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. 2014 Jul;88(13):7294-306.
doi: 10.1128/JVI.00591-14. Epub 2014 Apr 16.

Small-molecule probes targeting the viral PPxY-host Nedd4 interface block egress of a broad range of RNA viruses

Ziying Han  1 , Jianhong Lu  1 , Yuliang Liu  1 , Benjamin Davis  2 , Michael S Lee  3 , Mark A Olson  4 , Gordon Ruthel  1 , Bruce D Freedman  1 , Matthias J Schnell  2 , Jay E Wrobel  5 , Allen B Reitz  5 , Ronald N Harty  6
Affiliations

Small-molecule probes targeting the viral PPxY-host Nedd4 interface block egress of a broad range of RNA viruses

Ziying Han et al. J Virol. 2014 Jul.

Abstract

Budding of filoviruses, arenaviruses, and rhabdoviruses is facilitated by subversion of host proteins, such as Nedd4 E3 ubiquitin ligase, by viral PPxY late (L) budding domains expressed within the matrix proteins of these RNA viruses. As L domains are important for budding and are highly conserved in a wide array of RNA viruses, they represent potential broad-spectrum targets for the development of antiviral drugs. To identify potential competitive blockers, we used the known Nedd4 WW domain-PPxY interaction interface as the basis of an in silico screen. Using PPxY-dependent budding of Marburg (MARV) VP40 virus-like particles (VLPs) as our model system, we identified small-molecule hit 1 that inhibited Nedd4-PPxY interaction and PPxY-dependent budding. This lead candidate was subsequently improved with additional structure-activity relationship (SAR) analog testing which enhanced antibudding activity into the nanomolar range. Current lead compounds 4 and 5 exhibit on-target effects by specifically blocking the MARV VP40 PPxY-host Nedd4 interaction and subsequent PPxY-dependent egress of MARV VP40 VLPs. In addition, lead compounds 4 and 5 exhibited antibudding activity against Ebola and Lassa fever VLPs, as well as vesicular stomatitis and rabies viruses (VSV and RABV, respectively). These data provide target validation and suggest that inhibition of the PPxY-Nedd4 interaction can serve as the basis for the development of a novel class of broad-spectrum, host-oriented antivirals targeting viruses that depend on a functional PPxY L domain for efficient egress.

Importance: There is an urgent and unmet need for the development of safe and effective therapeutics against biodefense and high-priority pathogens, including filoviruses (Ebola and Marburg) and arenaviruses (e.g., Lassa and Junin) which cause severe hemorrhagic fever syndromes with high mortality rates. We along with others have established that efficient budding of filoviruses, arenaviruses, and other viruses is critically dependent on the subversion of host proteins. As disruption of virus budding would prevent virus dissemination, identification of small-molecule compounds that block these critical viral-host interactions should effectively block disease progression and transmission. Our findings provide validation for targeting these virus-host interactions as we have identified lead inhibitors with broad-spectrum antiviral activity. In addition, such inhibitors might prove useful for newly emerging RNA viruses for which no therapeutics would be available.

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Figures

FIG 1
FIG 1
Rationale and strategy for identifying PPxY budding inhibitors. The diagram shows arenavirus, filovirus, and rhabdovirus virions budding efficiently from the plasma membrane in the absence of inhibitors (A) or remaining tethered to the plasma membrane as a result of PPxY inhibitors blocking the interaction between host Nedd4 and the PPxY L domains present in the Z, VP40, and M viral matrix proteins (B). (C) Flow chart showing an in silico screen and SAR analysis to identify inhibitors of the viral PPxY-host Nedd4 interaction and PPxY-mediated budding. The in silico screen involved computational docking with AutoDock, version 4.0, energy minimization using CHARMM with the MMFF force field, and ranking with Accelrys LigScore2 of 4.8 million drug-like compounds from the ZINC database. The top 20 scoring compounds were tested, as indicated, leading to the identification of our initial lead compound 1. To understand how the structure of compound 1 affected its activity, the molecule was dissected into two fragments (see disconnection point in red), and 20 commercially available compounds having these two fragment substructures (10 compounds from each) were evaluated. Two compounds (2 and 3), one from each substructure search, were found to be more potent than compound 1. Further substructure searches of commercial databases were performed and led to the acquisition and testing of an additional 10 compounds (5 structurally related to compound 2 and 5 related to compound 3). Compound 4 showed improved potency over SAR analog 2, and compound 5 showed improved potency over SAR analog 3. Compound 6 is a structurally related inactive negative control.
FIG 2
FIG 2
Effect of 1 on PPxY-dependent budding of MARV VP40 VLPs and infectious VSV. (A) HEK293T cells transfected with pCAGGS vector (lane 1) or mVP40 (lanes 2 to 6) were treated with DMSO alone (lanes 1 and 2) or with the indicated concentrations of compound 1 (lanes 3 to 6). Cells and VLPs were harvested at 24 h posttransfection, and a representative Western blot to detect mVP40 is shown. A Western blot control for β-actin in cells is shown. Data in the bar graph showing mVP40 levels (ImageJ software) in VLPs from samples receiving 0 and 20 μM compound 1 represent the average from three independent experiments. (B) HEK293T cells were infected with VSV-WT or VSV-PY>A4 at an MOI of 0.1 in the absence (DMSO alone) or presence of 10 μM compound 1. Supernatants were harvested at 8 h postinfection, and virions were quantified by standard plaque assay on BHK-21 cells performed in triplicate and graphed as PFU/ml. *, P < 0.005; ns, not significant. Infected cell extracts were harvested at 8 h postinfection, and both VSV M and cellular actin were detected by Western blotting.
FIG 3
FIG 3
Effect of compounds 2 and 3 on budding of mVP40 and eVP40 VLPs. (A and B) HEK293T cells transfected with mVP40 were treated with DMSO alone (lanes 1) or with the indicated concentrations of compound 2 (A, lanes 2 to 5) or compound 3 (B, lanes 2 to 4). Cells and VLPs were harvested at 24 h posttransfection, and mVP40 was detected by Western blotting. A Western blot control for cellular GAPDH in cells is shown. (C and D) HEK293T cells transfected with eVP40 were treated with DMSO alone (lanes 1) or with the indicated concentrations of compound 2 (C, lanes 2 to 5) or compound 3 (D, lanes 2 to 6). Cells and VLPs were harvested at 24 h posttransfection, and eVP40 was detected by Western blotting. A Western blot control for cellular GAPDH in cells is shown. (E) HEK293T cells transfected with mVP40 were treated with DMSO alone (lane 1) or with the indicated concentrations of compound 6 (lanes 2 to 6) as a negative control. Cells and VLPs were harvested at 24 h posttransfection, and mVP40 was detected by Western blotting. Numbers in parentheses were determined using ImageJ software (NIH), and controls were set at 100%.
FIG 4
FIG 4
Compound 4 inhibits budding of mVP40 and eVP40 VLPs and blocks mVP40-Nedd4 protein-protein interaction. (A) HEK293T cells transfected with mVP40 were treated with DMSO alone (lane 1) or with the indicated concentrations of compound 4 (lanes 2 to 4). Cells and VLPs were harvested at 24 h posttransfection, and a representative Western blot for mVP40 is shown. Western blot controls for cellular GAPDH and HSP70 in cells are shown. The bar graph represents the average levels of mVP40 VLPs from three independent experiments. *, P < 0.001. Numbers in parentheses were determined using ImageJ software (NIH), and controls were set at 100%. (B) HEK293T cells transfected with eVP40 were treated with DMSO alone (lane 1) or with the indicated concentrations of compound 4 (lanes 2 and 3). Cells and VLPs were harvested at 24 h posttransfection, and a representative Western blot for eVP40 is shown. Western blot controls for cellular GAPDH and HSP70 in cells are shown. The bar graph represents the average levels of eVP40 VLPs from three independent experiments. *, P < 0.005. Numbers in parentheses were determined using ImageJ software (NIH), and controls were set at 100%. (C) BiMC assay and representative images of HEK293T cells coexpressing NYFP-Nedd4 and CYFP-mVP40 fusion proteins in the absence (DMSO alone) or presence of the indicated concentrations of compound 4 or 6. The green signal represents an interaction between mVP40 and Nedd4, and cell nuclei were stained blue with NucBlue. Scale bar, 200 μm. YFP-positive cells were quantified in triplicate using MetaMorph software.
FIG 5
FIG 5
BiMC assay of VP40-Nedd4 interactions. BiMC assay and representative images of HEK293T cells expressing NYFP-Nedd4 alone or with CYFP-eVP40, CYFP-eVP40-ΔPY/PY, CYFP-mVP40, or CYFP-mVP40-ΔPPPY are shown. The green signal represents an interaction between VP40 and Nedd4, and cell nuclei were stained blue with DAPI. A Western blot control is shown for endogenous Nedd4 and exogenous NYFP-Nedd4.
FIG 6
FIG 6
Compound 5 inhibits budding of mVP40 and eVP40 VLPs and blocks mVP40-Nedd4 protein-protein interaction. (A) HEK293T cells transfected with mVP40 were treated with DMSO alone (lane 1) or with the indicated concentrations of compound 5 (lanes 2 to 4). Cells and VLPs were harvested at 24 h posttransfection, and a representative Western blot for mVP40 is shown. Western blot controls for cellular GAPDH and HSP70 in cells are shown. The bar graph represents the average levels of mVP40 VLPs from three independent experiments. *, P < 0.001. Numbers in parentheses were determined using ImageJ software (NIH), and controls were set at 100%. (B) HEK293T cells transfected with eVP40 were treated with DMSO alone (lane 1) or with the indicated concentrations of compound 5 (lanes 2 to 4). Cells and VLPs were harvested at 24 h posttransfection, and a representative Western blot for eVP40 is shown. Western blot controls for cellular GAPDH and HSP70 in cells are shown. The bar graph represents the average levels of eVP40 VLPs from three independent experiments. *, P < 0.001. Numbers in parentheses were determined using ImageJ software (NIH), and controls were set at 100%. (C) BiMC assay and representative images of HEK293T cells coexpressing NYFP-Nedd4 and CYFP-mVP40 fusion proteins in the absence (DMSO alone) or presence of the indicated concentrations of compound 5. The green signal represents an interaction between mVP40 and Nedd4, and cell nuclei were stained blue with NucBlue. Scale bar, 200 μm. YFP-positive cells were quantified in triplicate using MetaMorph software.
FIG 7
FIG 7
Compounds 4 and 5 inhibit budding of LFV-Z VLPs. (A) VLP budding assay and Western blot demonstrating that the PPxY L-domain motif of LFV-Z protein is important for efficient VLP egress from HEK293T cells. Budding of LFV-Z-ΔPPPY VLPs was reduced by 4-fold compared to that of LFV-Z-WT. HEK293T cells transfected with LFV-Z-WT were treated with DMSO alone (0), or with the indicated concentrations of compound 4 (B) or 5 (C). Cells and VLPs were harvested at 24 h posttransfection, and LFV-Z-WT was detected by Western blotting. Western blot loading controls for cellular actin are shown. Numbers in parentheses were determined using ImageJ software (NIH), and controls were set at 100%.
FIG 8
FIG 8
Compounds 4 and 5 inhibit egress of infectious VSV and VSV recombinants in a PPxY-dependent and dose-dependent manner. (A) HEK293T cells were infected with VSV-WT, VSV-M40, or VSV-PY>A4 at an MOI of 0.1 in the absence (DMSO alone) or presence of 0.1 or 0.5 μM compound 4. Supernatants were harvested at 8 h postinfection, and virions were quantified by standard plaque assay on BHK-21 cells performed in triplicate. **, P < 0.01; ns, not significant (as determined by a one-way ANOVA test). Infected cell extracts were harvested at 8 h postinfection, and VSV-M, GAPDH, and HSP70 were detected by Western blotting. (B) HEK293T cells were infected with VSV-WT, VSV-M40, or VSV-PY>A4 at an MOI of 0.1 in the absence (DMSO alone) or presence of 0.1 or 0.5 μM compound 5. Supernatants were harvested at 8 h postinfection, and virions were quantified by standard plaque assay on BHK-21 cells performed in triplicate. **, P < 0.01; *, P < 0.5. Infected cell extracts were harvested at 8 h postinfection, and VSV-M, GAPDH, and HSP70 were detected by Western blotting. Numbers in parentheses were determined using ImageJ software (NIH), and controls were set at 100%.
FIG 9
FIG 9
Compounds 4 and 5 inhibit egress of infectious RABV in cell culture. Bar graphs representing multistep growth of RABV in HEK293T cells in the absence (DMSO alone) or presence of the indicated concentrations of compound 4 (A) or compound 5 (B). At the indicated time points, virus-containing supernatant was harvested and titrated in duplicate on BSR cells. **, P < 0.01; *, P < 0.05. (C) Western blot analysis of RABV-infected HEK293T cells in the absence (DMSO alone) or presence of the indicated concentrations of compounds 4 and 5. Cell extracts were harvested at 36 h p.i., and detection of RABV-M protein (24 kDa) by Western blotting is shown.
FIG 10
FIG 10
MTT cell viability assays. MTT cell viability assays were performed on HEK293T cells that were treated with DMSO or the indicated concentrations of compounds 1, 4, and 5 under conditions that mimicked those used for VLP transfection. Each concentration was tested in triplicate.
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References

    1. Feldmann H, Klenk HD. 1996. Filoviruses, p 877–888 In Baron S. (ed), Medical microbiology, 4th ed. University of Texas Medical Branch at Galveston, Galveston, TX - PubMed
    1. Feldmann H, Klenk HD. 1996. Marburg and Ebola viruses. Adv. Virus Res. 47:1–52. 10.1016/S0065-3527(08)60733-2 - DOI - PubMed
    1. Grant A, Seregin A, Huang C, Kolokoltsova O, Brasier A, Peters C, Paessler S. 2012. Junin virus pathogenesis and virus replication. Viruses 4:2317–2339. 10.3390/v4102317 - DOI - PMC - PubMed
    1. Peters CJ, Liu CT, Anderson GW, Jr, Morrill JC, Jahrling PB. 1989. Pathogenesis of viral hemorrhagic fevers: Rift Valley fever and Lassa fever contrasted. Rev. Infect. Dis. 11(Suppl 4):S743–S749. 10.1093/clinids/11.Supplement_4.S743 - DOI - PubMed
    1. Hartlieb B, Weissenhorn W. 2006. Filovirus assembly and budding. Virology 344:64–70. 10.1016/j.virol.2005.09.018 - DOI - PubMed

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