The overall goal of this program component is to use biochemical methods to identify small molecule inhibitors of Rev function. The main goal of this project will be to develop compound libraries for NMR screening, and use them to identify compounds that bind to RRE RNA and Rev-RRE complexes as potential Rev-directed inhibitors.
The Specific Aims are: 1) Develop small molecule compound libraries suitable for discovery of inhibitors by NMR screening. NMR provides a powerful tool for rapidly identifying weak binding ligands against any possible target. We will develop small diverse libraries from commercially available chemicals, or from combinatorial libraries, that are small soluble drug-like molecules with a variety of molecular frameworks. In addition, we will accumulate information that allows us to improve the RNA binding properties of our libraries in an iterative fasion. 2) Carry out NMR screening against the RRE RNA and Rev-RRE complexes to identify lead candidates. We will use the NMR libraries developed in Aim 1 to carry out a screen for small molecules that bind to the RRE, or to Rev-RRE complexes. The NMR screening will be carried out in an iterative fashion to develop structure-activity relationships, and to improve the potency of the compounds. 3) Quantitative analysis of the interactions of inhibitors with the RRE and Rev-RRE complexes. As a followup assay and alternative screening strategy, we will use gel shift assays to identify inhibitors of the Rev-RRE interaction. In addition, we will perform isothermal titration calorimetry to study the binding of inhibitors to the RNA. Using a variety of RNA and Rev constructs we can screen for compounds that block initial binding to the high affinity site, or to other RRE sites, or that block the oligomerization of Rev on the RRE. In addition, we will characterize the binding of the natural product harziphilone to the RRE.
| Marenchino, Marco; Armbruster, David W; Hennig, Mirko (2009) Rapid and efficient purification of RNA-binding proteins: application to HIV-1 Rev. Protein Expr Purif 63:112-9 |
| Pond, Stephanie J K; Ridgeway, William K; Robertson, Rae et al. (2009) HIV-1 Rev protein assembles on viral RNA one molecule at a time. Proc Natl Acad Sci U S A 106:1404-8 |
| Carlomagno, Teresa; Amata, Irene; Williamson, James R et al. (2008) NMR assignments of HIV-2 TAR RNA. Biomol NMR Assign 2:167-9 |
| Pljevaljcic, Goran; Millar, David P (2008) Single-molecule fluorescence methods for the analysis of RNA folding and ribonucleoprotein assembly. Methods Enzymol 450:233-52 |
| Edgcomb, Stephen P; Aschrafi, Angelique; Kompfner, Elizabeth et al. (2008) Protein structure and oligomerization are important for the formation of export-competent HIV-1 Rev-RRE complexes. Protein Sci 17:420-30 |
| Scott, Lincoln G; Hennig, Mirko (2008) RNA structure determination by NMR. Methods Mol Biol 452:29-61 |
| Hennig, Mirko; Scott, Lincoln G; Sperling, Edit et al. (2007) Synthesis of 5-fluoropyrimidine nucleotides as sensitive NMR probes of RNA structure. J Am Chem Soc 129:14911-21 |
| Hennig, Mirko; Munzarova, Marketa L; Bermel, Wolfgang et al. (2006) Measurement of long-range 1H-19F scalar coupling constants and their glycosidic torsion dependence in 5-fluoropyrimidine-substituted RNA. J Am Chem Soc 128:5851-8 |
