Community Research and Development Information Service - CORDIS


HIV LTR G-4 Report Summary

Project ID: 615879
Funded under: FP7-IDEAS-ERC
Country: Italy

Periodic Report Summary 2 - HIV LTR G-4 (G-quadruplexes in the HIV-1 genome: novel targets for the development of selective antiviral drugs)

In the first 30 months of the project, we have found and developed four small molecules that display anti-HIV-1 activity: one G-quadruplex (G4) ligand that inhibits HIV-1 with effective concentration (EC50) in the low micromolar range. To improve selectivity we have synthesize a new class of molecules whose lead is effective against HIV-1 in the nanomolar range. This compound selectively recognized the HIV-1 G4s by interacting with the unique long loops of the structures. The mechanism of action in infected cells is interference with the G4 in the LTR promoter of the provirus, which leads to inhibition of viral transcription and therefore of virus production. A third family of compounds was found by screening a commercial library: these molecules are selective for the HIV-1 G4s while they poorly bind cellular G4s. They display good antiviral properties with a G4-mediated mechanism of action. The fourth molecule is the aptamer AS1411, an oligonucleotide that folds into a stable G4 structure and has been used in Phase II clinical trials as anticancer agent. We found that AS1411 has a potent anti-HIV-1 activity with a mechanism that is independent of the LTR G4s as targets. AS1411 inhibits HIV-1 entry by binding to a viral co-receptor on the cell surface.
The photoreactive/fluorescence properties of some of our G4 ligands have been exploited to develop cleaver/marker molecules of HIV-1 infected cells. A G4 ligand was able to cleave T residues in the loops of G4s. A second compound effectively cleaved the loops of the HIV-1 G4s with a mechanism that depended on in situ production of radical oxygen species. An additional compound displayed mild fluorescence properties when free in solution: interestingly its fluorescence lighted up upon binding to G4s. This feature has been used to detect G4s in cells.
G4 ligands with peptide nucleic acids (PNA) moieties have been also developed: we have obtained good G4 stabilization coupled with much increased selectivity for the HIV-1 G4s. With this approach, we have also observed the possibility of stabilizing and unfold selected HIV-1 G4s. The possibility to maintain only one of the LTR G4s will enable us to deeply characterize the role of each G4 in infected cells and to finely tune the antiviral effect.

Structural analysis
In collaboration with the group of Anh Tuan Phan in Singapore, we have successfully characterized by NMR structural analysis the two main HIV-1 G4 structures. Their unique features, i.e. bulge within the G4 structure and a stem-hairpin in the loop, will likely allow the rational design of highly new selective binders.
Identification of binding partners
We successfully identified cellular proteins that specifically bind HIV-1 G4s: nucleolin, binds to most but not all HIV-1 LTR G4s, highlighting its selective binding, based on the recognition of G4s with long loops. This protein is highly effective in silencing HIV-1 transcription. A second cellular protein, hnRNP A2/B1, has been identified and shown to specifically bind the HIV-1 G4s. The activity of this protein is complementary to that of nucleolin: while the last stabilizes G4s, the former unfolds them. These data indicate that the HIV-1 G4s in the LTR promoter are finely regulated by multiple cellular proteins.
The importance of G4s in viruses has been also corroborated by the evidence of G4 massive presence in infected cells during the replication step of the Herpes simplex virus 1 and the possibility to inhibit virus production by G4 ligands.

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