Indolo[3,2-c]quinoline G-Quadruplex Stabilizers: a Structural Analysis of Binding to the Human Telomeric G-Quadruplex (original) (raw)
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Oxidative Medicine and Cellular Longevity
The upregulation telomerase activity is observed in over 85-90% of human cancers and provides an attractive target for cancer therapies. The high guanine content in the telomere DNA sequences and the hTERT promoter can form G-quadruplexes (G4s). Small molecules targeting G4s in telomeres and hTERT promoter could stabilize the G4s and inhibit hTERT expression and telomere extension. Several G4 ligands have shown inhibitory effects in cancer cells and xenograft mouse models, indicating these ligands have a potential for cancer therapies. The current review article describes the concept of the telomere, telomerase, and G4s. Moreover, the regulation of telomerase and G4s in telomeres and hTERT promoter is discussed as well. The summary of the small molecules targeting G4s in telomeric DNA sequences and the hTERT promoter will also be shown.
Angewandte Chemie International Edition, 2009
Telomere structures are essential for genome stability. [1] Telomere dysfunction can cause cell senescence, death, or genomic instability. [1] Human telomeric DNA consists of a duplex region composed of TTAGGG repeats, ending in a 100-200 nucleotide (nt) G-rich single-stranded overhang. [1] The telomeric overhang is recognized as a critical component of telomere structures, required for telomere end protection and extension. [1] It is also particularly attractive as a target for anticancer therapeutic development. [2] The detailed structural features and mechanisms of telomeric overhang that are responsible for protecting the chromosome ends are still insufficiently understood, although several alternative structures have been proposed for human telomeric DNA, such as G-quadruplex or T-loop. [3, 4] The T-loop and G-quadruplex structural models with the same human telomere sequence seem to leave many questions unanswered. Some important experimental findings are not adequately explained by these models. Among those findings are the following: 1) The T-loop structure, which is stabilized by the 3'-overhang strand encroaching into the double-stranded region, cannot explain the inhibition of telomerase activity by a G-quadruplex stabilizing molecule. [2] 2) Previous studies showed that only one TTAGGG repeat (6 nt) of 3'-overhang is sufficient for T-loop formation. [5] However, a recent study showed that, in most senescent cells, telomeric overhangs are still long, often averaging 50-100 nt. [6] This finding raises the question of why that DNA length, seemingly sufficient for T-loop formation, does not provide a protective structure for prevention of senescence in cells. For the G-quadruplex structure, most previous studies focused on individual G-quadruplexes formed by short telomeric DNA (especially 22 nt unimolecular G-quadruplexes). [3] However, it might be more biologically relevant to take into account the real length of the 3'-terminal singlestranded overhang of human telomeric DNA in vivo (100-200 nt) to directly reveal the structural features of long
Sequence specificity of inter- and intramolecular G-quadruplex formation by human telomeric DNA
Biopolymers, 2007
Human telomeric DNA consists of tandem repeats of the sequence 5′-d(TTAGGG)-3′. Guanine-rich DNA, such as that seen at telomeres, forms G-quadruplex secondary structures. Alternative forms of G-quadruplex structures can have differential effects on activities involved in telomere maintenance. With this in mind, we analyzed the effect of sequence and length of human telomeric DNA on G-quadruplex structures by native polyacrylamide gel electrophoresis and circular dichroism. Telomeric oligonucleotides shorter than four, 5′-d(TTAGGG)-3′ repeats formed intermolecular G-quadruplexes. However, longer telomeric repeats formed intramolecular structures. Altering the 5′-d(TTAGGG)-3′ to 5′-d(TTAGAG)-3′ in any one of the repeats of 5′-d(TTAGGG)4-3′ converted an intramolecular structure to intermolecular G-quadruplexes with varying degrees of parallel or anti-parallel-stranded character, depending on the length of incubation time and DNA sequence. These structures were most abundant in K+-containing buffers. Higher-order structures that exhibited ladders on polyacrylamide gels were observed only for oligonucleotides with the first telomeric repeat altered. Altering the sequence of 5′-d(TTAGGG)8-3′ did not result in the substantial formation of intermolecular structures even when the oligonucleotide lacked four consecutive telomeric repeats. However, many of these intramolecular structures shared common features with intermolecular structures formed by the shorter oligonucleotides. The wide variability in structure formed by human telomeric sequence suggests that telomeric DNA structure can be easily modulated by proteins, oxidative damage, or point mutations resulting in conversion from one form of G-quadruplex to another. © 2007 Wiley Periodicals, Inc. Biopolymers 87: 74–84, 2007This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
Nucleic Acids Research, 2007
Guanine-rich DNA sequences can form G-quadruplexes stabilized by stacked G-G-G-G tetrads in monovalent cation-containing solution. The length and number of individual G-tracts and the length and sequence context of linker residues define the diverse topologies adopted by G-quadruplexes. The review highlights recent solution NMR-based G-quadruplex structures formed by the four-repeat human telomere in K + solution and the guanine-rich strands of c-myc, c-kit and variant bcl-2 oncogenic promoters, as well as a bimolecular G-quadruplex that targets HIV-1 integrase. Such structure determinations have helped to identify unanticipated scaffolds such as interlocked G-quadruplexes, as well as novel topologies represented by doublechain-reversal and V-shaped loops, triads, mixed tetrads, adenine-mediated pentads and hexads and snap-back G-tetrad alignments. The review also highlights the recent identification of guanine-rich sequences positioned adjacent to translation start sites in 5'-untranslated regions (5'-UTRs) of RNA oncogenic sequences. The activity of the enzyme telomerase, which maintains telomere length, can be negatively regulated through G-quadruplex formation at telomeric ends. The review evaluates progress related to ongoing efforts to identify small molecule drugs that bind and stabilize distinct G-quadruplex scaffolds associated with telomeric and oncogenic sequences, and outlines progress towards identifying recognition principles based on several X-ray-based structures of ligand-Gquadruplex complexes.
Chromosomes in human cells are protected by telomeres. Telomere shortens during each round of cell division because of the DNA end-replication problem. Cancer cells maintain telomere length homeostasis by either telomerase or/and the alternative lengthening of telomere (ALT) mechanism to sustain their division potential. Telomeric DNA tends to form G-quadruplex preferentially at the extreme 3’ end. This unique feature prevents the 3’ end from being used as a substrate of telomerase and as a primer in the ALT. Therefore, stabilizing telomere G-quadruplex is expected to inhibit both pathways and limit the proliferation of cancer cells. Based on a mathematical modeling and experimental results, this mini-review proposes a hypothesis that the formation of G-quadruplex in telomere may constitute a significant contribution to the incomplete end-replication of telomere DNA by preventing the priming of DNA synthesis near the 3’ end during telomere replication. According to this, stabilization of telomere G-quadruplex by chemical ligand may promise to accelerate telomere shortening in proliferating cells.
Telomeric G-quadruplexes are a substrate and site of localization for human telomerase
Nature communications, 2015
It has been hypothesized that G-quadruplexes can sequester the 3' end of the telomere and prevent it from being extended by telomerase. Here we purify and characterize stable, conformationally homogenous human telomeric G-quadruplexes, and demonstrate that human telomerase is able to extend parallel, intermolecular conformations in vitro. These G-quadruplexes align correctly with the RNA template of telomerase, demonstrating that at least partial G-quadruplex resolution is required. A highly purified preparation of human telomerase retains this extension ability, establishing that the core telomerase enzyme complex is sufficient for partial G-quadruplex resolution and extension. The parallel-specific G-quadruplex ligand N-methyl mesoporphyrin IX (NMM) causes an increase in telomeric G-quadruplexes, and we show that telomerase colocalizes with a subset of telomeric G-quadruplexes in vivo. The ability of telomerase to partially unwind, extend and localize to these structures impli...
Journal of Biological Chemistry, 2012
Background: Telomeric repeat-containing RNA has recently been found in mammalian cells. Results: Oligonucleotide models of telomeric DNA and RNA form a hybrid G-quadruplex structure. Conclusion: We suggest a model system for understanding the structure and function of human telomeres. Significance: Our finding provides valuable information for understanding the structure and function of human telomeric DNA and RNA. Telomeric repeat-containing RNA, a non-coding RNA molecule, has recently been found in mammalian cells. The detailed structural features and functions of the telomeric RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In this study, using model human telomeric DNA and RNA sequences, we demonstrated that human telomeric RNA and DNA oligonucleotides form a DNA-RNA G-quadruplex. We next employed chemistry-based oligonucleotide probes to mimic the naturally formed telomeric DNA-RNA G-quadruplexes in living cells, suggesting that the process of DNA-RNA G-quadruplex formation with oligonucleotide models of telomeric DNA and RNA could occur in cells. Furthermore, we investigated the possible roles of this DNA-RNA G-quadruplex. The formation of the DNA-RNA G-quadruplex causes a significant increase in the clonogenic capacity of cells and has an effect on inhibition of cellular senescence. Here, we have used a model system to provide evidence about the formation of G-quadruplex structures involving telomeric DNA and RNA sequences that have the potential to provide a protective capping structure for telomere ends.
Ligand Binding to Tandem G Quadruplexes from Human Telomeric DNA
ChemBioChem, 2008
Ligand-induced stabilization of intramolecular telomeric G quadruplexes produced in the single-stranded overhang of the human telomere has become an attractive strategy for the development of anticancer drugs. [1] Several distinct solution conformations of human telomeric G quadruplexes have been elucidated in the presence of sodium [2] and potassium [3] cations. The K +-form, hybrid-type G-quadruplex structure has been considered to be a physiologically relevant conformation of the human telomeric sequence, and thus, can be specifically targeted by G-quadruplex-interactive, small-molecule A C H T U N G T R E N N U N G drugs. [3a, b] Recently, a beads-on-a-string model was proposed for the telomeric overhang, in which every four consecutive Grich repeats adopt an individual G-quadruplex structure, and two G-quadruplex units are connected by one TTA linker. [3a, c, 4] Ligand binding to the G quadruplex has mostly been investigated on telomere sequences producing a single G quadruplex, but few studies of ligand binding to beads-on-a-string G quadruplexes have been reported. To gain insight into the beads-on-a-string model and the nature of ligand binding, we undertook the polymerase stop assay on human telomere sequences of three to eight repeats (Table S1 in the Supporting Information) with TMPyP4, a G-quadruplex-interactive ligand, [5] and sanguinarine (Scheme 1), a natural isoquinoline alkaloid. The results described in this paper confirm the beads-on-astring structure of telomeric overhang and suggest a mode of ligand binding between tandem G-quadruplex beads. These observations should be taken into account for structure-based design of anticancer drugs targeting human telomeric DNA. Sanguinarine is a natural isoquinoline alkaloid isolated from the North American herb bloodroot (Sanguinaria canadensis). It was approved by the FDA in 2003 to be added to oral cleansing products as an antibacterial agent. Sanguinarine also possesses potent anticancer activity. [6] We have previously reported its DNA-binding activity and distinct sequence selectivity to double-stranded DNA, [7] which was proposed to be one of the molecular mechanisms of its anticancer activity. The structural similarity of sanguinarine with berberine, another isoquinoline alkaloid possessing G-quadruplex-binding activity, [8] prompted us to speculate that sanguinarine is probably a G-quadruplex binder. In this communication, we report its binding to the A C H T U N G T R E N N U N G telomeric overhang using DNA polymerase stop assays. [9] DNA templates Tem-3 and Tem-4 (Table S1), which contains three and four human telomeric repeats dA C H T U N G T R E N N U N G (TTAGGG), respectively, were employed, and TMPyP4 was used as the reference compound in the assay. [5, 9a, 10] Neither TMPyP4 nor sanguinarine blocked DNA synthesis on Tem-3, because an intramolecular Gquadruplex structure could not form with three human telomeric repeats on Tem-3 (Figure 1). In contrast, both sanguinarine and TMPyP4 produced paused bands in DNA synthesis on Tem-4. The position of the paused bands was the same for the two ligands. For each ligand, a series of concentration-dependent paused bands appeared at the beginning of the G-quadruplex-forming site, that is, the first site of G-rich repeats in Tem-4 (from 3' to 5'). In the presence of 3 mm TMPyP4, the polymerase reaction was totally suppressed to give no elongation of the primer. The tight binding of sanguinarine and TMPyP4 to the K +-form hybrid-type G-quadruplex structure was clearly indicated from the large increase in the melting temperature (DT m