Discovery of a Branched Peptide That Recognizes the Rev Response Element (RRE) RNA and Blocks HIV-1 Replication

Abstract

We synthesized and screened a unique 46,656-member library composed of unnatural amino acids that revealed several hits against RRE IIB RNA. Among the hit peptides identified, peptide 4A5 was found to be selective against competitor RNAs and inhibited HIV-1 Rev-RRE RNA interaction in cell culture in a p24 ELISA assay. Biophysical characterization in a ribonuclease protection assay suggested that 4A5 bound to the stem-loop region in RRE IIB while SHAPE MaP probing with 234 nt RRE RNA indicated additional interaction with secondary Rev binding sites. Taken together, our investigation suggests that HIV replication is inhibited by 4A5 blocking binding of Rev and subsequent multimerization.

Molecular recognition of a branched peptide with HIV-1 Rev Response Element (RRE) RNA

“…These results were consistent from our previous work, indicating the loop and bulge in the RRE SL-IIB construct, especially the upper stem region, are essential for peptide binding. 22,24 In addition, 4B3 showed lower selectivity for RRE SL-IIB in the presence of TAR RNA as the K d against RRE IIB increased approximately 10-fold. Since both TAR and RRE RNAs share a small stem-loop with a consensus sequence UGGG, this unique structure of the RNA may be necessary for 4B3 binding.…”

“…21 We recently reported the high-throughput screening and characterization of a fourth generation branched peptide library that consisted solely of unnatural amino acids at all variable positions. 22 Each unnatural amino acid offers a unique mode of binding (electrostatic interaction, hydrogen bonding, pi stacking, etc) and include L-Guanidinoproline (L), Daminoproline (D), guanine peptide nucleic acid (G), lysine boronic acid (B) and 1naphthylalanine (N) ( Fig. 1A).…”

“…29 SHAPE based footprinting has also been previously employed successfully to study ligand binding to RNA. 22,[30][31][32] Free RNA and RNA incubated with various concentrations of 4B3 were treated with electrophile l-methyl-7-nitroisatoic anhydride (IM7), and reactivity differences were plotted as a function of nucleotide position. A cutoff of +/− 0.3 was arbitrarily chosen to identify nucleotides that were rendered more flexible or constrained in the presence of 4B3.…”

RNA-Targeted Therapies and High-Throughput Screening Methods

“…The therapeutic effects are achieved through the regulation of RNA maturation, splicing, interference, translation and stabilisation. More possibilities of drug formats and therapeutic mechanisms have been discussed, including peptides [182], aptamers [183] and RNA editing with CRISPR-Cas13 [184]. Over the past five years, an increasing number of RNA-targeted drugs have been approved.…”

Structural Fluidity of the Human Immunodeficiency Virus Rev Response Element

“…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.…”