Synergistic binding of actinomycin D and echinomycin to DNA mismatch sites and their combined anti-tumour effects.

Combination cancer chemotherapy is one of the most useful treatment methods to achieve a synergistic effect and reduce the toxicity of dosing with a single drug. Here, we use a combination of two well-established anticancer DNA intercalators, actinomycin D (ActD) and echinomycin (Echi), to screen their binding capabilities with DNA duplexes containing different mismatches embedded within Watson-Crick base-pairs. We have found that combining ActD and Echi preferentially stabilised thymine-related T:T mismatches. The enhanced stability of the DNA duplex-drug complexes is mainly due to the cooperative binding of the two drugs to the mismatch duplex, with many stacking interactions between the two different drug molecules. Since the repair of thymine-related mismatches is less efficient in mismatch repair (MMR)-deficient cancer cells, we have also demonstrated that the combination of ActD and Echi exhibits enhanced synergistic effects against MMR-deficient HCT116 cells and synergy is maintained in a MMR-related MLH1 gene knockdown in SW620 cells. We further accessed the clinical potential of the two-drug combination approach with a xenograft mouse model of a colorectal MMR-deficient cancer, which has resulted in a significant synergistic anti-tumour effect. The current study provides a novel approach for the development of combination chemotherapy for the treatment of cancers related to DNA-mismatches.

Staggered intercalation of DNA duplexes with base-pair modulation by two distinct drug molecules induces asymmetric backbone twisting and structure polymorphism.

The use of multiple drugs simultaneously targeting DNA is a promising strategy in cancer therapy for potentially overcoming single drug resistance. In support of this concept, we report that a combination of actinomycin D (ActD) and echinomycin (Echi), can interact in novel ways with native and mismatched DNA sequences, distinct from the structural effects produced by either drug alone. Changes in the former with GpC and CpG steps separated by a A:G or G:A mismatch or in a native DNA with canonical G:C and C:G base pairs, result in significant asymmetric backbone twists through staggered intercalation and base pair modulations. A wobble or Watson-Crick base pair at the two drug-binding interfaces can result in a single-stranded 'chair-shaped' DNA duplex with a straight helical axis. However, a novel sugar-edged hydrogen bonding geometry in the G:A mismatch leads to a 'curved-shaped' duplex. Two non-canonical G:C Hoogsteen base pairings produce a sharply kinked duplex in different forms and a four-way junction-like superstructure, respectively. Therefore, single base pair modulations on the two drug-binding interfaces could significantly affect global DNA structure. These structures thus provide a rationale for atypical DNA recognition via multiple DNA intercalators and a structural basis for the drugs' potential synergetic use.

On Water Arrangements in Right- and Left-Handed DNA Structures.

DNA requires hydration to maintain its structural integrity. Crystallographic analyses have enabled patterns of water arrangements to be visualized. We survey these water motifs in this review, focusing on left- and right-handed duplex and quadruplex DNAs, together with the i-motif. Common patterns of linear spines of water organization in grooves have been identified and are widely prevalent in right-handed duplexes and quadruplexes. By contrast, a left-handed quadruplex has a distinctive wheel of hydration populating the almost completely circular single groove in this structure.

QN-302 demonstrates opposing effects between i-motif and G-quadruplex DNA structures in the promoter of the S100P gene.

GC-rich sequences can fold into G-quadruplexes and i-motifs and are known to control gene expression in many organisms. The potent G-quadruplex experimental anticancer drug QN-302 down-regulates a number of cancer-related genes, in particular S100P. Here we show this ligand has strong opposing effects with i-motif DNA structures and is one of the most potent i-motif destabilising agents reported to date. QN-302 down-regulates the expression of numerous cancer-related genes by pan-quadruplex targeting. QN-302 exhibits exceptional combined synergistic effects compared to many other G-quadruplex and i-motif interacting compounds. This work further emphasises the importance of considering G-quadruplex and i-motif DNA structures as one dynamic system.

Water spines and networks in G-quadruplex structures.

Quadruplex DNAs can fold into a variety of distinct topologies, depending in part on loop types and orientations of individual strands, as shown by high-resolution crystal and NMR structures. Crystal structures also show associated water molecules. We report here on an analysis of the hydration arrangements around selected folded quadruplex DNAs, which has revealed several prominent features that re-occur in related structures. Many of the primary-sphere water molecules are found in the grooves and loop regions of these structures. At least one groove in anti-parallel and hybrid quadruplex structures is long and narrow and contains an extensive spine of linked primary-sphere water molecules. This spine is analogous to but fundamentally distinct from the well-characterized spine observed in the minor groove of A/T-rich duplex DNA, in that every water molecule in the continuous quadruplex spines makes a direct hydrogen bond contact with groove atoms, principally phosphate oxygen atoms lining groove walls and guanine base nitrogen atoms on the groove floor. By contrast, parallel quadruplexes do not have extended grooves, but primary-sphere water molecules still cluster in them and are especially associated with the loops, helping to stabilize loop conformations.

Targeting the ALS/FTD-associated A-DNA kink with anthracene-based metal complex causes DNA backbone straightening and groove contraction.

The use of a small molecule compound to reduce toxic repeat RNA transcripts or their translated aberrant proteins to target repeat-expanded RNA/DNA with a G4C2 motif is a promising strategy to treat C9orf72-linked disorders. In this study, the crystal structures of DNA and RNA-DNA hybrid duplexes with the -GGGCCG- region as a G4C2 repeat motif were solved. Unusual groove widening and sharper bending of the G4C2 DNA duplex A-DNA conformation with B-form characteristics inside was observed. The G4C2 RNA-DNA hybrid duplex adopts a more typical rigid A form structure. Detailed structural analysis revealed that the G4C2 repeat motif of the DNA duplex exhibits a hydration shell and greater flexibility and serves as a 'hot-spot' for binding of the anthracene-based nickel complex, NiII(Chro)2 (Chro = Chromomycin A3). In addition to the original GGCC recognition site, NiII(Chro)2 has extended specificity and binds the flanked G:C base pairs of the GGCC core, resulting in minor groove contraction and straightening of the DNA backbone. We have also shown that Chro-metal complexes inhibit neuronal toxicity and suppresses locomotor deficits in a Drosophila model of C9orf72-associated ALS. The approach represents a new direction for drug discovery against ALS and FTD diseases by targeting G4C2 repeat motif DNA.

The Potent G-Quadruplex-Binding Compound QN-302 Downregulates S100P Gene Expression in Cells and in an In Vivo Model of Pancreatic Cancer.

The naphthalene diimide compound QN-302, designed to bind to G-quadruplex DNA sequences within the promoter regions of cancer-related genes, has high anti-proliferative activity in pancreatic cancer cell lines and anti-tumor activity in several experimental models for the disease. We show here that QN-302 also causes downregulation of the expression of the S100P gene and the S100P protein in cells and in vivo. This protein is well established as being involved in key proliferation and motility pathways in several human cancers and has been identified as a potential biomarker in pancreatic cancer. The S100P gene contains 60 putative quadruplex-forming sequences, one of which is in the promoter region, 48 nucleotides upstream from the transcription start site. We report biophysical and molecular modeling studies showing that this sequence forms a highly stable G-quadruplex in vitro, which is further stabilized by QN-302. We also report transcriptome analyses showing that S100P expression is highly upregulated in tissues from human pancreatic cancer tumors, compared to normal pancreas material. The extent of upregulation is dependent on the degree of differentiation of tumor cells, with the most poorly differentiated, from more advanced disease, having the highest level of S100P expression. The experimental drug QN-302 is currently in pre-IND development (as of Q1 2023), and its ability to downregulate S100P protein expression supports a role for this protein as a marker of therapeutic response in pancreatic cancer. These results are also consistent with the hypothesis that the S100P promoter G-quadruplex is a potential therapeutic target in pancreatic cancer at the transcriptional level for QN-302.

Machine learning shows torsion angle preferences in left-handed and right-handed quadruplex DNAs.

Left-handed G quadruplexes (LHG4) have been recently discovered as a new class of G quadruplexes. The biological functions of LHG4s are still unknown, but they share a striking resemblance to Z-DNA in their helicity and jagged phosphate backbone. To further understand structural features of the LHG4s that define their left handedness, we have employed human-interpretable machine-learning methods to classify right- and left-handed G4s purely based on torsional angle analysis. Our results reveal the importance of the α, ß, δ, and χ angles in left-handed structuring across both Z-DNAs and LHG4s. Our analysis may serve as the first step to understanding the conditions of formation for LHG4s and their potential biological relevance.

Beyond the double helix: DNA structural diversity and the PDB.

The determination of the double helical structure of DNA in 1953 remains the landmark event in the development of modern biological and biomedical science. This structure has also been the starting point for the determination of some 2000 DNA crystal structures in the subsequent 68 years. Their structural diversity has extended to the demonstration of sequence-dependent local structure in duplex DNA, to DNA bending in short and long sequences and in the DNA wound round the nucleosome, and to left-handed duplex DNAs. Beyond the double helix itself, in circumstances where DNA sequences are or can be induced to unwind from being duplex, a wide variety of topologies and forms can exist. Quadruplex structures, based on four-stranded cores of stacked G-quartets, are prevalent though not randomly distributed in the human and other genomes and can play roles in transcription, translation, and replication. Yet more complex folds can result in DNAs with extended tertiary structures and enzymatic/catalytic activity. The Protein Data Bank is the depository of all these structures, and the resource where structures can be critically examined and validated, as well as compared one with another to facilitate analysis of conformational and base morphology features. This review will briefly survey the major structural classes of DNAs and illustrate their significance, together with some examples of how the use of the Protein Data Bank by for example, data mining, has illuminated DNA structural concepts.

Polymorphic G:G mismatches act as hotspots for inducing right-handed Z DNA by DNA intercalation.

DNA mismatches are highly polymorphic and dynamic in nature, albeit poorly characterized structurally. We utilized the antitumour antibiotic CoII(Chro)2 (Chro = chromomycin A3) to stabilize the palindromic duplex d(TTGGCGAA) DNA with two G:G mismatches, allowing X-ray crystallography-based monitoring of mismatch polymorphism. For the first time, the unusual geometry of several G:G mismatches including syn-syn, water mediated anti-syn and syn-syn-like conformations can be simultaneously observed in the crystal structure. The G:G mismatch sites of the d(TTGGCGAA) duplex can also act as a hotspot for the formation of alternative DNA structures with a GC/GA-5' intercalation site for binding by the GC-selective intercalator actinomycin D (ActiD). Direct intercalation of two ActiD molecules to G:G mismatch sites causes DNA rearrangements, resulting in backbone distortion to form right-handed Z-DNA structures with a single-step sharp kink. Our study provides insights on intercalators-mismatch DNA interactions and a rationale for mismatch interrogation and detection via DNA intercalation.

A G-Quadruplex-Binding Small Molecule and the HDAC Inhibitor SAHA (Vorinostat) Act Synergistically in Gemcitabine-Sensitive and Resistant Pancreatic Cancer Cells.

The stabilisation of G-quadruplexes (G4s) by small-molecule compounds is an effective approach for causing cell growth arrest, followed by cell death. Some of these compounds are currently being developed for the treatment of human cancers. We have previously developed a substituted naphthalene diimide G4-binding molecule (CM03) with selective potency for pancreatic cancer cells, including gemcitabine-resistant cells. We report here that CM03 and the histone deacetylase (HDAC) inhibitor SAHA (suberanilohydroxamic acid) have synergistic effects at concentrations close to and below their individual GI50 values, in both gemcitabine-sensitive and resistant pancreatic cancer cell lines. Immunoblot analysis showed elevated levels of γ-H2AX and cleaved PARP proteins upon drug combination treatment, indicating increased levels of DNA damage (double-strand break events: DSBs) and apoptosis induction, respectively. We propose that the mechanism of synergy involves SAHA relaxing condensed chromatin, resulting in higher levels of G4 formation. In turn, CM03 can stabilise a greater number of G4s, leading to the downregulation of more G4-containing genes as well as a higher incidence of DSBs due to torsional strain on DNA and chromatin structure.

A naphthalene diimide G-quadruplex ligand inhibits cell growth and down-regulates BCL-2 expression in an imatinib-resistant gastrointestinal cancer cell line.

Gastro-intestinal tumours (GISTs) are driven by aberrant expression of the c-KIT oncoprotein. They can be effectively treated by the kinase inhibitor imatinib, which locks the c-KIT kinase domain into an inactive conformation. However resistance to imatinib, driven by active-site mutations, is a recurrent clinical challenge, which has been only partly met by the subsequent development of second and third-generation c-KIT inhibitors. It is reported here that a tetra-substituted naphthalene diimide derivative, which is a micromolar inhibitor of cell growth in a wild-type patient-derived GIST cell line, has a sub-micromolar activity in two distinct patient-derived imatinib-resistant cell lines. The compound has been previously shown to down-regulate expression of the c-KIT protein in a wild-type GIST cell line. It does not affect c-KIT protein expression in a resistant cell line to the same extent, whereas it profoundly down-regulates the expression of the anti-apoptopic protein BCL-2. It is proposed that the mechanism of action involves targeting quadruplex nucleic acid structures, and in particular those in the BCL-2 gene and its RNA transcript. The BCL-2 protein is up-regulated in the GIST-resistant cell line, and is strongly down-regulated after treatment. The compound strongly stabilises a range of G-quadruplexes including a DNA one from the BCL-2 promoter and an RNA quadruplex from its 5'-UTR region. A reporter assay construct incorporating the 5'-UTR quadruplex sequence demonstrates down-regulation of BCL-2 expression.

Folding of guanine quadruplex molecules-funnel-like mechanism or kinetic partitioning? An overview from MD simulation studies.

BACKGROUND: Guanine quadruplexes (GQs) play vital roles in many cellular processes and are of much interest as drug targets. In contrast to the availability of many structural studies, there is still limited knowledge on GQ folding. SCOPE OF REVIEW: We review recent molecular dynamics (MD) simulation studies of the folding of GQs, with an emphasis paid to the human telomeric DNA GQ. We explain the basic principles and limitations of all types of MD methods used to study unfolding and folding in a way accessible to non-specialists. We discuss the potential role of G-hairpin, G-triplex and alternative GQ intermediates in the folding process. We argue that, in general, folding of GQs is fundamentally different from funneled folding of small fast-folding proteins, and can be best described by a kinetic partitioning (KP) mechanism. KP is a competition between at least two (but often many) well-separated and structurally different conformational ensembles. MAJOR CONCLUSIONS: The KP mechanism is the only plausible way to explain experiments reporting long time-scales of GQ folding and the existence of long-lived sub-states. A significant part of the natural partitioning of the free energy landscape of GQs comes from the ability of the GQ-forming sequences to populate a large number of syn-anti patterns in their G-tracts. The extreme complexity of the KP of GQs typically prevents an appropriate description of the folding landscape using just a few order parameters or collective variables. GENERAL SIGNIFICANCE: We reconcile available computational and experimental studies of GQ folding and formulate basic principles characterizing GQ folding landscapes. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Flexibility and structural conservation in a c-KIT G-quadruplex.

A quadruplex sequence from the promoter region of the c-KIT gene forms a stable quadruplex, as characterized by crystallographic and NMR methods. Two new crystal structures are reported here, together with molecular dynamics simulation studies on these quadruplex crystal structures and an NMR structure. The new crystal structures, each in a distinct space group and lattice packing arrangement, together with the existing structures, demonstrate that the c-KIT quadruplex fold does not change with differing environments, suggesting that quadruplex topological dynamism is not a general phenomenon. The single and dinucleotide loops in these structures show a high degree of conformational flexibility within the three crystal forms and the NMR ensemble, with no evidence of clustering to particular conformers. This is in accord with the findings of high loop flexibility from the molecular dynamics studies. It is suggested that intramolecular quadruplexes can be grouped into two broad classes (i) those with at least one single-nucleotide loop, often showing singular topologies even though loops are highly flexible, and (ii) with all loops comprising at least two nucleotides, leading to topological dynamism. The loops can have more stable and less dynamic base-stacked secondary structures.

Loop flexibility in human telomeric quadruplex small-molecule complexes.

Quadruplex nucleic acids can be formed at the ends of eukaryotic chromosomes. Their formation and stabilisation by appropriate small molecules can be used as a means of inhibiting the telomere maintenance functions of telomerase in human cancer cells. The crystal structures have been determined for a number of complexes between these small molecules and human telomeric DNA and RNA quadruplexes. The detailed structural characteristics of these complexes have been surveyed here and the variations in conformation for the TTA and UUA loops have been explored. Loop conformations have been classified in terms of a number of discrete types and their distribution among the crystal structures. Sugar conformation and backbone angles have also been examined and trends highlighted. One particular loop class has been found to be most prevalent. Implications for in particular, rational drug design, are discussed.

Extended molecular dynamics of a c-kit promoter quadruplex.

The 22-mer c-kit promoter sequence folds into a parallel-stranded quadruplex with a unique structure, which has been elucidated by crystallographic and NMR methods and shows a high degree of structural conservation. We have carried out a series of extended (up to 10 µs long, ∼50 µs in total) molecular dynamics simulations to explore conformational stability and loop dynamics of this quadruplex. Unfolding no-salt simulations are consistent with a multi-pathway model of quadruplex folding and identify the single-nucleotide propeller loops as the most fragile part of the quadruplex. Thus, formation of propeller loops represents a peculiar atomistic aspect of quadruplex folding. Unbiased simulations reveal µs-scale transitions in the loops, which emphasizes the need for extended simulations in studies of quadruplex loops. We identify ion binding in the loops which may contribute to quadruplex stability. The long lateral-propeller loop is internally very stable but extensively fluctuates as a rigid entity. It creates a size-adaptable cleft between the loop and the stem, which can facilitate ligand binding. The stability gain by forming the internal network of GA base pairs and stacks of this loop may be dictating which of the many possible quadruplex topologies is observed in the ground state by this promoter quadruplex.

Induced-Fit Recognition of CCG Trinucleotide Repeats by a Nickel-Chromomycin Complex Resulting in Large-Scale DNA Deformation.

Small-molecule compounds targeting trinucleotide repeats in DNA have considerable potential as therapeutic or diagnostic agents against many neurological diseases. NiII (Chro)2 (Chro=chromomycin A3) binds specifically to the minor groove of (CCG)n repeats in duplex DNA, with unique fluorescence features that may serve as a probe for disease detection. Crystallographic studies revealed that the specificity originates from the large-scale spatial rearrangement of the DNA structure, including extrusion of consecutive bases and backbone distortions, with a sharp bending of the duplex accompanied by conformational changes in the NiII chelate itself. The DNA deformation of CCG repeats upon binding forms a GGCC tetranucleotide tract, which is recognized by NiII (Chro)2 . The extruded cytosine and last guanine nucleotides form water-mediated hydrogen bonds, which aid in ligand recognition. The recognition can be accounted for by the classic induced-fit paradigm.

Structural Insights into the Quadruplex-Duplex 3' Interface Formed from a Telomeric Repeat: A Potential Molecular Target.

We report here on an X-ray crystallographic and molecular modeling investigation into the complex 3' interface formed between putative parallel stranded G-quadruplexes and a duplex DNA sequence constructed from the human telomeric repeat sequence TTAGGG. Our crystallographic approach provides a detailed snapshot of a telomeric 3' quadruplex-duplex junction: a junction that appears to have the potential to form a unique molecular target for small molecule binding and interference with telomere-related functions. This unique target is particularly relevant as current high-affinity compounds that bind putative G-quadruplex forming sequences only rarely have a high degree of selectivity for a particular quadruplex. Here DNA junctions were assembled using different putative quadruplex-forming scaffolds linked at the 3' end to a telomeric duplex sequence and annealed to a complementary strand. We successfully generated a series of G-quadruplex-duplex containing crystals, both alone and in the presence of ligands. The structures demonstrate the formation of a parallel folded G-quadruplex and a B-form duplex DNA stacked coaxially. Most strikingly, structural data reveals the consistent formation of a TAT triad platform between the two motifs. This triad allows for a continuous stack of bases to link the quadruplex motif with the duplex region. For these crystal structures formed in the absence of ligands, the TAT triad interface occludes ligand binding at the 3' quadruplex-duplex interface, in agreement with in silico docking predictions. However, with the rearrangement of a single nucleotide, a stable pocket can be produced, thus providing an opportunity for the binding of selective molecules at the interface.

Atomic Force Microscopy and Voltammetric Investigation of Quadruplex Formation between a Triazole-Acridine Conjugate and Guanine-Containing Repeat DNA Sequences.

The interactions of the Tetrahymena telomeric repeat sequence d(TG4T) and the polyguanylic acid (poly(G)) sequence with the quadruplex-targeting triazole-linked acridine ligand GL15 were investigated using atomic force microscopy (AFM) at a highly oriented pyrolytic graphite and voltammetry at a glassy carbon electrode. GL15 interacted with both sequences, in a time dependent manner, and G-quadruplex formation was detected. AFM showed the adsorption of quadruplexes as small d(TG4T) and poly(G) spherical aggregates and large quadruplex-based poly(G) assemblies, and voltammetry showed the decrease and disappearance of GL15 and guanine oxidation peak currents and appearance of the G-quadruplex oxidation peak. The GL15 molecule strongly stabilized and accelerated G-quadruplex formation in both Na(+) and K(+) ion-containing solution, although only K(+) promoted the formation of perfectly aligned tetra-molecular G-quadruplexes. The small-molecule complex with the d(TG4T) quadruplex is discrete and approximately globular, whereas the G-quadruplex complex with poly(G) is formed at a number of points along the length of the polynucleotide, analogous to beads on a string.