**8. Exploiting RRE Conformational Flexibility with Branched Peptides**

The unique 3D architecture of RNA, often achieved by assuming a combination of local structures including bulges, stem-loops, pseudoknots, and turns, presents an attractive therapeutic opportunity. While exemplified by antimicrobial agents, such as aminoglycosides, macrolide, oxazolidinone, and tetracycline, that interfere with ribosomal RNA function, the field of small molecule RNA therapeutics, despite advances in high throughput screening technologies, has not fully matured and would benefit from innovative strategies.

As an alternative strategy that considers molecules of intermediate size, branched peptides offer the possibility of supporting multivalent interactions with different regions of a highly flexible RNA such as the RRE (Figure 9A) and would be predicted to enhance their selectivity and affinity. Support for branched peptides was provided by Bryson et al. [76], whose on-bead screening of a 4000 molecule

library identified ligands that spanned the bulge and apical loop of HIV-1 TAR RNA with binding affinities in the low micromolar range. Subsequently, to enhance selectivity/affinity Dai et al. [77] synthesized a ~46,000 compound on-bead library of branched peptides composed of unnatural amino acids (Figure 9B). These included L-guanidinoproline and D-aminoproline as electrostatic mimics of arginine and lysine, respectively, 1-naphthalene to promote π— π stacking with nucleobases and pyrazine which acts as a hydrogen bond donor/acceptor. High throughput screening identified the branched peptide 4A5 (Figure 8B) as a high affinity ligand for a synthetic RRE SL-IIB mimic (Kd = 0.88 ± 0.02 μM). The SHAPE analysis indicated that at a 1:1 RNA/4A5 ratio, nucleotides of a loop constituting the secondary SL-1 Rev binding site were protected from modification, while at a higher branched peptide/RNA ratio, nucleotides within SL-II became refractory to acylation. From this study, roles for 4A5 in occupying the active Rev binding sites and/or acting allosterically to prevent the SL-I-driven conformational change in the RRE were proposed. Although the exact mechanism remains to be elucidated, the SHAPE data lend credence to the notion of multivalent binding of branched peptides to the RRE. Finally, in the same study, 4A5 was demonstrated to inhibit Rev-RRE function in cell culture using HEK 293T cells transiently transfected with a Rev-expressing plasmid and a CMV promoter-driven GagPol-RRE plasmid (Figure 9C).

**Figure 9.** Restricting RRE conformational flexibility with multivalent branched peptides. (**A**) Cartoon depicting the branched peptide strategy, i.e., binding in a multivalent fashion to enhance affinity and selectivity toward the RNA target. (**B**) Structure of branched peptide 4A5. (**C**) Inhibition of Rev-RRE function in vivo using HEK 293T cells transiently transfected with a Rev-expressing plasmid and a CMV promoter-driven GagPol-RRE plasmid. Modified from Dai et al. [77].
