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

Abstract

Interaction of HIV-1 rev response element (RRE) RNA with its cognate protein. Rev, is critical for HIV-1 replication. Understanding the mode of interaction between RRE RNA and ligands at the binding site can facilitate RNA molecular recognition as well as provide a strategy for developing anti-HIV therapeutics. Our approach utilizes branched peptides as a scaffold for multivalent binding to RRE IIB (high affinity rev binding site) with incorporation of unnatural amino acids to increase affinity via non-canonical interactions with the RNA. Previous high throughput screening of a 46,656-member library revealed several hits that bound RRE IIB RNA in the sub-micromolar range. In particular, the lead compound, 4B3, displayed a K d value of 410 nM and demonstrated selectivity towards RRE. A ribonuclease protection assay revealed that 4B3 binds to the stem-loop structure of RRE IIB RNA, which was confirmed by SHAPE analysis with 234 nt long NL4-3 RRE RNA. Our studies further indicated interaction of 4B3 with both primary and secondary Rev binding sites.

Viral RNA structure analysis using DMS-MaPseq

“…The RRE is a stable structure that enables export of unspliced and incompletely spliced HIV-1 RNA from the nucleus of infected cells through interaction with the viral protein Rev [45]. Recent studies using SHAPE-Map were able to uncover the binding site of two new inhibitors on the RRE [46], [47]. Another study was able to track slight changes in structure of the RRE over the course of infection and correlate structural changes to activity of the RRE, thus showing that the RRE is under selection pressure to maintain or increase activity as HIV-1 evolves in the host [48].…”

The Rise of Boron-Containing Compounds: Advancements in Synthesis, Medicinal Chemistry, and Emerging Pharmacology

“…The most potent peptide in this series ((DLL) 2 KLGBY ( 44 , Figure ); K d = 0.41 μM) was subjected to an RNase protection assay which identified the loop and bulge in the upper stem region of RRE SL-IIB as essential for binding. SHAPE-MaP reactivity profiles of BPBA 44 with 234 nt RRE RNA revealed interactions with the internal loop and upper stem/apical loop regions of RRE SL-IIB and the SL-1 and SL-II regions RRE. − …”

HIV Rev-isited

“…Several of these agents bind to the RRE synonymously to Rev, inserting basic regions into the same wobble-base groove in the RRE. Peptide ligands have been developed which similarly adopt the same α-helicity as the Rev ARM; in some cases, these ligands are able to bind to the RRE with higher affinity than Rev itself (about sevenfold) and can successfully block HIV-1 replication [157][158][159][160][161]. Other small molecules, including 8-azaguanine, suppress viral gene expression by redirecting localization of Rev to the cytoplasm, impairing its function [162].…”