efficient than inhibition by analogous phosphorothioate (PS) oligomers. In contrast to high selectivity of inhibition by PNAs, PS oligomers inhibit telomerase in a non-sequence-selective fashion. These results demonstrate that PNAs can control the enzymatic activity of ribonucleoproteins and possess important advantages relative to PS oligomers in both the affinity and the specificity of their recognition. These observations should facilitate the development of effective inhibitors of telomerase activity and affinity probes of telomerase structure.
A number of 1,4- and 2,6-difunctionalized amidoanthracene-9, 10-diones have been prepared. We have examined their in vitro cytotoxicity in several tumor cell lines and their ability to inhibit the telomere-addition function of the human telomerase enzyme together with their inhibition of the Taq polymerase enzyme. Compounds with -(CH2)2- side chains terminating in basic groups such as piperidine show inhibition of telomerase at telIC50 levels of 4-11 microM. These are thus among the most potent nonnucleoside telomerase inhibitors reported to date. Cytotoxicity levels in human tumor cell lines were at comparable levels for several compounds. Implications for amidoanthracene-9,10-dione telomerase inhibitors as potential anticancer agents are discussed.