Demonstration that Small Molecules can Bind and Stabilize Low-abundance Short-lived RNA Excited Conformational States

Abstract

Many promising RNA drug targets have functions that require the formation of RNA-protein complexes, but inhibiting RNA-protein interactions can prove difficult using small molecules. Regulatory RNAs have been shown to transiently form excited conformational states (ESs) that remodel local aspects of secondary structure. In some cases, the ES conformation has been shown to be inactive and to be poorly recognized by protein binding partners. In these cases, specifically targeting and stabilizing the RNA ES using a small molecule provides a rational structure-based strategy for inhibiting RNA activity. However, this requires that a small molecule discriminates between two conformations of the same RNA to preferentially bind and stabilize the short-lived low-abundance ES relative to the long-lived more abundant ground state (GS). Here, we tested the feasibility of this approach by designing a mutant that inverts the conformational equilibrium of the HIV-1 transactivation response element (TAR) RNA, such that the native GS conformation becomes a low-abundance ES. Using this mutant and NMR chemical shift mapping experiments, we show that argininamide, a ligand mimic of TAR's cognate protein binding partner Tat, is able to restore a native-like conformation by preferentially binding and stabilizing the transient and low-populated ES. A synthetic small molecule optimized to bind the TAR GS also partially stabilized the ES, whereas an aminoglycoside molecule that binds RNAs nonspecifically did not preferentially stabilize the ES to a similar extent. These results support the feasibility of inhibiting RNA activity using small molecules that preferentially bind and stabilize the ES.

Keywords: HIV-1; RNA dynamics; TAR; drug discovery; ensemble.

Figure 1.

The ES-stabilizing mutant TAR^UUCG-ES experiences back exchange with a low-abundance and short-lived ES that has conformational features similar to the GS of wild-type TAR. a. Chemical exchange between the GS and ES in wtTAR and TAR^UUCG-ES. Shown are the populations (p_GS and p_ES) of the GS and ES, respectively and the rate constants (k₁ and k₋₁) for inter-conversion deduced from 2-state analysis of the R_1ρ RD data. Residues are labeled according to the wtTAR GS secondary structure: stem (blue), bulge (orange), apical loop (red). Residues that differ between the two TAR variants are in gray. b. R_1ρ RD profiles measured for TAR^UUCG-ES. Off-resonance profiles show the dependence of R₂ + R_ex on spinlock power (ω2π⁻¹) and offset (Ω2π⁻¹). Shown are the global fits (solid line) to a two-state model using the Bloch-McConnell equations assuming a two-state exchange process (Methods). On-resonance profiles show the dependence of R_1ρ on spinlock power (ω2π⁻¹). Error bars represent experimental error determined by propagation of error determined by Monte Carlo analysis of monoexponential decay curves and experimental signal to noise. Data was collected on a 600 MHz (¹H frequency) spectrometer. Buffer conditions are 15 mM sodium phosphate, 25 mM sodium chloride, 0.1 mM EDTA, 100% D₂O at pH 6.4 and 25 °C. c. Comparison of the difference between chemical shifts measured for the ES and GS (Δω = ω_ES - ω_GS) in TAR^UUCG-ES (green) and the inverse (Δω = ω_GS - ω_ES) in wtTAR (black) [13] using R_1ρ RD NMR.

Figure 2.

Argininamide (ARG) binds and stabilizes the ES of TAR^UUCG-ES. a. ARG binds the wtTAR GS secondary structure and stabilizes a unique base triple conformation. b. 2D [¹³C,¹H] aromatic SOFAST-HMQC [39] spectra of free TAR^UUCG-ES (blue), TAR^UUCG-ES + 128x ARG (red), free wtTAR (green), and wtTAR + 32x ARG (orange). Buffer conditions are 15 mM sodium phosphate, 25 mM sodium chloride, 0.1 mM EDTA, 10% D₂O at pH 6.4 and 25 °C.

Figure 3.

Small molecules can partially stabilize or non-specifically bind RNA. 2D [¹³C,¹H] aromatic SOFAST-HMQC [39] spectra of free TAR^UUCG-ES (blue), TAR^UUCG-ES + 16x small molecule (red), free wtTAR (green), and wtTAR + 8x small molecule (orange) for a. DMA-169 and b. neomycin. Buffer conditions are 15 mM sodium phosphate, 25 mM sodium chloride, 0.1 mM EDTA, 10% D₂O at pH 6.4 and 25 °C.