Fluorescence‐based methods for evaluating the RNA affinity and specificity of HIV‐1 Rev–RRE inhibitors

Abstract

RNA plays a pivotal role in the replication of all organisms, including viral and bacterial pathogens. The development of small molecules that 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. This article presents the HIV-1 Rev response element (RRE) as an important focus for the development of antiviral agents that target 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 evaluations of both the RRE affinity and specificity of numerous small molecule inhibitors are essential for deciphering the parameters that govern effective RRE recognition. This article discusses fluorescence-based techniques that are useful for probing a small molecule's RRE affinity and its ability to inhibit Rev-RRE binding. Rev displacement experiments can be conducted by observing the fluorescence anisotropy of a fluorescein-labeled Rev peptide, or by quantifying its displacement from a solid-phase immobilized RRE. Experiments conducted in the presence of competing nucleic acids are useful for evaluating the RRE specificity of Rev-RRE inhibitors. The discovery and characterization of new RRE ligands are described. Eilatin is a polycyclic aromatic heterocycle that has at least one binding site on the RRE (apparent Kd is approximately 0.13 microM), but it does not displace Rev upon binding the RRE (IC50 > 3 microM). In contrast, ethidium bromide and two eilatin-containing metal complexes show better consistency between their RRE affinity and their ability to displace a fluorescent Rev peptide from the RRE. These results highlight the importance of conducting orthogonal binding assays that establish both the RNA affinity of a small molecule and its ability to inhibit the function of the RNA target. Some Rev-RRE inhibitors, including ethidium bromide, Lambda-[Ru(bpy)(2)eilatin]2+, and Delta-[Ru(bpy)(2)eilatin]2+ also inhibit HIV-1 gene expression in cell cultures (IC50 = 0.2-3 microM). These (and similar) results should facilitate the future discovery and implementation of anti-HIV drugs that are targeted to viral RNA sites. In addition, a deeper general understanding of RNA-small molecule recognition will assist in the effective targeting of other therapeutically important RNA sites.

Downsizing human, bacterial, and viral proteins to short water-stable alpha helices that maintain biological potency

“…Full-length CSP-1 protein inhibits S. pneumoniae growth at only ≥10 μM (21). The 116 residue HIV-Rev protein binds to RNA with Kd approximately 1 nM (28). Nociceptin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) is an agonist at only nM concentrations (25).…”

Targeting RNA with Small Molecules

“…Re-evaluating the activity of each inhibitor under such stringent conditions yields essential information regarding the ligand's selectivity (Figure 8 b). [61] As part of the assay's validation process, we demonstrated its capability to faithfully reproduce the known affinity and selectivity trends among aminoglycoside antibiotics. [60] We also illustrated the ability of this novel assembly to readily handle binders that interfere with common biophysical assays (such as fluorescence anisotropy).…”

“…Challenging the immobilized fluorescent Rev ± RRE complex with potential inhibitors and determining the amount of RevFl displaced generates the necessary information regarding a ligand's affinity (Figure 8 a). [61] The strengths of this assay are its robustness and reliable performance in the presence of large amounts of competing nucleic acids. Re-evaluating the activity of each inhibitor under such stringent conditions yields essential information regarding the ligand's selectivity (Figure 8 b).…”

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

“…[9] This structure revealed that only the central part of the protein (residues 9-65) is structured, while the rest is intrinsically disordered. Motivated by the important role of Rev in the HIV replication cycle, and its potential as a drug target, [11,13,14] we developed two efficient synthetic and recombinant methods that allow the production of sufficient amounts of highly pure Rev, and provide the flexibility to prepare novel synthetic analogues of Rev for future studies to elucidate its structure-function relationship with spatial and temporal control.…”