Click Dimers To Target HIV TAR RNA Conformation

Abstract

A series of neomycin dimers have been synthesized using “Click chemistry” with varying functionality and length in the linker region to target the HIV-1 TAR RNA region of HIV virus. TAR (Transactivation Response) RNA region, a 59 base pair stem loop structure located at 5′-end of all nascent viral transcripts interacts with its target, a key regulatory protein, Tat, and necessitates the replication of HIV-1 virus. Neomycin, an aminosugar, has been shown to exhibit multiple binding sites on TAR RNA. This observation prompted us to design and synthesize a library of triazole linked neomycin dimers using click chemistry. The binding between neomycin dimers and TAR RNA was characterized using spectroscopic techniques including FID (Fluorescent Intercalator displacement), FRET (fluorescence resonance energy transfer) competitive assay, circular dichroism (CD) and UV-thermal denaturation. UV thermal denaturation studies demonstrate that neomycin dimers binding increase the melting temperature (Tm) of the HIV TAR RNA up to 10 °C. (Ethidium bromide) displacement (FID) and FRET competition assay revealed nanomolar binding affinity between neomycin dimers and HIV TAR RNA while in case of neomycin, only a weak binding was detected. More importantly, most of the dimers showed lower IC50s towards HIV TAR RNA, when compared to the fluorescent Tat peptide and show increased selectivity over mutant TAR RNA. Cytopathic effects investigated using MT-2 cells indicate a number of the dimers with high affinity towards TAR show promising anti HIV activity.

Characterization of Ribosomal Binding and Antibacterial Activities Using Two Orthogonal High-Throughput Screens

“…Neomycin sulfate, Hoechst stains 33258 and 33342, ampicillin sodium salt, penicillin G sodium salt, and bacitracin were all obtained from commercial sources. Synthesis of DPA 79 and all triazole-linked dimers has been reported previously by us (35)(36)(37). The thiourea linkers DPA 71, DPA 73, DPA 76, DPA 78, and DPA 81 and the urea linkers DPA 72, DPA 74, DPA 75, DPA 77, and DPA 80 were synthesized as previously described for the synthesis of DPA 79 (37), and the characterization details will be reported elsewhere.…”

The Design of RNA Binders: Targeting the HIV Replication Cycle as a Case Study

“…Interestingly, this compound shows increased selectivity over mutant TAR and a lower IC 50 than the fluorescent Tat peptide used for the FRET assay. [43] It also inhibits the release of reverse transcriptase into infected cell culture supernatant at a micromolar concentration. The design of peptides or peptidomimetics is a classic approach to inhibit interactions between macromolecules such as protein-protein or protein-RNA.…”

Screening for Protein Kinase C Ligands Using Fluorescence Resonance Energy Transfer

“…The aqueous layer was extracted three times with CHCl 3 . The combined organic layer was washed with brine, dried over MgSO 4 …”

“…The FRET phenomenon can be applied as a spectroscopic measure and a probe of conformational change of macro-biomolecules [1][2][3] and a FRET-mediated competitive assay can be used for studies of the binding between two molecules. 4,5) Radioisotopebased methods are extremely sensitive and are widely used for ligand binding assays, but fluorescent-based assays, which are suitable for high-throughput screening are free of hazards associated with radioactivity. Previously, we described fluorescence-based ligand screening assays as an alternative to radioisotope assays.…”