Binding Characteristics of Small Molecules That Mimic Nucleocapsid Protein-Induced Maturation of Stem-Loop 1 of HIV-1 RNA

Abstract

As a retrovirus, the human immunodeficiency virus (HIV-1) packages two copies of the RNA genome as a dimer in the infectious virion. Dimerization is initiated at the dimer initiation site (DIS) which encompasses stem-loop 1 (SL1) in the 5’-UTR of the genome. Study of genomic dimerization has been facilitated by the discovery that short RNA fragments containing SL1 can dimerize spontaneously without any protein factors. Based on the palindromic nature of SL1, a kissing loop model has been proposed. First, a metastable kissing dimer is formed via standard Watson-Crick base pairs and then converted into a more stable extended dimer by the viral nucleocapsid protein (NCp7). This dimer maturation in vitro is believed to mimic initial steps in the RNA maturation in vivo, which is correlated with viral infectivity. We previously discovered a small molecule activator, Lys-Ala-7-amido-4-methylcoumarin (KA-AMC), which facilitates dimer maturation in vitro, and determined aspects of its structure-activity relationship. In this report, we present measurements of the binding affinity of the activators and characterization of their interactions with the SL1 RNA. Guanidinium groups and increasing positive charge on the side chain enhance affinity and activity, but features in the aromatic ring at least partially decouple affinity from activity. Although KA-AMC can bind to multiple structural motifs, NMR study showed KA-AMC preferentially binds to unique structural motifs, such as the palindromic loop and the G-rich internal loop in the SL1 RNA. NCp7 binds to SL1 only an order of magnitude tighter than the best small molecule ligand tested. The study presented here provides guidelines for design of superior small molecule binders to the SL1 RNA that have the potential of being developed as an antiviral by either interfering with SL1-NCp7 interaction at the packaging and/or maturation stages.

Advances in targeting nucleocapsid–nucleic acid interactions in HIV-1 therapy

“…In their pursuit to identify compounds that bind to SL1 and inhibit SL1-NC interaction, Chung and colleagues have identified a peptide-based coumarin derivative compound, 19 (Fig 5), that binds the SL1 stem-bulge region, which appears to mimic the chaperoning effect of NC for SL1 (Chung et al, 2010). Although the identified compound is much less efficient at chaperoning than NC, the unique compounds and identification strategy opens a new avenue for identifying compounds that compete for NC and bind viral RNA.…”

NMR Studies of Retroviral Genome Packaging

“…In addition to small molecule inhibitors, 1 H NMR analysis showed that polypeptides rich in tryptophan also interact with the GGAG tetraloop of the Ψ-hairpin, and possibly block NC binding [ 390 ]. The DIS region might also be targeted, since dimerization is a prerequisite for retroviral genome packaging [ 391 ].…”

NCp7: targeting a multitask protein for next-generation anti-HIV drug development part 2. Noncovalent inhibitors and nucleic acid binders

“…The first stem-loop structure of the packaging site, SL1 forms the so-called kissing dimer, which is recognized by NCp7 and converted into a more stable extended duplex in the early stages of viral maturation. Using a virtual-screening-based procedure, James and colleagues compiled a list of potential kissing dimer binders [51,52] and, with a consecutive experimental screen based on a gel shift assay, they found that the coumarin derivative 28 (Fig. 4), instead of inhibiting, activated the conversion of the kissing dimer into the extended structure, thus unexpectedly acting as a NCp7 mimic.…”

“…4), instead of inhibiting, activated the conversion of the kissing dimer into the extended structure, thus unexpectedly acting as a NCp7 mimic. Compound 28 might serve as a small-molecule tool to study NCp7-mediated maturation or as the starting point for the development of antivirals that activate viral maturation prematurely [52].…”