G4 DNA Binding Research Articles

Structure-based discovery of first inhibitors targeting the helicase activity of human PIF1.

PIF1 is a conserved helicase and G4 DNA binding and unwinding enzyme, with roles in genome stability. Human PIF1 (hPIF1) is poorly understood, but its functions can become critical for tumour cell survival during oncogene-driven replication stress. Here we report the discovery, via an X-ray crystallographic fragment screen (XChem), of hPIF1 DNA binding and unwinding inhibitors. A structure was obtained with a 4-phenylthiazol-2-amine fragment bound in a pocket between helicase domains 2A and 2B, with additional contacts to Valine 258 from domain 1A. The compound makes specific interactions, notably through Leucine 548 and Alanine 551, that constrain conformational adjustments between domains 2A and 2B, previously linked to ATP hydrolysis and DNA unwinding. We next synthesized a range of related compounds and characterized their effects on hPIF1 DNA-binding and helicase activity in vitro, expanding the structure activity relationship (SAR) around the initial hit. A systematic analysis of clinical cancer databases is also presented here, supporting the notion that hPIF1 upregulation may represent a specific cancer cell vulnerability. The research demonstrates that hPIF1 is a tractable target through 4-phenylthiazol-2-amine derivatives as inhibitors of its helicase action, setting a foundation for creation of a novel class of anti-cancer therapeutics.

Tuning the push-pull dyes: Construction of polarity insensitive probes for G4 DNA

We report here the design and synthesis of two new polarity-insensitive push-pull dyes TCP-SO3 and TCP-COOH and developed as fluorescent probes for G-quadruplex (G4) DNAs. The strategy involved in this study is to use the traditional D-π-A building block of carbazole-pyridinium (CP), while introducing of a diphenylimidazole group as a secondary donor as well as molecular rotor to form the D-D-π-A configuration, thus ascertaining the effect of the auxiliary group on the photophysics of the dyes. Additionally, N-sulfobutylpyridinium and N-(carboxyethyl) pyridinium bromide moieties are employed as electron-withdrawing anchoring groups, respectively, due to their hydrophilicity and electronic characters, thereby regulating the G4 DNA binding affinity and photophysical properties. It was found that the introduction of diphenylimidazole group to form the D-D-π-A configuration brought superior performance, in terms of polarity-insensitive property with a fluorescence-off state in various polarity solvents. Both dyes exhibited a clear-cut off-on sensing preference for G4 over non-G4 DNA structures, dye TCP-SO3 having a hydrophilic sulfonate group at electron-acceptor part displayed superior G4 DNA binding and selectivity compared to TCP-COOH with a carboxylic acid group. Detailed investigation on the relationship between the structure, photophysical properties, and G4 DNA sensing performance was described.

D-π-A type fluorescent dyes: Effect of π-bridge units on optical and G4 DNA binding properties

Fluorescent solvatochromic dyes that are sensitive to the nature of local microenvironmental, have been explored as probes in applications ranging from the imaging biomolecules to understanding of basic biomolecule functions. To expand the scope of fluorescent solvatochromic dyes for G-quadruplex (G4) DNA structures, and to illustrate the relationship between structure and properties, three newly designed D-π-A type fluorescent dyes were synthesized by introducing diarylimidazole to carbazole skeleton linked to benzene, furan or thiophene π-conjugated bridge and connected with pyridinium acceptor, respectively. Their structural characteristics, optical properties, and G4 DNA binding properties were discussed in detail. In general, the incorporation of furan and thiophene as π-conjugated bridges leads the better conjugation and molecular coplanarity with more efficient intramolecular charge transfer (ICT) effect compared with benzene bridge. The fluorescence intensities induced upon interaction were found that TP-6 with thiophene π-conjugated bridge had the strongest response toward G4 DNAs. In addition, the application of this dye as a fluorescent agent for living cell imaging was also demonstrated.

DEAD-box RNA helicase Dbp2 binds to G-quadruplex nucleic acids and regulates different conformation of G-quadruplex DNA

G-quadruplexes (G4s) are important in regulating DNA replication, repair and RNA transcription through interactions with specialized proteins. Dbp2 has been identified as a G4 DNA binding protein from Saccharomyces cerevisiae cell lysates. The majority of G4 motifs in Saccharomyces cerevisiae display 5–50 nt loops, only a few have 1–2 nt loops. Human DDX5 could unfold MycG4 DNA, whether Dbp2 also participates in remodeling G4 motifs with short loops in Saccharomyces cerevisiae remains elusive. Here we find that Dbp2 prefers G-rich substrates and binds MycG4 with a high affinity. Dbp2 possesses a dual function for different conformations of MycG4, destabilizing the folded MycG4 and inducing further folding of the unfolded MycG4. Similarly, DDX5 can unfold MycG4, but it exhibits a weaker MycG4 folding-promoting activity relative to Dbp2. Furthermore, Dbp2 facilitates DNA annealing activity in the absence of ATP, suggesting that Dbp2 can work on DNA substrates and possibly participate in DNA metabolism. Our results demonstrate that Dbp2 plays an important role in regulating the folding and unfolding activities of MycG4.

Cleavage-defective Topoisomerase I mutants sharply increase G-quadruplex-associated genomic instability.

Topoisomerase 1 (Top1) removes transcription-associated helical stress to suppress G4-formation and its induced recombination at genomic loci containing guanine-run containing sequences. Interestingly, Top1 binds tightly to G4 structures, and its inhibition or depletion can cause elevated instability at these genomic loci. Top1 is targeted by the widely used anti-cancer chemotherapeutic camptothecin (CPT) and its derivatives, which stabilize Top1 covalently attached on a DNA nick and prevent the re-ligation step. Here we investigated how CPT-resistance conferring Top1 mutants, which emerge in cancer patients and cells treated with CPT, affect G4-induced genomic instability in S. cerevisiae. We found that Top1 mutants form stable complexes with G4 DNA and that expression of Top1 cleavage-defective mutants but not a DNA-binding-defective mutant lead to significantly elevated instability at a G4-forming genomic locus. Elevated recombination rates were partly suppressed by their proteolytic removal by SPRTN homolog Wss1 SUMO-dependent metalloprotease in vivo. Furthermore, interaction between G4-DNA binding protein Nsr1, a homolog to clinically-relevant human nucleolin, and Top1 mutants lead to a synergistic increase in G4-associated recombination. These results in the yeast system are strengthened by our cancer genome data analyses showing that functionally detrimental mutations in Top1 correlate with an enrichment of mutations at G4 motifs. Our collective experimental and computational findings point to cooperative binding of Top1 cleavage-defective mutants and Nsr1 as promoting DNA replication blockage and exacerbating genomic instability at G4-motifs, thus complicating patient treatment.

A DNA small molecular probe with increasing K+ concentration promoted selectivity.

DNA small molecular probe study was considered as a promising approach to achieve DNA related disease diagnosis. Most related reports were performed under specific salinity. Herein, 4-imino-3-(pyridin-2-yl)-4H-quinolizine-1-carbonitrile (IPQC) was generated via a facile procedure with high yield (85%). It is found that IPQC could act as a universal probe for most tested ssDNA, dsDNA and G4 DNA in low [K+] concentration (less than 20 mM). However, IPQC showed highly selective G4 DNA binding via UV-vis and fluorescence response in increasing [K+] (e.g., 150 mM) conditions. The ion atmosphere effects are instructive for DNA probe exploration. This provides guidance for the design, selection and optimization of the probes for target DNA sensing.

Topoisomerase IB interacts with genome segregation proteins and is involved in multipartite genome maintenance in Deinococcus radiodurans

Deinococcus radiodurans, an extremophile, resistant to many abiotic stresses including ionizing radiation, has 2 type I topoisomerases (drTopo IA and drTopo IB) and one type II topoisomerase (DNA gyrase). The role of drTopo IB in guanine quadruplex DNA (G4 DNA) metabolism was demonstrated earlier in vitro. Here, we report that D. radiodurans cells lacking drTopo IB (ΔtopoIB) show sensitivity to G4 DNA binding drug (NMM) under normal growth conditions. The activity of G4 motif containing promoters like mutL and recQ was reduced in the presence of NMM in mutant cells. In mutant, the percentage of anucleate cells was more while the copy number of genome elements were less as compared to wild type. Protein-protein interaction studies showed that drTopo IB interacts with genome segregation and DNA replication initiation (DnaA) proteins. The typical patterns of cellular localization of GFP-PprA were affected in the mutant cells. Microscopic examination of D. radiodurans cells expressing drTopo IB-RFP showed its localization on nucleoid forming a streak parallel to the old division septum and perpendicular to newly formed septum. These results together suggest the role of drTopo IB in genome maintenance in this bacterium.

The Relation Between Position and Chemical Composition of Bis-Indole Substituents Determines Their Interactions with G-Quadruplex DNA.

G‐quadruplex (G4) DNA structures are linked to fundamental biological processes and human diseases, which has triggered the development of compounds that affect these DNA structures. However, more knowledge is needed about how small molecules interact with G4 DNA structures. This study describes the development of a new class of bis‐indoles (3,3‐diindolyl‐methyl derivatives) and detailed studies of how they interact with G4 DNA using orthogonal assays, biophysical techniques, and computational studies. This revealed compounds that strongly bind and stabilize G4 DNA structures, and detailed binding interactions which for example, show that charge variance can play a key role in G4 DNA binding. Furthermore, the structure–activity relationships generated opened the possibilities to replace or introduce new substituents on the core structure, which is of key importance to optimize compound properties or introduce probes to further expand the possibilities of these compounds as tailored research tools to study G4 biology.

Structural modification of nonspecific thiazole orange for ligand-DNA interaction study: Understanding the ligand recognition selectivity towards G4-DNA over duplex-DNA

The study of ligand interactions with nucleic acid structures such as G-quadruplexes versus double-stranded DNA is important because these interactions are fundamental for many intracellular processes. In the investigation of ligand-DNA binding process, achieving high fluorescent signal discrimination with strong binding affinity is challenging. To develop binding ligands with excellent recognition ability towards G4-DNA over duplex-DNA, the design of appropriate molecular scaffolds that are able to match with the G4-DNA binding pocket (G-quartet) is crucial. In the present study, the new fluorescent DNA binding ligands were designed and synthesized through the integration of a small and conjugated substituent group at the 2-position of the 1-methylquinolinium moiety of the nonspecific thiazole orange scaffold. The ligands were investigated in fluorescence binding assays and showed different interaction properties and significant fluorescent recognition selectivity towards G4-DNA over duplex-DNA in vitro. Molecular docking study of the ligands in complex with telo21 G4-DNA and ds26 duplex DNA also revealed different binding modes. In addition, the cytotoxicity of the fluorescent DNA binding ligands was evaluated in MTT assays against two selected cancer cell lines (human prostate cancer cell (PC3) and human hepatoma cell (hepG2)). The IC50 was found in the range of 6.3–12.5 μM, indicating a relatively high cytotoxicity of the ligands towards the cancer cells examined.

Spectroscopic and SEM evidences for G4-DNA binding by a synthetic alkyne-containing amino acid with anticancer activity.

Herein, we present a spectroscopic (CD and UV) and SEM study of a phenylalanine derivative carrying a terminal alkyne moiety and indicated by us CF3IIIPhe, with particular attention to its interaction with Cu(II) cation and some biological macromolecules, as well as a preliminary evaluation of its effect on cancerous cells. CD spectroscopy evidenced the ability of CF3IIIPhe to interact with tel26 and c-myc, two quadruplex DNA (G4 DNA) models explored in this study. Other CD and UV studies revealed the ability of the unnatural amino acid to form aggregates in aqueous solution, to bind Cu(II) cation, and to interact with bovine serum albumin (BSA). Cellular studies demonstrated CF3IIIPhe antiproliferative activity on PC3 cells. Its ability to bind telomeric DNA was verified with tel26 by CD investigation and SEM analysis, that revealed a noteworthy change in DNA morphology (mainly based on nanosphere structures) by CF3IIIPhe, confirming its G4-DNA binding ability already evidenced by spectroscopy.

DrRecQ regulates guanine quadruplex DNA structure dynamics and its impact on radioresistance in Deinococcus radiodurans.

The GC-rich genome of Deinococcus radiodurans contains a very high density of putative guanine quadruplex (G4) DNA motifs and its RecQ (drRecQ) was earlier characterized as a 3'→5' dsDNA helicase. We saw that N-Methyl mesoporphyrin IX (NMM), a G4 DNA binding drug affected normal growth as well as the gamma radiation resistance of the wild-type bacterium. Interestingly, NMM treatment and recQ deletion showed additive effect on normal growth but there was no effect of NMM on gamma radiation resistance of recQ mutant. The recombinant drRecQ showed~400 times higher affinity to G4 DNA (Kd =11.74±1.77nM) as compared to dsDNA (Kd =4.88±1.30µM). drRecQ showed ATP independent helicase function on G4 DNA, which was higher than ATP-dependent helicase activity on dsDNA. Unlike wild-type cells that sparingly stained for G4 structure with Thioflavin T (ThT), recQ mutant showed very high-density of ThT fluorescence foci on DNA indicating an important role of drRecQ in regulation of G4 DNA structure dynamics in vivo. These results together suggested that drRecQ is an ATP independent G4 DNA helicase that plays an important role in the regulation of G4 DNA structure dynamics and its impact on radioresistance in D. radiodurans.

One-step synthesis of methylene-bridged bis-carbazole and evaluation of its antitumor activity and G-quadruplex DNA binding property

Most reported carbazolyl G-quadruplex DNA (G4-DNA) ligands possess a rigid structure rather than a flexible one. The conformationally flexible ligands are paid much less attention. In this study, we report a novel class of non-rigid methylene-bridged biscarbazolyl ligand and their G4-DNA binding properties. Moreover, the antitumor activities of all these oligomers have been evaluated. The results show that this family of oligomers could be facilely synthesized via solely one step. Among them, compound 2, the bis-carbazole derivative, displays the best antitumor activity and IC50 values against HT-29, HepG2, A375 and MCF-7 cells are 0.69, 5.09, 3.15 and 3.8 μ mol/L, respectively. Although conformationally flexible, 2 is still capable of binding to as well as stabilizing G4-DNA via π-π stacking interaction. Moreover, 2 selectively binds to G4-DNA over duplex DNA. The current study enriches the category of carbazolyl G4-DNA ligands and paves the way for the search of more efficient G4-DNA ligands and antitumor leads.

Topoisomerase IB of Deinococcus radiodurans resolves guanine quadruplex DNA structures in vitro.

Deinococcus radiodurans genome contains a large number of guanine repeats interrupted by a few non-guanine bases, termed G motifs. Some of these G motifs were shown forming guanine quadruplex (G4) DNA structure in vitro. How is the formation and relaxation of G4 DNA regulated in the genome of D. radiodurans is not known and is worth investigating. Here, we showed that the topoisomerase Ib of D. radiodurans (DraTopoIB) could change the electrophoretic mobility of fast migrating intramolecular recF-G4 DNA into the slow migrating species. DraTopoIB also reduced the positive ellipticity in circular diachroism (CD) spectra of intramolecular recF-G4 DNA structures stabilized by K+. On the contrary, when DraTopoIB is incubated with G-motifs annealed without K+, it showed neither any change in electrophoretic mobility nor was ellipticity of the CD spectra affected. DNA synthesis by Taq DNA polymerase through G4 DNA structure was attenuated in the presence of G4 DNA binding drugs, which was abrogated by DraTopoIB. This implies that DraTopoIB could destabilize the G4 DNA structure, which is required for G4 drugs binding and stabilization. Camptothecin treatment inhibited DraTopoIB activity on intramolecular G4 DNA structures. These results suggested that DraTopoIB can relax intramolecular G4 DNA structure in vitro and it may be one such protein that could resolve G4 DNA under normal growth conditions in D. radiodurans.

Synthesis, G-quadruplexes DNA binding, and photocytotoxicity of novel cationic expanded porphyrins

Intensive reports allowed the conclusion that molecules with extended aromatic surfaces always do good jobs in the DNA interactions. Inspired by the previous successful researches, herein, we designed a series of cationic porphyrins with expanded planar substituents, and evaluated their binding behaviors to G-quadruplex DNA using the combination of surface-enhanced raman, circular dichroism, absorption spectroscopy and fluorescence resonance energy transfer melting assays. Asymmetrical tetracationic porphyrin with one phenyl-4-N-methyl-4-pyridyl group and three N-methyl-4-pyridyl groups exhibit the best G4-DNA binding affinities among all the designed compounds, suggesting that the bulk of the substituents should be matched to the width of the grooves they putatively lie in. Theoretical calculations applying the density functional theory have been carried out and explain the binding properties of these porphyrins reasonably. Meanwhile, these porphyrins were proved to be potential photochemotherapeutic agents since they have photocytotoxic activities against both myeloma cell (Ag8.653) and gliomas cell (U251) lines.

The maize (Zea mays L.) nucleoside diphosphate kinase1 (ZmNDPK1) gene encodes a human NM23-H2 homologue that binds and stabilizes G-quadruplex DNA.

Noncanonical forms of DNA like the guanine quadruplex (G4) play important roles in regulating transcription and translation through interactions with their protein partners. Although potential G4 elements have been identified in or near genes from species diverse as bacteria, mammals, and plants, little is known about how they might function as cis-regulatory elements or as binding sites for trans-acting protein partners. In fact, until now no G4 binding partners have been identified in the plant kingdom. Here, we report on the cloning and characterization of the first plant-kingdom gene known to encode a G4-binding protein, maize (Zea mays L.) nucleoside diphosphate kinase1 (ZmNDPK1). Structural characterization by X-ray crystallography reveals that it is a homohexamer, akin to other known NDPKs like the human homologue NM23-H2. Further probing into the G4-binding properties of both NDPK homologues suggests that ZmNDPK1 possesses properties distinct from that of NM23-H2, which is known to interact with a G-rich sequence element upstream of the c-myc gene and, in doing so, modulate its expression. Indeed, ZmNDPK1 binds the folded G4 with low nanomolar affinity but corresponding unfolded G-rich DNA more weakly, whereas NM23-H2 binds both folded and unfolded G4 with low nanomolar affinities; nonetheless, both homologues appear to stabilize folded DNAs whether they were prefolded or not. We also demonstrate that the G4-binding activity of ZmNDPK1 is independent of nucleotide binding and kinase activity, suggesting that the G4-binding region and the enzyme active sites are separate. Together, these findings establish a broad evolutionary conservation of some NDPKs as G4-DNA binding enzymes, but with potentially distinct biochemical properties that may reflect divergent evolution or species-specific deployment of these elements in gene regulatory processes.

The intrinsically disordered amino-terminal region of human RecQL4: multiple DNA-binding domains confer annealing, strand exchange and G4 DNA binding.

Human RecQL4 belongs to the ubiquitous RecQ helicase family. Its N-terminal region represents the only homologue of the essential DNA replication initiation factor Sld2 of Saccharomyces cerevisiae, and also participates in the vertebrate initiation of DNA replication. Here, we utilized a random screen to identify N-terminal fragments of human RecQL4 that could be stably expressed in and purified from Escherichia coli. Biophysical characterization of these fragments revealed that the Sld2 homologous RecQL4 N-terminal domain carries large intrinsically disordered regions. The N-terminal fragments were sufficient for the strong annealing activity of RecQL4. Moreover, this activity appeared to be the basis for an ATP-independent strand exchange activity. Both activities relied on multiple DNA-binding sites with affinities to single-stranded, double-stranded and Y-structured DNA. Finally, we found a remarkable affinity of the N-terminus for guanine quadruplex (G4) DNA, exceeding the affinities for other DNA structures by at least 60-fold. Together, these findings suggest that the DNA interactions mediated by the N-terminal region of human RecQL4 represent a central function at the replication fork. The presented data may also provide a mechanistic explanation for the role of elements with a G4-forming propensity identified in the vicinity of vertebrate origins of DNA replication.

Binding of Gemini Bisbenzimidazole Drugs with Human Telomeric G-Quadruplex Dimers: Effect of the Spacer in the Design of Potent Telomerase Inhibitors

The study of anticancer agents that act via stabilization of telomeric G-quadruplex DNA (G4DNA) is important because such agents often inhibit telomerase activity. Several types of G4DNA binding ligands are known. In these studies, the target structures often involve a single G4 DNA unit formed by short DNA telomeric sequences. However, the 3′-terminal single-stranded human telomeric DNA can form higher-order structures by clustering consecutive quadruplex units (dimers or n-mers). Herein, we present new synthetic gemini (twin) bisbenzimidazole ligands, in which the oligo-oxyethylene spacers join the two bisbenzimidazole units for the recognition of both monomeric and dimeric G4DNA, derived from d(T2AG3)4 and d(T2AG3)8 human telomeric DNA, respectively. The spacer between the two bisbenzimidazoles in the geminis plays a critical role in the G4DNA stability. We report here (i) synthesis of new effective gemini anticancer agents that are selectively more toxic towards the cancer cells than the corresponding normal cells; (ii) formation and characterization of G4DNA dimers in solution as well as computational construction of the dimeric G4DNA structures. The gemini ligands direct the folding of the single-stranded DNA into an unusually stable parallel-stranded G4DNA when it was formed in presence of the ligands in KCl solution and the gemini ligands show spacer length dependent potent telomerase inhibition properties.

Screening of a Chemical Library by HT-G4-FID for Discovery of Selective G-quadruplex Binders

Due to the lack of structural guidelines about G-quadruplex ligands, rational design cannot be the only approach to discover potent G4-ligands. As a complementary approach, screening of chemical library may provide interesting scaffolds known as hits provided that specific tools are available. In this work, the Institut Curie-CNRS chemical library was firstly screened by chemoinformatics methods. Similarity estimations by comparison with reference compounds (Phen-DC3, 360A, MMQ12) provided a set of molecules, which were then evaluated by high-throughput G4-FID (HT-G4-FID) against various G-quadruplex DNA. A full investigation of the most interesting molecules, using the HT-G4-FID assay and molecular modeling, supplied an interesting structure-activity relationship confirming the efficiency of this general approach. Overall, we demonstrated that HT-G4-FID coupled with screening of chemical libraries is a powerful tool to identify new G4-DNA binding scaffolds.

Put on your thinking cap: G-quadruplexes, helicases, and telomeres

Understanding how G-quadruplex (G4) DNA structures that form in G-rich tracts of the genome affect chromosomal stability and processes such as copying the genetic information (DNA replication) or decoding the information (RNA transcription) has posed a significant challenge to researchers in the field. Although historically there has been some controversy over the existence of G4 DNA structures in vivo, emerging evidence suggests that they are indeed likely to form and have cellular consequences. In a recent study, Smith et al. investigated a role of G4 DNA in telomere capping [1], i.e., the adaptation of a nucleoprotein structure that prevents the chromosomal DNA ends from being recognized as DNA breaks and protects them from becoming degraded or fused. Telomere capping is a fairly complex process since a number of proteins have been shown to bind telomeric single-stranded or double-stranded DNA at the chromosome end. Moreover, the ability of telomeric DNA to form a variety of conformations including t-loops and G-quadruplexes adds to the complexity of how competing proteins and DNA structures influence the structural topology and metabolism of chromosome ends [2]. Using genetic and pharmacological approaches, Smith et al. showed that under conditions that stabilize G4 DNA structures, which form from a guanine-rich telomeric ssDNA exposed in a yeast temperature sensitive cdc13-1 mutant, telomere capping is in turn enhanced and phenotypes associated with capping defects are suppressed [1]. Conversely, telomere uncapping occurs under conditions that dissuade the formation of telomeric G4 DNA in the cdc13-1 mutant. The authors proposed a model in which G4 DNA structure enables G4 DNA binding proteins to further stabilize the telomere end by binding to G-quadruplex DNA, thereby preventing 5′ to 3′ exonucleolytic resection when the normal protein that blocks telomeric end processing is defective. Rad53-mediated checkpoint activation is also dampened, permitting suppression of the growth defects characteristic of the cdc13-1 mutant at the nonpermissive temperature.

Rudimentary G-quadruplex–based telomere capping in Saccharomyces cerevisiae

Telomere capping conceals chromosome ends from exonucleases and checkpoints, but the full range of capping mechanisms is not well defined. Telomeres have the potential to form G-quadruplex (G4) DNA, although evidence for telomere G4 DNA function in vivo is limited. In budding yeast, capping requires the Cdc13 protein and is lost at nonpermissive temperatures in cdc13-1 mutants. Here, we use several independent G4 DNA-stabilizing treatments to suppress cdc13-1 capping defects. These include overexpression of three different G4 DNA binding proteins, loss of the G4 DNA unwinding helicase Sgs1, or treatment with small molecule G4 DNA ligands. In vitro, we show that protein-bound G4 DNA at a 3' overhang inhibits 5'→3' resection of a paired strand by exonuclease I. These findings demonstrate that, at least in the absence of full natural capping, G4 DNA can play a positive role at telomeres in vivo.