G-quadruplex DNA Motifs Research Articles

APTO-253 Interaction with G-Quadruplex DNA Is Linked to Inhibition of c-Myc Expression, Induction of DNA Damage, and Generation of Synthetic Lethality in Cells with BRCA1/2 Impairment

APTO-253 is a Phase 1 clinical stage small molecule that selectively kills acute myeloid leukemia (AML) cells in the absence of toxicity to normal peripheral blood mononuclear (PBMCs) and bone marrow cells in various animal species and humans. Previously, APTO-253 was reported to exhibit concentration and time dependent inhibition of c-MYC oncogene expression in AML cells and consequently to induce G0/G1 cell cycle arrest and apoptosis, but not affect normal PBMCs (58th ASH abstract #1716). Current mechanistic studies demonstrated that APTO-253 modulates c-MYC at the transcriptional level by decreasing acetylated H3K27 at its promoters and additionally by destabilizing c-MYC mRNA. In addition, differential gene expression analysis of RNA-seq and reverse phase protein array (RPPA) data highlighted a role for c-MYC in the mechanism of APTO-253 (GO terms - Down regulated by c-MYC p-value 6E-26, Gene promoters bound by c-MYC p-value 4.2 E-10, ChIP targets of c-MYC p-value 3.3E-8). Furthermore, from the RPPA data we observed an increase in p-Chk1, p-Chk2, γH2Ax, and total p53 and E2F1, all of which are indicative of activation of DNA damage response pathways. This was accompanied by elevated levels of XBP1, GRP78, and p-p38 that point towards cellular stress response signaling (GO term Regulation of Cell Stress, p-value 1.89E-8). Subsequent work demonstrated that APTO-253 induced DNA damage as documented by gH2AX foci formation and the neutral COMET assay, and exhibited synthetic lethality in cells with BRCA1/2 mutations that have impaired ability to repair DNA damage by homologous recombination (abstract submitted to 59th ASH annual meeting). This amalgam of data, coupled with structural informatics analyses, suggested a mechanism of action involving the binding and/or stabilization of G-quadruplex (G4) DNA motifs. Intracellular pharmacokinetic studies revealed that APTO-253 converts from a monomer to a ferrous complex. PCR stop and modified FRET assays demonstrated that the APTO-253 ferrous complex Fe(253)3 has activity similar to that of TMPyP4, a well-established G4 ligand, to bind/stabilize G4 structures in telomere sequences (∆T1/2 vs concentration slope 7.7 ±2.7 and 10.67 ±1.23 for Fe(253)3 and TMPyP4, respectively), in MYC promoter sequences (5.07 ±1.56 and 5.62 ±0.75, respectively) and in rRNA sequences (4.6 ±1.1 and 5.6 ±0.88, respectively). Although APTO-253 may participate in multiple mechanistic events, the effect of APTO-253 on c-MYC expression, cell cycle arrest and DNA damage, as well as synthetic lethality in cells with compromised DNA repair mechanisms, can be explained by the action of the Fe(253)3 complex on G-quadruplex DNA motifs. DisclosuresLocal:Aptose Biosciences, Inc.: Employment. Zhang:Aptose Biosciences, Inc.: Employment. Benbatoul:Aptose Biosciences, Inc.: Employment. Folger:Aptose Biosciences, Inc.: Employment. Sheng:Aptose Biosciences, Inc.: Employment. Howell:Aptose Biosciences, Inc.: Membership on an entity's Board of Directors or advisory committees. Rice:Aptose Biosciences, Inc.: Employment, Equity Ownership, Patents & Royalties.

Amplified collection of binary G-quadruplex on a binary C-rich functionalized palindromic hairpin probe for label-free detection of a molecular cancer biomarker of microRNA

Detection of well-studied cancer biomarkers of microRNAs (miRNAs) is very valuable for early cancer diagnosis and therapy in clinical medicine. Herein, we designed a binary C-rich functionalized palindromic hairpin probe (BC-FPHP) and employed it as the only species to amplify the common ovarian cancer biomarker of miRNA-21. The BC-FPHP is concurrently proposed with a miRNA-21 recognition loop region, a self-complementary stem region, a binary C-rich fragment at its 5′ end, and a palindromic sequence at its 3′ end. The presence of target miRNA-21 induces the configuration change of BC-FPHP. It makes the amplification event on BC-FPHP occurred without the aid of any other probes, leading to a substantial collection of binary G-quadruplex DNA motifs that can be inserted with thioflavin T (ThT) for remarkable fluorescence emission. In this system, the BC-FPHP is smartly designed with the capabilities to be self-primered, self-templated, and self-amplified, ensuring the isothermal amplification system a simple, low cost, and user-friendly detection platform for sensitive and specific detection of miRNA-21. In addition, the BC-FPHP based amplification system has been successfully used for the detection of miRNA-21 in diluted human serum samples. We envision that this work holds great potential in the early assisting screening of ovarian cancer.

APTO-253 Stabilizes G-quadruplex DNA, Inhibits MYC Expression, and Induces DNA Damage in Acute Myeloid Leukemia Cells.

APTO-253 is a phase I clinical stage small molecule that selectively induces CDKN1A (p21), promotes G0-G1 cell-cycle arrest, and triggers apoptosis in acute myeloid leukemia (AML) cells without producing myelosuppression in various animal species and humans. Differential gene expression analysis identified a pharmacodynamic effect on MYC expression, as well as induction of DNA repair and stress response pathways. APTO-253 was found to elicit a concentration- and time-dependent reduction in MYC mRNA expression and protein levels. Gene ontogeny and structural informatic analyses suggested a mechanism involving G-quadruplex (G4) stabilization. Intracellular pharmacokinetic studies in AML cells revealed that APTO-253 is converted intracellularly from a monomer to a ferrous complex [Fe(253)3]. FRET assays demonstrated that both monomeric APTO-253 and Fe(253)3 stabilize G4 structures from telomeres, MYC, and KIT promoters but do not bind to non-G4 double-stranded DNA. Although APTO-253 exerts a host of mechanistic sequelae, the effect of APTO-253 on MYC expression and its downstream target genes, on cell-cycle arrest, DNA damage, and stress responses can be explained by the action of Fe(253)3 and APTO-253 on G-quadruplex DNA motifs. Mol Cancer Ther; 17(6); 1177-86. ©2018 AACR.

G-Quadruplex DNA Motifs in the Malaria Parasite Plasmodium falciparum and Their Potential as Novel Antimalarial Drug Targets

G-quadruplexes are DNA or RNA secondary structures that can be formed from guanine-rich nucleic acids. These four-stranded structures, composed of stacked quartets of guanine bases, can be highly stable and have been demonstrated to occur in vivo in the DNA of human cells and other systems, where they play important biological roles, influencing processes such as telomere maintenance, DNA replication and transcription, or, in the case of RNA G-quadruplexes, RNA translation and processing. We report for the first time that DNA G-quadruplexes can be detected in the nuclei of the malaria parasite Plasmodium falciparum, which has one of the most A/T-biased genomes sequenced and therefore possesses few guanine-rich sequences with the potential to form G-quadruplexes. We show that despite this paucity of putative G-quadruplex-forming sequences, P. falciparum parasites are sensitive to several G-quadruplex-stabilizing drugs, including quarfloxin, which previously reached phase 2 clinical trials as an anticancer drug. Quarfloxin has a rapid initial rate of kill and is active against ring stages as well as replicative stages of intraerythrocytic development. We show that several G-quadruplex-stabilizing drugs, including quarfloxin, can suppress the transcription of a G-quadruplex-containing reporter gene in P. falciparum but that quarfloxin does not appear to disrupt the transcription of rRNAs, which was proposed as its mode of action in both human cells and trypanosomes. These data suggest that quarfloxin has potential for repositioning as an antimalarial with a novel mode of action. Furthermore, G-quadruplex biology in P. falciparum may present a target for development of other new antimalarial drugs.

G4PromFinder: an algorithm for predicting transcription promoters in GC-rich bacterial genomes based on AT-rich elements and G-quadruplex motifs

BackgroundOver the last few decades, computational genomics has tremendously contributed to decipher biology from genome sequences and related data. Considerable effort has been devoted to the prediction of transcription promoter and terminator sites that represent the essential “punctuation marks” for DNA transcription. Computational prediction of promoters in prokaryotes is a problem whose solution is far from being determined in computational genomics. The majority of published bacterial promoter prediction tools are based on a consensus-sequences search and they were designed specifically for vegetative σ70 promoters and, therefore, not suitable for promoter prediction in bacteria encoding a lot of σ factors, like actinomycetes.ResultsIn this study we investigated the possibility to identify putative promoters in prokaryotes based on evolutionarily conserved motifs, and focused our attention on GC-rich bacteria in which promoter prediction with conventional, consensus-based algorithms is often not-exhaustive. Here, we introduce G4PromFinder, a novel algorithm that predicts putative promoters based on AT-rich elements and G-quadruplex DNA motifs. We tested its performances by using available genomic and transcriptomic data of the model microorganisms Streptomyces coelicolor A3(2) and Pseudomonas aeruginosa PA14. We compared our results with those obtained by three currently available promoter predicting algorithms: the σ70consensus-based PePPER, the σ factors consensus-based bTSSfinder, and PromPredict which is based on double-helix DNA stability. Our results demonstrated that G4PromFinder is more suitable than the three reference tools for both the genomes. In fact our algorithm achieved the higher accuracy (F1-scores 0.61 and 0.53 in the two genomes) as compared to the next best tool that is PromPredict (F1-scores 0.46 and 0.48). Consensus-based algorithms produced lower performances with the analyzed GC-rich genomes.ConclusionsOur analysis shows that G4PromFinder is a powerful tool for promoter search in GC-rich bacteria, especially for bacteria coding for a lot of σ factors, such as the model microorganism S. coelicolor A3(2). Moreover consensus-based tools and, in general, tools that are based on specific features of bacterial σ factors seem to be less performing for promoter prediction in these types of bacterial genomes.

Self-assembled Pt2L2 boxes strongly bind G-quadruplex DNA and influence gene expression in cancer cells.

Supramolecular Pt(ii) quadrangular boxes bind native and G-quadruplex DNA motifs in a size-dependent fashion. Three Pt molecular squares of distinct size show biological activity against cancer cells and heavily influence the expression of genes known to form G-quadruplexes in their promoter regions. The smallest Pt-box displays less activity but more selectivity for a quadruplex formed in the c-Kit gene.

Regulation of gene expression by the BLM helicase correlates with the presence of G-quadruplex DNA motifs.

Bloom syndrome is a rare autosomal recessive disorder characterized by genetic instability and cancer predisposition, and caused by mutations in the gene encoding the Bloom syndrome, RecQ helicase-like (BLM) protein. To determine whether altered gene expression might be responsible for pathological features of Bloom syndrome, we analyzed mRNA and microRNA (miRNA) expression in fibroblasts from individuals with Bloom syndrome and in BLM-depleted control fibroblasts. We identified mRNA and miRNA expression differences in Bloom syndrome patient and BLM-depleted cells. Differentially expressed mRNAs are connected with cell proliferation, survival, and molecular mechanisms of cancer, and differentially expressed miRNAs target genes involved in cancer and in immune function. These and additional altered functions or pathways may contribute to the proportional dwarfism, elevated cancer risk, immune dysfunction, and other features observed in Bloom syndrome individuals. BLM binds to G-quadruplex (G4) DNA, and G4 motifs were enriched at transcription start sites (TSS) and especially within first introns (false discovery rate ≤ 0.001) of differentially expressed mRNAs in Bloom syndrome compared with normal cells, suggesting that G-quadruplex structures formed at these motifs are physiologic targets for BLM. These results identify a network of mRNAs and miRNAs that may drive the pathogenesis of Bloom syndrome.

Synthesis and Binding Studies of Novel Diethynyl‐Pyridine Amides with Genomic Promoter DNA G‐Quadruplexes

Herein, we report the design, synthesis and biophysical evaluation of novel 1,2,3-triazole-linked diethynyl-pyridine amides and trisubstituted diethynyl-pyridine amides as promising G-quadruplex binding ligands. We have used a Cu(I)-catalysed azide-alkyne cycloaddition click reaction to prepare the 1,2,3-triazole-linked diethynyl-pyridine amides. The G-quadruplex DNA binding properties of the ligands have been examined by using a Förster resonance energy transfer (FRET) melting assay and surface plasmon resonance (SPR) experiments. The investigated compounds are conformationally flexible, having free rotation around the triple bond, and exhibit enhanced G-quadruplex binding stabilisation and specificity between intramolecular promoter G-quadruplex DNA motifs compared to the first generation of diaryl-ethynyl amides (J. Am. Chem. Soc. 2008, 130, 15950-15956). The ligands show versatility in molecular recognition and promising G-quadruplex discrimination with 2-50-fold selectivity exhibited between different intramolecular promoter G-quadruplexes. Circular dichroism (CD) spectroscopic analysis suggested that at higher concentration these ligands disrupt the c-kit2 G-quadruplex structure. The studies validate the design concept of the 1,3-diethynyl-pyridine-based scaffold and demonstrate that these ligands exhibit not only significant selectivity over duplex DNA but also variation in G-quadruplex interaction properties based on small chemical changes in the scaffold, leading to unprecedented differential recognition of different DNA G-quadruplex sequences.