Normal and pathogenic variation of RFC1 repeat expansions: implications for clinical diagnosis.

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) is an autosomal recessive neurodegenerative disease, usually caused by biallelic AAGGG repeat expansions in RFC1. In this study, we leveraged whole genome sequencing data from nearly 10 000 individuals recruited within the Genomics England sequencing project to investigate the normal and pathogenic variation of the RFC1 repeat. We identified three novel repeat motifs, AGGGC (n = 6 from five families), AAGGC (n = 2 from one family) and AGAGG (n = 1), associated with CANVAS in the homozygous or compound heterozygous state with the common pathogenic AAGGG expansion. While AAAAG, AAAGGG and AAGAG expansions appear to be benign, we revealed a pathogenic role for large AAAGG repeat configuration expansions (n = 5). Long-read sequencing was used to characterize the entire repeat sequence, and six patients exhibited a pure AGGGC expansion, while the other patients presented complex motifs with AAGGG or AAAGG interruptions. All pathogenic motifs appeared to have arisen from a common haplotype and were predicted to form highly stable G quadruplexes, which have previously been demonstrated to affect gene transcription in other conditions. The assessment of these novel configurations is warranted in CANVAS patients with negative or inconclusive genetic testing. Particular attention should be paid to carriers of compound AAGGG/AAAGG expansions when the AAAGG motif is very large (>500 repeats) or the AAGGG motif is interrupted. Accurate sizing and full sequencing of the satellite repeat with long-read sequencing is recommended in clinically selected cases to enable accurate molecular diagnosis and counsel patients and their families.

The MDM2 inducible promoter folds into four-tetrad antiparallel G-quadruplexes targetable to fight malignant liposarcoma.

Well-differentiated liposarcoma (WDLPS) is a malignant neoplasia hard to diagnose and treat. Its main molecular signature is amplification of the MDM2-containing genomic region. The MDM2 oncogene is the master regulator of p53: its overexpression enhances p53 degradation and inhibits apoptosis, leading to the tumoral phenotype. Here, we show that the MDM2 inducible promoter G-rich region folds into stable G-quadruplexes both in vitro and in vivo and it is specifically recognized by cellular helicases. Cell treatment with G-quadruplex-ligands reduces MDM2 expression and p53 degradation, thus stimulating cancer cell cycle arrest and apoptosis. Structural characterization of the MDM2 G-quadruplex revealed an extraordinarily stable, unique four-tetrad antiparallel dynamic conformation, amenable to selective targeting. These data indicate the feasibility of an out-of-the-box G-quadruplex-targeting approach to defeat WDLPS and all tumours where restoration of wild-type p53 is sought. They also point to G-quadruplex-dependent genomic instability as possible cause of MDM2 expansion and WDLPS tumorigenesis.

Lights on 2,5-diaryl tetrazoles: applications and limits of a versatile photoclick reaction.

Recently, photoclick chemistry emerged as a powerful tool employed in several research fields, from medicinal chemistry and biology to material sciences. The growing interest in this type of chemical process is justified by the possibility to produce complex molecular systems using mild reaction conditions. However, the elevated spatio-temporal control offered by photoclick chemistry is highly intriguing, as it expands the range of applications. In this context, the light-triggered reaction of 2,5-diaryl tetrazoles with dipolarophiles emerged for its interesting features: excellent stability of the substrates, fast reaction kinetic, and the formation of a highly fluorescent product, fundamental for sensing applications. In the last years, 2,5-diaryl tetrazoles have been extensively employed, especially for bioorthogonal ligations, to label biomolecules and nucleic acids. In this review, we summarized recent applications of this interesting photoclick reaction, with a particular focus on biological fields. Moreover, we described the main limits that affect this system and current strategies proposed to overcome these issues. The general discussion here presented could prompt further optimization of the process and pave the way for the development of new original structures and innovative applications.

Selective targeting of mutually exclusive DNA G-quadruplexes: HIV-1 LTR as paradigmatic model.

Targeting of G-quadruplexes, non-canonical conformations that form in G-rich regions of nucleic acids, has been proposed as a novel therapeutic strategy toward several diseases, including cancer and infections. The unavailability of highly selective molecules targeting a G-quadruplex of choice has hampered relevant applications. Herein, we describe a novel approach, based on naphthalene diimide (NDI)-peptide nucleic acid (PNA) conjugates, taking advantage of the cooperative interaction of the NDI with the G-quadruplex structure and hybridization of the PNA with the flanking region upstream or downstream the targeted G-quadruplex. By biophysical and biomolecular assays, we show that the NDI-PNA conjugates are able to specifically recognize the G-quadruplex of choice within the HIV-1 LTR region, consisting of overlapping and therefore mutually exclusive G-quadruplexes. Additionally, the conjugates can induce and stabilize the least populated G-quadruplex at the expenses of the more stable ones. The general and straightforward design and synthesis, which readily apply to any G4 target of choice, together with both the red-fluorescent emission and the possibility to introduce cellular localization signals, make the novel conjugates available to selectively control G-quadruplex folding over a wide range of applications.

High Flexibility of RNaseH2 Catalytic Activity with Respect to Non-Canonical DNA Structures.

Ribonucleotides misincorporated in the human genome are the most abundant DNA lesions. The 2'-hydroxyl group makes them prone to spontaneous hydrolysis, potentially resulting in strand breaks. Moreover, their presence may decrease the rate of DNA replication causing replicative fork stalling and collapse. Ribonucleotide removal is initiated by Ribonuclease H2 (RNase H2), the key player in Ribonucleotide Excision Repair (RER). Its absence leads to embryonic lethality in mice, while mutations decreasing its activity cause Aicardi-Goutières syndrome. DNA geometry can be altered by DNA lesions or by peculiar sequences forming secondary structures, like G-quadruplex (G4) and trinucleotide repeats (TNR) hairpins, which significantly differ from canonical B-form. Ribonucleotides pairing to lesioned nucleotides, or incorporated within non-B DNA structures could avoid RNase H2 recognition, potentially contributing to genome instability. In this work, we investigate the ability of RNase H2 to process misincorporated ribonucleotides in a panel of DNA substrates showing different geometrical features. RNase H2 proved to be a flexible enzyme, recognizing as a substrate the majority of the constructs we generated. However, some geometrical features and non-canonical DNA structures severely impaired its activity, suggesting a relevant role of misincorporated ribonucleotides in the physiological instability of specific DNA sequences.

DNA Binding Mode Analysis of a Core-Extended Naphthalene Diimide as a Conformation-Sensitive Fluorescent Probe of G-Quadruplex Structures.

G-quadruplex existence was proved in cells by using both antibodies and small molecule fluorescent probes. However, the G-quadruplex probes designed thus far are structure- but not conformation-specific. Recently, a core-extended naphthalene diimide (cex-NDI) was designed and found to provide fluorescent signals of markedly different intensities when bound to G-quadruplexes of different conformations or duplexes. Aiming at evaluating how the fluorescence behaviour of this compound is associated with specific binding modes to the different DNA targets, cex-NDI was here studied in its interaction with hybrid G-quadruplex, parallel G-quadruplex, and B-DNA duplex models by biophysical techniques, molecular docking, and biological assays. cex-NDI showed different binding modes associated with different amounts of stacking interactions with the three DNA targets. The preferential binding sites were the groove, outer quartet, or intercalative site of the hybrid G-quadruplex, parallel G-quadruplex, and B-DNA duplex, respectively. Interestingly, our data show that the fluorescence intensity of DNA-bound cex-NDI correlates with the amount of stacking interactions formed by the ligand with each DNA target, thus providing the rationale behind the conformation-sensitive properties of cex-NDI and supporting its use as a fluorescent probe of G-quadruplex structures. Notably, biological assays proved that cex-NDI mainly localizes in the G-quadruplex-rich nuclei of cancer cells.

Effects of the Combined Treatment with a G-Quadruplex-Stabilizing Ligand and Photon Beams on Glioblastoma Stem-like Cells: A Magnetic Resonance Study.

Glioblastoma multiforme is a malignant primary brain tumor with a poor prognosis and high rates of chemo-radiotherapy failure, mainly due to a small cell fraction with stem-like properties (GSCs). The mechanisms underlying GSC response to radiation need to be elucidated to enhance sensitivity to treatments and to develop new therapeutic strategies. In a previous study, two GSC lines, named line #1 and line #83, responded differently to carbon ions and photon beams, with the differences likely attributable to their own different metabolic fingerprint rather than to radiation type. Data from the literature showed the capability of RHPS4, a G-quadruplex stabilizing ligand, to sensitize the glioblastoma radioresistant U251MG cells to X-rays. The combined metabolic effect of ligand #190, a new RHPS4-derivative showing reduced cardiotoxicity, and a photon beam has been monitored by magnetic resonance (MR) spectroscopy for the two GSC lines, #1 and #83, to reveal whether a synergistic response occurs. MR spectra from both lines were affected by single and combined treatments, but the variations of the analysed metabolites were statistically significant mainly in line #1, without synergistic effects due to combination. The multivariate analysis of ten metabolites shows a separation between control and treated samples in line #1 regardless of treatment type, while separation was not detected in line #83.

Selective Binding and Redox-Activity on Parallel G-Quadruplexes by Pegylated Naphthalene Diimide-Copper Complexes.

G-quadruplexes (G4s) are higher-order supramolecular structures, biologically important in the regulation of many key processes. Among all, the recent discoveries relating to RNA-G4s, including their potential involvement as antiviral targets against COVID-19, have triggered the ever-increasing need to develop selective molecules able to interact with parallel G4s. Naphthalene diimides (NDIs) are widely exploited as G4 ligands, being able to induce and strongly stabilize these structures. Sometimes, a reversible NDI-G4 interaction is also associated with an irreversible one, due to the cleavage and/or modification of G4s by functional-NDIs. This is the case of NDI-Cu-DETA, a copper(II) complex able to cleave G4s in the closest proximity to the target binding site. Herein, we present two original Cu(II)-NDI complexes, inspired by NDI-Cu-DETA, differently functionalized with 2-(2-aminoethoxy)ethanol side-chains, to selectively drive redox-catalyzed activity towards parallel G4s. The selective interaction toward parallel G4 topology, controlled by the presence of 2-(2-aminoethoxy)ethanol side chains, was already firmly demonstrated by us using core-extended NDIs. In the present study, the presence of protonable moieties and the copper(II) cavity, increases the binding affinity and specificity of these two NDIs for a telomeric RNA-G4. Once defined the copper coordination relationship and binding constants by competition titrations, ability in G4 stabilization, and ROS-induced cleavage were analyzed. The propensity in the stabilization of parallel topology was highlighted for both of the new compounds HP2Cu and PE2Cu. The results obtained are particularly promising, paving the way for the development of new selective functional ligands for binding and destructuring parallel G4s.

More is not always better: finding the right trade-off between affinity and selectivity of a G-quadruplex ligand.

Guanine-rich nucleic acid sequences can fold into four-stranded G-quadruplex (G4) structures. Despite growing evidence for their biological significance, considerable work still needs to be done to detail their cellular occurrence and functions. Herein, we describe an optimized core-extended naphthalene diimide (cex-NDI) to be exploited as a G4 light-up sensor. The sensing mechanism relies on the shift of the aggregate-monomer equilibrium towards the bright monomeric state upon G4 binding. In contrast with the majority of other ligands, this novel cex-NDI is able to discriminate among G4s with different topologies, with a remarkable fluorescent response for the parallel ones. We investigate this sensing by means of biophysical methods, comparing the lead compound to a non-selective analogue. We demonstrate that mitigating the affinity of the binding core for G4s results in an increased selectivity and sensitivity of the fluorescent response. This is achieved by replacing positively charged substituents with diethylene glycol (DEG) side chains. Remarkably, the limit of detection values obtained for parallel G4s are more than one order of magnitude lower than those of the parallel-selective ligand N-methyl mesoporphyrin IX (NMM). Interestingly, the classical fluorescent intercalator displacement (FID) assay failed to reveal binding of cex-NDI to G4 because of the presence a ternary complex (G4-TO-cex-NDI) revealed by electrospray-MS. Our study thus provides a rational basis to design or modify existent scaffolds to redirect the binding preference of G4 ligands.

Groundbreaking Anticancer Activity of Highly Diversified Oxadiazole Scaffolds.

Nowadays, an increasing number of heterocyclic-based drugs found application in medicinal chemistry and, in particular, as anticancer agents. In this context, oxadiazoles-five-membered aromatic rings-emerged for their interesting biological properties. Modification of oxadiazole scaffolds represents a valid strategy to increase their anticancer activity, especially on 1,2,4 and 1,3,4 regioisomers. In the last years, an increasing number of oxadiazole derivatives, with remarkable cytotoxicity for several tumor lines, were identified. Structural modifications, that ensure higher cytotoxicity towards malignant cells, represent a solid starting point in the development of novel oxadiazole-based drugs. To increase the specificity of this strategy, outstanding oxadiazole scaffolds have been designed to selectively interact with biological targets, including enzymes, globular proteins, and nucleic acids, showing more promising antitumor effects. In the present work, we aim to provide a comprehensive overview of the anticancer activity of these heterocycles, describing their effect on different targets and highlighting how their structural versatility has been exploited to modulate their biological properties.

Trifunctionalized Naphthalene Diimides and Dimeric Analogues as G-Quadruplex-Targeting Anticancer Agents Selected by Affinity Chromatography.

A focused library of newly designed monomeric and dimeric naphthalene diimides (NDIs) was analyzed in its ability to recognize specific G-quadruplex (G4) structures discriminating duplex DNA. The best G4 ligands-according to an affinity chromatography-based screening method named G4-CPG-were tested on human cancer and healthy cells, inducing DNA damage at telomeres, and in parallel, showing selective antiproliferative activity on HeLa cancer cells with IC50 values in the low nanomolar range. CD and fluorescence spectroscopy studies allowed detailed investigation of the interaction in solution with different G4 and duplex DNA models of the most promising NDI of the series, as determined by combining the biophysical and biological assays' data.

Design of Disruptors of the Hsp90-Cdc37 Interface.

The molecular chaperone Hsp90 is a ubiquitous ATPase-directed protein responsible for the activation and structural stabilization of a large clientele of proteins. As such, Hsp90 has emerged as a suitable candidate for the treatment of a diverse set of diseases, such as cancer and neurodegeneration. The inhibition of the chaperone through ATP-competitive inhibitors, however, was shown to lead to undesirable side effects. One strategy to alleviate this problem is the development of molecules that are able to disrupt specific protein-protein interactions, thus modulating the activity of Hsp90 only in the particular cellular pathway that needs to be targeted. Here, we exploit novel computational and theoretical approaches to design a set of peptides that are able to bind Hsp90 and compete for its interaction with the co-chaperone Cdc37, which is found to be responsible for the promotion of cancer cell proliferation. In spite of their capability to disrupt the Hsp90-Cdc37 interaction, no important cytotoxicity was observed in human cancer cells exposed to designed compounds. These findings imply the need for further optimization of the compounds, which may lead to new ways of interfering with the Hsp90 mechanisms that are important for tumour growth.

Dyads of G-Quadruplex Ligands Triggering DNA Damage Response and Tumour Cell Growth Inhibition at Subnanomolar Concentration.

Naphthalene diimide (NDI) dyads exhibiting a different substitution pattern and linker length have been synthesised and evaluated as G-quadruplex (G4) ligands, by investigating their cytotoxicity in selected cell lines. The dyads with the long C7 linker exhibit extremely low IC50 values, below 10 nm, on different cancer cell lines. Contrary, the dyads with the shorter C4 linker were much less effective, with IC values increasing up to 1 µm. Among the three dyads with the longest linker, small differences in the IC50 values emerge, suggesting that the linker length plays a more important role than the substitution pattern. We have further shown that the dyads are able to induce cellular DNA damage response, which is not limited to the telomeric regions and is likely the origin of their cytotoxicity. Both absorption titration and dynamic light scattering of the most cytotoxic dyads in the presence of hTel22 highlight their ability to induce effective G4 aggregation, acting as non-covalent cross-linking agents.

Naphthalene Diimides as Multimodal G-Quadruplex-Selective Ligands.

G-quadruplexes are four-stranded nucleic acids structures that can form in guanine-rich sequences. Following the observation that G-quadruplexes are particularly abundant in genomic regions related to cancer, such as telomeres and oncogenes promoters, several G-quadruplex-binding molecules have been developed for therapeutic purposes. Among them, naphthalene diimide derivatives have reported versatility, consistent selectivity and high affinity toward the G-quadruplex structures. In this review, we present the chemical features, synthesis and peculiar optoelectronic properties (absorption, emission, redox) that make naphtalene diimides so versatile for biomedical applications. We present the latest developments on naphthalene diimides as G-quadruplex ligands, focusing on their ability to bind G-quadruplexes at telomeres and oncogene promoters with consequent anticancer activity. Their different binding modes (reversible versus irreversible/covalent) towards G-quadruplexes and their additional use as antimicrobial agents are also presented and discussed.

A Catalytic and Selective Scissoring Molecular Tool for Quadruplex Nucleic Acids.

A copper complex embedded in the structure of a water-soluble naphthalene diimide has been designed to bind and cleave G-quadruplex DNA. We describe the properties of this ligand, including its catalytic activity in the generation of ROS. FRET melting, CD, NMR, gel sequencing, and mass spectrometry experiments highlight a unique and unexpected selectivity in cleaving G-quadruplex sequences. This selectivity relies both on the binding affinity and structural features of the targeted G-quadruplexes.

Oxadiazole/Pyridine-Based Ligands: A Structural Tuning for Enhancing G-Quadruplex Binding.

Non-macrocyclic heteroaryls represent a valuable class of ligands for nucleic acid recognition. In this regard, non-macrocyclic pyridyl polyoxazoles and polyoxadiazoles were recently identified as selective G-quadruplex stabilizing compounds with high cytotoxicity and promising anticancer activity. Herein, we describe the synthesis of a new family of heteroaryls containing oxadiazole and pyridine moieties targeting DNA G-quadruplexes. To perform a structure⁻activity analysis identifying determinants of activity and selectivity, we followed a convergent synthetic pathway to modulate the nature and number of the heterocycles (1,3-oxazole vs. 1,2,4-oxadiazole and pyridine vs. benzene). Each ligand was evaluated towards secondary nucleic acid structures, which have been chosen as a prototype to mimic cancer-associated G-quadruplex structures (e.g., the human telomeric sequence, c-myc and c-kit promoters). Interestingly, heptapyridyl-oxadiazole compounds showed preferential binding towards the telomeric sequence (22AG) in competitive conditions vs. duplex DNA. In addition, G4-FID assays suggest a different binding mode from the classical stacking on the external G-quartet. Additionally, CD titrations in the presence of the two most promising compounds for affinity, TOxAzaPy and TOxAzaPhen, display a structural transition of 22AG in K-rich buffer. This investigation suggests that the pyridyl-oxadiazole motif is a promising recognition element for G-quadruplexes, combining seven heteroaryls in a single binding unit.

A Fragment-Based Approach for the Development of G-Quadruplex Ligands: Role of the Amidoxime Moiety.

G-quadruplex (G4) nucleic acid structures have been reported to be involved in several human pathologies, including cancer, neurodegenerative disorders and infectious diseases; however, G4 targeting compounds still need implementation in terms of drug-like properties and selectivity in order to reach the clinical use. So far, G4 ligands have been mainly identified through high-throughput screening methods or design of molecules with pre-set features. Here, we describe the development of new heterocyclic ligands through a fragment-based drug discovery (FBDD) approach. The ligands were designed against the major G4 present in the long terminal repeat (LTR) promoter region of the human immunodeficiency virus-1 (HIV-1), the stabilization of which has been shown to suppress viral gene expression and replication. Our method is based on the generation of molecular fragment small libraries, screened against the target to further elaborate them into lead compounds. We screened 150 small molecules, composed by structurally and chemically different fragments, selected from commercially available and in-house compounds; synthetic elaboration yielded several G4 ligands and two final G4 binders, both embedding an amidoxime moiety; one of these two compounds showed preferential binding for the HIV-1 LTR G4. This work presents the discovery of a novel potential pharmacophore and highlights the possibility to apply a fragment-based approach to develop G4 ligands with unexpected chemical features.

Copper-Aß Peptides and Oxidation of Catecholic Substrates: Reactivity and Endogenous Peptide Damage.

The oxidative reactivity of copper complexes with Aß peptides 1-16 and 1-28 (Aß16 and Aß28) against dopamine and related catechols under physiological conditions has been investigated in parallel with the competitive oxidative modification undergone by the peptides. It was found that both Aß16 and Aß28 markedly increase the oxidative reactivity of copper(II) towards the catechol compounds, up to a molar ratio of about 4:1 of peptide/copper(II). Copper redox cycling during the catalytic activity induces the competitive modification of the peptide at selected amino acid residues. The main modifications consist of oxidation of His13/14 to 2-oxohistidine and Phe19/20 to ortho-tyrosine, and the formation of a covalent His6-catechol adduct. Competition by the endogenous peptide is rather efficient, as approximately one peptide molecule is oxidized every 10 molecules of 4-methylcatechol.

Copper(I) Forms a Redox-Stable 1:2 Complex with α-Synuclein N-Terminal Peptide in a Membrane-Like Environment.

α-Synuclein (αS) is the main protein component of Lewy bodies, characterizing the pathogenesis of Parkinson's disease. αS is unstructured in solution but adopts a helical structure in its extended N-terminal segment upon association with membranes. In vitro the protein binds avidly Cu(II), but in vivo the protein is N-acetylated, and Cu(II) binding is lost. We have now clarified the binding characteristics of the Cu(I) complex with the truncated αS peptide 1-15, both in N-acetylated and free amine forms, in a membrane mimetic environment and found that complexation occurs with a 1:2 Cu(I)-αS stoichiometry, where Cu(I) is bound to Met1 and Met5 residues of two helical peptide chains. The resulting tetrahedral Cu(I) center is redox-stable, does not form reactive oxygen species, and is unreactive against dopamine in the presence of O2. This suggests that, unlike cytosolic Cu(I)-αS, which retains the capacity to activate O2 and promote oxidative reactions, membrane-bound Cu(I)-αS may serve as a sink for unreactive copper.

Naphthalene Diimide-Tetraazacycloalkane Conjugates Are G-Quadruplex-Based HIV-1 Inhibitors with a Dual Mode of Action.

Human immunodeficiency virus 1 (HIV-1) therapeutic regimens consist of three or more drugs targeting different steps of the viral life cycle to limit the emergence of viral resistance. In line with the multitargeting strategy, here we conjugated a naphthalene diimide (NDI) moiety with a tetraazacycloalkane to obtain novel naphthalene diimide (NDI)-tetraazacycloalkane conjugates. The NDI inhibits the HIV-1 promoter activity by binding to LTR G-quadruplexes, and the tetraazacycloalkane mimics AMD3100, which blocks HIV entry into cells by interfering with the CXCR4 coreceptor. We synthesized, purified, and tested the metal-free NDI-tetraazacycloalkane conjugate and the two derived metal-organic complexes (MOCs) that incorporate Cu2+ and Zn2+. The NDI-MOCs showed enhanced binding to LTR G4s as assessed by FRET and CD assays in vitro. They also showed enhanced activity in cells where they dose-dependently reduced LTR promoter activity and inhibited viral entry only of the HIV-1 strain that exploited the CXCR4 coreceptor. The time of addition assay confirmed the dual targeting at the different HIV-1 steps. Our results indicate that the NDI-MOC conjugates can simultaneously inhibit viral entry, by targeting the CXCR4 coreceptor, and LTR promoter activity, by stabilizing the LTR G-quadruplexes. The approach of combining multiple targets in a single compound may streamline treatment regimens and improve the overall patient outcomes.