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G-quadruplex (G4) sequences, which can fold into higher-order G4 structures, are abundant in the human genome and are over-represented in the promoter regions of many genes involved in human cancer initiation, progression, and metastasis. They are plausible targets for G4-binding small molecules, which would, in the case of promoter G4s, result in the transcriptional downregulation of these genes. However, structural information is currently available on only a very small number of G4s and their ligand complexes. This limitation, coupled with the currently restricted information on the G4-containing genes involved in most complex human cancers, has led to the development of a phenotypic-led approach to G4 ligand drug discovery. This approach was illustrated by the discovery of several generations of tri- and tetra-substituted naphthalene diimide (ND) ligands that were found to show potent growth inhibition in pancreatic cancer cell lines and are active in in vivo models for this hard-to-treat disease. The cycles of discovery have culminated in a highly potent tetra-substituted ND derivative, QN-302, which is currently being evaluated in a Phase 1 clinical trial. The major genes whose expression has been down-regulated by QN-302 are presented here: all contain G4 propensity and have been found to be up-regulated in human pancreatic cancer. Some of these genes are also upregulated in other human cancers, supporting the hypothesis that QN-302 is a pan-G4 drug of potential utility beyond pancreatic cancer. [ABSTRACT FROM AUTHOR]

The tetrasubstituted naphthalene diimide compound QN-302 binds to G-quadruplex (G4) DNA structures. It shows high potency in pancreatic ductal adenocarcinoma (PDAC) cells and inhibits the transcription of cancer-related genes in these cells and in PDAC animal models. It is currently in Phase 1a clinical evaluation as an anticancer drug. A study of structure–activity relationships of QN-302 and two related analogues (CM03 and SOP1247) is reported here. These have been probed using comparisons of transcriptional profiles from whole-genome RNA-seq analyses, together with molecular modelling and molecular dynamics simulations. Compounds CM03 and SOP1247 differ by the presence of a methoxy substituent in the latter: these two compounds have closely similar transcriptional profiles. Whereas QN-302 (with an additional benzyl-pyrrolidine group), although also showing down-regulatory effects in the same cancer-related pathways, has effects on distinct genes, for example in the hedgehog pathway. This distinctive pattern of genes affected by QN-302 is hypothesized to contribute to its superior potency compared to CM03 and SOP1247. Its enhanced ability to stabilize G4 structures has been attributed to its benzyl-pyrrolidine substituent fitting into and filling most of the space in a G4 groove compared to the hydrogen atom in CM03 or the methoxy group substituent in SOP1247. [ABSTRACT FROM AUTHOR]

The tetrasubstituted naphthalene diimide compound QN-302 binds to G-quadruplex (G4) DNA structures. It shows high potency in pancreatic ductal adenocarcinoma (PDAC) cells and inhibits the transcription of cancer-related genes in these cells and in PDAC animal models. It is currently in Phase 1a clinical evaluation as an anticancer drug. A study of structure–activity relationships of QN-302 and two related analogues (CM03 and SOP1247) is reported here. These have been probed using comparisons of transcriptional profiles from whole-genome RNA-seq analyses, together with molecular modelling and molecular dynamics simulations. Compounds CM03 and SOP1247 differ by the presence of a methoxy substituent in the latter: these two compounds have closely similar transcriptional profiles. Whereas QN-302 (with an additional benzyl-pyrrolidine group), although also showing down-regulatory effects in the same cancer-related pathways, has effects on distinct genes, for example in the hedgehog pathway. This distinctive pattern of genes affected by QN-302 is hypothesized to contribute to its superior potency compared to CM03 and SOP1247. Its enhanced ability to stabilize G4 structures has been attributed to its benzyl-pyrrolidine substituent fitting into and filling most of the space in a G4 groove compared to the hydrogen atom in CM03 or the methoxy group substituent in SOP1247. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The appY gene has been characterised as conferring resistance to a novel series of antimicrobial benzimidazole derivatives in E. coli MC1061 cells when expressed in high copy-number. A microarray approach was used to identify genes involved in the mechanism of appY-mediated antibacterial resistance, that were up- or down-regulated following induction of the gene in the appY knockout strain JW0553. In total, expression of 90 genes was induced and 48 repressed greater than 2.5-fold (P < 0.05), 45 min after appY induction. Over half the genes up-regulated following appY expression had confirmed or putative roles in acid resistance (AR) and response to oxidative and antibiotic stresses. These included the genes for MdtE and MdtF, which form a multi-drug transporter with TolC and have been implicated in resistance to several antibiotics including erythromycin. Amongst the acid resistance genes were gadAB and adiAC encoding the glutamate-dependant (AR2) and arginine-dependant (AR3) acid resistance systems respectively, in addition to the transcriptional activators of these systems gadE and gadX. In agreement with earlier studies, appA, appCB and hyaA-F were also up-regulated following induction of appY. This study has also confirmed that over-expression of mdtEF confers resistance to these antibacterial benzimidazoles, indicating that the observation of appY conferring resistance to these compounds, proceeds through an appY-mediated up-regulation of this efflux transporter. To assess the importance of the AppY enzyme to acid stress responses, the percentage survival of bacteria in acidified media (pH ≤ 2) was measured. From an initial input of 1 × 106 CFU/ml, the wild-type strain MG1655 showed 7.29% and 0.46% survival after 2 and 4 h, respectively. In contrast, strain JW0553 in which appY is deleted was completely killed by the treatment. Transformation of JW0553 with a plasmid carrying appY returned survival to wild-type levels (7.85% and 1.03% survival at 2 and 4 h). Further dissection of the response by prior induction of each of the three AR systems has revealed that AR1 and AR3 were most affected by the absence of appY. This work highlights an important and previously unidentified role for the AppY enzyme in mediating the responses to several stress conditions. It is likely that the appY gene fits into a complex transcriptional regulatory network involving σS and gadE and gadX. Further work to pinpoint its position in such a hierarchy and to assess the contribution of appY to oxidative stress responses should help determine its full significance. This work is also consistent with recent studies in C. difficile showing that the mechanism of action of ridinilazole involves AT-rich DNA minor groove binding. [ABSTRACT FROM AUTHOR]

Gemcitabine is a drug of choice in the treatment of human pancreatic cancer. Chemo-resistance to this drug is common and has been attributed to a variety of distinct mechanisms, involving > 100 genes. A recently developed small-molecule G-quadruplex ligand, the trisubstituted naphthalene diimide compound CM03, has previously been shown to have equivalent potency to gemcitabine in the pancreatic cancer cell line MIA PaCa-2. We report here on cell lines of increased resistance to gemcitabine that have been generated from this line, with the most resistant having 1,000-fold reduced sensitivity to gemcitabine. These resistant lines retain nM sensitivity to CM03. The molecular basis for the retention of potency by this G-quadruplex ligand has been examined using whole transcriptome data analysis with RNA-seq. This has revealed that the pattern of pathways down regulated by CM03 in the parental MIA PaCa-2 cell line is largely unaffected in the gemcitabine-resistant line. The analysis has also shown that the expression patterns of numerous genes involved in gemcitabine sensitivity are down regulated in the resistant line upon CM03 treatment. These results are supportive of the concept that G-quadruplex small molecules such as CM03 have potential for clinical use in the treatment of gemcitabine-resistant human pancreatic cancer. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A recent paper in Drug and Alcohol Review analysed the information on cancer disseminated by 27 alcohol industry funded organisations. The independent UK alcohol education charity Drinkaware was among the organisations whose information was studied, and based on the analysis claims were made of misrepresentation of evidence about the alcohol-related risk of cancer and alcohol industry influence. This commentary challenges the validity of these findings in respect to the evidence relating to the Drinkaware information, as the analysis is found to be misrepresenting the information by both disregarding the wider information content provided and the order and prominence with which alcohol-related cancer risk is presented. Furthermore, it is argued that the public has a right to be provided with relevant evidence-based information about cancer risk. It is critical that Drinkaware's important public health function is not compromised by unjustified allegations of inaccuracy and by unwarranted attacks on its independence and integrity. [ABSTRACT FROM AUTHOR]

The story behind some of the early studies in the laboratory of Stephen Neidle on quadruplex-binding small molecules and the structural studies on quadruplexes and their complexes is presented and discussed in the context of his earlier work on drug-DNA interactions. More recent studies and future directions in the rational design of small molecules targeting telomeric and gene promoter quadruplexes are also described. [ABSTRACT FROM AUTHOR]

G-rich sequences in DNA and RNA have a propensity to fold into stable secondary structures termed G-quadruplexes. G-quadruplex forming sequences are widespread throughout the human genome, within both, protein coding and non-coding genes, and regulatory regions. G-quadruplexes have been implicated in multiple cellular functions including chromatin epigenetic regulation, DNA recombination, transcriptional regulation of gene promoters and enhancers, and translation. Here we will review the evidence for the occurrence of G-quadruplexes both in vitro and in vivo; their role in neurological diseases including G-quadruplex-forming repeat expansions in the C9orf72 gene in frontotemporal dementia and amyotrophic lateral sclerosis and loss of the G-quadruplex binding protein FMRP in the intellectual disability fragile X syndrome. We also review mounting evidence that supports a role for G-quadruplexes in regulating the processing or function of a range of non-coding RNAs. Finally we will highlight current perspectives for therapeutic interventions that target G-quadruplexes. [ABSTRACT FROM AUTHOR]

Background: A guanine-rich strand within the promoter of the KRAS gene can fold into an intra-molecular G-quadruplex structure (G4), which has an important role in the regulation of KRAS transcription. We have previously identified indolo[3,2-b]quinolines with a 7-carboxylate group and three alkylamine side chains (IQ3A) as effective G4 stabilizers and promising selective anticancer leads. Herein we investigated the anticancer mechanism of action of these compounds, which we hypothesized due to stabilization of the G4 sequence in the KRAS promoter and subsequent down-regulation of gene expression. Methodology/Principal Findings: IQ3A compounds showed greater stabilization of G4 compared to duplex DNA structures and reduced KRAS promoter activity in a dual luciferase reporter assay. Moreover, IQ3A compounds showed high anti-proliferative activity in HCT116 and SW620 colon cancer cells (IC50 < 2.69 μM), without eliciting cell death in non-malignant HEK293T human embryonic kidney, and human colon fibroblasts CCD18co. IQ3A compounds significantly reduced KRAS mRNA and protein steady-state levels at IC50 concentrations, and increased p53 protein steady-state levels and cell death by apoptosis in HCT116 cells (mut KRAS, wt p53). Furthermore, KRAS silencing in HCT116 p53 wild-type (p53(+/+)) and null (p53(-/-)) isogenic cell lines induced a higher level of cell death, and a higher IQ3A-induced cell death in HCT116 p53(+/+) compared to HCT116 p53(-/-). Conclusions: Herein we provide evidence that G4 ligands such as IQ3A compounds can target G4 motifs present in KRAS promoter, down-regulate the expression of the mutant KRAS gene through inhibition of transcription and translation, and induce cell death by apoptosis in colon cancer cell lines. Thus, targeting KRAS at the genomic level with G4 ligands may be a new anticancer therapy strategy for colon cancer. [ABSTRACT FROM AUTHOR]

CCG triplet repeats can fold into tetraplex structures, which are associated with the expansion of (CCG) n trinucleotide sequences in certain neurological diseases. These structures are stabilized by intertwining i-motifs. However, the structural basis for tetraplex i-motif formation in CCG triplet repeats remains largely unknown. We report the first crystal structure of a CCG-repeat sequence, which shows that two dT(CCG)3A strands can associate to form a tetraplex structure with an i-motif core containing four C:C+ pairs flanked by two G:G homopurine base pairs as a structural motif. The tetraplex core is attached to a short parallel-stranded duplex. Each hairpin itself contains a central CCG loop in which the nucleotides are flipped out and stabilized by stacking interactions. The helical twists between adjacent cytosine residues of this structure in the i-motif core have an average value of 30°, which is greater than those previously reported for i-motif structures. [ABSTRACT FROM AUTHOR]

CCG triplet repeats can fold into tetraplex structures, which are associated with the expansion of (CCG) n trinucleotide sequences in certain neurological diseases. These structures are stabilized by intertwining i-motifs. However, the structural basis for tetraplex i-motif formation in CCG triplet repeats remains largely unknown. We report the first crystal structure of a CCG-repeat sequence, which shows that two dT(CCG)3A strands can associate to form a tetraplex structure with an i-motif core containing four C:C+ pairs flanked by two G:G homopurine base pairs as a structural motif. The tetraplex core is attached to a short parallel-stranded duplex. Each hairpin itself contains a central CCG loop in which the nucleotides are flipped out and stabilized by stacking interactions. The helical twists between adjacent cytosine residues of this structure in the i-motif core have an average value of 30°, which is greater than those previously reported for i-motif structures. [ABSTRACT FROM AUTHOR]

We report here on the screening of a fragment library against a G-quadruplex element in the human c-MYC promoter. The ten fragment hits had significant concordance between a biophysical assay, in silico modelling and c-MYC expression inhibition, highlighting the feasibility of applying a fragment-based approach to the targeting of a quadruplex nucleic acid. [ABSTRACT FROM AUTHOR]

We report here on the discovery and preliminary evaluation of a novel non-macrocyclic low molecular weight quadruplex-stabilizing chemotype. The lead compounds, based on a furan core, show high G-quadruplex stabilisation and selectivity as well as potent in vitro anti-proliferative activity. [ABSTRACT FROM AUTHOR]

ABSTRACT G-quadruplexes are higher-order four-stranded structures formed from repetitive guanine-containing tracts in nucleic acids. They comprise a core of stacked guanine-quartets linked by loops of length and sequence that vary with the context in which the quadruplex sequence occurs. Such sequences can be found in a number of genomic environments; at the telomeric ends of eukaryotic chromosomes, in promoter regions, in untranslated sequences and in open reading frames. Quadruplex formation can inhibit telomere maintenance, transcription and translation, especially when enhanced by quadruplex-binding small molecules, and quadruplex targeting is currently of considerable interest. The available experimental structural data shows that quadruplexes can have high conformational flexibility, especially in loop regions, which has hampered attempts to use high-throughput docking to find quadruplex-binding small-molecules with new scaffolds or to optimize existing ones with structure-based design methods. An approach to overcome the challenge of quadruplex conformational flexibility is presented here, which uses a combined multiple molecular dynamics and sampling approach. Two test small molecules have been used, RHPS4 and pyridostatin, which themselves have contrasting degrees of conformational flexibility. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 989-1005, 2013. [ABSTRACT FROM AUTHOR]

Following previous studies on anthraquinone and acridine-based G-quadruplex ligands, here we present a study of similar aromatic cores, with the specific aim of increasing G-quadruplex binding and selectivity with respect to duplex DNA. Synthesized compounds include two and three-side chain xanthone and xanthene derivatives, as well as a dimeric "bridged" form. ESI and FRET measurements suggest that all the studied molecules are good G-quadruplex ligands, both at telomeres and on G-quadruplex forming sequences of oncogene promoters. The dimeric compound and the three-side chain xanthone derivative have been shown to represent the best compounds emerging from the different series of ligands presented here, having also high selectivity for G-quadruplex structures with respect to duplex DNA. Molecular modeling simulations are in broad agreement with the experimental data. [ABSTRACT FROM AUTHOR]

Abstract: The STAT3 transcription factor plays a central role in a wide range of cancer types where it is over-expressed. Previously, phosphorylation of this protein was thought to be a prerequisite for direct binding to DNA. However, we have now shown complete binding of a purified unphosphorylated STAT3 (uSTAT3) core directly to M67 DNA, the high affinity STAT3 target DNA sequence, by a protein electrophoretic mobility shift assay (PEMSA). Binding to M67 DNA was inhibited by addition of increasing concentrations of a phosphotyrosyl peptide. X-ray crystallography demonstrates one mode of binding that is similar to that known for the STAT3 core phosphorylated at Y705. Structured summary of protein interactions: pSTAT3βtc and pSTAT3βtc bind by molecular sieving (View interaction) [Copyright &y& Elsevier]

In our continuing efforts to find new antiherpetic agents from plants, an extract prepared from the stems of Carissa spinarum L. was found to possess appreciable activity against herpes simplex viruses (HSV I and II). A chemical study of this plant was then initiated, and this led to the isolation of 12 compounds, including a coumarin, two cardiac glycosides and nine lignans. These isolated compounds were evaluated for several biological activities, including antiherpetic, cytotoxic, antioxidant and antibacterial effects. The cardiac glycoside evomonoside was found to be the only antiherpetic principle, showing moderate activity against herpes simplex virus types I and II in the inactivation method. The lignans (−)-carinol, (−)-carissanol and (−)-nortrachelogenin exhibited cytotoxicity against breast (MCF7) and lung (A549) cancer cells. Moderate anti-DPPH free radical activity was observed for all the lignans. None of the isolates showed antibacterial activity. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

G-quadruplexes are four-stranded DNA structures that are over-represented in gene promoter regions and are viewed as emerging therapeutic targets in oncology, as transcriptional repression of oncogenes through stabilization of these structures could be a novel anticancer strategy. Many gene promoter G-quadruplexes have physicochemical properties and structural characteristics that might make them druggable, and their structural diversity suggests that a high degree of selectivity might be possible. Here, we describe the evidence for G-quadruplexes in gene promoters and discuss their potential as therapeutic targets, as well as progress in the development of strategies to harness this potential through intervention with small-molecule ligands. [ABSTRACT FROM AUTHOR]

The 3′-ends of human chromosomal DNA terminate in short single-stranded guanine-rich tandem-repeat sequences. In cancer cells, these are associated with the telomere-maintenance enzyme telomerase together with the end-binding protein hPOT1. Small molecules that can compete with these proteins and induce the single-stranded DNA to form quadruplex–ligand complexes are, in effect, able to expose these 3′-ends, which results in the activation of a DNA damage response and selective inhibition of cell growth. Several of these G-quadruplex binding molecules have shown promising anticancer activity in tumour xenograft models, which indicate that the approach may be applicable to the treatment of a wide range of human cancers. This minireview summarizes the available data on these compounds and the challenges posed for drug discovery. [ABSTRACT FROM AUTHOR]