RNA molecules fold into complex tertiary structures that dictate biological function. These RNA molecules also represent an untapped class of potential drug targets. Specific binding of small molecules remains a significant hurdle in the validation of RNA as a drug target. To address this question, we chose to focus on a model RNA:protein interaction between the HIV-1 Rev protein and the Rev-responsive element (RRE) RNA.

The RRE is a 351-nucleotide element present on all intron-containing RNAs generated from the HIV-1 genome, able to drive nuclear RNA export through interaction with the viral Rev protein. The insertion of an α-helical arginine-rich motif (ARM) of Rev into the widened major groove of a stem-loop within the RRE, known as RRE IIB, provides most of the driving force for this interaction.

To further study the specific components necessary for RRE recognition and determine the "druggability" of this RNA target, we set out to synthesize and discover molecules that would be able to specifically recognize the RRE and compete off the Rev peptide. We utilized two strategies to elucidate these molecules: the use of macrolactam bridged α-helical peptidomimetics and a high-throughput screening campaign for Rev:RRE inhibitors.

In chapter II we describe our efforts to synthesize an a-helical peptidomimetic derived from an ARM known as R6QR7. We were able to utilize one specific location and orientation of macrolactam linkage that was compatible with both a-helical secondary structure as well as RRE binding. This peptidomimetic consists of fourteen amino acids and binds more specifically than the twenty-two amino acid peptide derived from the wild-type Rev protein sequence.

Chapter III focuses on the use of this a-helical peptidomimetic scaffold to study the specific components involved in RRE recognition. These studies illustrate that this high level of specific recognition by the peptidomimetic is accomplished through only a minimal interaction motif, namely one glutamine and one arginine which are located on the same helical face.

Finally, Chapter IV contains information regarding a validated high-throughput screening effort using an Alphascreen biochemical assay. This screen elucidated several small molecules able to bind the RRE some with a relatively good specificity.

Human African Trypanosomiasis (HAT), caused by the eukaryotic parasite Trypanosoma brucei, is a serious health problem in much of central Africa. The only validated molecular target for treatment of HAT is ornithine decarboxylase (ODC), which catalyzes the first step in polyamine metabolism. Herein, we describe the optimization of high throughput screening protocols at St. Jude Children's Research Hospital as well as the development of a high-throughput compatible assay for ODC. We have used this assay to screen 316,114 unique molecules to identify potent and selective inhibitors of ODC. This effort identified four novel families of ODC inhibitors, including the first inhibitors selective for the parasitic enzyme. These compounds display unique binding modes, suggesting the presence of allosteric regulatory sites on the enzyme. Docking of a subset of these inhibitors, coupled with mutagenesis, also supports the existence of these allosteric sites.

In response to glucocorticoids, the glucocorticoid receptor induces a cell type specific transcriptional program through both direct and indirect interactions with glucocorticoid response elements (GREs). The association between GREs and regulated genes is often unclear, because a GRE can be tens of thousands of base pairs from the gene it regulates. To help understand the mechanisms driving GR-DNA binding and transcription, we looked at the genome-wide binding and transcriptional response to glucocorticoids in three cell lines. Consistent with previous reports, we found that binding is more closely associated with upregulation than with downregulation. Interestingly, we found that GR binding regions (GRBRs) that contain a canonical GR motif, called a GR binding sequence (GBS), are better predictors of upregulation than those lacking a GBS, while the reverse is true of downregulation. To help understand the determinants of cell type specific binding, we compared the sequences of the GRBRs in the three different cell types and found that binding motifs for both the glucocorticoid receptor and for other transcription factors are overrepresented to different degrees in each cell type. The canonical GR motif is present in 66% of the U2OS sites, verses 14% of the Nalm6 sites; similarly, the AP-1 binding motif is present in 22% of A549 sites and 6% of Nalm6 sites. This difference in the sequence composition of binding sites suggests that the determinants of binding may be encompassed in the local sequence of a binding site. Supporting this, we found that the cell type specificity of binding could be recapitulated in reporters containing approximately 400 base pairs of DNA.