Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription

Folding of the LTR promoter into dynamic G-quadruplex conformations has been shown to suppress its transcriptional activity in HIV-1. Here we sought to identify the proteins that control the folding of this region of proviral genome by inducing/stabilizing G-quadruplex structures. The implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize G-quadruplex structures present in the LTR promoter. Nucleolin recognized with high affinity and specificity the majority, but not all the possible G-quadruplexes folded by this sequence. In addition, it displayed greater binding preference towards DNA than RNA G-quadruplexes, thus indicating two levels of selectivity based on the sequence and nature of the target. The interaction translated into stabilization of the LTR G-quadruplexes and increased promoter silencing activity; in contrast, disruption of nucleolin binding in cells by both siRNAs and a nucleolin binding aptamer greatly increased LTR promoter activity. These data indicate that nucleolin possesses a specific and regulated activity toward the HIV-1 LTR promoter, which is mediated by G-quadruplexes. These observations provide new essential insights into viral transcription and a possible low mutagenic target for antiretroviral therapy

Major G-Quadruplex Form of HIV-1 LTR Reveals a (3 + 1) Folding Topology Containing a Stem-Loop

Nucleic acids can form noncanonical four-stranded structures called G-quadruplexes. G-quadruplex-forming sequences are found in several genomes including human and viruses. Previous studies showed that the G-rich sequence located in the U3 promoter region of the HIV-1 long terminal repeat (LTR) folds into a set of dynamically interchangeable G-quadruplex structures. G-quadruplexes formed in the LTR could act as silencer elements to regulate viral transcription. Stabilization of LTR G-quadruplexes by G-quadruplex-specific ligands resulted in decreased viral production, suggesting the possibility of targeting viral G-quadruplex structures for antiviral purposes. Among all the G-quadruplexes formed in the LTR sequence, LTR-III was shown to be the major G-quadruplex conformation in vitro. Here we report the NMR structure of LTR-III in K+ solution, revealing the formation of a unique quadruplex-duplex hybrid consisting of a three-layer (3 + 1) G-quadruplex scaffold, a 12-nt diagonal loop containing a conserved duplex-stem, a 3-nt lateral loop, a 1-nt propeller loop, and a V-shaped loop. Our structure showed several distinct features including a quadruplex-duplex junction, representing an attractive motif for drug targeting. The structure solved in this study may be used as a promising target to selectively impair the viral cycle

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

Synthesis, Binding and Antiviral Properties of Potent Core-Extended Naphthalene Diimides Targeting the HIV-1 Long Terminal Repeat Promoter G-Quadruplexes

We have previously reported that stabilization of the G-quadruplex structures in the HIV-1 long terminal repeat (LTR) promoter suppresses viral transcription. Here we sought to develop new G-quadruplex ligands to be exploited as antiviral compounds by enhancing binding toward the viral G-quadruplex structures. We synthesized naphthalene diimide derivatives with a lateral expansion of the aromatic core. The new compounds were able to bind/stabilize the G-quadruplex to a high extent, and some of them displayed clear-cut selectivity toward the viral G-quadruplexes with respect to the human telomeric G-quadruplexes. This feature translated into low nanomolar anti-HIV-1 activity toward two viral strains and encouraging selectivity indexes. The selectivity depended on specific recognition of LTR loop residues; the mechanism of action was ascribed to inhibition of LTR promoter activity in cells. This is the first example of G-quadruplex ligands that show increased selectivity toward the viral G-quadruplexes and display remarkable antiviral activity

G-quadruplexes in the HIV-1 genome: structure and targeting

Nucleic acids may form non-canonical tetraplex secondary structures called G-quadruplexes. G-quadruplexes have been found in eukaryotic and prokaryotic genomes, including viruses. Located in key functional regions of genomes, G-quadruplexes play important regulatory roles in transcription, replication and translation processes. Particularly, G-quadruplex-mediated transcription regulation of oncogene promoters has been widely described. Previous studies demonstrated that a set of dynamic G-quadruplex structures in the promoter region of the HIV-1 Long Terminal Repeat (LTR) regulates viral transcription acting as repressor elements. G-quadruplex-directed targeting with stabilizing ligands enhances their inhibitory effect, resulting in decrease of viral production and suggesting viral G4 structures as potential antiviral targets. We aimed at 1) develop antiviral compounds selective for viral G-quadruplexes over cellular structures. We screened a newly designed series of NDI-core based G-quadruplex ligands and pointed out structural features of the compounds that led to improve the selectivity. 2) We identified by nuclear magnetic resonance the deep structural coordinates of the G-quadruplex targets as the preliminary necessary step for rational drug design approach. We described the singular hybrid quadruplex/duplex topology of the major G-quadruplex component within the LTR region, which allows novel implication for selective recognition of viral structures. 3) We also explored the formation of G-quadruplex structures at the HIV-1 RNA genome level, which emerged as a pre-integration target for the antiviral activity of a well-known G-quadruplex ligand. We investigated the formation, stability and involvement of RNA G-quadruplexes in the reverse transcription process and the role of the HIV-1 nucleocapsid protein in controlling folding of these structures.Acidi nucleici ricchi in guanine possono formare strutture secondarie alternative chiamate G-quadruplex. I G-quadruplex sono stati caratterizzati in diversi tipi di genomi, tra cui genomi virali e umano. Nel genoma umano le strutture G-quadruplex sono prinicipalmente localizzate in importanti regioni funzionali, dove possono assumere ruoli regolatori dei processi come trascrizione, replicazione a traduzione. In particolare, la regolazione della trascrizione dei promotori degli oncogeni mediata dalle strutture G-quadruplex è stata ampiamente descritta, evidenziando che i G-quadruplex promotoriali agiscono principalmente da silenziatori del processo trascrizionale. I precedenti studi, condotti dal nostro gruppo di ricerca hanno dimostrato che una serie di strutture G-quadruplex al livello del Long Terminal repeat (LTR) del genoma provirale di HIV-1 è coinvolta nella regolazione della trascrizione virale. La stabilizzazione di queste strutture con i ligandi specifici si traduce in repressione dell’attività promotoriale e in inibizione della produzione del virus in cellule infettate, suggerendo che i G-quadruplex nella regione LTR di HIV-1 possono essere dei promettenti target antivirali. Lo scopo principale di questa tesi è stato quello di individuare composti con attività antivirale che mostrano un legame preferenziale verso le strutture G-quadruplex virali. Abbiamo testato una nuova serie di composti leganti G-quadruplex sviluppata a partire dal NDI-core e abbiamo identificato dei componenti strutturali responsabili della maggiore affinità verso le strutture G-quadruplex virali che possono guidare verso ulteriore miglioramento della selettività. L’obiettivo di utilizzare un approccio razionale per lo sviluppo dei composti selettivi ha richiesto di individuare le coordinate strutturali del target. Abbiamo identificato che il componente G-quadruplex prevalente nella regione LTR considerata è foldato in una topologia molto particolare, descritta come struttura ibrida quadruplex/duplex e presenta interessanti implicazioni per il riconoscimento selettivo da parte delle piccole molecole

Strutture G-quadruplex con funzione regolatoria nel genoma di HIV-1.

I G-quadruplex sono strutture secondarie alternative degli acidi nucleici che si possono formare in sequenze di DNA o RNA ricche di guanine. Le guanine formano quartetti che, impilandosi tra di loro, danno origine a strutture secondarie stabili, dette G-quadruplex. In questa tesi vengono prese in considerazione sequenze ricche di guanine presenti a livello della regione promotoriale del genoma provirale di HIV-1 a dsDNA e della regione corrispondente a livello del genoma virale a ssRNA

G-quadruplexes in the HIV-1 genome: structure and targeting

Nucleic acids may form non-canonical tetraplex secondary structures called G-quadruplexes. G-quadruplexes have been found in eukaryotic and prokaryotic genomes, including viruses. Located in key functional regions of genomes, G-quadruplexes play important regulatory roles in transcription, replication and translation processes. Particularly, G-quadruplex-mediated transcription regulation of oncogene promoters has been widely described. Previous studies demonstrated that a set of dynamic G-quadruplex structures in the promoter region of the HIV-1 Long Terminal Repeat (LTR) regulates viral transcription acting as repressor elements. G-quadruplex-directed targeting with stabilizing ligands enhances their inhibitory effect, resulting in decrease of viral production and suggesting viral G4 structures as potential antiviral targets. We aimed at 1) develop antiviral compounds selective for viral G-quadruplexes over cellular structures. We screened a newly designed series of NDI-core based G-quadruplex ligands and pointed out structural features of the compounds that led to improve the selectivity. 2) We identified by nuclear magnetic resonance the deep structural coordinates of the G-quadruplex targets as the preliminary necessary step for rational drug design approach. We described the singular hybrid quadruplex/duplex topology of the major G-quadruplex component within the LTR region, which allows novel implication for selective recognition of viral structures. 3) We also explored the formation of G-quadruplex structures at the HIV-1 RNA genome level, which emerged as a pre-integration target for the antiviral activity of a well-known G-quadruplex ligand. We investigated the formation, stability and involvement of RNA G-quadruplexes in the reverse transcription process and the role of the HIV-1 nucleocapsid protein in controlling folding of these structures

The G-quadruplex-forming aptamer AS1411 potently inhibits HIV-1 attachment to the host cell

AS1411 is a G-rich aptamer that forms a stable G-quadruplex structure and displays antineoplastic properties both in vitro and in vivo. This oligonucleotide has undergone phase 2 clinical trials. The major molecular target of AS1411 is nucleolin (NCL), a multifunctional nucleolar protein also present in the cell membrane where it selectively mediates the binding and uptake of AS1411. Cell-surface NCL has been recognised as a low-affinity co-receptor for human immunodeficiency virus type 1 (HIV-1) anchorage on target cells. Here we assessed the anti-HIV-1 properties and underlying mechanism of action of AS1411. The antiviral activity of AS1411 was determined towards different HIV-1 strains, host cells and at various times post-infection. Acutely, persistently and latently infected cells were tested, including HIV-1-infected peripheral blood mononuclear cells from a healthy donor. Mechanistic studies to exclude modes of action other than virus binding via NCL were performed. AS1411 efficiently inhibited HIV-1 attachment/entry into the host cell. The aptamer displayed antiviral activity in the absence of cytotoxicity at the tested doses, therefore displaying a wide therapeutic window and favourable selectivity indexes. These findings, besides validating cell-surface-expressed NCL as an antiviral target, open the way for the possible use of AS1411 as a new potent and promisingly safe anti-HIV-1 agent