Oligonucleotide inhibition of the interaction of HIV-1 Tat protein with the trans-activation responsive region (TAR) of HIV RNA

The interaction of HIV-1 Tat protein with its recognition sequence, the trans-activation responsive region TAR is a potential target for drug discovery against HIV infection. We show by use of an in vitro competition filter binding interference assay that synthetic oligodeoxyribonucleotides complementary to the HIV-1 TAR RNA apical stem-loop and bulge region inhibit the binding of Tat protein or a Tat peptide (residues 37-72) better than two small molecules that have been shown to bind TAR RNA, Hoechst 33258 and neomycin B. The inhibition is not sensitive to length between 13 and 16 residues or precise positioning but shorter oligonucleotides are less effective. Enhanced inhibition was obtained for a 16-mer 2'-O-methyl oligoribonucleotide but not for C5-propyne pyrimidine-substituted oligonucleotides. Control non-antisense oligonucleotides were occasionally also effective in filter binding interference but only the complementary antisense 2'-O-methyl oligoribonucleotide was effective in gel mobility shift assays in direct TAR binding or in interference with Tat peptide binding to the TAR stem-loop. This is the first demonstration of effective inhibition of the Tat-TAR interaction by nuclease-stabilized oligonucleotide analogues.     

Dynamic ensemble view of the conformational landscape of HIV-1 TAR RNA and allosteric recognition

RNA conformational dynamics and the resulting structural heterogeneity play an important role in RNA functions, e.g., recognition. Recognition of HIV-1 TAR RNA has been proposed to occur via a conformational capture mechanism. Here, using ultrafast time-resolved fluorescence spectroscopy, we have probed the complexity of the conformational landscape of HIV-1 TAR RNA and monitored the position-dependent changes in the landscape upon binding of a Tat protein-derived peptide and neomycin B. In the ligand-free state, the TAR RNA samples multiple families of conformations with various degrees of base stacking around the three-nucleotide bulge region. Some subpopulations partially resemble those ligand-bound states, but the coaxially stacked state is below the detection limit. When Tat or neomycin B binds, the bulge region as an ensemble undergoes a conformational transition in a position-dependent manner. Tat and neomycin B induce mutually exclusive changes in the TAR RNA underlying the mechanism of allosteric inhibition at an ensemble level with residue-specific details. Time-resolved anisotropy decay measurements revealed picosecond motions of bases in both ligand-free and ligand-bound states. Mutation of a base pair at the bulge--stem junction has differential effects on the conformational distributions of the bulge bases. A dynamic model of the ensemble view of the conformational landscape for HIV-1 TAR RNA is proposed, and the implication of the general mechanism of RNA recognition and its impact on RNA-based therapeutics are discussed.     

Neomycin B-arginine conjugate, a novel HIV-1 Tat antagonist: synthesis and anti-HIV activities.

HIV-1 transactivating protein Tat is essential for virus replication and progression of HIV disease. HIV-1 Tat stimulates transactivation by binding to HIV-1 transactivator responsive element (TAR) RNA, and while secreted extracellularly, it acts as an immunosuppressor, an activator of quiescent T-cells for productive HIV-1 infection, and by binding to CXC chemokine receptor type 4 (CXCR4) as a chemokine analogue. Here we present a novel HIV-1 Tat antagonist, a neomycin B-hexaarginine conjugate (NeoR), which inhibits Tat transactivation and antagonizes Tat extracellular activities, such as increased viral production, induction of CXCR4 expression, suppression of CD3-activated proliferation of lymphocytes, and upregulation of the CD8 receptor. Moreover, Tat inhibits binding of fluoresceine isothiocyanate (FITC)-labeled NeoR to human peripheral blood mononuclear cells (PBMC), indicating that Tat and NeoR bind to the same cellular target. This is further substantiated by the finding that NeoR competes with the binding of monoclonal Abs to CXCR4. Furthermore, NeoR suppresses HIV-1 binding to cells. Importantly, NeoR accumulates in the cell nuclei and inhibits the replication of M- and T-tropic HIV-1 laboratory isolates (EC(50) = 0.8-5.3 microM). A putative model structure for the TAR-NeoR complex, which complies with available experimental data, is presented. We conclude that NeoR is a multitarget HIV-1 inhibitor; the structure, and molecular modeling and dynamics, suggest its binding to TAR RNA. NeoR inhibits HIV-1 binding to cells, partially by blocking the CXCR4 HIV-1 coreceptor, and it antagonizes Tat functions. NeoR is therefore an attractive lead compound, capable of interfering with different stages of HIV infection and AIDS pathogenesis.     

Structural rearrangements of HIV-1 Tat-responsive RNA upon binding of neomycin B

Binding of human immunodeficiency virus type 1 (HIV-1) transactivator (Tat) protein to Tat-responsive RNA (TAR) is essential for viral replication and is considered a promising starting point for the design of anti-HIV drugs. NMR spectroscopy indicated that the aminoglycosides neomycin B and ribostamycin bind to TAR and that neomycin is able to inhibit Tat binding to TAR. The solution structure of the neomycin-bound TAR has been determined by NMR spectroscopy. Chemical shift mapping and intermolecular nuclear Overhauser effects define the binding region of the aminoglycosides on TAR and give strong evidence for minor groove binding. Based on 15 nuclear Overhauser effect-derived intermolecular distance restraints, a model structure of the TAR-neomycin complex was calculated. Neomycin is bound in a binding pocket formed by the minor groove of the lower stem and the uridine-rich bulge of TAR, which adopts a conformation different from those known. The neamine core of the aminoglycoside (rings I and II) is covered with the bulge, explaining the inhibition of Tat by an allosteric mechanism. Neomycin reduces the volume of the major groove in which Tat is bound and thus impedes essential protein-RNA contacts.     

A high-throughput screening utilizing intramolecular fluorescence resonance energy transfer for the discovery of the molecules that bind HIV-1 TAR RNA specifically

A 16-residue peptide, including the Tat(49-57) sequence was labeled with a fluorescein and a tetramethylrhodamine at its N- and C-terminus, respectively. This double dye-labeled peptide was prepared as a tracer for high-throughput screening utilizing intramolecular fluorescence resonance energy transfer (FRET). The binding of the competitor molecules for HIV-1 TAR RNA were monitored and dissociation constants of those molecule were determined by using this tracer. This novel screening system might be useful to discover the drug for HIV-1 TAR RNA.     

Recognition of HIV-TAR RNA using neomycin–benzimidazole conjugates

Synthesis of a novel class of compounds and their biophysical studies with TAR-RNA are presented. The synthesis of these compounds was achieved by conjugating neomycin, an aminoglycoside, with benzimidazoles modeled from a B-DNA minor groove binder, Hoechst 33258. The neomycin–benzimidazole conjugates have varying linkers that connect the benzimidazole and neomycin units. The linkers of varying length (5–23 atoms) in these conjugates contain one to three triazole units. The UV thermal denaturation experiments showed that the conjugates resulted in greater stabilization of the TAR-RNA than either neomycin or benzimidazole used in the synthesis of conjugates. These results were corroborated by the FID displacement and tat-TAR inhibition assays. The binding of ligands to the TAR-RNA is affected by the length and composition of the linker. Our results show that increasing the number of triazole groups and the linker length in these compounds have diminishing effect on the binding to TAR-RNA. Compounds that have shorter linker length and fewer triazole units in the linker displayed increased affinity towards the TAR RNA.