Orientation and affinity of HIV-1 Tat fragments in Tat-TAR complex determined by fluorescence resonance energy transfer.

The human immunodeficiency virus (HIV-1) encodes a transcriptional activator protein, Tat, which is expressed early in the viral life cycle and is essential for viral gene expression, replication, and pathogenesis. Tat interacts with the transactivation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. Tat-derived peptides that contain the basic arginine-rich region of Tat are able to form in vitro complexes with TAR RNA, and these peptides provide a well-characterized system for understanding the mechanism of RNA-protein recognition. It is not known how RNA-binding Tat peptides are folded or docked in the Tat-TAR complex, and to what extent structural reorganization occurs upon TAR binding. To address these questions, we developed a fluorescence resonance energy transfer (FRET) system to analyze the interactions between TAR RNA and a Tat protein fragment (aa 38-72) uniquely labeled with donor and acceptor dye molecules, respectively. Using this FRET assay, we determined the binding affinity of Tat (47-58) and Tat (38-72) for TAR RNA under physiological conditions. We also delineated the distance between the N- and C-terminus of Tat (38-72) and the distance between the two termini and the 5' end of TAR when Tat (38-72) is bound to TAR. Our results suggest that the N- and C-termini of Tat (38-72) are close to each other when the peptide is folded and that the peptide does not go through a large structural change upon TAR binding.

A new class of RNA-binding oligomers: peptoid amide and ester analogues.

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. Here we report the design, synthesis and in vitro activities of oligopeptoid amide and ester analogues which bind TAR RNA with high affinities. These results show that we have identified a new class of unnatural oligomers for RNA targeting.

Discovery of a small molecule Tat-trans-activation-responsive RNA antagonist that potently inhibits human immunodeficiency virus-1 replication.

Antiretroviral therapy to treat AIDS uses molecules that target the reverse transcriptase and protease enzymes of human immunodeficiency virus, type 1 (HIV-1). A major problem associated with these treatments, however, is the emergence of drug-resistant strains. Thus, there is a compelling need to find drugs against other viral targets. One such target is the interaction between Tat, an HIV-1 regulatory protein essential for viral replication, and trans-activation-responsive (TAR) RNA. Here we describe the design and synthesis of an encoded combinatorial library containing 39,304 unnatural small molecules. Using a rapid high through-put screening technology, we identified 59 compounds. Structure-activity relationship studies led to the synthesis of 19 compounds that bind TAR RNA with high affinities. In the presence of a representative Tat-TAR inhibitor (5 microM TR87), we observed potent and sustained suppression of HIV replication in cultured cells over 24 days. The same concentration of this inhibitor did not exhibit any toxicity in cell cultures or in mice. TR87 was also shown to specifically disrupt Tat-TAR binding in vitro and inhibit Tat-mediated transcriptional activation in vitro and in vivo, providing a strong correlation between its activities and inhibition of HIV-1 replication. These results provide a structural scaffold for further development of new drugs, alone or in combination with other drugs, for treatment of HIV-1-infected individuals. Our results also suggest a general strategy for discovering pharmacophores targeting RNA structures that are essential in progression of other infectious, inflammatory, and genetic diseases.