Generation of host-directed and virus-specific antivirals using targeted protein degradation promoted by small molecules and viral RNA mimics

Abstract

Targeted protein degradation (TPD), as exemplified by proteolysis-targeting chimera (PROTAC), is an emerging drug discovery platform. PROTAC molecules, which typically contain a target protein ligand linked to an E3 ligase ligand, recruit a target protein to the E3 ligase to induce its ubiquitination and degradation. Here, we applied PROTAC approaches to develop broad-spectrum antivirals targeting key host factors for many viruses and virus-specific antivirals targeting unique viral proteins. For host-directed antivirals, we identified a small-molecule degrader, FM-74-103, that elicits selective degradation of human GSPT1, a translation termination factor. FM-74-103-mediated GSPT1 degradation inhibits both RNA and DNA viruses. Among virus-specific antivirals, we developed viral RNA oligonucleotide-based bifunctional molecules (Destroyers). As a proof of principle, RNA mimics of viral promoter sequences were used as heterobifunctional molecules to recruit and target influenza viral polymerase for degradation. This work highlights the broad utility of TPD to rationally design and develop next-generation antivirals.

Keywords: CMV; GSPT1; PROTAC; SARS-CoV-2; antiviral therapeutics; influenza virus; oligonucleotide; small molecule.

Figures 1.. 103 inhibits IAV replication.

A. Structures of the indicated compounds and their cartoon illustrations. B. (Top) Fluorescence images (scale bars = 400 μm): A549 cells infected by NS1-GFP PR8 virus (MOI = 1) and treated with 1 μM of the indicated compounds for 24 hours post infection. (Bottom) GFP intensities in top panel (green bars and right axis). Mean ± sd, n = 3; CellTiter-Glo luminescence assays: ATP levels in A549 cells treated with 1 μM of the indicated compounds for 24 hours (blue bars and left axis). Mean ± sd, n = 3. C. (Top) Plaque assays: viral titers in supernatants of A549 cells infected by WT PR8 virus (MOI = 1) and treated with 1 μM of the indicated compounds for 24 hours post infection (purple bars and right axis). Mean ± sd, n = 2; CellTiter-Glo luminescence assays: ATP levels in A549 cells treated with 1 μM of the indicated compounds for 24 hours (blue bars and left axis). Mean ± sd, n = 3. (Bottom) Western blots: A549 cells from the experiments described in top panel. D. Plaque assays: viral titers in supernatants of HBTE cells infected by A/Puerto Rico/8/34 (H1N1) virus (WT PR8 virus) (MOI=1) (Left) or A/Netherlands/602/2009 (H1N1) virus (MOI=1) (Right) and treated with the indicated concentrations of Nucleozin (black bars) or 103 (grey bars) for 24 hours post infection. IC₅₀ are indicated. Mean ± sd, n = 3. E. Proteomics assay: (Left) Volcano plot of proteomics data from uninfected A549 cells treated with DMSO or 1 μM 103 for 4 hours (No infection, 4h). (Right) Volcano plot of proteomics data from A549 cells infected by WT PR8 virus (MOI = 1) and treated with DMSO or 1 μM 103 for 24 hours post infection (WT PR8 virus, 24hpi). GSPT1/eRF1 and IAV proteins are highlighted in blue and red, respectively. n = 3. F. Proteomics assay: absolute abundance of the indicated proteins from experiments described in Figure 1E. Mean ± sd, n = 3. All p-values were calculated by Student’s T test. p-value: * < 0.05, ** < 0.01, *** < 0.001, and **** < 0.0001. D: DMSO, Nuc: Nucleozin, Len: Lenalidomide, NA: Not available. See also Figure S1.

Figures 2.. 103 selectively depletes human GSPT1.

A. Western blots: A549 cells treated with DMSO or 0.1 μM 103 for the indicated periods of time. B. Western blots: A549 cells treated with the indicated compounds at the indicated concentrations for 24 hours. C. Western blots: A549 cells treated with DMSO or 0.1 μM 103 for 24 hours (−). The medium containing the compounds was then removed and cells were cultured in fresh medium for the indicated periods of time (washout). D. Western blots: A549 cells transfected with 100 nM non-targeting siRNA (siNT) or siRNA against CRBN (siCRBN) for 48 hours, followed by treatment with DMSO or 1 μM 103 for 8 hours. E. Western blots: A549 cells treated with 10 μM of the indicated competitor compounds for 1 hour, followed by co-treatment with 1 μM 103 for 6 hours. ★: overdeveloped region as defined by the dashed box. F. Western blots: A549 cells were co-transfected with HA-Ub and FLAG-GSPT1 plasmids for 24 hours, followed by treatment with DMSO or 1 μM 103 for 8 hours. FLAG-GSPT1 with HA-Ub modification was pulled down from cell lysates. G. Fluorescence images (scale bars = 400 μm) (Left) and western blots (Right): A549 cells were simultaneously transfected with FLAG-GFP plasmid and treated with DMSO or 1 μM of the indicated compounds for 24 hours. Cartoon illustrations are indicated, see also Figure 1A. D: DMSO, Nuc: Nucleozin, Len: Lenalidomide, MLN: MLN4924, MG: MG-132. See also Figure S2.

Figures 3.. 103-induced degradation of GSPT1 confers anti-IAV effect.

A. Plaque assays: viral titers in supernatants of A549 cells infected by WT PR8 virus (MOI = 1) and treated with DMSO or 1 μM 103 for 24 hours post infection. 103 was added into the medium at the indicated time points before or after infection. Mean ± sd, n = 2. B. Plaque assays: A549 cells were treated with DMSO or 0.1 μM 103 for 24 hours. The medium containing the compounds were then removed and cells were infected by WT PR8 virus (MOI = 1) and re-treated with DMSO or 1 μM 103 for 24 hours post infection (−) or without re-treatment of the compounds (washout (24h)). Mean ± sd, n = 2. C. Plaque assays: A549 cells were transfected with 100 nM siNT or siCRBN for 48 hours, followed by being infected by WT PR8 virus (MOI = 1) and treated with DMSO or 1 μM 103 for 3 hours before infection and 24 hours post infection. Mean ± sd, n = 2. D. Plaque assays: viral titers in supernatants of A549 cells infected by WT PR8 virus (MOI = 1) and treated with DMSO or 1 μM 103 for 3 hours before infection and 24 hours post infection. Where indicated, A549 cells were concurrently treated with 10 μM of the indicated competitor compounds. Mean ± sd, n = 2. E. Plaque assays: viral titers in supernatants of A549 cells infected by WT PR8 virus (MOI = 1) and treated with DMSO or 1 μM of the indicated compounds for 3 hours before infection and 24 hours post infection. Cartoon illustrations are indicated, see also Figure 1A. Mean ± sd, n = 2. F. Plaque assays (Left) and Western blots (Right): A549 cells were transfected with 100 nM siNT, siRNAs against GSPT1 (siGSPT1), ETF1 (siETF1), or EIF2S1 (siEIF2S1) for 72 hours, followed by being infected by WT PR8 virus (MOI = 1) for 24 hours post infection. Mean ± sd, n = 2. All p-values are calculated by Student’s T-test. P-value: ns (not significant) > 0.05, * < 0.05, ** < 0.01, and *** < 0.001. D: DMSO, Nuc: Nucleozin, Len: Lenalidomide, MLN: MLN4924, MG: MG-132.

Figures 4.. 103 elicits anti-IAV activity in lung organoid model.

A. (Left) Fluorescence images (scale bars = 400 μm): lung organoid-derived epithelial cells infected by NS1-GFP PR8 virus (MOI=1) and treated with DMSO or 1 μM of the indicated compounds for 3 hours before infection and 24 hours post infection. (Middle) GFP intensities of images in left panel (green bars and right axis). Mean ± sd, n = 3; CellTiter-Glo luminescence assays: ATP levels in lung organoid cells treated with 1 μM of the indicated compounds for 24 hours (blue bars and left axis). Mean ± sd, n = 3. All p-values are calculated by Student’s T-test. P-value: *** < 0.001. (Right) Western blots: lung organoid cells from the experiments described in left and middle panels. ←: nonspecific bands. B. Immunofluorescence images (scale bars = 100 μm and 25 μm): 3D lung organoids infected by WT PR8 virus (MOI = 1) and treated with DMSO or 1 μM of the indicated compounds for 3 hours before infection and 24 hours post infection. Yellow solid boxes: regions of interest (ROI) on overlaying images. D: DMSO, Nuc: Nucleozin, Len: Lenalidomide.

Figures 5.. 103 has antiviral activity against SARS-CoV-2.

A. (Top) plaque assays: viral titers in supernatants of A549-ACE2 cells infected by SARS-CoV-2 (MOI = 1) and treated with DMSO or 1 μM of the indicated compounds for 3 hours before infection and 24 hours post infection (purple bars and right axis). Mean ± sd, n = 2; CellTiter-Glo luminescence assays: ATP levels in A549-ACE2 cells treated with 1 μM of the indicated compounds for 24 hours (blue bars and left axis). Mean ± sd, n = 3. (Bottom). Western blots: A549-ACE2 cells from the experiments described in top panel. B. Proteomics assay: (Top) Volcano plot of proteomics data from uninfected A549-ACE2 cells treated with DMSO or 1 μM 103 for 4 hours (No infection, 4h); (Middle) Volcano plot of proteomics data from A549-ACE2 cells infected by SARS-CoV-2 (MOI = 1) and treated with DMSO or 1 μM 103 for 3 hours before infection and 24 hours post infection (SARS-CoV-2, 3hbi+24hpi); (Bottom) Volcano plot of proteomics data from A549-ACE2 cells infected by SARS-CoV-2 (MOI = 1) and treated with DMSO or 1 μM 103 for 24 hours post infection (SARS-CoV-2, 24hpi). GSPT1/eRF1 and SARS-CoV-2 proteins are highlighted in blue and purple, respectively. n = 3. C. (Top) Plaque assays: A549-ACE2 cells were transfected with 100 nM siNT or siCRBN for 48 hours, followed by being infected by SARS-CoV-2 (MOI = 1) and treated with DMSO or 1 μM 103 for 3 hours before infection and 24 hours post infection. Mean ± sd, n = 2. (Bottom) Western blots: A549-ACE2 cells from the experiments described in top panel. D. Immunofluorescence images (scale bars = 100 μm): 3D lung organoids infected by SARS-CoV-2 (MOI = 1) and treated with DMSO or 1 μM 103 for 3 hours before infection and 24 hours post infection. All p-values were calculated by Student’s T test. P-value: ns (not significant) > 0.05, ** < 0.01. D: DMSO, Nuc: Nucleozin, Len: Lenalidomide. See also Figure S3.

Figures 6.. 103 has antiviral activity against CMV.

A. (Left) Fluorescence images (scale bars = 400 μm): ARPE-19 cells infected by AD169^BADrUL131 virus (MOI = 1) and treated with DMSO or 1 μM of the indicated compounds for 3 hours before infection and 24 hours post infection. (Middle) GFP intensities of images in left panel (green bars and right axis). Mean ± sd, n = 3; CellTiter-Glo luminescence assays: ATP levels in ARPE-19 cells treated with 1 μM of the indicated compounds for 24 hours (blue bars and left axis). Mean ± sd, n = 3. (Right) Western blots: ARPE-19 cells from the experiments described in left and middle panels. B. (Left) Fluorescence images (scale bars = 400 μm): ARPE-19 cells were transfected with 100 nM siNT or siCRBN for 48 hours, followed by being infected by AD169^BADrUL131 virus (MOI = 1) and treated with DMSO or 1 μM 103 for 3 hours before infection and 24 hours post infection. (Middle) GFP intensities of images in left panel (green bars and right axis). Mean ± sd, n = 3; CellTiter-Glo luminescence assays: ATP levels in ARPE-19 cells treated with 1 μM of the indicated compounds for 24 hours (blue bars and left axis). Mean ± sd, n = 3. (Right) Western blots: ARPE-19 cells from the experiments described in left and middle panels. All p-values were calculated by Student’s T test. P-value: ns (not significant) > 0.05, *** < 0.001, and **** < 0.0001. D: DMSO, Nuc: Nucleozin, Len: Lenalidomide. See also Figure S4.

Figures 7.. TPD promoted by RNA mimics exhibits anti-IAV activity.a

A. Conserved terminal sequences of IAV segments 1 through 3. Publicly available and unique full-length UTR sequences of the indicated viral segments (vRNA) were aligned. Total number of sequences, the breakdown of contributing strains (H1N1, H1N2, H3N2, H5N1, and others) and host species (Avian, Human, Swine and others) are indicated in the pie charts. B. Sequences and modifications of Biotin-RNAs and Destroyers. *: phosphorothioate backbones; m: 2’O-methyl modifications; Phos: phosphorylation. C. Western blots: A549 cells were infected by WT PR8 virus (MOI = 1) for 24 hours. IAV NP was pulled down by 1 μM of the indicated Biotin-RNA oligonucleotides from cell lysates. D. Visualization of RNA3-A in orange (Left) and RNA4-A in purple (Right) on top of the crystal structure of vPOL and vRNAs (5’ vRNA in purple; 3’ vRNA in orange) (PDB 4WSB) using Pymol. The original vRNA oligonucleotide sequences used in PDB 4WSB were retained for this analysis. Black dashed boxes: AHPC; Yellow dashed lines: polar contacts. E. Western blots: A549 cells were infected by FLAG-PB1 PR8 virus (MOI = 1) for 24 hours. IAV PB1 and VHL were pull down by 1 μM RNA3-A or RNA4-A from cell lysates. F. Flow cytometry: HEK293T cells were infected by NS1-GFP PR8 virus (MOI = 1) and transfected with 1 μM RNA3-A or RNA4-A for 24 hours post infection in the presence or absence of 100 nM MG132. Mean ± sd, n = 3–4. All p-values were calculated by one-way ANOVA followed by Sidak multiple comparisons test. p-value: ns (not significant) > 0.1234, * < 0.0332, **** p < 0.0001. UT: untreated. See also Figure S5.

Scheme 1

Synthetic route of compound FM-123–96, FM-123–142 and FM-74–103