Human Telomeric G-quadruplex Research Articles

Targeting human telomeric and c-myc G-quadruplexes with alkynylplatinum(II) terpyridine complexes under molecular crowding conditions

The interactions between alkynylplatinum(II) terpyridine complexes 1–3 and the G-quadruplex DNA, including c-myc and telomeric quadruplex DNA, are investigated both in dilute solution and under molecular crowding conditions. The UV–vis absorption spectroscopy, circular dichroism and molecular docking studies suggest that 1–3 associate with telomeric and c-myc G-quadruplexes via groove binding, and electrostatic interactions. Experimental studies indicate that under molecular crowding conditions (in the presence of 40wt% PEG 200), 1–2 show weak affinity for c-myc, while 3 still displays high affinity and selectivity for c-myc. On the other hand, 1–3 act as efficient and selective ligand for telomeric quadruplex DNA under molecular crowding conditions. The complex 3 exhibits excellent cytotoxicity against A549, K562 and SGC-7901, with IC50 values that are 35.0-fold, 10.0-fold, and 12.1-fold lower than the values of cisplatin under the same conditions, respectively.

Anti-tumor activity and mechanism of apoptosis of A549 induced by ruthenium complex.

Two new ruthenium (II) polypyridyl complexes [Ru(MeIm)4(pip)]2+ (1) and [Ru(MeIm)4(4-npip)]2+ (2) were synthesized under the guidance of computational studies (DFT). Their binding property to human telomeric G-quadruplex studied by UV-Vis absorption spectroscopy, the fluorescent resonance energy transfer (FRET) melting assay and circular dichroism (CD) spectroscopy for validating the theoretical prediction. Both of them were evaluated for their potential anti-proliferative activity against four human tumor cell lines. Complex 2 shows growth inhibition against all the cell lines tested, especially the human lung tumor cell (A549). The RTCA analysis not only validated the inhibition activity but also showed the ability of reducing A549 cells' migration. DNA-flow cytometric analysis, mitochondrial membrane potential (ΔΨm) and the scavenger measurements of reactive oxygen species (ROS) analysis carried out to investigate the mechanism of cell growth inhibition and apoptosis-inducing effect of complex 2. The results demonstrated that complex 2 induces tumor cells apoptosis by acting on both mitochondrial homeostasis destruction and death receptor signaling pathways. And those suggested that complex 2 could be a candidate for further evaluation as a chemotherapeutic agent against human tumor.

Folding Kinetics of Single Human Telomeric G-Quadruplex Affected by Cisplatin.

G-Quadruplex DNA structure has been proven to be a binding target for small molecular organic compounds and hence regarded as a promising pharmacological target. Cisplatin is a widely used chemotherapy drug that targets duplex DNA and was recently shown to react also with G-quadruplex, implying that cisplatin actually may also target G-quadruplex. In this work, we employed magnetic tweezers to investigate the influence of cisplatin on the folding kinetics of single human telomeric G-quadruplex. It was revealed that cisplatin and G-quadruplex interact in two different and competitive ways that depend on cisplatin concentration.

Selective recognition and stabilization of new ligands targeting the potassium form of the human telomeric G-quadruplex DNA

The development of a ligand that is capable of distinguishing among the wide variety of G-quadruplex structures and targeting telomeres to treat cancer is particularly challenging. In this study, the ability of two anthraquinone telomerase inhibitors (NSC749235 and NSC764638) to target telomeric G-quadruplex DNA was probed. We found that these ligands specifically target the potassium form of telomeric G-quadruplex DNA over the DNA counterpart. The characteristic interaction with the telomeric G-quadruplex DNA and the anticancer activities of these ligands were also explored. The results of this present work emphasize our understanding of the binding selectivity of anthraquinone derivatives to G-quadruplex DNA and assists in future drug development for G-quadruplex-specific ligands.

Ru(L)2(3-tppp)]2 + (L = bpy, phen) stabilizes two different forms of the human telomeric G-quadruplex DNA

The binding properties of new complexes [Ru(L)2(3-tppp)]2+ (L=bpy, phen) towards two different forms of the human telomeric G-quadruplexes DNA have been investigated by UV–Vis spectroscopy, fluorescent indicator displacement (FID) assays, fluorescence resonance energy transfer (FRET) melting assays and molecular docking studies. The molecular docking studies indicated that both complexes interacted with 22AG with the stoichiometric ratio of 1:1, but the two complexes showed different G-quadruplexed DNA binding affinity. Complex 2 bound to the G-quadruplexes DNA more tightly than complex 1 did. Moreover, the FRET melting assay revealed that both complexes could be potential stabilizers for G-quadruplex architectures. These studies are useful not only for better understanding of the interaction between the target G-quadruplexes DNA and metal complexes but also valuable in defining the best strategy to prepare metal complexes as potential anticancer drugs.

Abstract 1357: In silico design and biological evaluation of benzofused polyamides targeting G-quadruplex DNA structures

Abstract Guanine-rich nucleic acids can fold into distinctive four-stranded G-quadruplex structures which are found in telomeric DNA repeats as well as in sequences in the promoter and other regulatory regions of genes, especially those involved in cellular proliferation. Small molecules that can selectively bind and stabilize the G-quadruplex structure have become of significant interest to researchers, and are gaining momentum as a possible new class of anticancer agents. We recently reported a series of novel biaryl polyamides with significant selectively toward G-quadruplex compared to duplex DNA. Using a distamycin scaffold as a starting point, we introduced biaryl building blocks in place of pyrroles to switch preference from duplex to G-quadruplex DNA. This alteration in shape ensured that the molecules had low affinity for duplex DNA while increasing their interaction with a G-quadruplex structure. We have now used a molecular modeling approach to modify the structure of the previously reported biaryl polyamides by incorporating benzofused building blocks to improve affinity for G-quadruplexes while further reducing affinity for duplex DNA to enhance selectivity for G-quadruplex versus duplex DNA. A small, focused benzofused-polyamide library (17 molecules) was initially synthesized and evaluated for the ability of members to stabilize G-quadruplex structures using a FRET-based DNA thermal denaturation assay and molecular dynamics (MD) simulations. However, these compounds failed to stabilize the human telomeric G-quadruplex and c-Kit quadruplexes, and MD simulations suggested that the shape of the molecules required further modification to facilitate G-quadruplex interaction. A second library of molecules (50 in total) was then designed and synthesized using a molecular modeling based approach. In this series, the shape of the polyamide fragment was changed, while retaining the original scaffold, by introducing benzofused moieties with 3,5-substitutions. Evaluation of these molecules in the same FRET assay showed a notable increase in stabilization of the F21T quadruplex for many library members. For example, compounds AR-130 and AR-168 stabilized the G-quadruplex by 15°C and 17°C, respectively (at 1 μM concentration), while showing insignificant affinity for duplex DNA. Cytotoxicity in the micromolar region is anticipated based on recent studies on related compounds, and a full growth inhibition evaluation is presently underway in MiaPaCa2, MDA-MB 231, HeLa and NCIH1975 cell lines. Given their low molecular weight (e.g., between 422-646 Daltons), reasonable water solubility and good cellular penetration properties compared to other known G-quadruplex inhibitors which are mostly non-drug-like, molecules of this type have the potential to be developed into reagents that can probe DNA structure, and novel therapeutic agents based on G-quadruplex targeting. Citation Format: Khondaker M. Rahman, AKM Azadur Rahman, Paul J. M. Jackson, David E. Thurston. In silico design and biological evaluation of benzofused polyamides targeting G-quadruplex DNA structures. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1357.

Toward the Development of Specific G-Quadruplex Binders: Synthesis, Biophysical, and Biological Studies of New Hydrazone Derivatives.

G-Quadruplex-binding compounds are currently perceived as possible anticancer therapeutics. Here, starting from a promising lead, a small series of novel hydrazone-based compounds were synthesized and evaluated as G-quadruplex binders. The in vitro G-quadruplex-binding properties of the synthesized compounds were investigated employing both human telomeric and oncogene promoter G-quadruplexes with different folding topologies as targets. The present investigation led to the identification of potent G-quadruplex stabilizers with high selectivity over duplex DNA and preference for one G-quadruplex topology over others. Among them, selected derivatives have been shown to trap G-quadruplex structures in the nucleus of cancer cells. Interestingly, this behavior correlates with efficient cytotoxic activity in human osteosarcoma and colon carcinoma cells.

Selective and Cooperative Ligand Binding to Antiparallel Human Telomeric DNA G-Quadruplexes.

The quest for ligands that specifically bind to particular G-quadruplex nucleic acid structures is particularly important to conceive molecules with specific effects on gene expression or telomere maintenance, or conceive structure-specific molecular probes. Using electrospray mass spectrometry in native conditions, we reveal a highly cooperative and selective 2:1 binding of Cu(II) -tolylterpyridine complexes to human telomeric G-quadruplexes. Circular dichroism and comparisons of affinities for different sequences reveal a marked preference for antiparallel structures with diagonal loops and/or wide-medium-narrow-medium groove-width order. The cooperativity is attributed to conformational changes in the polymorphic telomeric G-quadruplex sequences, which convert preferably into an antiparallel three-quartet topology upon binding of two ligands.

A Comparative Study of G-Quadruplex Unfolding and DNA Reeling Activities of Human RECQ5 Helicase

RECQ5 is one of five members of the RecQ family of helicases in humans, which include RECQ1, Bloom (BLM), Werner (WRN), RECQ4, and RECQ5. Both WRN and BLM have been shown to resolve G-quadruplex (GQ) structures. Deficiencies in unfolding GQ are known to result in DNA breaks and genomic instability, which are prominent in Werner and Bloom syndromes. RECQ5 is significant in suppressing sister chromatid exchanges during homologous recombination but its GQ unfolding activity are not known. We performed single-molecule studies under different salt (50–150 mM KCl or NaCl) and ATP concentrations on different GQ constructs including human telomeric GQ (with different overhangs and polarities) and GQ formed by thrombin-binding aptamer to investigate this activity. These studies demonstrated a RECQ5-mediated GQ unfolding activity that was an order of magnitude weaker than BLM and WRN. On the other hand, BLM and RECQ5 demonstrated similar single-stranded DNA (ssDNA) reeling activities that were not coupled to GQ unfolding. These results demonstrate overlap in function between these RecQ helicases; however, the relatively weak GQ destabilization activity of RECQ5 compared to BLM and WRN suggests that RECQ5 is not primarily associated with GQ destabilization, but could substitute for the more efficient helicases under conditions where their activity is compromised. In addition, these results implicate a more general role for helicase-promoted ssDNA reeling activity such as removal of proteins at the replication fork, whereas the association of ssDNA reeling with GQ destabilization is more helicase-specific.

X-ray characterization of mesophases of human telomeric G-quadruplexes and other DNA analogues.

Observed in the folds of guanine-rich oligonucleotides, non-canonical G-quadruplex structures are based on G-quartets formed by hydrogen bonding and cation-coordination of guanosines. In dilute 5′-guanosine monophosphate (GMP) solutions, G-quartets form by the self-assembly of four GMP nucleotides. We use x-ray diffraction to characterize the columnar liquid-crystalline mesophases in concentrated solutions of various model G-quadruplexes. We then probe the transitions between mesophases by varying the PEG solution osmotic pressure, thus mimicking in vivo molecular crowding conditions. Using the GMP-quadruplex, built by the stacking of G-quartets with no covalent linking between them, as the baseline, we report the liquid-crystalline phase behaviors of two other related G-quadruplexes: (i) the intramolecular parallel-stranded G-quadruplex formed by the 22-mer four-repeat human telomeric sequence AG3(TTAG3)3 and (ii) the intermolecular parallel-stranded G-quadruplex formed by the TG4T oligonucleotides. Finally, we compare the mesophases of the G-quadruplexes, under PEG-induced crowding conditions, with the corresponding mesophases of the canonical duplex and triplex DNA analogues.

Computational Insights into the Stability and Folding Pathways of Human Telomeric DNA G-Quadruplexes.

G-quadruplex is a noncanonical yet crucial secondary structure of nucleic acids, which has proven its importance in cell aging, anticancer therapies, gene expression, and genome stability. In this study, the stability and folding dynamics of human telomeric DNA G-quadruplexes were investigated via enhanced sampling techniques. First, temperature-replica exchange MD (REMD) simulations were employed to compare the thermal stabilities among the five established folding topologies. The hybrid-2 type adopted by extended human telomeric sequence is revealed to be the most stable conformation in our simulations. Next, the free energy landscapes and folding intermediates of the hybrid-1 and -2 types were investigated with parallel tempering metadynamics simulations in the well-tempered ensemble. It was observed that the N-glycosidic conformations of guanines can flip over to accommodate into the cyclic Hoogsteen H-bonding on G-tetrads in which they were not originally involved. Furthermore, a hairpin and a triplex intermediate were identified for the folding of the hybrid-1 type conformation, whereas for the hybrid-2 type, there were no folding intermediates observed from its free energy surface. However, the energy barrier from its native topology to the transition structure is found to be extremely high compared to that of the hybrid-1 type, which is consistent with our stability predictions from the REMD simulations. We hope the insights presented in this work can help to complement current understanding on the stability and dynamics of G-quadruplexes, which is necessary not only to stabilize the structures but also to intervene their formation in genome.

Inside Cover: Ultrafast Electron Transfer in Complexes of Doxorubicin with Human Telomeric G‐Quadruplexes and GC Duplexes Probed by Femtosecond Fluorescence Spectroscopy (ChemPhysChem 9/2016)

The Inside Cover picture illustrates how femtosecond fluorescence spectroscopy provides evidence of fast photoinduced electron transfer in complexes of doxorubicin with d(GC) duplexes and human telomeric G-quadruplexes. More information can be found in the Full Paper by P. Changenet-Barret et al. on page 1264 in Issue 9, 2016 (DOI: 10.1002/cphc.201501091).

Hit Identification of a Novel Dual Binder for h-telo/c-myc G-Quadruplex by a Combination of Pharmacophore Structure-Based Virtual Screening and Docking Refinement.

It is well known that G-quadruplexes are targets of great interest for their roles in crucial biological processes, such as aging and cancer. Hence, a promising strategy for anticancer drug therapy is the stabilization of these structures by small molecules. We report a high-throughput in silico screening of commercial libraries from several different vendors by means of a combined structure-based pharmacophore model approach followed by docking simulations. The compounds selected by the virtual screening procedure were then tested for their ability to interact with human telomeric G-quadruplex folding by circular dichroism, fluorescence spectroscopy, and fluorescence intercalator displacement. Our approach resulted in the identification of a 13-[(dimethylamino)methyl]-12-hydroxy-8H-benzo[c]indolo[3,2,1-ij][1,5]naphthyridin-8-one derivative as a novel promising stabilizer of G-quadruplex structures within the human telomeric and the c-myc promoter sequences.

A novel transition pathway of ligand-induced topological conversion from hybrid forms to parallel forms of human telomeric G-quadruplexes.

The folding topology of DNA G-quadruplexes (G4s) depends not only on their nucleotide sequences but also on environmental factors and/or ligand binding. Here, a G4 ligand, 3,6-bis(1-methyl-4-vinylpyridium iodide)-9-(1-(1-methyl-piperidinium iodide)-3,6,9-trioxaundecane) carbazole (BMVC-8C3O), can induce topological conversion of non-parallel to parallel forms in human telomeric DNA G4s. Nuclear magnetic resonance (NMR) spectroscopy with hydrogen-deuterium exchange (HDX) reveals the presence of persistent imino proton signals corresponding to the central G-quartet during topological conversion of Tel23 and Tel25 G4s from hybrid to parallel forms, implying that the transition pathway mainly involves local rearrangements. In contrast, rapid HDX was observed during the transition of 22-CTA G4 from an anti-parallel form to a parallel form, resulting in complete disappearance of all the imino proton signals, suggesting the involvement of substantial unfolding events associated with the topological transition. Site-specific imino proton NMR assignments of Tel23 G4 enable determination of the interconversion rates of individual guanine bases and detection of the presence of intermediate states. Since the rate of ligand binding is much higher than the rate of ligand-induced topological conversion, a three-state kinetic model was evoked to establish the associated energy diagram for the topological conversion of Tel23 G4 induced by BMVC-8C3O.

Calorimetric investigation on the interaction of proflavine with human telomeric G-quadruplex DNA

The interaction of an acridine dye, proflavine, with human telomeric G-quadruplex DNA was characterized by isothermal titration calorimetry and differential scanning calorimetry. The equilibrium constant of binding was deduced to be (1.36±0.09)·106M−1 at T=298.15K. The binding reaction was driven by both negative enthalpy and positive entropy contributions. The equilibrium constant decreased and the reaction became increasingly enthalpy driven with rise in temperature. However, the standard molar Gibbs energy change exhibited only marginal alterations suggesting the occurrence of enthalpy–entropy compensation. Negative heat capacity values were also obtained from the temperature dependence of enthalpy change. Parsing of the standard molar Gibbs energy using the salt dependent calorimetric data revealed that the binding was dominated by non-polyelectrolytic forces which remained virtually unaltered with changing salt concentration. Proflavine binding also significantly enhanced the thermal stability of G-quadruplex DNA against thermal unfolding.

Can We Execute Reliable MM-PBSA Free Energy Computations of Relative Stabilities of Different Guanine Quadruplex Folds?

The self-assembly and stability of DNA G-quadruplexes (GQs) are affected by the intrinsic stability of different GpG base steps embedded in their G-quartet stems. We have carried out MD simulations followed by MM-PBSA (molecular mechanics Poisson-Boltzmann surface area) free energy calculations on all the experimentally observed three-quartet intramolecular human telomeric GQ topologies. We also studied antiparallel GQ models with alternative syn-anti patterns of the G-quartets. We tested different ions, dihedral variants of the DNA force field, water models, and simulation lengths. In total, ∼35 μs of simulations have been carried out. The systems studied here are considerably more complete than the previously analyzed two-quartet stems. Among other effects, our computations included the stem-loop coupling and ion-ion interactions inside the stem. The calculations showed a broad agreement with the earlier predictions. However, the increase in the completeness of the system was associated with increased noise of the free energy data which could be related, for example, to the presence of long-lived loop substates and rather complex dynamics for the two bound ions inside the G-stem. As a result, the MM-PBSA data were noisy and we could not improve their quantitative convergence even by expanding the simulations to 2.5 μs long trajectories. We also suggest that the quality of MM-based free energy computations based on MD simulations of complete GQs is more sensitive to the neglect of explicit polarization effects, which, in real systems, are associated with the presence of multiple closely spaced ions inside the GQs. Thus, although the MM-PBSA procedure provides very useful insights that complement the structural-dynamics data from MD trajectories of GQs, the method is far from reaching quantitative accuracy. Our conclusions are in agreement with critical assessments of the MM-PBSA methodology available in contemporary literature for other types of problems.

Synthesis, characterization and DNA binding studies of two Ru(II) complexes containing guanidinium ligands

Two new Ru(II) complexes containing guanidinium groups have been synthesized, characterized and analyzed according to their interactions with different G-quadruplexes and duplex DNA. A FRET assay and a competitive FRET assay showed that both complexes promote the formation and stabilization of the human telomeric (h-telo) G-quadruplex and exhibit higher selectivity compared to promoters (such as c-myc, c-kit and bcl2) or duplex DNA. After binding to G-quadruplex, the two complexes have shown different DNA affinity and fluorescence enhancement. CD analyses further indicate that the two complexes display the ability to induce and stabilize the formation of antiparallel G-quadruplex structures in K+, Na+ or ion-free buffers. The binding stoichiometry with h-telo was of the order of three ruthenium complexes per quadruplex.

Dynamics of Human Telomeric G-Quadruplex Probed by Single Molecule Fluorescence-Force Spectroscopy

G-quadruplexes (GQs) are constituted by stacks of G-quartets formed in guanine rich sequences in nucleic acids. Among its many functions, GQ plays a pivotal role in regulating telomere length in cells. Mechanistic understanding of dynamics of GQ is key to understanding its biological functions. Integrated single molecule force-fluorescence spectroscopy has been used herein to study the dynamics of human telomeric GQs under tension. GQ unfolding at physiological K+ concentration showed three features: a significant population resisted perturbation by force (∼35 pN) and remained in the folded state, while unfolding culminated into either a partially or completely unfolded state. Complete unfolding of GQs occurred stochastically within a wide spectrum of forces between 3 and 35 pN, even within the same molecule across different stretching cycles. Correspondingly, although GQs exhibited diverse refolding behavior, most of the refolding occurred within 5-10 pN. Such diversity in the forces required for complete unfolding of GQs was also observed with Na+. Replacement of a single guanine precluded GQ formation at lower metal ion concentrations (up to 100 mM) and only partially folded GQ-like species were induced which unfolded/refolded at lower forces with no evident hysteresis. At high metal ion concentrations (1M of Na+/K+) GQ with single point mutation showed an unfolding/refolding behavior redolent of GQ, suggesting formation of GQ-like species. The understanding of different levels of stability in human telomeric GQ can be leveraged into developing anti-cancer drugs.

A Comparative Study on G-Quadruplex Unfolding Activity of RecQ Helicases

The RecQ family has five member helicases in humans: RecQL1, RecQL2 or WRN, RecQL3 or BLM, RecQL4, and RecQL5. WRN, BLM, and RecQL4 helicases are associated with Werner, Bloom, and Rothmund-Thomson syndromes, respectively. RecQL1 and RecQL5 have not been directly linked with a specific syndrome. Both of WRN and BLM helicases resolve non-canonical DNA structures including the G-quadruplex (GQ), which otherwise result in DNA breaks and genomic instability. Whether RecQL5 also possesses GQ unfolding activity may potentially provide insight about why RecQL5 is not associated with a specific syndrome, assuming GQ structures play a significant role in aforementioned syndromes. We recently demonstrated that BLM unfolds human telomeric GQ (hGQ) in the presence of ATP and also to a lesser extent upon binding to the vicinity of hGQ in the absence of ATP, both under physiologically germane salt and pH conditions. In the current study, GQ unfolding activity of RecQL5 was examined using single molecule FRET using hGQ and other GQ constructs. Our data show that RecQL5 unfolds hGQ at a significantly weaker rate compared to BLM in the presence of ATP, and does not unfold GQ in the absence of ATP. RecQL5-mediated GQ unfolding is strongly dependent on GQ stability, as modulated by salt or GQ structure. Significant differences were observed between the dynamics of BLM and RecQL5 interactions with the DNA, in particular in terms of the ‘reeling’ activity previously observed in several GQ unfolding helicases. In conclusion, RecQL5 is a significantly less efficient GQ destabilizer compared to BLM and WRN, but may assist these helicases in this task. These results are consistent with GQs being significant components of Bloom and Werner syndromes, and might explain why deficiencies in RecQL5 are not directly associated with such a syndrome.

Probing the Folding Dynamics of Human Telomeric G-Quadruplex with Single-Molecule FRET

Guanine-rich sequences consisting of several tandem repeats of TTAGGG are abundant in human telomeric DNA. They have been shown to fold into unique secondary structures under physiological conditions. One of these is the G-quadruplex structure that can undergo complex folding pathways [1-3]. Single-molecule fluorescence microscopy combined with Forster resonance energy transfer (FRET) allows for probing the conformation and dynamics of individual G-quadruplex molecules without time and population averaging and thus resolving their structural heterogeneity that is otherwise hidden in ensemble experiments [4,5].Here we utilize single-molecule FRET microscopy to probe the K+-induced folding dynamics of human telomeric G-quadruplex DNA. Our single-molecule experiments identify several distinct FRET states populated along the folding of G-quadruplexes. We find that initial dynamic interconversion occurs between three states - the unfolded state and two transient folded states, followed by transition into a dominant long-lived folded G-quadruplex conformation. Additional single-molecule FRET measurements, employing specific chemically modified G-quadruplex sequences, and advanced molecular modeling enabled linking the experimentally observed FRET states to particular G-quadruplex conformations. Our results thus allow uncovering a possible folding pathway of the human telomeric G-quadruplex structures.(1) Bochman, M. L.; Paeschke, K.; Zakian, V. A. Nature Reviews Genetics 2012, 13, 770.(2) Biffi, G.; Tannahill, D.; McCafferty, J.; Balasubramanian, S. Nature Chemistry 2013, 5, 182.(3) Gray, R. D.; Trent, J. O.; Chaires, J. B. J. Mol. Biol. 2014, 426, 1629.(4) Single-Molecule Techniques - A Laboratory Manual; Selvin, P. R.; Ha, T., Eds.; Cold Spring Harbor, NY, 2008.(5) Lee, J. Y.; Okumus, B.; Kim, D. S.; Ha, T. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 18938.