Abstract. Innovative approaches are needed to create therapeutics that target HIV. Existing drugs can prolong
patient lifespan by targeting multiple facets of the viral life cycle, but next-generation therapies are needed that
act on new targets — especially those that resist mutation — to improve long-term therapeutic compliance and
outcome. HIV-1 TAR RNA is a validated drug target that resists mutations to interact with the viral protein Tat,
thus giving rise to an RNA-protein complex essential for proviral transcription and HIV-1 propagation. So far,
TAR has evaded discovery of compounds with sufficient affinity and selectivity to warrant pharmaceutical
development. To address this challenge, we undertook a ‘semi-design and protein evolution’ approach that
yielded many novel, high-affinity (KDs ~ 1.3 to 0.5 nM) TAR Binding Proteins (TBPs) using yeast display
maturation. We then determined the 1.80 Å resolution co-crystal structure of one variant, TBP6.7, in complex
with TAR, revealing that the major binding interface consists of evolved loop ß2-ß3, which reads out the TAR
RNA major groove. We hypothesize that cyclic peptides comprising the TBP6.7 ß2-ß3 loop, or other TBP loops
evolved in our lab, will be entry points to create a novel class of TAR binders. Indeed, the TBP6.7 ß2-ß3
hairpin retains affinity and specificity for TAR when fused to the small protein SUMO, signifying that the ß2-ß3
loop is necessary and sufficient for TAR recognition. Structural identification of the ß-hairpin motif, and our use
of semi-design and evolution make our approach fundamentally different from prior efforts to block the Tat-TAR
interaction, while providing a robust experimental premise to pursue our aims: (Aim 1) Validate the observed
TBP6.7-TAR interface and determine additional novel co-crystal structures of other TBPs evolved in our lab;;
(Aim 2) synthesize and optimize cyclic peptides derived from Aim 1 that bind TAR and inhibit its interaction with
Tat;; (Aim 3) Test cyclic peptides from Aim 2 using viral infectivity assays to investigate mechanisms of action,
therapeutic indices, and pharmacological properties in animals. To our knowledge, no other group has used
protein evolution and structural biology to develop HIV-1 TAR-targeted reagents. We are a team of experts,
comprising two P.I.s, with strong records in protein evolution, peptide-based drug discovery, HIV therapeutic
discovery, measuring cell penetration and toxicity of biologics (McNaughton), and structural biology of
therapeutically-relevant RNAs, protein-RNA complexes, and biophysical analysis of protein-RNA interactions
(Wedekind), as well as two collaborators: Harold Smith (University of Rochester), a leader in drug discovery
and development, and CEO of OyaGen Inc., a private company developing anti-HIV drugs, and Dan Gustafson
(Colorado State University), a clinician and pharmacologist with expertise in measuring pharmacological
profiles of therapeutics. We are uniquely qualified and well suited to perform this work. High-value outcomes
include: (i) identification of novel lead inhibitors of HIV, and (ii) validation of our ‘semi-design’ and structural
approach, which has the potential for sustained impact on the drug discovery and inhibitor design fields.