Host targeted antiviral (HTA): functional inhibitor compounds of scaffold protein RACK1 inhibit herpes simplex virus proliferation

Figures

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  Figure 1. (A) Shown are sample two receptor-based three-point pharmacophore models generated on the RACK1A phosphorylation site with exclusion spheres colored pink, geometric and distance constraints (flexible) shown as lines and filled white circles as centers. HP-hydrophobic; D-donor; A-acceptor. (B) Ligand-based pharmacophore model generated on SD-29 with pharmacophore constraints acceptor, donor, hydrophobic ring, and hydrophilic sites represented filled circles. Structures of compounds SD-29-12 and SD-29-14 are shown.

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  Figure 2. (A) Docked Model of RACK1A with SD-29 at the Y248 phosphorylation site. (left panel) Modeled structure of RACK1A with SD-29 (carbon in green) docked into it. The targeted binding pocket is highlighted in green. RACK1A is shown as ribbon model (white). (right panel) Detailed view of the SD-29 (carbon in green) interaction with RACK1A site pocket. The residues interacting with SD-29 are shown in a ball-and-stick model. Hydrogen bonds are shown as red broken lines. SD-29 binding site is surrounded by both hydrophobic (HP1) and hydrophilic residues (HP2). The structural model of ‘SD-29’ with RACK1A showing hydrogen bonds with Ser244, Trp249 and hydrophobic interactions with Tyr248, Phe243, Pro204, Leu 263and Trp249 residues. (B) RACK1 functional inhibitor compounds inhibit stress hormone induced RACK1A Y248 phosphorylation. One-week old Arabidopsis seedlings were treated with 10 μM of stress hormone Abscisic acid (ABA) in the presence/absence of the inhibitor compounds for 12 hours in a growth chamber (overnight) at 22°C. Lysates were probed with an antibody raised to detect phosphorylated Y248 residue of RACK1A protein in Arabidopsis. Lysates from a rack1a-1 knock-out mutant…

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  Figure 3. Effects of the compounds on HSV-1 protein expression. (A) Time and concentration dependent virus protein expression to deduce optimum drug treatment regimen. Left panel shows the time dependent HSV-1 virus protein expression after infection with HSV-1 17 at MOI 1.25. Right panel shows the same virus protein expression under different concentrations of virus starting from 0.01 to 6.25 MOI. (B) Hep-2 cells were pre-treated with the compounds for 24 h and three different virus proteins (ICP0, ICP8, and gD) were assayed 24 h post infection with different titers of HSV-1. Red arrows show the downregulated virus proteins and blue arrow shows the downregulated RACK1 protein. Tubulin expression was used as the loading control for the blots.

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  Figure 4. Real-time PCR analysis of HSV-1 ICP0 expression in the Hep-2 primary treated with the indicated inhibitor compounds and DMSO (as control) for the indicated time points. The total transcript levels were quantified using actin expression as an internal control. The cDNA obtained immediately after the virus and drugs transfection was set as a baseline value. The normalized expression values were transformed to the 1og10 value. The data plot represents mean ± standard error calculated from three replicates.

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  Figure 5. Co-treatment of drugs with virus increases efficacy. (A) The effect of drugs of indicated concentration after 24-hour post-infection with HSV1-Luc (4 × 106 pfu/ml media) and the same treatments are shown in duplicates. (B) The HSV-1 major structural protein ICP0 expression was assayed from the 48-hour post infection samples as shown in panel (A). The expression of tubulin was used as loading control (lower panel).

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  Figure 6. Visualization of the RACK1 inhibitor induced inhibition of HSV-1 proliferation in the HEp-2 cells. The HSV-1-Luc virus and the indicated concentration of compounds were added and incubated for 24 h (panels A and B) samples. The luciferase signals were imaged and quantified in a Perkin Elmer IVIS Spectrum Imaging system. (A) The inhibitor compounds effectively inhibited the HSV-1 proliferation as can be seen in a dose dependent reduction in the luciferase signal (red). (B) Quantification of the luciferase signal from the samples in panel (A). Three replicates from two separate experiments were combined to generate the average and the standard error bar.

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  Figure 7. Immunofluorescent evidence for HSV-1 proliferation inhibition by the RACK1 inhibitor compound SD29-14. The HEp-2 cells were infected with HSV-1 at an MOI of 1.0 for 30h along with the indicated compounds and control. The no-virus control (A), vehicle DMSO treated (B) and SD29-14 10 μM treated (C) cells were fixed with 4% paraformaldehyde and stained with anti-ICP0-FITC (green) and DAPI for DNA (blue) in the nucleus. The slides were observed under a Nikon confocal microscope (60 × magnification lens) and pictures were taken to show infected cells (green) and total cells (DAPI). All scale bars correspond to 20 μm. The imaging experiments were performed three independent times, and the results shown represent one of the three experiments. (D) Quantification of fluorescent cells by Image based cell sorting in a Cellometer (Nexcelom Vision). The Y-axis shows the total fluorescent cells per ml. The cells were prepared by immunostaining with ICP0 antibody after 24-h incubation with the indicated compounds. The total cell counts were 1.01 × 106/ml, 4.69 × 106/ml, and 5.36 × 106/ml for DMSO, SD29-14 (10 μM), and SD29-14 (100 μM) respectively. The numbers show the mean ± SE of three…

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  Figure 8. Efficacy of SD29-14 compared with anti-herpes drug acyclovir. (A) Hep-2 cells were incubated with the indicated compounds in the presence of HSV1-Luc virus at a concentration of 4 × 106 pfu/ml media. (B) Quantification of the luciferase signal from the samples in panel (A). Signals from three replicates from two separate experiments were combined to generate the average and the standard error bar.