Interaction of porphyrin with G-quadruplex structures

Isothermal titration calorimetry (ITC) is a sensitive technique for probing bimolecular processes and can provide direct information about the binding affinity and stoichiometry and the key thermodynamic parameters involved. ITC has been used to investigate the interaction of the ligand H2TMPyP to the two DNA quadruplexes, [d(AGGGT)]4 and [d(TGGGGT)]. Analysis of the ITC data reveals that porphyrin/quadruplex binding stoichiometry under saturating conditions is 1:2 for [d(AGGGT)]4 and 2:1 for [d(TGGGGT)], respectively.     

Probing the interaction of distamycin A with S100β: the “unexpected” ability of S100β to bind to DNA‐binding ligands

DNA‐minor‐groove‐binding ligands are potent antineoplastic molecules. The antibiotic distamycin A is the prototype of one class of these DNA‐interfering molecules that have been largely used in vitro. The affinity of distamycin A for DNA is well known, and the structural details of the complexes with some B‐DNA and G‐quadruplex‐forming DNA sequences have been already elucidated. Here, we show that distamycin A binds S100β, a protein involved in the regulation of several cellular processes. The reported affinity of distamycin A for the calcium(II)‐loaded S100β reinforces the idea that some biological activities of the DNA‐minor‐groove‐binding ligands arise from the binding to cellular proteins. Copyright © 2015 John Wiley & Sons, Ltd.     

Aminoglycoside binding to Oxytricha nova telomeric DNA

Telomeric DNA sequences have been at the center stage of drug design for cancer treatment in recent years. The ability of these DNA structures to form four-stranded nucleic acid structures, called G-quadruplexes, has been perceived as target for inhibiting telomerase activity vital for the longevity of cancer cells. Being highly diverse in structural forms, these G-quadruplexes are subjects of detailed studies of ligand-DNA interactions of different classes, which will pave the way for logical design of more potent ligands in future. The binding of aminoglycosides was investigated with Oxytricha nova quadruplex forming DNA sequence (GGGGTTTTGGGG)(2). Isothermal titration calorimetry (ITC) determined ligand to quadruplex binding ratio shows 1:1 neomycin:quadruplex binding with association constants (K(a)) ～ 10(5) M(-1) while paromomycin was found to have a 2-fold weaker affinity than neomycin. The CD titration experiments with neomycin resulted in minimal changes in the CD signal. FID assays, performed to determine the minimum concentration required to displace half of the fluorescent probe bound, showed neomycin as the best of the all aminoglycosides studied for quadruplex binding. Initial NMR footprint suggests that ligand-DNA interactions occur in the wide groove of the quadruplex. Computational docking studies also indicate that aminoglycosides bind in the wide groove of the quadruplex.     

Studies on the binding of 5-N-methylated quindoline derivative to human telomeric G-quadruplex

Quindoline derivatives as telomeric quadruplex ligands have shown good biological activity for telomerase inhibition. In the present study, we used spectroscopic and calorimetric methods to investigate the interactions between a quindoline derivative (5-methyl-11-(2-morpholinoethylamino)-10-H-indolo-[3,2-b]quinolin-5-ium iodide, compound 1) and human telomeric G-quadruplex. The thermodynamic studies using isothermal titration calorimetry (ITC) indicated that their binding process was temperature-dependent and enthalpy–entropy co-driven. The significant negative heat capacity was obtained experimentally from the temperature dependence of enthalpy changes, which was consistent with that from theoretical calculation, and all suggesting significant hydrophobic contribution to the molecular recognition process. Based on the results from UV–vis, ITC, steady-state and time-resolved fluorescence, their binding mode was determined as two ligand molecules stacking on the quartets on both ends of the quadruplex. These results shed light on rational design and development of quindoline derivatives as G-quadruplex binding ligands.     

INTERACTION OF PORPHYRIN WITH G-QUADRUPLEX STRUCTURES

Isothermal titration calorimetry (ITC) is a sensitive technique for probing bimolecular processes and can provide direct information about the binding affinity and stoichiometry and the key thermodynamic parameters involved. ITC has been used to investigate the interaction of the ligand H₂TMPyP to the two DNA quadruplexes, [d(AGGGT)]₄ and [d(TGGGGT)]₄. Analysis of the ITC data reveals that porphyrin/quadruplex binding stoichiometry under saturating conditions is 1:2 for [d(AGGGT)]₄ and 2:1 for [d(TGGGGT)]₄, respectively.     

Stability and binding properties of a modified thrombin binding aptamer

Aptamer-based drugs represent an attractive approach in pharmacological therapy. The most studied aptamer, thrombin binding aptamer (TBA), folds into a well-defined quadruplex structure and binds to its target with good specificity and affinity. Modified aptamers with improved biophysical properties could constitute a new class of therapeutic aptamers. In this study we show that the modified thrombin binding aptamer (mTBA), ^3′GGT^5′-^5′TGGTGTGGTTGG^3′, which also folds into a quadruplex structure, is more stable than its unmodified counterpart and shows a higher thrombin affinity. The stability of the modified aptamer was investigated using differential scanning calorimetry, and the energetics of mTBA and TBA binding to thrombin was characterized by means of isothermal titration calorimetry (ITC). ITC data revealed that TBA/thrombin and mTBA/thrombin binding stoichiometry is 1:2 for both interactions. Structural models of the two complexes of thrombin with TBA and with mTBA were also obtained and subjected to molecular dynamics simulations in explicit water. Analysis of the models led to an improvement of the understanding of the aptamer-thrombin recognition at a molecular level.     

Recognition of human telomeric G‐quadruplex DNA by berberine analogs: effect of substitution at the 9 and 13 positions of the isoquinoline moiety

G‐quadruplex forming sequences are widely distributed in human genome and serve as novel targets for regulating gene expression and chromosomal maintenance. They offer unique targets for anticancer drug development. Here, the interaction of berberine (BC) and two of its analogs bearing substitution at 9 and 13‐position with human telomeric G‐quadruplex DNA sequence has been investigated by biophysical techniques. Both the analogs exhibited several‐fold higher binding affinity than berberine. The Scatchard binding isotherms revealed non‐cooperative binding. 9‐ω‐amino hexyl ether analog (BC1) showed highest affinity (1.8 × 10⁶ M^?1) while the affinity of the 13‐phenylpropyl analog (BC2) was 1.09 × 10⁶ M^?1. Comparative fluorescence quenching and polarization anisotropy of the emission spectra gave evidence for a stronger stacking interaction of the analogs compared to berberine. The thiazole orange displacement assay has clearly established that the analogs were more effective in displacing the end stacked dye in comparison to berberine. However, the binding of the analogs did not induce any major structural perturbation in the G‐quadruplex structure, but led to higher thermal stability. Energetics of the binding indicated that the association of the analogs was exothermic and predominantly entropy driven phenomenon. Increasing the temperature resulted in weaker binding; the enthalpic contribution increased and the entropic contribution decreased. A small negative heat capacity change with significant enthalpy–entropy compensation established the involvement of multiple weak noncovalent interactions in the binding process. The 9‐ω‐amino hexyl ether analog stabilized the G‐quadruplex structure better than the 13‐phenyl alkyl analog. Copyright © 2015 John Wiley & Sons, Ltd.     

Targeting DNA quadruplexes with distamycin A and its derivatives: an ITC and NMR study

The use of small molecules that bind and stabilize G-quadruplex structures is emerging as a promising way to inhibit telomerase activity in tumor cells. In this paper, isothermal titration calorimetry (ITC) and (1)H NMR studies have been conducted to examine the binding of distamycin A and its two carbamoyl derivatives (compounds 1 and 2) to the target [d(TGGGGT)](4) and d[AG(3)(T(2)AG(3))(3)] quadruplexes from the Tetrahymena and human telomeres, respectively. The interactions were examined using two different buffered solutions containing either K(+) or Na(+) at a fixed ionic strength, to evaluate any influence of the ions present in solution on the binding behaviour. Experiments reveal that distamycin A and compound 1 bind the investigated quadruplexes in both solution conditions; conversely, compound 2 appears to have a poor affinity in any case. Moreover, these studies indicate that the presence of different cations in solution affects the stoichiometry and thermodynamics of the interactions.     

Binding properties of human telomeric quadruplex multimers: a new route for drug design

Human telomeric G-quadruplex structures are known to be promising targets for an anticancer therapy. In the past decade, several research groups have been focused on the design of new ligands trying to optimize the interactions between these small molecules and the G-quadruplex motif. In most of these studies, the target structures were the single quadruplex units formed by short human DNA telomeric sequences (typically 21-26 nt). However, the 3′-terminal single-stranded human telomeric DNA is actually 100-200 bases long and can form higher-order structures by clustering several consecutive quadruplex units (multimers). Despite the increasing number of structural information on longer DNA telomeric sequences, very few data are available on the binding properties of these sequences compared with the shorter DNA telomeric sequences.In this paper we use a combination of spectroscopic (CD, UV and fluorescence) and calorimetric techniques (ITC) to compare the binding properties of the (TTAGGG)₈TT structure formed by two adjacent quadruplex units with the binding properties of the (AG₃TT)₄ single quadruplex structure. The three side-chained triazatruxene derivative azatrux and TMPyP4 cationic porphyrin were used as quadruplex ligands. We found that, depending on the drug, the number of binding sites per quadruplex unit available in the multimer structure was smaller or greater than the one expected on the basis of the results obtained from individual quadruplex binding studies. This work suggests that the quadruplex units along a multimer structure do not behave as completely independent. The presence of adjacent quadruplexes results in a diverse binding ability not predictable from single quadruplex binding studies. The existence of quadruplex-quadruplex interfaces in the full length telomeric overhang may provide an advantageous factor in drug design to enhance both affinity and selectivity for DNA telomeric quadruplexes.     

Structure-specific recognition of quadruplex DNA by organic cations: influence of shape, substituents and charge

Combining structure-specific recognition of nucleic acids with limited sequence reading is a promising method to reduce the size of the recognition unit required to achieve the necessary selectivity and binding affinity to control function. It has been demonstrated recently that G-quadruplex DNA structures can be targeted by organic cations in a structure-specific manner. Structural targets of quadruplexes include the planar end surfaces of the G-tetrad stacked columns and four grooves. These provide different geometries and functional groups relative to duplex DNA. We have used surface plasmon resonance and isothermal titration calorimetry to show that binding affinity and selectivity of a series of quadruplex end-stacking molecules to human telomeric DNA are sensitive to compound shape as well as substituent type and position. ITC results indicate that binding is largely enthalpy driven. Circular dichroism was also used to identify a group of structurally related compounds that selectively target quadruplex grooves.     

A more detailed picture of the interactions between virtual screening-derived hits and the DNA G-quadruplex: NMR, molecular modelling and ITC studies

The growing amount of literature about G-quadruplex DNA clearly demonstrates that such a structure is no longer viewed as just a biophysical strangeness but it is instead being considered as an important target for the treatment of various human disorders such as cancers or venous thrombosis. In this scenario, with the aim of finding brand new molecular scaffolds able to interact with the groove of the DNA quadruplex [d(TGGGGT)]₄, we recently performed a successful structure-based virtual screening (VS) campaign. As a result, six molecules were found to be somehow groove binders. Herein, we report the results of novel NMR titration experiments of these VS-derived ligands with modified quadruplexes, namely [d(TGG^BrGGT)]₄ and [d(TGGGG^BrT)]₄. The novel NMR spectroscopy experiments combined with molecular modelling studies, allow for a more detailed picture of the interaction between each binder and the quadruplex DNA. Noteworthy, isothermal titration calorimetry (ITC) measurements on the above-mentioned compounds revealed that 2, 4, and 6 besides their relatively small dimensions bind the DNA quadruplex [d(TGGGGT)]₄ with higher affinity than distamycin A, to the best of our knowledge, the most potent groove binder identified thus far.     

Interaction of pyrrolobenzodiazepine (PBD) ligands with parallel intermolecular G-quadruplex complex using spectroscopy and ESI-MS

Studies on ligand interaction with quadruplex DNA, and their role in stabilizing the complex at concentration prevailing under physiological condition, has attained high interest. Electrospray ionization mass spectrometry (ESI-MS) and spectroscopic studies in solution were used to evaluate the interaction of PBD and TMPyP4 ligands, stoichiometry and selectivity to G-quadruplex DNA. Two synthetic ligands from PBD family, namely pyrene-linked pyrrolo[2,1-c][1,4]benzodiazepine hybrid (PBD1), mixed imine-amide pyrrolobenzodiazepine dimer (PBD2) and 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) were studied. G-rich single-stranded oligonucleotide d(5'GGGGTTGGGG3') designated as d(T(2)G(8)), from the telomeric region of Tetrahymena Glaucoma, was considered for the interaction with ligands. ESI-MS and spectroscopic methods viz., circular dichroism (CD), UV-Visible, and fluorescence were employed to investigate the G-quadruplex structures formed by d(T(2)G(8)) sequence and its interaction with PBD and TMPyP4 ligands. From ESI-MS spectra, it is evident that the majority of quadruplexes exist as d(T(2)G(8))(2) and d(T(2)G(8))(4) forms possessing two to ten cations in the centre, thereby stabilizing the complex. CD band of PBD1 and PBD2 showed hypo and hyperchromicity, on interaction with quadruplex DNA, indicating unfolding and stabilization of quadruplex DNA complex, respectively. UV-Visible and fluorescence experiments suggest that PBD1 bind externally where as PBD2 intercalate moderately and bind externally to G-quadruplex DNA. Further, melting experiments using SYBR Green indicate that PBD1 unfolds and PBD2 stabilizes the G-quadruplex complex. ITC experiments using d(T(2)G(8)) quadruplex with PBD ligands reveal that PBD1 and PBD2 prefer external/loop binding and external/intercalative binding to quadruplex DNA, respectively. From experimental results it is clear that the interaction of PBD2 and TMPyP4 impart higher stability to the quadruplex complex.     

Molecular basis for the inhibition of HMGA1 proteins by distamycin A

The molecular mechanism for the displacement of HMGA1 proteins from DNA is integral to disrupting their cellular function, which is linked to many metastatic cancers. Chemical shift and NOESY NMR experiments provide structural evidence for the displacement of an AT hook peptide (DNA binding motif of HMGA1 proteins) by both monomeric and dimeric distamycin. However, the displaced AT hook alters distamycin binding by weakening the distamycin:DNA complex, while slowing monomeric distamycin dissociation when AT hook is in excess. The central role of the AT hook was evaluated by monitoring full-length HMGA1a protein binding using fluorescence anisotropy. HMGA1a was effectively displaced by distamycin, but the cooperative binding exhibited by distamycin was eliminated by displaced HMGA1a. Additionally, these studies indicate that HMGA1a is displaced from the DNA by 1 equiv of distamycin, suggesting the ability to develop therapeutics that take advantage of the positively cooperative nature of HMGA1a binding.     

The role of positive charges on G-quadruplex binding small molecules: Learning from bisaryldiketene derivatives

Background

G-quadruplexes are promising therapeutic targets for small molecules. In general, the introduction of steady positive charges through the in situ alkylation of nitrogen atoms within potential G-quadruplex ligands can significantly improve their quadruplex binding and stabilization abilities. However, our previous studies on bisaryldiketene derivatives showed that the derivative M4, whose central piperidone moiety is quaternized, exhibits a poor G-quadruplex stabilization ability.

Methods

To clarify this unusual finding, CD, ITC, UV and NMR analyses were performed to determine the binding behaviors of M4 and its non-quaternized analog M2 to G-quadruplex DNA [d(TGGGT)]₄. Molecular modeling approaches were also employed to help illustrate ligand–quadruplex DNA interactions.

Results

The CD melting and ITC analyses revealed that M2 exhibited much stronger stabilization and binding abilities to [d(TGGGT)]₄ compared to M4. Moreover, the CD and ITC analyses in combination with UV, NMR and MD simulations revealed that M2 tended to be end-stacked on the G-quartet, whereas M4 tended to be bound in the groove region. Analysis of the electrostatic potential showed that the charged surface of M4 was more positive than that of M2 and other reported ligands that bind to the G-quadruplex via end-stacking interactions.

Conclusions

The results indicated that the different positively charged surfaces of M2 and M4 might be the key reason for their different binding modes. These different binding modes also lead to different binding affinities and stabilization abilities for [d(TGGGT)]₄.

General significance

These results provide new clues for the rational design of G-quadruplex-binding small molecules with steady positive charges.     

Binding of distamycin A and netropsin to the 12mer DNA duplexes containing mixed AT.GC sequences with at most five or three successive AT base pairs

Circular dichroism (CD), isothermal calorimetric titrations (ITC), and temperature-dependent UV spectroscopy were used to investigate binding of the minor groove-directed ligands distamycin A (Dst) and netropsin (Net) to the following duplexes: d(GTTAGTATTTGG). d(CCAAATACTAAC), d(GTTAGTATATGG).d(CCATATACTAAC), d(GTTAGTACTTGG). d(CCAAGTACTAAC), and d(GTTAGTAGTTGG).d(CCAACTACTAAC). Our results reveal that Dst binds within the minor grooves of these dodecamers that contain five-AT and/or four-AT.GC binding sites exclusively in a dimeric high-affinity 2:1 binding mode (K approximately 10(16) M(-)(2)). By contrast, Net exhibits high-affinity binding only when it binds in a 1:1 mode (K(1) approximately 10(9) M(-)(1)) to the two duplexes that contain five-AT sites (5'-TATTT-3' and 5'-TATAT-3'). Its further binding to these two duplexes occurs in a low-affinity mode (K(2) approximately 10(6) M(-)(1)) and results in the formation of 2:1 Net-DNA complexes. To the other two duplexes that contain sequences with at most three AT consecutive base pairs Net binds in two distinctive low-affinity 1:1 binding modes (K(1) approximately 10(7) M(-)(1), K(2) approximately 10(6) M(-)(1)). Competition experiments (CD and ITC titrations) reveal that Dst entirely displaces Net from its 1:1 and 2:1 complexes with any of the four duplexes. We discuss and interpret our optical and calorimetric results in the context of the available structural information about the complexes between DNA and the sequence-specific minor groove binders Dst and Net.     

Interactions between distamycin A analogs bound to DNA

The experimental binding isotherms of the distamycin A analog to 8 natural and synthetic DNAs were analyzed. The shapes of binding isotherms suggest that the bound ligand molecule induces transitions of DNA (B-form) into two perturbated conformation states. These transitions are responsible for the existence of positive and negative cooperative effects on binding of distamycin analogs to DNA. At low levels of binding positive cooperative effects play a dominating role whereas at high levels of binding negative cooperative effects are observed. These cooperative effects can be described by the aid of a potential of pairwise interactions between nearest neighbour bound antibiotic molecules. A detailed analysis of experimental binding isotherms shows that characteristic distances over which these interactions are extended depend on the AT content of DNA. The energetical and structural parameters characterising the allosteric transitions of DNA to the perturbated states are obtained.     

Heat capacity changes associated with guanine quadruplex formation: an isothermal titration calorimetry study

This study addresses the temperature dependence of the enthalpy of formation for several unimolecular quadruplexes in the presence of excess monovalent salt. We examined a series of biologically significant guanine-rich DNA sequences: thrombin binding aptamer (TBA) (d(G(2)T(2)G(2)TGTG(2)T(2)G(2)), PS2.M, a catalytically active aptamer (d(GTG(3)TAG(3)CG(3)T(2)G(2))), and the human telomere repeat (HT) (d(AG(3)(T(2)AG(3))(3))). Using CD spectra and UV melting, we confirmed the presence of quadruplex structures and established the temperature range in which quadruplex conformation is stable. We then performed ITC experiments, adding DNA to a solution containing excess NaCl or KCl. In this approach, only several additions are made, and only the enthalpy of quadruplex formation is measured. This measurement was repeated at different temperatures to determine the temperature dependence of the enthalpy change accompanying quadruplex formation. To control for the effect of nonspecific salt interactions during DNA folding, we repeated the experiment by replacing the quadruplex-forming sequences with a similar but nonfolding sequence. Dilution enthalpies were also subtracted to obtain the final enthalpy value involving only the quadruplex folding process. For all sequences studied, quadruplex formation was exothermic but with an increasing magnitude with increasing temperature. These results are discussed in terms of the change in heat capacity associated with quadruplex formation.     

Distamycin A and derivatives as synergic drugs in cisplatin-sensitive and -resistant ovarian cancer cells

Acquired resistance to cisplatin (cDDP) is a multifactorial process that represents one of the main problems in ovarian cancer therapy. Distamycin A is a minor groove DNA binder whose toxicity has limited its use and prompted the synthesis of derivatives such as NAX001 and NAX002, which have a carbamoyl moiety and different numbers of pyrrolamidine groups. Their interaction with a B-DNA model and with an extended-TATA box model, [Polyd(AT)], was investigated using isothermal titration calorimetry (ITC) to better understand their mechanism of interaction with DNA and therefore better explain their cellular effects. Distamycin A interactions with Dickerson and Poly[d(AT)(6)] oligonucleotides show a different thermodynamic with respect to NAX002. The bulkier distamycin A analogue shows a non optimal binding to DNA due to its additional pyrrolamidine group. Cellular assays performed on cDDP-sensitive and -resistant cells showed that these compounds, distamycin A in particular, affect the expression of folate cycle enzymes even at cellular level. The optimal interaction of distamycin A with DNA may account for the down-regulation of both dihydrofolate reductase (DHFR) and thymidylate synthase (TS) and the up-regulation of spermidine/spermine N1-acetyltransferase (SSAT) caused by this compound. These effects seem differently modulated by the cDDP-resistance phenotype. NAX002 which presents a lower affinity to DNA and slightly affected these enzymes, showed a synergic inhibition profile in combination with cDDP. In addition, their combination with cDDP or polyamine analogues increased cell sensitivity to the drugs suggesting that these interactions may have potential for development in the treatment of ovarian carcinoma.     

Determination of the number and location of the manganese binding sites of DNA quadruplexes in solution by EPR and NMR.

The paramagnetic metal ion Mn2+ has been used to probe the electrostatic potentials of a DNA quadruplex that has two quartets with an overall fold of the chair type. A quadruplex with a basket type structure has also been examined. The binding of the paramagnetic ion manganese to these quadruplex DNAs has been investigated by solution state electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopies. The EPR results indicate that the DNA aptamer, d(GGTTGGTGTGGTTGG), binds two manganese ions and that the binding constants for each of these sites is approximately 10(5) M-1. The NMR results indicate that the binding sites of the manganese are in the narrow grooves of this quadruplex DNA. The binding sites of the DNA quadruplex formed by dimers of d(GGGGTTTTGGGG) which forms a basket structure are also in the narrow groove. These results indicate that the close approach of phosphates in the narrow minor grooves of the quadruplex structures provide strong binding sites for the manganese ions and that EPR and NMR monitoring of manganese binding can be used to distinguish between the different types of quadruplex structures.