IRES-targeting small molecule inhibits enterovirus 71 replication via allosteric stabilization of a ternary complex

Figures

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  Fig. 1. RNA-biased small molecules bind to the EV71 SLII IRES domain. a Schematic of the consensus secondary structure of the EV71 5′UTR determined using phylogenetic comparisons. b Secondary structure of EV71 SLII as validated by NMR spectroscopy9. c 3D structure (PDB code 5V16) of SLII refined against NOE-derived distant restraints and RDCs9. d Screening data for small molecules vs the EV71 SLII (wt) IRES domain at 10 and 50 µm concentrations. Error bars shown are standard errors of mean calculated from three individual replicates. Each replicate data point is the average of nine reads from three parallel readings on three wells (technical replicates). The chemical structure of DMA-135 is shown within the inset. Screening conditions were carried out in 50 mm Tris, 50 mm KCl, 0.01% Triton-X-100, 5% DMSO, pH = 7.4; Tat peptide: 60 nm; RNA: 90 nm; small molecule: 10 or 50 µm; excitation λ = 485 nm, emission λ = 590 nm; incubation time: 30 min.

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  Fig. 2. DMAs bind to the EV71 SLII bulge via an entropically driven mechanism. a Single-point 1H-13C TROSY HSQC titrations of A(13C)-selectively labeled SLII constructs with the respective DMAs added at fivefold excess. The blue correlation peaks across each spectrum correspond to that of free SLII. The spectra were collected at 900 MHz in 10 mm K2HPO4, 20 mm KCl, 0.5 mm EDTA, and 4 mm BME (pH 6.5) D2O buffer at 298 K. b Calorimetric titrations of DMA-001, DMA-135, and DMA-155 reveal a single transition on the binding isotherm characterized by an entropically favored and enthalpically disfavored molecular recognition event. All titration data were processed and analyzed using Affinimeter25. The processed thermograms were fit to a 1:1 stoichiometric binding model, represented by the red lines. Reported values for dissociation constants (KD) and corresponding standard deviation are from triplicate experiments. Goodness of fit (χ2) of the experimental data to the 1:1 binding model are reported for each titration. The experiments were performed in 10 mm K2HPO4, 20 mm KCl, 0.5 mm EDTA, and 4 mm DTT (pH 6.5) buffer at 298 K. Data represented as mean ±SD of n = 3 experimental replicates.…

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  Fig. 3. DMA-135 inhibits EV71 IRES-dependent translation and replication. a The diagram depicts the bicistronic reporter plasmid used to synthesize RNA for transfections and dual-luciferase assays. b Effect of DMA-135 on EV71 IRES activity. SF268 cells were transfected with RLuc-EV71-5′UTR-FLuc RNA and cultured with various concentrations of DMA-135. Luciferase activity was measured 2 days later. Mean values ± standard deviations from three independent experiments (N = 3) are shown in the bar graphs. P values were determined by unpaired two-tailed Student’s t test. ***P < 0.001; **P < 0.01; N.S., not significant. 0.001 µm: P = 0.867669; 0.1 µm: P = 0.006658; 0.25 µm: P = 0.000011; 0.5 µm: P = 0.000305; 1 µm: P = 0.000031; 10 µm: P < 0.00001; 50 µm: P < 0.00001; 100 µm: P < 0.00001; 250 µm: P < 0.00001; 500 µm: P < 0.00001; 1000 µm: P < 0.00001. c Effect of DMA-135 on EV71 replication. SF268 cells were infected with EV71 at an moi = 1. Various concentrations of DMA-135 were added to the cells. Media were harvested 24 hr post infection and assayed for infectious virus by plaque formation in Vero cells. Mean values ± standard deviations from three independent experiments (N = 3) are shown.…

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  Fig. 4. DMA-135 changes the local and global structure of the EV71 SLII IRES domain. a Overlay of 1H-1H NOESY spectra (tm = 200 ms) of free SLII (red) and the (DMA-135)-SLII complex (black). The orange lines (solid and dashed) indicate NOE spin systems and chemical shifts that are identical between free SLII and the (DMA-135)-SLII complex. The black lines (solid and dashed) indicate NOE spin systems and chemical shifts of SLII within the complex that are significantly different from those of free SLII (indicated by red solid and dashed lines). Selected region of the 1H-1H NOESY spectrum of the (DMA-135)-SLII complex showing intermolecular and intramolecular NOEs between methyl protons on DMA-135 and nucleobases of SLII. b 1D 19F NMR spectra of DMA-197 in the absence (red) and presence of SLII at 1:1 complex (blue). c Correlation plot showing agreement between measured RDCs of free SLII and SLII within the DMA-135 complex. All NMR spectra were collected at 900 MHz in 10 mm K2HPO4, 20 mm KCl, 0.5 mm EDTA, and 4 mm BME (pH 6.5) D2O buffer at 298 K.

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  Fig. 5. DMA-135 changes the local and global structure of SLII. a Superimposition of 10 lowest energy SLII-(DMA-135) complex structures refined in AMBER using NOE-derived distance restraints and RDCs. The overall RMSD for the stably base paired regions is 2.4 Å. b Comparison of the bulge stacking interactions of (left) free SLII to that of the SLII-(DMA-135) complex (right). c Correlation plot between measured and back-calculated RDCs from a representative low energy SLII-(DMA-135) structure and to back-calculated RDCs from the free SLII structure. d Superimposition of the lower helices of free and bound SLII reveals that DMA-135 induces a global change to the SLII structure.

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  Fig. 6. DMA-135 allosterically stabilizes a AUF1-SLII-(DMA-135) ternary complex. a Calorimetric titrations of AUF1-RRM1,2 to SLII or to SLII:DMA-135 complex at different molar ratios reveal a single transition in the binding isotherm characterized by an enthalpically driven molecular recognition event. All data were processed and analyzed using Affinimeter25. The processed thermograms were fitted to a 1:1 stoichiometric binding model, represented by the red lines. Values for dissociation constants (KD) and corresponding standard deviation are reported for each titration. The experiments were performed in 10 mm K2HPO4, 20 mm KCl, 0.5 mm EDTA, and 4 mm BME (pH 6.5) buffer at 298 K. b Graphical representation of the dissociation constants for the AUF1-RRM1,2-SLII complex as a function of DMA-135 concentration. Reported KD values and corresponding standard deviations are from n = 3 experimental replicates. Data represented as mean ± SD of triplicate experiments. Uncertainties in individual binding isotherms are calculated using Affinimeter and are determined by considering the noise and quality of raw data and the dispersion of the integrated signal as a function of concentration.

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  Fig. 7. Allosteric effects of DMA-135 within the cellular environment. a Protein-biotinylated RNA pull-down experiments were performed to examine the effect of DMA-135 on the interaction between AUF1 and the EV71 SLII IRES domain. Biotinylated SLII was incubated with SF268 cell lysate to which was added the indicated concentrations of DMA-135. Streptavidin-linked beads were used to pull-down biotin-labeled SLII and its associated cellular proteins. Samples were analyzed by western blot using anti-AUF1 antibody. The experiment was performed twice yielding similar results. b Biotinylated wild-type SLII or the CCC-bulge mutant SLII were transfected into SF268 cells. Cells were cultured with various concentrations of DMA-135. Cell lysates were used for pull-down assays of SLII-associated proteins as described above. AUF1 bound to SLII RNA was detected by western blotting. The experiment was performed twice yielding similar results. c RIP assays for AUF1-EV71 Luc RNA interactions in cells. SF268 cells were transfected with RLuc-EV71 5′UTR-FLuc RNA, harboring the wild-type SLII, or with CCC-bulge mutant SLII and cultured with various concentrations of DMA-135. Lysates were prepared and…