Threading Intercalators Research Articles

Synthesis and biological evaluation of novel tetranuclear cyclopalladated complex bearing thiosemicarbazone scaffold ligand: Interactions with double‐strand DNA, coronavirus, and molecular modeling studies

AbstractThreading intercalators are a novel class of materials that carry two substituents along the diagonal positions of an aromatic ring. When bound to DNA, these substituents project out in DNA grooves. Tetranuclear complexes appear to be promising threading intercalators for developing therapeutics against cancer and viral infections that require high nucleic acid binding affinity. The objective of this work was to prepare the thiosemicarbazone scaffold ligand [4‐ClC6H4CHN=NC(S)NHPh] and tetranuclear cyclopalladated complex [Pd(4‐ClC6H4CHN=NC(S)NHPh)4] and to characterize the compounds by elemental analysis, 1D and 2D NMR, HRMS, and IR spectroscopy. The calf thymus DNA (CT‐DNA) binding properties of the compounds were investigated in vitro under simulated physiological conditions using UV–vis spectroscopy, emission spectral titration, methylene blue competitive binding, circular dichroism, DNA thermal denaturation, DNA binding, and coronavirus interactions using molecular simulation. The compounds showed cytotoxic effect against both human breast (MCF‐7) and colorectal (HCT116) cancer cells in a dose‐dependent manner. We demonstrated that the compounds are promising for DNA threading intercalation binders with large DNA binding constants on the order of 107 M−1 magnitude.

A Minimal Load-and-Lock RuII Luminescent DNA Probe.

Threading intercalators bind DNA with high affinities. Here, we describe single‐molecule studies on a cell‐permeant luminescent dinuclear ruthenium(II) complex that has been previously shown to thread only into short, unstable duplex structures. Using optical tweezers and confocal microscopy, we show that this complex threads and locks into force‐extended duplex DNA in a two‐step mechanism. Detailed kinetic studies reveal that an individual stereoisomer of the complex exhibits the highest binding affinity reported for such a mono‐intercalator. This stereoisomer better preserves the biophysical properties of DNA than the widely used SYTOX Orange. Interestingly, threading into torsionally constrained DNA decreases dramatically, but is rescued on negatively supercoiled DNA. Given the “light‐switch” properties of this complex on binding DNA, it can be readily used as a long‐lived luminescent label for duplex or negatively supercoiled DNA through a unique “load‐and‐lock” protocol.

Effect of Chirality on the Elastic Properties of the DNA-Threading Binuclear Ruthenium Complex

Transition metal-based small molecules have been promising candidates for cancer treatments. A certain type of these molecules falls into a category known as threading intercalators, that have a dumbbell shape with a flat intercalating section in between bulky side chains. In order to bind to DNA, they must thread one of their bulky side chains through the DNA base pairs. The ruthenium-based molecule, ΛΛ-[μ-bidppz(phen)4Ru2]4+ (ΛΛ-P for short), is a transition metal-based threading intercalator. We use optical tweezers to study the interactions of ΛΛ-P with DNA to compare it with the previously studied ΔΔ-P, a complex that has the same chemical components but an opposite chirality. In these studies, we use the optical tweezers to trap a single DNA molecule and stretch it in the presence of various concentrations of ΛΛ-P. The DNA stretches obtained at saturated concentrations of ΛΛ-P at various forces allows us to obtain the effective elastic properties of the DNA-ΛΛ-P complex. This allows us to compare these properties to the previously studied ΔΔ-P complex to determine whether chirality has an effect. This type of comparison may lead us towards a better understanding of the role chirality has towards DNA binding.

Investigating How Chirality of a Threading Binuclear Ruthenium Complex Affects the DNA Threading Intercalation using Optical Tweezers

Using optical tweezers, we have been able to study the interactions of small molecules and prospective cancer drugs with DNA. One type of these molecules, known as threading intercalators, has a flat planar moiety in between the molecule's bulky side chains. In order to bind with DNA they have to thread their bulky side chains in between the base pairs. Due to this requirement for binding, these molecules tend to have high binding affinities and slow kinetics. We have been investigating the binding properties of the ruthenium-based threading intercalator ∧∧-[μ-bidppz(phen)4Ru2]4+, or ∧∧-P for short. This complex has the exact chemical components but an opposite chirality to the previously studied ΔΔ-P complex. Preliminary data show that the change in chirality affects the DNA binding kinetics, the association and dissociation rates, to at least a threefold in favor of the left-handed ∧∧-P complex. The data also confirm that both the left-handed and right-handed molecule have a similar binding affinity as a result of their common intercalating moiety. This comparison will give us a better understanding of how chirality affects the binding to DNA and will contribute towards improved designs of potential cancer treatment drugs.

Reshaping the Energy Landscape Transforms the Mechanism and Binding Kinetics of DNA Threading Intercalation.

Molecules that bind DNA via threading intercalation show high binding affinity as well as slow dissociation kinetics, properties ideal for the development of anticancer drugs. To this end, it is critical to identify the specific molecular characteristics of threading intercalators that result in optimal DNA interactions. Using single-molecule techniques, we quantify the binding of a small metal-organic ruthenium threading intercalator (Δ,Δ-B) and compare its binding characteristics to a similar molecule with significantly larger threading moieties (Δ,Δ-P). The binding affinities of the two molecules are the same, while comparison of the binding kinetics reveals significantly faster kinetics for Δ,Δ-B. However, the kinetics is still much slower than that observed for conventional intercalators. Comparison of the two threading intercalators shows that the binding affinity is modulated independently by the intercalating section and the binding kinetics is modulated by the threading moiety. In order to thread DNA, Δ,Δ-P requires a "lock mechanism", in which a large length increase of the DNA duplex is required for both association and dissociation. In contrast, measurements of the force-dependent binding kinetics show that Δ,Δ-B requires a large DNA length increase for association but no length increase for dissociation from DNA. This contrasts strongly with conventional intercalators, for which almost no DNA length change is required for association but a large DNA length change must occur for dissociation. This result illustrates the fundamentally different mechanism of threading intercalation compared with conventional intercalation and will pave the way for the rational design of therapeutic drugs based on DNA threading intercalation.

Discriminating Intercalative Effects of Threading Intercalator Nogalamycin, from Classical Intercalator Daunomycin, Using Single Molecule Atomic Force Spectroscopy.

DNA threading intercalators are a unique class of intercalating agents, albeit little biophysical information is available on their intercalative actions. Herein, the intercalative effects of nogalamycin, which is a naturally-occurring DNA threading intercalator, have been investigated by high-resolution atomic force microscopy (AFM) and spectroscopy (AFS). The results have been compared with those of the well-known chemotherapeutic drug daunomycin, which is a non-threading classical intercalator bearing structural similarity to nogalamycin. A comparative AFM assessment revealed a greater increase in DNA contour length over the entire incubation period of 48 h for nogalamycin treatment, whereas the contour length increase manifested faster in case of daunomycin. The elastic response of single DNA molecules to an externally applied force was investigated by the single molecule AFS approach. Characteristic mechanical fingerprints in the overstretching behaviour clearly distinguished the nogalamycin/daunomycin-treated dsDNA from untreated dsDNA—the former appearing less elastic than the latter, and the nogalamycin-treated DNA distinguished from the daunomycin-treated DNA—the classically intercalated dsDNA appearing the least elastic. A single molecule AFS-based discrimination of threading intercalation from the classical type is being reported for the first time.

Dissecting the Dynamic Pathways of Stereoselective DNA Threading Intercalation

DNA intercalators that have high affinity and slow kinetics are developed for potential DNA-targeted therapeutics. Although many natural intercalators contain multiple chiral subunits, only intercalators with a single chiral unit have been quantitatively probed. Dumbbell-shaped DNA threading intercalators represent the next order of structural complexity relative to simple intercalators, and can provide significant insights into the stereoselectivity of DNA-ligand intercalation. We investigated DNA threading intercalation by binuclear ruthenium complex [μ-dppzip(phen)4Ru2]4+ (Piz). Four Piz stereoisomers are defined by the chirality of the intercalating subunit (Ru(phen)2dppz) and the distal subunit (Ru(phen)2ip), respectively, each of which can be either right-handed (Δ) or left-handed (Λ). We used optical tweezers to measure single DNA molecule elongation due to threading intercalation, revealing force-dependent DNA intercalation rates and equilibrium dissociation constants. The force spectroscopy analysis provided the zero-force DNA binding affinity, the equilibrium DNA-ligand elongation Δxeq, and the dynamic DNA structural deformations during ligand association xon and dissociation xoff. We found that Piz stereoisomers exhibit over 20-fold differences in DNA binding affinity, from a Kd of 27 ± 3 nM for (Δ,Λ)-Piz to a Kd of 622 ± 55 nM for (Λ,Δ)-Piz. The striking affinity decrease is correlated with increasing Δxeq from 0.30 ± 0.02 to 0.48 ± 0.02 nm and xon from 0.25 ± 0.01 to 0.46 ± 0.02 nm, but limited xoff changes. Notably, the affinity and threading kinetics is 10-fold enhanced for right-handed intercalating subunits, and 2- to 5-fold enhanced for left-handed distal subunits. These findings demonstrate sterically dispersed transition pathways and robust DNA structural recognition of chiral intercalators, which are critical for optimizing DNA binding affinity and kinetics.

Role of the Moiety Chirality in Determining the DNA Binding Characteristics of Threading Intercalators

Threading intercalators are small molecules that bind to DNA by threading their ancillary groups through the DNA bases to intercalate their middle planar section between the DNA base pairs. The high binding affinity and slow dissociation rates of threading intercalators have put them in the class of prospective anti-cancer drugs. In this study we explore the binding of a specific threading intercalator, the binuclear ruthenium complex ΛΛ-P (ΛΛ-[µ-bidppz(phen)4Ru2]4+) using optical tweezers. A single DNA molecule is held at a constant force and ΛΛ-P is introduced to the system in varying concentrations until equilibrium is achieved. Measurements of DNA extension at various concentrations of ΛΛ-P as a function of time provide the DNA equilibrium binding affinity and binding kinetics for this molecule. Preliminary data analysis at constant force suggests that ΛΛ-P exhibits significantly faster binding kinetics compared to the very similar ΔΔ-P [μ-bidppz(phen)4Ru2]4+. These complexes have the same chemical structure and only differ in their chirality, which suggests that the left handed (ΛΛ) threading moieties require less DNA structural distortion for threading compared with the right handed (ΔΔ) threading moieties.

Resolving the DNA Binding Mode of a Rotationally Flexible Binuclear Ruthenium Complex

Binuclear ruthenium complexes demonstrate slow dissociation upon binding to DNA, and they are considered as potential DNA-targeted therapeutic drugs. Quantitative investigations of their properties, such as binding affinity, binding mode and kinetics, require approaches beyond traditional experimental techniques. In particular, previous reports showed that the rotationally flexible binuclear ruthenium complex Δ,Δ-[µ-bipb(phen)4Ru2]4+ (Δ,Δ-Pi) strongly condenses DNA. However, the strong DNA condensation poses a challenge in bulk experiments such that even the DNA-ligand binding mode could not be resolved. We examined the kinetics of ΔΔ-Pi interactions with single λ-DNA molecule as a function of a constant applied force of 30 pN and ligand concentration of 5 nM using dual-beams optical tweezers. We find that Δ,Δ-Pi exhibits characteristics of threading intercalation into DNA base pairs. DNA elongation measurements, as monitored over tens of minutes, illustrate two distinct phases during association; rapid intercalation that is analogous to classic intercalation, followed by very slow intercalation that approaches equilibrium with a rate that is comparable to that observed for other threading intercalators. We investigated ΔΔ-Pi dissociation after reaching the equilibrium extension by rinsing the binding ligands from the surrounding solution, and observed that the DNA-ligand complex extension decreases to the DNA-only extension over a timescale longer than the association process. Interestingly, the dissociation measurements fit well to a single rate that is slower than the dissociation rate measured for the previously reported binuclear ruthenium complex Δ,Δ-[µ-bidppz-(phen)4Ru2]4+ (Δ,Δ-P), which is a threading intercalator. Further measurements of force-dependent intercalation thermodynamics and kinetics will allow us to fully quantitatively characterize the complex binding mechanism of this unusual intercalator.

Single Molecule DNA Stretching Studies of DNA Intercalation

Many proteins and ligands bind double stranded (ds) DNA by inserting their hydrophobic residues in between the DNA bases, either partially or completely, thereby locally elongating, bending and unwinding the dsDNA. This intercalative binding mode is typical of many proteins that control transcription by locally deforming the dsDNA, such as HMG-type proteins or TATA box binding proteins. Also intercalative binding is typical of many small aromatic molecules that are used either as the research tool for dsDNA imaging (EtBr or Yo-Yo), or as anticancer drugs (ActinomysinD or Ruthenium threading intercalators). Conventional approaches to the study of intercalative binding are often limited as many of these molecules also have the non-intercalative binding modes, may cause DNA aggregation, intercalate weakly, or too slowly. Recently the single molecule stretching of polymeric dsDNA with Optical Tweezers in the presence of several intercalating molecules was employed to characterize their equilibrium dissociation constant, DNA elongation and the binding site size. In this theoretical work we discuss how the complete stretching curves of the DNA-intercalator complexes can be used to study the proteins with weak intercalative ability and slow binding kinetics, the effect of intercalators on the dsDNA duplex stability, flexibility and elasticity. In some cases, even the protein-induced dsDNA bending angle and elongation can be quantified based on the intercalator - dsDNA titrations coupled to DNA stretching. This approach also offers a new way to study the contributions of individual intercalating and non-intercalating groups of the protein to its DNA intercalative ability. For some proteins, such as nucleocapsid proteins of retroviruses, discovery of their intercalative nature by DNA stretching may suggest their novel physiological roles.

Bridging Ligand Length Controls AT Selectivity and Enantioselectivity of Binuclear Ruthenium Threading Intercalators

The slow dissociation of DNA threading intercalators makes them interesting as model compounds in the search for new DNA targeting drugs, as there appears to be a correlation between slow dissociation and biological activity. Thus, it would be of great value to understand the mechanisms controlling threading intercalation, and for this purpose we have investigated how the length of the bridging ligand of binuclear ruthenium threading intercalators affects their DNA binding properties. We have synthesised a new binuclear ruthenium threading intercalator with slower dissociation kinetics from ct-DNA than has ever been observed for any ruthenium complex with any type of DNA, a property that we attribute to the increased distance between the ruthenium centres of the new complex. By comparison with previously studied ruthenium complexes, we further conclude that elongation of the bridging ligand reduces the sensitivity of the threading interaction to DNA flexibility, resulting in a decreased AT selectivity for the new complex. We also find that the length of the bridging ligand affects the enantioselectivity with increasing preference for the ΔΔ enantiomer as the bridging ligand becomes longer.

Design and synthesis of threading intercalators to target DNA

Threading intercalators are high affinity DNA binding agents that bind by inserting a chromophore into the duplex and locating one group in each groove. The first threading intercalators that can be conjugated to acids, sulfonic acids and peptides to target them to duplex DNA are described, based upon the well studied acridine-3- or 4-carboxamides. Cellular uptake of the parent acridine is rapid and it can be visualized in the nucleus of cells. Both the parent compounds and their conjugates maintain antitumor activity.

Synthesis of 4,8‐Substitiutednaphtho[2,1‐b]thiophene Threading Intercalators

Abstractmagnified imageEight 4,8‐substitiutednaphtho[2,1‐b]thiophenes with cationic containing side chains were synthesized as potential threading intercalators and their DNA binding affinity was evaluated. The key step in the synthesis of these systems was oxidative‐photocyclizatJon of α‐(2‐thienyl)‐β‐(4‐methoxycarbonylphenyl) acrylic acid. Various N ,N‐dimethylaminoalkylammes were coupled With naphtho[2,1‐b]thiophene 4,8‐dicarboxylic acid to yield the title compounds.

Binding of a homologous series of anthraquinones to DNA

The DNA binding characteristics of a series of homologous 2,6-disubstituted anthraquinone threading intercalators bearing one to four ethylene glycol units in their side chains have been studied. Binding constants were measured via surface plasmon resonance (SPR). These compounds bind to an AT-rich hairpin with slightly higher affinity than to a GC-rich hairpin. The binding constants decrease as the length of the side chain increases.

Solid-phase synthesis of acridine-based threading intercalator peptides

The preparation of a novel acridine-based amino acid is reported. This N-Alloc-protected monomer can be coupled and deprotected under solid-phase peptide synthesis procedures to create acridine–peptide conjugates as potential threading intercalators. A peptide containing this novel amino acid undergoes spectral changes in the presence of duplex DNA and RNA consistent with intercalative binding.

Threading Intercalators as a New DNA Structural Probe

Abstract Nogalamycin is a threading intercalator and when it binds with double stranded DNA, one of the two sugar substituents goes between adjacent base pairs of DNA to form a stable complex in which the two substituents of nogalamycin are projecting out in each groove of DNA. Since the molecular volume of the substituents of nogalamycin is larger than that of the space created through the motion of DNA strands constituting the double helix, a cleavage of hydrogen bonds in the base pairs of DNA duplex is necessary to form such a complex. The structure of double-stranded DNA is dynamic, and the base pair cleavage (base pair opening) and re-formation (closing) occur frequently at ambient temperature. A wider variety of threading intercalators is obtainable synthetically, and they include several families such as 1,5-disubstituted anthraquinones, 9,10-disubstituted anthracenes, and naphthalene bis(dicarboximide) derivatives. Kinetic study of the complex formation and dissociation of naphthalene bis(dicarboximide) carrying substituents of different size have made one recognize the dynamic character of duplex DNA in intercalation reaction. A cyclic bis-intercalator was also able to form a threading complex of a catenane-like structure. A thorough study of the binding behavior of natural and synthetic threading intercalators with DNA revealed that a proper design of substituents on threading intercalators can produce ligands which specifically bind either to single- or double-stranded DNA. These ligands, synthetic ones in particular, will hopefully serve not only as new DNA structural probes but also as chemotherapeutic agents.

POLY-INTERCALATORS CARRYING THREADING INTERCALATOR MOIETIES AS NOVEL DNA TARGETING LIGANDS

Threading intercalators are a novel class of intercalators that carry two substituents along the diagonal positions of an aromatic ring. These substituents are projecting out in DNA grooves when bound to DNA. Poly-intercalators carrying threading intercalating parts are quite novel and were recently found to show a unique DNA binding behavior. We review herein two types of poly-intercalators. First, tris-intercalators carrying a threading intercalator part in the middle of the molecule are described. These intercalators appear to intercalate into double stranded DNA in a special binding manner, which we call the penetrating mode, in which all the three intercalating units are arranged linearly with one of them penetrating into the DNA ladder. We synthesized two tris-intercalators ( 3 and 4) of this type and studied their binding behavior for double stranded DNA. All the experimental results were consistent with the proposed penetrating mode. Another type of threading poly-intercalators is a macrocyclic bis-threading intercalator ( 5). We found that this compound can bis-intercalate to double stranded DNA when the base pairing is disrupted temporarily to form a complex with a unique structure like a catenane. On the basis of a study of the interaction of such intercalators we envisage that DNA is a flexible and dynamic entity. These novel families of poly-intercalators will expand the scope of DNA poly-intercalation chemistry with possible medicinal applications.

DNA threading agents: effect of sidechain bulk on DNA binding and cytotoxicity of 9-anilinoacridine-4-carboxamides

A series of 9-anilinoacridine-4-carboxamides with cycloalkyl sidechains has been synthesised to study the effect of sidechain bulk on the DNA binding properties and biological activity of these potential threading intercalators. With sidechains larger than cyclohexane the pKa of the acridine is exceptionally low, so that DNA binding is restricted to pHs below 5. The compounds are cytotoxic to human colon carcinoma cells in the μM range irrespective of their ability to bind to DNA.

Kinetic and equilibrium analysis of a threading intercalation mode: DNA sequence and ion effects

The interaction of a symmetric naphthalene diimide with alkylamino substituents at each imide position was investigated with the alternating sequence polymers, poly[d(A-T)]2 and poly[d(G-C)]2. Spectrophotometric binding studies indicate strong binding of the diimide to both sequences although the GC binding constant is 20-25 times larger than the AT binding constant. Analysis of the effects of salt concentration on the binding equilibria shows that the diimide forms two ion pairs in its complex with both polymers as expected for a simple dication. Stopped-flow kinetics experiments demonstrate that the diimide both associates and dissociates from DNA more slowly than classical intercalators with similar binding constants. Analysis of salt concentration effects on dissociation kinetics rate constants (kd) reveals that slopes in log kd versus log [Na+] plots are only approximately half the value obtained for classical dicationic intercalators that have both charged groups in the same groove. These kinetics results support a threading intercalation model, with one charged diimide substituent in each of the DNA grooves rather than with both side chains in the same groove, for the diimide complex with DNA. In the rate-determining step of the mechanism for dissociation of a threading complex only one ion pair is broken; the free side chain can then slide between base pairs to put both diimide side chains in the same groove, and this is followed by rapid full dissociation of the diimide. This sequential release of ion pairs makes the dissociation slope for dicationic threading intercalators more similar to the slope for classical monocationic intercalating ligands.(ABSTRACT TRUNCATED AT 250 WORDS)