Disrupting HIV replication with structure-based design of Tat inhibitors

Interaction of the HIV-1 protein Tat with its RNA response element TAR is an essential step in viral replication and an attractive target for development of anti-HIV drugs with new mechanism of action. Compounds have been identified that bind to the three nucleotide RNA bulge where the interaction occurs, but none have had sufficient potency to warrant pharmaceutical development. This work explores two novel approaches toward the development of potent and selective ligands for HIV-1 TAR RNA.;My primary focus has been the development of conformationally-constrained cyclic peptide mimetics of Tat, an approach that has yielded specific sub-nM inhibitors of the Tat-TAR interaction that are nearly as active in human lymphocytes as current anti-HIV drugs against a variety of clinical isolates. The NMR structures of two lead peptide-RNA complexes reveal that these molecules interfere with TAR function by binding simultaneously at the RNA bulge and apical loop, forming an unusually deep pocket in the RNA. Certain peptide-RNA contacts are highly conserved between the two complexes, despite significant differences in the peptide sequences. These define crucial interaction elements for peptide design and a basis for optimizing their activities. The complexes illustrate that RNA-binding molecules can achieve specificity by simultaneously interacting with multiple secondary structure elements.;My second approach to the development of TAR ligands is fragment based drug-design. Our in-house fragment library was screened against TAR RNA bound to a pre-selected arginine derivative, based on the hypothesis that arginine is a minimum motif for recognition of the bulge region of TAR. The structure of the Arg-TAR complex and the results of our fragment screen reveal that the arginine deriviative acts as a molecular switch to create a pocket in the RNA that is capable of binding additional fragments. Importantly, these fragments do not appreciably bind the free RNA. Lead fragments were docked with the arginine-TAR complex using interligand NOE data and show that the pocket created by the arginine derivative is in the RNA major groove just above the UCU bulge where Tat binds. The docked structures offer insight into development of a Tat inhibitor based on linking the arginine derivative to these drug-like fragments.