Discovery of selective bioactive small molecules by targeting an RNA dynamic ensemble.

Department of Chemistry and Biophysics, University of Michigan, Ann Arbor, Michigan, USA.
Nature Chemical Biology (Impact Factor: 13.22). 06/2011; 7(8):553-9. DOI: 10.1038/nchembio.596
Source: PubMed

ABSTRACT Current approaches used to identify protein-binding small molecules are not suited for identifying small molecules that can bind emerging RNA drug targets. By docking small molecules onto an RNA dynamic ensemble constructed by combining NMR spectroscopy and computational molecular dynamics, we virtually screened small molecules that target the entire structure landscape of the transactivation response element (TAR) from HIV type 1 (HIV-1). We quantitatively predict binding energies for small molecules that bind different RNA conformations and report the de novo discovery of six compounds that bind TAR with high affinity and inhibit its interaction with a Tat peptide in vitro (K(i) values of 710 nM-169 μM). One compound binds HIV-1 TAR with marked selectivity and inhibits Tat-mediated activation of the HIV-1 long terminal repeat by 81% in T-cell lines and HIV replication in an HIV-1 indicator cell line (IC(50) ∼23.1 μM).


Available from: Aaron T Frank, Mar 19, 2015
  • [Show abstract] [Hide abstract]
    ABSTRACT: African trypanosomes cause a parasitic disease known as sleeping sickness. Mitochondrial transcript maturation in these organisms requires a RNA editing reaction that is characterized by the insertion and deletion of U-nucleotides into otherwise non-functional mRNAs. Editing represents an ideal target for a parasite-specific therapeutic intervention since the reaction cycle is absent in the infected host. In addition, editing relies on a macromolecular protein complex, the editosome, that only exists in the parasite. Therefore, all attempts to search for editing interfering compounds have been focused on molecules that bind to proteins of the editing machinery. However, in analogy to other RNA-driven biochemical pathways it should be possible to stall the reaction by targeting its substrate RNAs. Here we demonstrate inhibition of editing by specific aminoglycosides. The molecules bind into the major groove of the gRNA/pre-mRNA editing substrates thereby causing a stabilization of the RNA molecules through charge compensation and an increase in stacking. The data shed light on mechanistic details of the editing process and identify critical parameters for the development of new trypanocidal compounds.
    PLoS ONE 03/2015; 10(3):e0118940. DOI:10.1371/journal.pone.0118940 · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Higher sensitivity of NMR spectrometers and novel isotopic labeling schemes have ushered the development of rapid data acquisition methodologies, improving the time resolution with which NMR data can be acquired. For nucleic acids, longitudinal relaxation optimization in conjunction with Ernst angle excitation (SOFAST-HMQC) for imino protons, in addition to rendering rapid pulsing, has been demonstrated to yield significant improvements in sensitivity per unit time. Extending such methodology to other spins offers a viable prospect to measure additional chemical shifts, thereby broadening their utilization for various applications. Here, we introduce the 2D [(13)C, (1)H] aromatic SOFAST-HMQC that results in overall sensitivity gain of 1.4- to 1.7-fold relative to the conventional HMQC and can also be extended to yield long-range heteronuclear chemical shifts such as the adenine imino nitrogens N1, N3, N7 and N9. The applications of these experiments range from monitoring real-time biochemical processes, drug/ligand screening, and to collecting data at very low sample concentration and/or in cases where isotopic enrichment cannot be achieved.
    Journal of Biomolecular NMR 09/2014; 60(2-3). DOI:10.1007/s10858-014-9856-9 · 3.31 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: HIV-1 TAR RNA is a two-helix bulge motif that plays a critical role in HIV viral replication and is an important drug target. However, efforts at designing TAR inhibitors have been challenged by its high degree of structural flexibility, which includes slow large-amplitude reorientations of its helices with respect to one another. Here, we use the recently introduced algorithm WExplore in combination with Euler angles to achieve unprecedented sampling of the TAR conformational ensemble. Our ensemble achieves similar agreement with experimental NMR data when compared with previous TAR computational studies, and is generated at a fraction of the computational cost. It clearly emerges from configuration space network analysis that the intermittent formation of the A22-U40 base pair acts as a reversible switch that enables sampling of interhelical conformations that would otherwise be topologically disallowed. We find that most previously determined ligand-bound structures are found in similar location in the network, and we use a sample-and-select approach to guide the construction of a set of novel conformations which can serve as the basis for future drug development efforts. Collectively, our findings demonstrate the utility of WExplore in combination with suitable order parameters as a method for exploring RNA conformational space.
    Nucleic Acids Research 10/2014; DOI:10.1093/nar/gku799 · 8.81 Impact Factor