Potentiometric titrations to study ligand interactions with DNA i-motifs.

i-Motifs are non-canonical secondary structures of DNA formed by mutual intercalation of hemi-protonated cytosine-cytosine base pairs, most typically in slightly acidic conditions (pH<7.0). These structures are well-studied in vitro and have recently been suggested to exist in cells. Despite nearly a decade of active research, the quest for small-molecule ligands that could selectively bind to and stabilize i-motifs continues, and no reference, bona fide i-motif ligand is currently available. This is, at least in part, due to the lack of robust methods to assess the interaction of ligands with i-motifs, since many techniques well-established for studies of other secondary structures (such as CD-, UV-, and FRET-melting) may generate artifacts when applied to i-motifs. Here, we describe an implementation of automated, potentiometric (pH) titrations as a robust isothermal method to assess the impact of ligands or cosolutes on thermodynamic stability of i-motifs. This approach is validated through the use of a cosolute previously known to stabilize i-motifs (PEG2000) and three small-molecule ligands that are able to stabilize, destabilize, or have no effect on the stability of i-motifs, respectively.

Integrated High-Throughput Screening and Large-Scale Isobolographic Analysis to Accelerate the Discovery of Radiosensitizers With Greater Selectivity for Cancer Cells.

PURPOSE: High-throughput screening (HTS) platforms have been widely used to identify candidate anticancer drugs and drug-drug combinations; however, HTS-based identification of new drug-ionizing radiation (IR) combinations has rarely been reported. Herein, we developed an integrated approach including cell-based HTS and computational large-scale isobolographic analysis to accelerate the identification of radiosensitizing compounds acting strongly and more specifically on cancer cells. METHODS AND MATERIALS: In a 384-well plate format, 160 compounds likely to interfere with the cell response to radiation were screened on human glioblastoma (U251-MG) and cervix carcinoma (ME-180) cell lines, as well as on normal fibroblasts (CCD-19Lu). After drug exposure, cells were irradiated or not and short-term cell survival was assessed by high-throughput cell microscopy. Computational large-scale dose-response and isobolographic approach were used to identify promising synergistic drugs radiosensitizing cancer cells rather than normal cells. Synergy of a promising compound was confirmed on ME-180 cells by an independent 96-well assay protocol, and finally, by the gold-standard colony forming assay. RESULTS: We retained 4 compounds synergistic at 2 isoeffects in U251-MG and ME-180 cell lines and 11 compounds synergistically effective in only one cancer cell line. Among these 15 promising radiosensitizers, 5 compounds showed limited toxicity combined or not with IR on normal fibroblasts. CONCLUSIONS: Overall, this study demonstrated that HTS chemoradiation screening together with large-scale computational analysis is an efficient tool to identify synergistic drug-IR combinations, with concomitant assessment of unwanted toxicity on normal fibroblasts. It sparks expectations to accelerate the discovery of highly desired agents improving the therapeutic index of radiation therapy.

Unprecedented reactivity of polyamines with aldehydic DNA modifications: structural determinants of reactivity, characterization and enzymatic stability of adducts.

Apurinic/apyrimidinic (AP) sites, 5-formyluracil (fU) and 5-formylcytosine (fC) are abundant DNA modifications that share aldehyde-type reactivity. Here, we demonstrate that polyamines featuring at least one secondary 1,2-diamine fragment in combination with aromatic units form covalent DNA adducts upon reaction with AP sites (with concomitant cleavage of the AP strand), fU and, to a lesser extent, fC residues. Using small-molecule mimics of AP site and fU, we show that reaction of secondary 1,2-diamines with AP sites leads to the formation of unprecedented 3'-tetrahydrofuro[2,3,4-ef]-1,4-diazepane ('ribodiazepane') scaffold, whereas the reaction with fU produces cationic 2,3-dihydro-1,4-diazepinium adducts via uracil ring opening. The reactivity of polyamines towards AP sites versus fU and fC can be tuned by modulating their chemical structure and pH of the reaction medium, enabling up to 20-fold chemoselectivity for AP sites with respect to fU and fC. This reaction is efficient in near-physiological conditions at low-micromolar concentration of polyamines and tolerant to the presence of a large excess of unmodified DNA. Remarkably, 3'-ribodiazepane adducts are chemically stable and resistant to the action of apurinic/apyrimidinic endonuclease 1 (APE1) and tyrosyl-DNA phosphoesterase 1 (TDP1), two DNA repair enzymes known to cleanse a variety of 3' end-blocking DNA lesions.

Alternative splicing of BCL-x is controlled by RBM25 binding to a G-quadruplex in BCL-x pre-mRNA.

BCL-x is a master regulator of apoptosis whose pre-mRNA is alternatively spliced into either a long (canonical) anti-apoptotic Bcl-xL isoform, or a short (alternative) pro-apoptotic Bcl-xS isoform. The balance between these two antagonistic isoforms is tightly regulated and overexpression of Bcl-xL has been linked to resistance to chemotherapy in several cancers, whereas overexpression of Bcl-xS is associated to some forms of diabetes and cardiac disorders. The splicing factor RBM25 controls alternative splicing of BCL-x: its overexpression favours the production of Bcl-xS, whereas its downregulation has the opposite effect. Here we show that RBM25 directly and specifically binds to GQ-2, an RNA G-quadruplex (rG4) of BCL-x pre-mRNA that forms at the vicinity of the alternative 5' splice site leading to the alternative Bcl-xS isoform. This RBM25/rG4 interaction is crucial for the production of Bcl-xS and depends on the RE (arginine-glutamate-rich) motif of RBM25, thus defining a new type of rG4-interacting domain. PhenDC3, a benchmark G4 ligand, enhances the binding of RBM25 to the GQ-2 rG4 of BCL-x pre-mRNA, thereby promoting the alternative pro-apoptotic Bcl-xS isoform and triggering apoptosis. Furthermore, the screening of a combinatorial library of 90 putative G4 ligands led to the identification of two original compounds, PhenDH8 and PhenDH9, superior to PhenDC3 in promoting the Bcl-xS isoform and apoptosis. Thus, favouring the interaction between RBM25 and the GQ-2 rG4 of BCL-x pre-mRNA represents a relevant intervention point to re-sensitize cancer cells to chemotherapy.

Solution and Gas-Phase Stability of DNA Junctions from Temperature-Controlled Electrospray Ionization and Surface-Induced Dissociation.

DNA three-way junction (TWJ) structures transiently form during key cellular processes such as transcription, replication, and DNA repair. Despite their significance, the thermodynamics of TWJs, including the influence of strand length, base pair composition, and ligand binding on TWJ stability and dissociation mechanisms, are poorly understood. To address these questions, we interfaced temperature-controlled nanoelectrospray ionization mass spectrometry (TC-nESI-MS) with a cyclic ion mobility spectrometry (cIMS) instrument that was also equipped with a surface-induced dissociation (SID) stage. This novel combination allowed us to investigate the structural intermediates of three TWJ complexes and examine the effects of GC base pairs on their dissociation pathways. We found that two TWJ-specific ligands, 2,7-tris-naphthalene (2,7-TrisNP) and tris-phenoxybenzene (TrisPOB), lead to TWJ stabilization, revealed by an increase in the melting temperature (Tm) by 13 or 26 °C, respectively. To gain insights into conformational changes in the gas phase, we employed cIMS and SID to analyze TWJs and their complexes with ligands. Analysis of IM arrival distributions suggested a single-step dissociation of TWJs and their intermediates for the three studied TWJ complexes. Upon ligand binding, a higher SID energy by 3 V (2,7-TrisNP) and 5 V (TrisPOB) was required to induce 50% dissociation of TWJ, compared to 38 V in the absence of ligands. Our results demonstrate the power of utilizing TC-nESI-MS in combination with cIMS and SID for thermodynamic characterization of TWJ complexes and investigation of ligand binding. These techniques are essential for the TWJ design and development as drug targets, aptamers, and structural units for functional biomaterials.

The hide-and-seek game of the oncogenic Epstein-Barr virus-encoded EBNA1 protein with the immune system: An RNA G-quadruplex tale.

The Epstein-Barr virus (EBV) is the first oncogenic virus described in human. EBV infects more than 90% of the human population worldwide, but most EBV infections are asymptomatic. After the primary infection, the virus persists lifelong in the memory B cells of the infected individuals. Under certain conditions the virus can cause several human cancers, that include lymphoproliferative disorders such as Burkitt and Hodgkin lymphomas and non-lymphoid malignancies such as 100% of nasopharyngeal carcinoma and 10% of gastric cancers. Each year, about 200,000 EBV-related cancers emerge, hence accounting for at least 1% of worldwide cancers. Like all gammaherpesviruses, EBV has evolved a strategy to escape the host immune system. This strategy is mainly based on the tight control of the expression of its Epstein-Barr nuclear antigen-1 (EBNA1) protein, the EBV-encoded genome maintenance protein. Indeed, EBNA1 is essential for viral genome replication and maintenance but, at the same time, is also highly antigenic and T cells raised against EBNA1 exist in infected individuals. For this reason, EBNA1 is considered as the Achilles heel of EBV and the virus has seemingly evolved a strategy that employs the binding of nucleolin, a host cell factor, to RNA G-quadruplex (rG4) within EBNA1 mRNA to limit its expression to the minimal level required for function while minimizing immune recognition. This review recapitulates in a historical way the knowledge accumulated on EBNA1 immune evasion and discusses how this rG4-dependent mechanism can be exploited as an intervention point to unveil EBV-related cancers to the immune system.

Guidelines for G-quadruplexes: I. In vitro characterization.

Besides the well-known DNA double-helix, non-canonical nucleic acid structures regulate crucial biological activities. Among these oddities, guanine-rich DNA sequences can form unusual four-stranded secondary structures called G-quadruplexes (G4s). G4-prone sequences have been found in the genomes of most species, and G4s play important roles in essential processes such as transcription, replication, genome integrity and epigenetic regulation. Here, we present a short overview of G-quadruplexes followed by a detailed description of the biophysical and biochemical methods used to characterize G4s in vitro. The principles, experimental details and possible shortcomings of each method are discussed to provide a comprehensive view of the techniques used to study these structures. We aim to provide a set of guidelines for standardizing research on G-quadruplexes; these guidelines are not meant to be a dogmatic set of rules, but should rather provide useful information on the methods currently used to study these fascinating motifs.

Novel Synthesis of IMC-48 and Affinity Evaluation with Different i-Motif DNA Sequences.

During the last decade, the evidence for the biological relevance of i-motif DNA (i-DNA) has been accumulated. However, relatively few molecules were reported to interact with i-DNA, and a controversy concerning their binding mode, affinity, and selectivity persists in the literature. In this context, the cholestane derivative IMC-48 has been reported to modulate bcl-2 gene expression by stabilizing an i-motif structure in its promoter. In the present contribution, we report on a novel, more straightforward, synthesis of IMC-48 requiring fewer steps compared to the previous approach. Furthermore, the interaction of IMC-48 with four different i-motif DNA sequences was thoroughly investigated by bio-layer interferometry (BLI) and circular dichroism (CD) spectroscopy. Surprisingly, our results show that IMC-48 is a very weak ligand of i-DNA as no quantifiable interaction or significant stabilization of i-motif structures could be observed, stimulating a quest for an alternative mechanism of its biological activity.

Optimization of G-Quadruplex Ligands through a SAR Study Combining Parallel Synthesis and Screening of Cationic Bis(acylhydrazones).

G-quadruplexes (G4s), secondary structures adopted by guanine-rich DNA and RNA sequences, are implicated in numerous biological processes and have been suggested as potential drug targets. Accordingly, there is an increasing interest in developing high-throughput methods that allow the generation of congeneric series of G4-targeting molecules ("ligands") and investigating their interactions with the targets. We have developed an operationally simple method of parallel synthesis to generate "ready-to-screen" libraries of cationic acylhydrazones, a motif that we have previously identified as a promising scaffold for potent, biologically active G4 ligands. Combined with well-established screening techniques, such as fluorescence melting, this method enables the rapid synthesis and screening of combinatorial libraries of potential G4 ligands. Following this protocol, we synthesized a combinatorial library of 90 bis(acylhydrazones) and screened it against five different nucleic acid structures. This way, we were able to analyze the structure-activity relationships within this series of G4 ligands, and identified three novel promising ligands whose interactions with G4-DNAs of different topologies were studied in detail by a combination of several biophysical techniques, including native mass spectrometry, and molecular modeling.

Assessment of presumed small-molecule ligands of telomeric i-DNA by biolayer interferometry (BLI).

Biolayer interferometry (BLI) and circular dichroism (CD) spectroscopy were used to investigate the interaction between previously reported i-motif DNA (i-DNA) ligands and folded or unfolded i-DNA in acidic (pH 5.5) and near-neutral (pH 6.5) conditions. We observed that although several ligands, in particular macrocyclic bis-acridine (BisA) and pyridostatin (PDS), showed good affinities for the telomeric i-motif forming sequence, none of the ligands displayed selective interactions with the i-DNA structure nor was able to promote its formation.

Harnessing an emissive guanine surrogate to design small-molecule fluorescent chemosensors of O6-methylguanine-DNA-methyltransferase (MGMT).

The fluorescence properties of an emissive guanine surrogate, thienoguanine (thGN, 2-aminothieno[3,4-d]pyrimidin-4(3H)-one), were exploited to design two real-time chemosensors of O6-methylguanine-DNA-methyltransferase (MGMT), a key DNA repair enzyme involved in the resistance to DNA-alkylating anti-cancer drugs though direct reversal of O6-alkylated guanine adducts.

The different activities of RNA G-quadruplex structures are controlled by flanking sequences.

The role of G-quadruplex (G4) RNA structures is multifaceted and controversial. Here, we have used as a model the EBV-encoded EBNA1 and the Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded LANA1 mRNAs. We have compared the G4s in these two messages in terms of nucleolin binding, nuclear mRNA retention, and mRNA translation inhibition and their effects on immune evasion. The G4s in the EBNA1 message are clustered in one repeat sequence and the G4 ligand PhenDH2 prevents all G4-associated activities. The RNA G4s in the LANA1 message take part in similar multiple mRNA functions but are spread throughout the message. The different G4 activities depend on flanking coding and non-coding sequences and, interestingly, can be separated individually. Together, the results illustrate the multifunctional, dynamic and context-dependent nature of G4 RNAs and highlight the possibility to develop ligands targeting specific RNA G4 functions. The data also suggest a common multifunctional repertoire of viral G4 RNA activities for immune evasion.

Acridine-O6-benzylguanine hybrids: Synthesis, DNA binding, MGMT inhibition and antiproliferative activity.

O6-Methylguanine-DNA-methyltransferase (MGMT) is a key DNA repair enzyme involved in chemoresistance to DNA-alkylating anti-cancer drugs such as Temozolomide (TMZ) through direct repair of drug-induced O6-methylguanine residues in DNA. MGMT substrate analogues, such as O6-benzylguanine (BG), efficiently inactivate MGMT in vitro and in cells; however, these drugs failed to reach the clinic due to adverse side effects. Here, we designed hybrid drugs combining a BG residue covalently linked to a DNA-interacting moiety (6-chloro-2-methoxy-9-aminoacridine). Specifically, two series of hybrids, encompassing three compounds each, were obtained by varying the position of the attachment point of BG (N9 of guanine vs. the benzyl group) and the length and nature of the linker. UV/vis absorption and fluorescence data indicate that all six hybrids adopt an intramolecularly stacked conformation in aqueous solutions in a wide range of temperatures. All hybrids interact with double-stranded DNA, as clearly evidenced by spectrophotometric titrations, without intercalation of the acridine ring and do not induce thermal stabilization of the duplex. All hybrids, as well as the reference DNA intercalator (6-chloro-2-methoxy-9-aminoacridine 8), irreversibly inhibit MGMT in vitro with variable efficiency, comparable to that of BG. In a multidrug-resistant glioblastoma cell line T98G, benzyl-linked hybrids 7a-c and the N9-linked hybrid 19b are moderately cytotoxic (GI50 ≥ 15 µM after 96 h), while N9-linked hybrids 19a and 19c are strongly cytotoxic (GI50 = 1-2 µM), similarly to acridine 8 (GI50 = 0.6 µM). Among all compounds, hybrids 19a and 19c, similarly to BG, display synergic cytotoxic effect upon co-treatment with subtoxic doses of TMZ, with combination index (CI) values as low as 0.2-0.3. In agreement with in vitro results, compound 19a inactivates cellular MGMT but, unlike BG, does not induce significant levels of DNA damage, either alone or in combination with TMZ, as indicated by the results of γH2AX immunostaining experiments. Instead, and unlike BG, compound 19a alone induces significant apoptosis of T98G cells, which is not further increased in a combination with TMZ. These results indicate that molecular mechanisms underlying the cytotoxicity of 19a and its combination with TMZ are distinct from that of BG. The strongly synergic properties of this combination represent an interesting therapeutic opportunity in treating TMZ-resistant cancers.

Dual targeting of higher-order DNA structures by azacryptands induces DNA junction-mediated DNA damage in cancer cells.

DNA is intrinsically dynamic and folds transiently into alternative higher-order structures such as G-quadruplexes (G4s) and three-way DNA junctions (TWJs). G4s and TWJs can be stabilised by small molecules (ligands) that have high chemotherapeutic potential, either as standalone DNA damaging agents or combined in synthetic lethality strategies. While previous approaches have claimed to use ligands that specifically target either G4s or TWJs, we report here on a new approach in which ligands targeting both TWJs and G4s in vitro demonstrate cellular effects distinct from that of G4 ligands, and attributable to TWJ targeting. The DNA binding modes of these new, dual TWJ-/G4-ligands were studied by a panel of in vitro methods and theoretical simulations, and their cellular properties by extensive cell-based assays. We show here that cytotoxic activity of TWJ-/G4-ligands is mitigated by the DNA damage response (DDR) and DNA topoisomerase 2 (TOP2), making them different from typical G4-ligands, and implying a pivotal role of TWJs in cells. We designed and used a clickable ligand, TrisNP-α, to provide unique insights into the TWJ landscape in cells and its modulation upon co-treatments. This wealth of data was exploited to design an efficient synthetic lethality strategy combining dual ligands with clinically relevant DDR inhibitors.

Dye-functionalized phosphate-binding macrocycles: from nucleotide to G-quadruplex recognition and "turn-on" fluorescence sensing.

A novel strategy to design "turn-on" fluorescent receptors for G-quadruplexes of DNA is presented, which relies on the connection of phosphate binding macrocycles (PBM) with naphthalimide dyes. A new PBM-dye family was synthesized and evaluated in terms of binding and detection of nucleotides and DNA G-quadruplexes of different topologies.

Identifying G-Quadruplex-DNA-Disrupting Small Molecules.

The quest for small molecules that strongly bind to G-quadruplex-DNA (G4), so-called G4 ligands, has invigorated the G4 research field from its very inception. Massive efforts have been invested to discover or rationally design G4 ligands, evaluate their G4-interacting properties in vitro through a series of now widely accepted and routinely implemented assays, and use them as innovative chemical biology tools to interrogate cellular networks that might involve G4s. In sharp contrast, only uncoordinated efforts aimed at developing small molecules that destabilize G4s have been invested to date, even though it is now recognized that such molecular tools would have tremendous application in neurobiology as many genetic and age-related diseases are caused by an overrepresentation of G4s. Herein, we report on our efforts to develop in vitro assays to reliably identify molecules able to destabilize G4s. This workflow comprises the newly designed G4-unfold assay, adapted from the G4-helicase assay implemented with Pif1, as well as a series of biophysical and biochemical techniques classically used to study G4/ligand interactions (CD, UV-vis, PAGE, and FRET-melting), and a qPCR stop assay, adapted from a Taq-based protocol recently used to identify G4s in the genomic DNA of Schizosaccharomyces pombe. This unique, multipronged approach leads to the characterization of a phenylpyrrolocytosine (PhpC)-based G-clamp analog as a prototype of G4-disrupting small molecule whose properties are validated through many different and complementary in vitro evaluations.

SARS-CoV-2 Nsp3 unique domain SUD interacts with guanine quadruplexes and G4-ligands inhibit this interaction.

The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds.

Quadruplex DNA-guided ligand selection from dynamic combinatorial libraries of acylhydrazones.

Dynamic combinatorial libraries of acylhydrazones were prepared from diacylhydrazides and several cationic or neutral aldehydes in the presence of 5-methoxyanthranilic acid catalyst. Pull-down experiments with magnetic beads functionalized with a G-quadruplex (G4)-forming oligonucleotide led to the identification of putative ligands, which were resynthesized or emulated by close structural analogues. G4-binding properties of novel derivatives were assessed by fluorimetric titrations, mass spectrometry and thermal denaturation experiments, giving evidence of strong binding (Kd < 10 nM) for two compounds.

FRET-MC: A fluorescence melting competition assay for studying G4 structures in vitro.

G-quadruplexes (G4) play crucial roles in biology, analytical chemistry and nanotechnology. The stability of G4 structures is impacted by the number of G-quartets, the length and positions of loops, flanking motifs, as well as additional structural elements such as bulges, capping base pairs, or triads. Algorithms such as G4Hunter or Quadparser may predict if a given sequence is G4-prone by calculating a quadruplex propensity score; however, experimental validation is still required. We previously demonstrated that this validation is not always straightforward, and that a combination of techniques is often required to unambiguously establish whether a sequence forms a G-quadruplex or not. In this article, we adapted the well-known FRET-melting assay to characterize G4 in batch, where the sequence to be tested is added, as an unlabeled competitor, to a system composed of a dual-labeled probe (F21T) and a specific quadruplex ligand. PhenDC3 was preferred over TMPyP4 because of its better selectivity for G-quadruplexes. In this so-called FRET-MC (melting competition) assay, G4-forming competitors lead to a marked decrease of the ligand-induced stabilization effect (∆Tm ), while non-specific competitors (e.g., single- or double-stranded sequences) have little effect. Sixty-five known sequences with different typical secondary structures were used to validate the assay, which was subsequently employed to assess eight novel sequences that were not previously characterized.

Harnessing intrinsic fluorescence for typing of secondary structures of DNA.

High-throughput investigation of structural diversity of nucleic acids is hampered by the lack of suitable label-free methods, combining fast and cheap experimental workflow with high information content. Here, we explore the use of intrinsic fluorescence emitted by nucleic acids for this scope. After a preliminary assessment of suitability of this phenomenon for tracking conformational changes of DNA, we examined steady-state emission spectra of an 89-membered set of oligonucleotides with reported conformation (G-quadruplexes (G4s), i-motifs, single- and double-strands) by means of multivariate analysis. Principal component analysis of emission spectra resulted in successful clustering of oligonucleotides into three corresponding conformational groups, without discrimination between single- and double-stranded structures. Linear discriminant analysis was exploited for the assessment of novel sequences, allowing the evaluation of their G4-forming propensity. Our method does not require any labeling agent or dye, avoiding the related bias, and can be utilized to screen novel sequences of interest in a high-throughput and cost-effective manner. In addition, we observed that left-handed (Z-) G4 structures were systematically more fluorescent than most other G4 structures, almost reaching the quantum yield of 5'-d[(G3T)3G3]-3' (G3T, the most fluorescent G4 structure reported to date).