RNA Affinity and Specificity of Modified Aminoglycosides, Metal Complexes, and Intercalating Agents That Target the HIV-1 Rev Response Element
RNA plays a pivotal role in the replication of all organisms, including viral and bacterial pathogens. The development of small molecules that can selectively interfere with undesired RNA activity is a promising new direction for drug design. Currently, there are no anti-HIV treatments that target nucleic acids. The HIV-1 Rev Response Element
(RRE) is presented as an important target for the development of anti-viral agents that bind to RNA. The Rev binding site on the RRE is highly conserved, even between different groups of HIV-1 isolates. Compounds that inhibit HIV replication by binding to the RRE and displacing Rev are, therefore, expected to retain activity across groups of genetically diverse HIV infections.
Systematic studies of the binding interactions between small molecules and the RRE are essential for deciphering the parameters that govern effective RRE recognition. Novel techniques for determining the RRE affinity and specificity of Rev-RRE inhibitors are described. Rev displacement experiments can be conducted by monitoring the fluorescence anisotropy of a fluorescein-labeled Rev protein fragment, or by quantifying its displacement from a solid-phase immobilized RRE. Experiments conducted in the presence of competitor nucleic acids are used to evaluate the RRE specificity of Rev-RRE inhibitors.
The design, synthesis, and characterization of new RRE ligands are described. Derivatives based upon aminoglycosides, octahedral ruthenium complexes, and ethidium bromide are highlighted. These compounds are evaluated for their affinity to the RRE as well as non-targeted nucleic acids, including: calf thymus DNA, a yeast tRNA mixture, and numerous duplex and single- stranded DNAs and RNAs. We have concluded that synthesizing new ligands with high RRE affinity is relatively easy compared to discovering new ligands with high specificity for this RNA target.
A number of the new RRE ligands are found to possess significant anti-viral activities in HIV-1 infected cell cultures. The RRE affinity and specificity of every small molecule is not always proportional to its anti-HIV activity. Issues related to cellular uptake, localization, and the non-specific binding of other cellular components can dramatically affect the biological activities of small molecules.
These results (and those like them) should facilitate the future discovery and implementation of anti-HIV drugs that are targeted to the HIV-1 RRE. In addition, our expanded basic understanding of RNA molecular recognition should assist in the future identification and targeting of other therapeutically important RNA sites.
Introduction: The Central Dogma of Biology and RNA Ligands
Background: The HIV Lifecycle and Rev-RRE Interaction
Methods for Measuring RNA-Ligand Affinity and Specificity
New RRE Ligands: [Ru(bpy)2Eilatin]2+
Ethidium Bromide: Biological Activities and Nucleic Acid Binding
Aminoglycoside-Based RNA Ligands