CD Titration Research Articles

Exploring the Interaction of G-quadruplex Binders with a (3 + 1) Hybrid G-quadruplex Forming Sequence within the PARP1 Gene Promoter Region.

The enzyme PARP1 is an attractive target for cancer therapy, as it is involved in DNA repair processes. Several PARP1 inhibitors have been approved for clinical treatments. However, the rapid outbreak of resistance is seriously threatening the efficacy of these compounds, and alternative strategies are required to selectively regulate PARP1 activity. A noncanonical G-quadruplex-forming sequence within the PARP1 promoter was recently identified. In this study, we explore the interaction of known G-quadruplex binders with the G-quadruplex structure found in the PARP gene promoter region. The results obtained by NMR, CD, and fluorescence titration, also confirmed by molecular modeling studies, demonstrate a variety of different binding modes with small stabilization of the G-quadruplex sequence located at the PARP1 promoter. Surprisingly, only pyridostatin produces a strong stabilization of the G-quadruplex-forming sequence. This evidence makes the identification of a proper (3+1) stabilizing ligand a challenging goal for further investigation.

DNA and BSA Interaction Studies and Antileukemic Evaluation of Polyaromatic Thiosemicarbazones and Their Copper Complexes

Some ten million cancer deaths occurred in 2020, highlighting the fact that the search for new anticancer drugs remains extremely topical. In the search for new coordination compounds with relevant biological properties, the choice of a metal ion is important for the design of the complex. In this regard, copper plays a peculiar role, thanks to its distinct properties. Thiosemicarbazones are, analogously, a unique class of ligands because they are easily modifiable, and therefore, extremely versatile in terms of modulating molecular properties. In this work, we synthesized and characterized, by means of X-ray diffraction, four new naphthaldehyde and anthraldehyde thiosemicarbazone derivatives and their copper complexes to be used in interaction studies with biological systems. The objective was to evaluate the antileukemic activity of these compounds. Reactions of these ligands with Cu(II) salts produced unexpected oxidation products and the isolation of Cu(I) metal complexes. One ligand and its related Cu(I) complex, which is stable in physiological conditions, were subjected to in vitro biological tests (UV-Vis and CD titration). An important interaction with DNA and an affinity toward BSA were observed in FT-IR experiments. Preliminary in vitro biological tests against a histiocytic lymphoma cell line revealed an interestingly low IC50 value, i.e., 5.46 µM, for the Cu(I) complex.

Amidine- and Amidoxime-Substituted Heterocycles: Synthesis, Antiproliferative Evaluations and DNA Binding.

The novel 1,2,3-triazolyl-appended N- and O-heterocycles containing amidine 4–11 and amidoxime 12–22 moiety were prepared and evaluated for their antiproliferative activities in vitro. Among the series of amidine-substituted heterocycles, aromatic diamidine 5 and coumarine amidine 11 had the most potent growth-inhibitory effect on cervical carcinoma (HeLa), hepatocellular carcinoma (HepG2) and colorectal adenocarcinoma (SW620), with IC50 values in the nM range. Although compound 5 was toxic to non-tumor HFF cells, compound 11 showed certain selectivity. From the amidoxime series, quinoline amidoximes 18 and 20 showed antiproliferative effects on lung adenocarcinoma (A549), HeLa and SW620 cells emphasizing compound 20 that exhibited no cytostatic effect on normal HFF fibroblasts. Results of CD titrations and thermal melting experiments indicated that compounds 5 and 10 most likely bind inside the minor groove of AT-DNA and intercalate into AU-RNA. Compounds 6, 9 and 11 bind to AT-DNA with mixed binding mode, most probably minor groove binding accompanied with aggregate binding along the DNA backbone.

Novel insights on nucleopeptide binding: A spectroscopic and in silico investigation on the interaction of a thymine-bearing tetrapeptide with a homoadenine DNA

Nucleopeptides are a class of molecules with numerous applications in the field of therapy, diagnostics and biomaterials development. Despite their nucleobase-decorated nature, their binding to natural nucleic acid targets does not necessarily involve all nucleopeptide bases, as we showed in this study. Here, we present a CD study on the interaction of a dithymine-functionalized tetra-L-serine with a homoadenine DNA (dA12) reporting an interpretation of the experimental data in light of our computational studies based on molecular docking and molecular dynamics (MD), as well as computer-assisted CD interpretation and simulation of the predicted complex structure. The stoichiometry of the complex, emerged by CD titration, accounted for a 1:2 T:A ratio. Hence, we supposed that binding did not involve a full pairing of the complementary bases but a partial thymines engagement. This hypothesis was sustained by the docking and MD simulations performed on the selected ligand and the complementary target of DNA and RNA, used for comparison. The nucleopeptide bound the DNA through a single A-T recognition involving complementary base-pairing, as well as by some interactions between its backbone (and in particular L-serine OH) and the nucleic acid. Overall, this confirmed that nucleopeptides can interact with nucleic acids leaving some of their nucleobases free for establishing further interactions with other biomolecules or for crosslinking in supramolecular structures in aqueous solution. Nevertheless, even though no typical DNA secondary structure is formed after nucleopeptide-binding, this ligand is able to induce a higher degree of structuration in the random deoxyoligonucleotide target as evidenced by CD, MD and CD simulation.

Investigation of the Complexes Formed between PARP1 Inhibitors and PARP1 G-Quadruplex at the Gene Promoter Region.

DNA repair inhibitors are one of the latest additions to cancer chemotherapy. In general, chemotherapy produces DNA damage but tumoral cells may become resistant if enzymes involved in DNA repair are overexpressed and are able to reverse DNA damage. One of the most successful drugs based on modulating DNA repair are the poly(ADP-ribose) polymerase 1 (PARP1) inhibitors. Several PARP1 inhibitors have been recently developed and approved for clinical treatments. We envisaged that PARP inhibition could be potentiated by simultaneously modulating the expression of PARP 1 and the enzyme activity, by a two-pronged strategy. A noncanonical G-quadruplex-forming sequence within the PARP1 promoter has been recently identified. In this study, we explored the potential binding of clinically approved PARP1 inhibitors to the G-quadruplex structure found at the gene promoter region. The results obtained by NMR, CD, and fluorescence titration confirmed by molecular modeling demonstrated that two out the four PARP1 inhibitors studied are capable of forming defined complexes with the PARP1 G-quadruplex. These results open the possibility of exploring the development of better G-quadruplex binders that, in turn, may also inhibit the enzyme.

Polycationic Monomeric and Homodimeric Asymmetric Monomethine Cyanine Dyes with Hydroxypropyl Functionality-Strong Affinity Nucleic Acids Binders.

New analogs of the commercial asymmetric monomethine cyanine dyes thiazole orange (TO) and thiazole orange homodimer (TOTO) with hydroxypropyl functionality were synthesized and their properties in the presence of different nucleic acids were studied. The novel compounds showed strong, micromolar and submicromolar affinities to all examined DNA ds-polynucleotides and poly rA–poly rU. The compounds studied showed selectivity towards GC-DNA base pairs over AT-DNA, which included both binding affinity and a strong fluorescence response. CD titrations showed aggregation along the polynucleotide with well-defined supramolecular chirality. The single dipyridinium-bridged dimer showed intercalation at low dye-DNA/RNA ratios. All new cyanine dyes showed potent micromolar antiproliferative activity against cancer cell lines, making them promising theranostic agents.

Imide Condensation as a Strategy for the Synthesis of Core-Diversified G-Quadruplex Ligands with Anticancer and Antiparasitic Activity*.

A facile imide coupling strategy for the one‐step preparation of G‐quadruplex ligands with varied core chemistries is described. The G‐quadruplex stabilization of a library of nine compounds was examined using FRET melting experiments, and CD, UV‐Vis, fluorescence and NMR titrations, identifying several compounds that were capable of stabilizing G‐quadruplex DNA with interesting selectivity profiles. The best G4 ligand was identified as compound 3, which was based on a perylene scaffold and exhibited 40‐fold selectivity for a telomeric G‐quadruplex over duplex DNA. Surprisingly, a tetra‐substituted flexible core, compound 11, also exhibited selective stabilization of G4 DNA over duplex DNA. The anticancer and antiparasitic activity of the library was also examined, with the lead compound 3 exhibiting nanomolar inhibition of Trypanosoma brucei with 78‐fold selectivity over MRC5 cells. The cellular localization of this compound was also studied via fluorescence microscopy. We found that uptake was time dependant, with localization outside the nucleus and kinetoplast that could be due to strong fluorescence quenching in the presence of small amounts of DNA.

Complexation of Chiral Zinc(II)Porphyrin Tweezer with Chiral Guests: Control, Discrimination and Rationalization of Supramolecular Chirality.

A series of 1:1 sandwich complexes consisting of chiral zinc(II) bisporphyrin hosts and a series of chiral guests has been synthesized and has rationalized the underpinning mechanism of chirality transfer in the host-guest supramolecular assemblies. The number of stereogenic centers is also varied in both the host and guests, which provides insight into the overall helicity of the assembly. The interactions between the chiral host and chiral guests have been investigated by UV-visible, CD, and 1H NMR spectroscopic titrations along with extensive DFT studies. Interestingly, CD spectral changes are very different between chiral guests with one and two chiral centers. It has been observed that the sign of the CD couplet of the host-guest complexes is dictated by the chirality of the host only with guests having one chiral center. The match and mismatch of the chirality of the guest only affects the amplitude of the CD signal; the sign, however, remains intact. In sharp contrast, the helicity of the 1:1 sandwich complex is dictated by the chirality of the guests having two chiral centers. However, amplification of the CD couplet is observed upon matching of the chirality between the host and guest, while a mismatch leads to an inversion of the CD couplet. The enantiomeric host also displays similar trends with chiral guests but with opposite sign. The enantioselective host also displays excellent chiral discrimination ability toward enantiomeric guests with two chiral centers. The guest with the same chirality as the host binds much stronger as compared to its enantiomer. Remarkable enantiodiscrimination effects were also detected in the 1H NMR spectra of the diastereomeric complexes in which well-resolved separation of the signals is clearly visible. The theoretical calculations are consistent with those of the experiment.

Cadmium and proton adsorption onto a halophilic archaeal species: The role of cell envelope sulfhydryl sites

The adsorption of metal onto archaea could affect metal behavior and bioavailability in hypersaline environments where archaea represent the dominant life form. However, the mechanism and thermodynamics of metal adsorption onto halophilic archaea are not well understood. In this study, Cd adsorption experiments and potentiometric titrations were conducted to model metal adsorption by the halophilic archaeon Halobacterium sp.. Cd adsorption onto the archaeal cells exhibits slow adsorption kinetics, taking 8 h to reach steady-state. The extent of Cd adsorption depends strongly on pCH+ and Cd loading. Desorption of Cd is even slower than adsorption, but exposure of the Cd-adsorbed biomass to high concentrations of cysteine promotes desorption and demonstrates that the distribution of Cd is controlled by reversible adsorption reactions, likely involving sulfhydryl binding of Cd on the cell surface. We conducted potentiometric titration experiments with and without sulfhydryl site blocking to yield a total sulfhydryl site concentration of 91 ± 28 μmol/g within the archaeal cell envelope. Using a combination of the X-ray absorption fine structure analysis results from Showalter et al. (2016) and the Cd adsorption measurements and titration data from this study, we construct a surface complexation model of the Cd adsorption behavior. Because the Cd adsorption behavior is likely similar to that of a range of chalcophilic micronutrients, the importance of Cd-sulfhydryl binding suggests that the production of high affinity sulfhydryl binding sites by the archaea may represent an adaptive strategy for halophiles to obtain metal nutrients in environments in which aqueous metal-chloride complexation dominates the speciation for many metals.

PNA as Hybrid Catalyst Scaffold Catalyzed Asymmetric Friedel–Crafts Alkylation

Double-stranded DNA can form efficient hybrid catalysts for asymmetric catalysis upon binding of small metal complexes, which has attracted significant interest. Catalytic microenvironment and chiral transfer are still a matter of concern. Here, we introduced peptide nucleic acid (PNA) chains to design oligo-dsPNA and hybrid PNA/DNA as chiral scaffolds to catalyze Friedel–Crafts reaction. Though with the same sequence as DNA control, dsPNA and hybrid duplexes are less suited for use in asymmetric catalysis due to ligand-Cu2+ complex does not form highly active and enantioselective hybrid catalysts with PNA-containing scaffolds. CD and UV titration assays indicated that the complex bound to suitable groove and the resulting preferential conformation are the key to catalytic performance, rather than just binding affinity. The negative charge of backbone and the resulting groove size of DNA are the key to act as a more active and highly selective catalyst than PNA.

Photocontrolled DNA minor groove interactions of imidazole/pyrrole polyamides.

Azobenzenes are photoswitchable molecules capable of generating significant structural changes upon E-to-Z photoisomerization in peptides or small molecules, thereby controlling geometry and functionality. E-to-Z photoisomerization usually is achieved upon irradiation at 350 nm (π–π* transition), while the Z-to-E isomerization proceeds photochemically upon irradiation at >400 nm (n–π* transition) or thermally. Photoswitchable compounds have frequently been employed as modules, e.g., to control protein–DNA interactions. However, their use in conjunction with minor groove-binding imidazole/pyrrole (Im/Py) polyamides is yet unprecedented. Dervan-type Im/Py polyamides were equipped with an azobenzene unit, i.e., 3-(3-(aminomethyl)phenyl)azophenylacetic acid, as the linker between two Im/Py polyamide strands. Only the (Z)-azobenzene-containing polyamides bound to the minor groove of double-stranded DNA hairpins. Photoisomerization was exemplarily evaluated by 1H NMR experiments, while minor groove binding of the (Z)-azobenzene derivatives was proven by CD titration experiments. The resulting induced circular dichroism (ICD) bands of the bound ligands, together with the photometric determination of the dsDNA melting temperature, revealed a significant stabilization of the DNA upon association with the ligand. The (Z)-azobenzene acted as a building block inducing a reverse turn, which favored hydrogen bonds between the pyrrole/imidazole amide and the DNA bases. In contrast, the E-configured polyamides did not induce any ICD characteristic for minor groove binding. The incorporation of the photoswitchable azobenzene unit is a promising strategy to obtain photoswitchable Im/Py hairpin polyamides capable of interacting with the dsDNA minor groove only in the Z-configuration.

PPEF: A bisbenzimdazole potent antimicrobial agent interacts at acidic triad of catalytic domain of E. coli topoisomerase IA

BackgroundTopoisomerase is a well known target to develop effective antibacterial agents. In pursuance of searching novel antibacterial agents, we have established a novel bisbenzimidazole (PPEF) as potent E. coli topoisomerase IA poison inhibitor. MethodsIn order to gain insights into the mechanism of action of PPEF and understanding protein-ligand interactions, we have produced wild type EcTopo 67 N-terminal domain (catalytic domain) and its six mutant proteins at acidic triad (D111, D113, E115). The DDE motif is replaced by alanine (A) to create three single mutants: D111A, D113A, E115A and three double mutants: D111A-D113A, D113A-E115A and D111A-E115A. ResultsCalorimetric study of PPEF with single mutants showed 10 fold lower affinity than that of wild type EcTopo 67 (7.32 × 106 M−1for wild type, 0.89 × 106 M−1for D111A) and 100 fold lower binding with double mutant D113A-E115A (0.02 × 106 M−1) was observed. The mutated proteins showed different CD signature as compared to wild type protein. CD and fluorescence titrations were done to study the interaction between EcTopo 67 and ligands. Molecular docking study validated that PPEF has decreased binding affinity towards mutated enzymes as compared to wild type. ConclusionThe overall study reveals that PPEF binds to D113 and E115 of acidic triad of EcTopo 67. Point mutations decrease binding affinity of PPEF towards DDE motif of topoisomerase. General significanceThis study concludes PPEF as poison inhibitor of E. coli Topoisomerase IA, which binds to acidic triad of topoisomerase IA, responsible for its function. PPEF can be considered as therapeutic agent against bacteria.

Oxadiazole/Pyridine-Based Ligands: A Structural Tuning for Enhancing G-Quadruplex Binding

Non-macrocyclic heteroaryls represent a valuable class of ligands for nucleic acid recognition. In this regard, non-macrocyclic pyridyl polyoxazoles and polyoxadiazoles were recently identified as selective G-quadruplex stabilizing compounds with high cytotoxicity and promising anticancer activity. Herein, we describe the synthesis of a new family of heteroaryls containing oxadiazole and pyridine moieties targeting DNA G-quadruplexes. To perform a structure–activity analysis identifying determinants of activity and selectivity, we followed a convergent synthetic pathway to modulate the nature and number of the heterocycles (1,3-oxazole vs. 1,2,4-oxadiazole and pyridine vs. benzene). Each ligand was evaluated towards secondary nucleic acid structures, which have been chosen as a prototype to mimic cancer-associated G-quadruplex structures (e.g., the human telomeric sequence, c-myc and c-kit promoters). Interestingly, heptapyridyl-oxadiazole compounds showed preferential binding towards the telomeric sequence (22AG) in competitive conditions vs. duplex DNA. In addition, G4-FID assays suggest a different binding mode from the classical stacking on the external G-quartet. Additionally, CD titrations in the presence of the two most promising compounds for affinity, TOxAzaPy and TOxAzaPhen, display a structural transition of 22AG in K-rich buffer. This investigation suggests that the pyridyl-oxadiazole motif is a promising recognition element for G-quadruplexes, combining seven heteroaryls in a single binding unit.

Supramolecular recognition of A-tracts DNA by calix[4]carbazole

Naturally occurring biomolecules such as transcription factors recognize specific sequences of DNA by binding to its major groove whereas the synthetic sequence-specific major-groove binders are rare. We herein report that a synthetic calix[4]carbazole could serve as the optical ligand of A-tracts (B*-form DNA) by binding to its major-groove, elucidated by UV-vis, fluorescence, CD and NMR titrations.

A Bis(guanidinium)alcohol Attached to a Hairpin Polyamide: Synthesis, DNA Binding, and Plasmid Cleavage.

Bis(guanidinium)alcohols have been designed to react with phosphodiester substrates in a fast transphosphorylation step, a quasi-intramolecular process taking place in contact ion pairs. Here the attachment of such compounds to Dervan-type hairpin polyamides is described. The resulting conjugate 1 binds to AT-rich DNA duplexes with affinity similar to that of the parent polyamide as shown by UV melting experiments and CD titrations. Conjugate 1 nicks plasmid DNA at concentrations ranging from micromolar to high nanomolar.

A triphenylamine-based colorimetric and fluorescent probe with donor–bridge–acceptor structure for detection of G-quadruplex DNA.

In this Letter, three triphenylamine-based dyes (TPA-1, TPA-2a and TPA-2b) with donor–bridge–acceptor (D–p–A) structure were designed and synthesized for the purpose of G-quadruplexes recognition. In aqueous conditions, the interactions of the dyes with G-quadruplexes were studied with the aim to establish the influence of the geometry of the dyes on their binding and probing properties. Results indicate that TPA-2b displays significant selective colorimetric and fluorescent changes upon binding of G-quadruplex DNA. More importantly, its distinct color change enables visual detection and differentiation of G-quadruplexes from single and duplex DNA structures. CD titration date reveals that TPA-2b could induce and stabilize the formation of G-quadruplex structure. All these remarkable properties of TPA-2b suggest that it should have promising application in the field of G-quadruplexes research.

Reactivity of a Zn(Cys)2(His)2 Zinc Finger with Singlet Oxygen: Oxidation Directed toward Cysteines but not Histidines.

Singlet oxygen ((1)O2) is an important reactive oxygen species in biology that has deleterious effects. Proteins constitute the main target of (1)O2 in cells. Several organisms are able to mount a transcriptional defense against (1)O2. ChrR and MBS are two proteins with Zn(Cys)2(His)2 zinc finger sites that are involved in the regulation of the defense against (1)O2. In this article, we investigate the reactivity of Zn⋅CPF, a Zn(Cys)2(His)2 classical ββα zinc finger, with (1)O2. We show that Zn⋅CPF interacts with (1)O2 mainly by physical quenching using a combination of (1)O2 luminescence quenching and kinetic competition experiments. The chemical reaction, which accounts for 5% of the interaction, leads to oxidation of cysteines but not histidines. Primary photooxidation products, identified by HPLC and mass spectrometry, are sulfinate (75±5%) and disulfides (25±5%). The peptides that have a single cysteine thiolate oxidized into a sulfinate are still able to bind one equivalent Zn(2+) but with a dramatic reduction of the binding constant compared to Zn⋅CPF despite the preservation of the ββα fold, as shown by NMR and CD titrations. Finally, Zn⋅CPF is compared to Zn⋅LTC, a treble clef Zn(Cys)4 zinc finger, to gain further insight into the behavior of zinc fingers toward (1)O2.

Human telomeric RNA G-quadruplex response to point mutation in the G-quartets.

Many putative G-quadruplex forming sequences have been predicted to exist in the human genome and transcriptome. As these sequences are subject to point mutations or SNPs (single nucleotide polymorphisms) during the course of evolution, we attempt to understand impact of these mutations in context of RNA G-quadruplex formation using human telomeric RNA (TERRA) as a model sequence. Our studies suggest that G-quadruplex stability is sensitive to substitution of the guanines comprising G-quartets. While central G-quartet plays a crucial role in maintaining the DNA G-quadruplex stability as evident in literature, there is equal importance of three G-quartets in the stability of RNA quadruplex structure. The work here highlights the alterations in the G-quartet are detrimental to the integrity of overall RNA G-quadruplex structure. Furthermore, TmPyP4 molecules are shown to exhibit similar binding behavior toward telomeric RNA G-quadruplex harboring base substitutions employing CD titrations and isothermal titration calorimetry; well indicating that mutation does not influence TmPyP4 recognition ability as it affects the stability of RNA G-quadruplex. Thus, our study implicates that mutation in G-quartets causes destabilization of RNA G-quadruplex without affecting its trans factor binding ability.

Interactions with polynucleotides and antitumor activity of amidino and imidazolinyl substituted 2-phenylbenzothiazole mesylates

Based on previously reported antiproliferative activity screening, four most promising disubstituted 2-phenylbenzothiazole hydrochlorides were chosen for detailed study. Water solubility, as well as liphophilicity/hydrophilicity balance of organic core were modified by conversion to mesylate salts. For purpose of structure/activity studies their structures were determined by X-ray structure analysis. Detailed analysis of interactions of new compounds with double stranded (ds-) DNA/RNA by UV/Vis and CD titrations, thermal melting and viscometry experiments revealed that most of studied compounds intercalate into ds-RNA but bind into minor groove of AT-DNA, and agglomerate along GC-DNA. Furthermore, compounds also interact with ss-RNA, but only amino-imidazolinyl 2-phenylbenzothiazole, 4b displayed well defined orientation and dominant binding mode (by induced CD signals) with poly A and poly G. Besides, in vitro investigations revealed moderate to high antiproliferative activity of benzothiazoles against seven human cancer cell lines, while in some cases (HTC 116, SW620, MIA PaCa-2) high correlation between the type of the amidino group and cytotoxic activity was observed.

Ru-indoloquinoline complex as a selective and effective human telomeric G-quadruplex binder

Indoloquinoline and its derivatives have been reported to be a kind of efficient G-quadruplex binder and have been found to interact preferentially to intramolecular G-quadruplex and inhibit telomerase activity in human K562 cells and SW620 cells. In contrast to indoloquinoline derivatives, much less is known about the metal complex based on indoloquinoline or its derivative. In this report, we studied the interaction of ruthenium complex [Ru(bpy)2(itatp)]2+ containing indoloquinoline moiety with human telomeric G-quadruplex DNA (Telo22) and c-myc G-quadruplex DNA (Pu27) by UV–visible (UV–Vis), fluorescence spectroscopy, fluorescent intercalator displacement (FID), thermal denaturation studies and CD spectroscopy. The results suggest that [Ru(bpy)2(itatp)]2+ displays a strong π–π stacking interaction with human telomeric G-quadruplex with a high binding constant (∼107M−1), but just exhibits moderate binding affinity to c-myc G-quadruplex, thus showing significant selectivity to human telomeric G-quadruplex. The CD titration results indicate that [Ru(bpy)2(itatp)]2+ could effectively convert Telo22 into antiparallel G-quadruplex conformation, while in the c-myc G-quadruplex case, instead of promoting Pu27 to fold into G-quadruplex, [Ru(bpy)2(itatp)]2+ destroys the parallel G-quadruplex structure of Pu27.