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.

TERRA G-quadruplex stabilization as a new therapeutic strategy for multiple myeloma.

Background: Multiple myeloma (MM) is a hematologic malignancy characterized by high genomic instability, and telomere dysfunction is an important cause of acquired genomic alterations. Telomeric repeat-containing RNA (TERRA) transcripts are long non-coding RNAs involved in telomere stability through the interaction with shelterin complex. Dysregulation of TERRAs has been reported across several cancer types.

Quindoline-derivatives display potent G-quadruplex-mediated antiviral activity against herpes simplex virus 1.

G-quadruplexes (G4s) are non-canonical nucleic acid structures that regulate key biological processes, from transcription to genome replication both in humans and viruses. The herpes simplex virus-1 (HSV-1) genome is prone to form G4s that, along with proteins, regulate its viral cycle. General G4 ligands have been shown to hamper the viral cycle, pointing to viral G4s as original antiviral targets.

Chromene Derivatives as Selective TERRA G-Quadruplex RNA Binders with Antiproliferative Properties.

In mammalian cells, telomerase transcribes telomeres in large G-rich non-coding RNA, known as telomeric repeat-containing RNA (TERRA), which folds into noncanonical nucleic acid secondary structures called G-quadruplexes (G4s). Since TERRA G4 has been shown to be involved in telomere length and translation regulation, it could provide valuable insight into fundamental biological processes, such as cancer growth, and TERRA G4 binders could represent an innovative strategy for cancer treatment. In this work, the three best candidates identified in our previous virtual screening campaign on bimolecular DNA/RNA G4s were investigated on the monomolecular Tel DNA and TERRA G4s by means of molecular modelling simulations and in vitro and in cell analysis.

Selective Recognition of a Single HIV-1 G-Quadruplex by Ultrafast Small-Molecule Screening.

G-quadruplexes (G4s) are implicated in pathological processes such as cancer and infective diseases. Their targeting with G4-ligands has shown therapeutic capacity. Most of the current G4-ligands are planar molecules, do not discriminate among G4s, and have poor druglike properties.

Promoter G-quadruplexes and transcription factors cooperate to shape the cell type-specific transcriptome.

Cell identity is maintained by activation of cell-specific gene programs, regulated by epigenetic marks, transcription factors and chromatin organization. DNA G-quadruplex (G4)-folded regions in cells were reported to be associated with either increased or decreased transcriptional activity. By G4-ChIP-seq/RNA-seq analysis on liposarcoma cells we confirmed that G4s in promoters are invariably associated with high transcription levels in open chromatin.

Parallel G-quadruplexes recruit the HSV-1 transcription factor ICP4 to promote viral transcription in herpes virus-infected human cells.

G-quadruplexes (G4s) are four-stranded nucleic acid structures abundant at gene promoters. They can adopt several distinctive conformations. G4s have been shown to form in the herpes simplex virus-1 (HSV-1) genome during its viral cycle.

Antiviral Activity of the G-Quadruplex Ligand TMPyP4 against Herpes Simplex Virus-1.

The herpes simplex virus 1 (HSV-1) genome is extremely rich in guanine tracts that fold into G-quadruplexes (G4s), nucleic acid secondary structures implicated in key biological functions. Viral G4s were visualized in HSV-1 infected cells, with massive virus cycle-dependent G4-formation peaking during viral DNA replication. Small molecules that specifically interact with G4s have been shown to inhibit HSV-1 DNA replication.

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.

Novel monomolecular derivatives of the anti-HIV-1 G-quadruplex-forming Hotoda's aptamer containing inversion of polarity sites.

Here we report on the design, preparation and investigation of four analogues of the anti-HIV G-quadruplex-forming Hotoda's aptamer, based on an unprecedented linear topology. In these derivatives, four TGGGAGT tracts have been joined together by exploiting 3'-3' and 5'-5' inversion of polarity sites formed by canonical phosphodiester bonds or a glycerol-based linker. Circular dichroism data suggest that all oligodeoxynucleotides fold in monomolecular G-quadruplex structures characterized by a parallel strand orientation and three side loops connecting 3'- or 5'-ends.

A dynamic i-motif with a duplex stem-loop in the long terminal repeat promoter of the HIV-1 proviral genome modulates viral transcription.

I-motifs are non-canonical nucleic acids structures characterized by intercalated H-bonds between hemi-protonated cytosines. Evidence on the involvement of i-motif structures in the regulation of cellular processes in human cells has been consistently growing in the recent years. However, i-motifs within non-human genomes have never been investigated.

HIV-1 Nucleocapsid Protein Unfolds Stable RNA G-Quadruplexes in the Viral Genome and Is Inhibited by G-Quadruplex Ligands.

The G-quadruplexes that form in the HIV-1 RNA genome hinder progression of reverse transcriptase in vitro, but not in infected cells. We investigated the possibility that the HIV-1 nucleocapsid protein NCp7, which remains associated with the viral RNA during reverse transcription, modulated HIV-1 RNA G-quadruplex stability. By electrophoresis, circular dichroism, mass spectrometry, and reverse transcriptase stop assays, we demonstrated that NCp7 binds and unfolds the HIV-1 RNA G-quadruplexes and promotes DNA/RNA duplex formation, allowing reverse transcription to proceed.

G-Quadruplex Visualization in Cells via Antibody and Fluorescence Probe.

G-quadruplexes (G4s) are noncanonical nucleic acids structures involved in key regulatory and pathological roles in eukaryotes, prokaryotes, and viruses: the development of specific antibodies and fluorescent probes represent an invaluable tool to understand their biological relevance. We here present three protocols for the visualization of G4s in cells, both uninfected and HSV-1 infected, using a specific antibody and a fluorescent G4 ligand, and the effect of the fluorescent ligand on a G4 binding protein, nucleolin, upon binding of the molecule to the nucleic acids structure.

Conserved G-Quadruplexes Regulate the Immediate Early Promoters of Human .

Human comprise three members, herpes simplex virus (HSV) 1 and 2 and varicella zoster virus (VZV). These viruses are characterized by a lytic cycle in epithelial cells and latency in the nervous system, with lifelong infections that may periodically reactivate and lead to serious complications, especially in immunocompromised patients. The mechanisms that regulate viral transcription have not been fully elucidated, but the master role of the immediate early (IE) genes has been established.

Stable and Conserved G-Quadruplexes in the Long Terminal Repeat Promoter of Retroviruses.

Retroviruses infect almost all vertebrates, from humans to domestic and farm animals, from primates to wild animals, where they cause severe diseases, including immunodeficiencies, neurological disorders, and cancer. Nonhuman retroviruses have also been recently associated with human diseases. To date, no effective treatments are available; therefore, finding retrovirus-specific therapeutic targets is becoming an impelling issue.

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.

Synthesis and photocytotoxic activity of [1,2,3]triazolo[4,5-h][1,6]naphthyridines and [1,3]oxazolo[5,4-h][1,6]naphthyridines.

[1,2,3]Triazolo[4,5-h][1,6]naphthyridines and [1,3]oxazolo[5,4-h][1,6]naphthyridines were synthesized with the aim to investigate their photocytotoxic activity. Upon irradiation, oxazolo-naphtapyridines induced light-dependent cell death at nanomolar/low micromolar concentrations (EC 0.01-6.

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.

Down-Regulation of the Androgen Receptor by G-Quadruplex Ligands Sensitizes Castration-Resistant Prostate Cancer Cells to Enzalutamide.

Stabilization of the G-quadruplexes (G4s) within the androgen receptor (AR) gene promoter to block transcription may represent an innovative approach to interfere with aberrant AR signaling in castration resistant prostate cancer (CRPC). A library of differently functionalized naphthalene diimides (NDIs) was screened for their ability to stabilize AR G4s: the core-extended NDI (7) stood out as the most promising ligand. AR-positive cells were remarkably sensitive to 7 in comparison to AR-negative CRCP or normal prostate epithelial cells; 7 induced remarkable impairment of AR mRNA and protein amounts and significant perturbations in the expression levels of KLK3 and of genes involved in the activation of AR program via feedback mechanisms.

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.

Surface Plasmon Resonance kinetic analysis of the interaction between G-quadruplex nucleic acids and an anti-G-quadruplex monoclonal antibody.

Background: G-quadruplexes (G4s) are nucleic acids secondary structures formed in guanine-rich sequences. Anti-G4 antibodies represent a tool for the direct investigation of G4s in cells. Surface Plasmon Resonance (SPR) is a highly sensitive technology, suitable for assessing the affinity between biomolecules.

Identification of G-quadruplex DNA/RNA binders: Structure-based virtual screening and biophysical characterization.

Background: Recent findings demonstrated that, in mammalian cells, telomere DNA (Tel) is transcribed into telomeric repeat-containing RNA (TERRA), which is involved in fundamental biological processes, thus representing a promising anticancer target. For this reason, the discovery of dual (as well as selective) Tel/TERRA G-quadruplex (G4) binders could represent an innovative strategy to enhance telomerase inhibition.

Methods: Initially, docking simulations of known Tel and TERRA active ligands were performed on the 3D coordinates of bimolecular G4 Tel DNA (Tel) and TERRA (TERRA).

The cellular protein nucleolin preferentially binds long-looped G-quadruplex nucleic acids.

Background: G-quadruplexes (G4s) are four-stranded nucleic acid structures that form in G-rich sequences. Nucleolin (NCL) is a cellular protein reported for its functions upon G4 recognition, such as induction of neurodegenerative diseases, tumor and virus mechanisms activation. We here aimed at defining NCL/G4 binding determinants.