A Limited Structural Modification Results in a Significantly More Efficacious Diazachrysene-Based Filovirus Inhibitor

Ebola (EBOV) and Marburg (MARV) filoviruses are highly infectious pathogens causing deadly hemorrhagic fever in humans and non-human primates. Promising vaccine candidates providing immunity against filoviruses have been reported. However, the sporadic nature and swift progression of filovirus disease underlines the need for the development of small molecule therapeutics providing immediate antiviral effects. Herein we describe a brief structural exploration of two previously reported diazachrysene (DAAC)-based EBOV inhibitors. Specifically, three analogs were prepared to examine how slight substituent modifications would affect inhibitory efficacy and inhibitor-mediated toxicity during not only EBOV, but also MARV cellular infection. Of the three analogs, one was highly efficacious, providing IC50 values of 0.696 mu M +/- 0.13 mu M and 2.76 mu M +/- 0.21 mu M against EBOV and MARV infection, respectively, with little or no associated cellular toxicity. Overall, the structure-activity and structure-toxicity results from this study provide a framework for the future development of DAAC-based filovirus inhibitors that will be both active and non-toxic in vivo

Machupo Virus Glycoprotein Determinants for Human Transferrin Receptor 1 Binding and Cell Entry

Machupo virus (MACV) is a highly pathogenic New World arenavirus that causes hemorrhagic fever in humans. MACV, as well as other pathogenic New World arenaviruses, enter cells after their GP1 attachment glycoprotein binds to their cellular receptor, transferrin receptor 1 (TfR1). TfR1 residues essential for this interaction have been described, and a co-crystal of MACV GP1 bound to TfR1 suggests GP1 residues important for this association. We created MACV GP1 variants and tested their effect on TfR1 binding and virus entry to evaluate the functional significance of some of these and additional residues in human and simian cells. We found residues R111, D123, Y122, and F226 to be essential, D155, and P160 important, and D114, S116, D140, and K169 expendable for the GP1-TfR1 interaction and MACV entry. Several MACV GP1 residues that are critical for the interaction with TfR1 are conserved among other New World arenaviruses, indicating a common basis of receptor interaction. Our findings also open avenues for the rational development of viral entry inhibitors

High content image-based screening of a protease inhibitor library reveals compounds broadly active against Rift Valley fever virus and other highly pathogenic RNA viruses.

High content image-based screening was developed as an approach to test a protease inhibitor small molecule library for antiviral activity against Rift Valley fever virus (RVFV) and to determine their mechanism of action. RVFV is the causative agent of severe disease of humans and animals throughout Africa and the Arabian Peninsula. Of the 849 compounds screened, 34 compounds exhibited ≥ 50% inhibition against RVFV. All of the hit compounds could be classified into 4 distinct groups based on their unique chemical backbone. Some of the compounds also showed broad antiviral activity against several highly pathogenic RNA viruses including Ebola, Marburg, Venezuela equine encephalitis, and Lassa viruses. Four hit compounds (C795-0925, D011-2120, F694-1532 and G202-0362), which were most active against RVFV and showed broad-spectrum antiviral activity, were selected for further evaluation for their cytotoxicity, dose response profile, and mode of action using classical virological methods and high-content imaging analysis. Time-of-addition assays in RVFV infections suggested that D011-2120 and G202-0362 targeted virus egress, while C795-0925 and F694-1532 inhibited virus replication. We showed that D011-2120 exhibited its antiviral effects by blocking microtubule polymerization, thereby disrupting the Golgi complex and inhibiting viral trafficking to the plasma membrane during virus egress. While G202-0362 also affected virus egress, it appears to do so by a different mechanism, namely by blocking virus budding from the trans Golgi. F694-1532 inhibited viral replication, but also appeared to inhibit overall cellular gene expression. However, G202-0362 and C795-0925 did not alter any of the morphological features that we examined and thus may prove to be good candidates for antiviral drug development. Overall this work demonstrates that high-content image analysis can be used to screen chemical libraries for new antivirals and to determine their mechanism of action and any possible deleterious effects on host cellular biology

High Content Image Based Analysis Identifies Cell Cycle Inhibitors as Regulators of Ebola Virus Infection

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Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)^FBXW11-NSs E3 Ligase

<div><p>Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)^FBXW11 E3 ligase. NSs binds to the F-box protein, FBXW11, <i>via</i> the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through an alternate binding site to the SCF^FBXW11 E3 ligase. We further show that disrupting the assembly of the SCF^FBXW11-NSs E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCF^FBXW11-NSs E3 ligase is sufficient to inhibit RVFV infection. Furthermore, <i>FBXW11</i> and <i>BTRC</i> are the two homologues of the <i>βTrCP</i> (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCF^FBXW11 complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection.</p></div

MLN4924 inhibits RVFV infection.

<p><b>(A)</b> IFA of RVFV N or G expression (green) in HeLa cells (cytoplasm in red) that were infected with ZH501 (MOI = 1, 24h) or MP-12 (MOI = 1, 24h) and were either treated with control DMSO (0.5%) or MLN4924 (1 μM) from 1h prior to start of infection to the end of the infection. <b>(B-C)</b> Dose response curve analyses of MLN4924’s antiviral activity against ZH501 (B) and MP-12 (C) virus infection in HeLa cells. Virus infections were measured by HCA of G or N expressing cells or by plaque assay. The values in the brackets next to the name of the dose response curve indicates the average infection in mock (DMSO) treated cells. The infection rates of MLN4924 treated cells were normalized with the corresponding values derived from DMSO treated controls, which were considered as 100% and expressed as the mean ± SD. The final values were expressed as the mean ± SD. <b>(D)</b> Dose response curve analyses of the MLN4924’s antiviral activity against RVFV ZH501 in different cell lines. Infections were measured by HCA of N expressing cells. The values in the brackets next to the names of each cell line, indicate the average infection rate of virus infected cells that were mock (DMSO) treated. Data were normalized as in B-C <b>(E-F)</b> MLN4924 activity against different viruses: VEEV (MOI = 0.1, 24h), MARV (MOI = 3, 48h) and LASV (MOI = 1, 48h) when compared to RVFV ZH501 and RVFV MP-12 infections in HeLa cells. The virus infections were measured by HCA of viral antigen expressing cells (E) or by plaque assay (F): the relative infection in E was calculated by normalizing infection rates of compound treated cells with mock treated cells. The values in the brackets below the virus names indicate the average infection rate in mock (DMSO) treated cells.</p

MLN4924 regulates NSs-dependent early PKR activation in RVFV-infected cells.

<p><b>(A)</b> Western blot analysis demonstrating MLN4924 regulation of PKR activation and inhibition of viral gene expression during the course of a single cycle of viral replication (~ 12h). HeLa cells treated with control (DMSO) or MLN4924 (1 μM) were either mock infected or infected with rMP12-NSs-V5. At the indicated time points, cell lysates were harvested and analyzed for gene expression by Western blot analysis. <b>(B)</b> NSs regulates MLN4924 mediated PKR activation during RVFV infection. HeLa cells expressing control non-targeting siRNA or PKR siRNA were treated with DMSO (vehicle control) or MLN4924 (1 μM) and infected with either wildtype viruses including RVFV ZH501 or rMP-12 or NSs deficient viruses including clone 13 or rMP-12ΔNSs::Luci at an MOI = 10 for 12h. The infection rates were determined by enumerating G expressing cells (rMP-12 and rMP-12ΔNSs::Luci) or N expressing cells (RVFV ZH501 and clone13) by HCA. The data derived from MLN4924 treated cells was normalized with the corresponding DMSO treated controls (average infection rates are indicated under the bar graph), which were considered as 100% and expressed as the mean ± SD. The inset shows a decrease in PKR expression levels in PKR siRNA transfected cells by Western blot analysis. <b>(C)</b> Western blot analysis demonstrating the regulation of the PKR activation pathway and viral gene expression kinetics by NSs deficient virus (rMP-12ΔNSs::Luci virus) during single cycle of virus infection. (<b>D</b>) Western blot analysis which shows that MLN4924 treatment of HeLa cells that were transiently transfected with plasmid DNA expressing the NSs gene was sufficient to activate PKR.</p

F-box protein FBXW11 binds to NSs and regulates RVFV.

<p><b>(A)</b> siRNA screening assay targeting 70 out of 72 human F-box genes to determine RVFV infection inhibitors. HeLa cells were transfected with non-targeting control siRNA or with a pool of four different siRNA targeting each of the 70 F-box genes for 48h followed by incubation with ZH501 (MOI = 1) for 24h. Percentage of infected cells was determined by HCA of N expressing cells. Each data point in panels A to D is an average of three replicates ±SD and is representative of 2 independent experiments. <b>(B)</b> Validation of FBXW11 as a true hit from the siRNA screening: Each siRNA from the pool of 4 siRNAs was individually tested for its ability to inhibit RVFV (ZH501 or MP-12) infection and reduce FBXW11 mRNA levels. Infection was quantified by HCA of viral antigen expressing cells as described in A, while mRNA levels were determined by real time PCR. The relative infection or mRNA levels were calculated by normalizing with the values derived from controls cells that were transfected with non-targeting siRNA and infected with the corresponding viruses. The infection rates of control siRNA treated cells are indicated in the brackets next to the virus names. <b>(C)</b> FBXW11 is the major homologue of βTrCP gene regulating RVFV infection, but its activity is enhanced by BTRC. Same as in B except siRNA targeting BTRC was used either singly or combined with FBXW11 siRNA. <b>(D)</b> Real-Time PCR analysis of BTRC and FBXW11 mRNA levels in HeLa cells that were transfected with the siRNAs are indicated on the X-axis. Relative mRNA levels were derived by normalizing mRNA levels in cells transfected with non-targeting siRNA. <b>(E)</b> Western blot analysis shows that βTrCP siRNA knockdown induces PKR activation and inhibition of viral gene expression similar to MLN4924 treated cells. HeLa cells were transfected with different siRNAs as indicated for 48h and then either mock infected or infected with rMP-12-NSs-V5 (MOI = 10, 8h), and were either treated with DMSO or MLN4924 at 2h, PI. Cells lysates were harvested at 8h, PI for Western blot analysis. <b>(F)</b> Co-immunoprecipitation (co-IP) assay is consistent with the WD40 domain regulating βTrCP binding to NSs: Top panel, depicts the domain structure of the BTRC and FBXW11 protein which contains D or the Dimerization domain, F or F-box motif, and the seven WD40 repeats. In the bottom panel is data from co-IP assay showing the binding of NSs or CUL1 to the various deletion mutants of BTRC and FBXW11. 293T cell lysates overexpressing myc-CUL1 and the vector alone or flag tagged wildtype βTrCP or deletion mutants of βTrCP were combined with rMP-12-NSs-V5 (MOI = 10, 8h) infected cell lysates and immunoprecipitated with anti-Flag antibody. The bound proteins were detected by Western blot analysis as described in the figure. Lysate controls represent 5% of extract used for co-IP.</p

Antiviral activity of the four hit compounds and reference compounds against RVFV-infected HeLa cells or SAEC primary cells.

<p>HeLa cells and SAEC were infected at MOIs of 1.0 and 0.5, respectively for 24 h.</p><p>SI is determined by the formula: CC₅₀/EC₅₀.</p><p>ND, not determined.</p

Hit compounds target virus replication and/or virus egress, but not virus entry.

<p>(<b>A</b>) Kinetics of N, Gn or S-Gn expression in RVFV-infected HeLa cells (MOI = 10) during a single replication cycle. Cells were fixed at various time points post-infection (PI) (0 h) as indicated in the x-axis. (<b>B</b>) Kinetics of N, Gn or S-Gn expression in RVFV-infected HeLa cells with hit compound treatment. (<b>C</b>) Time-of-addition assay in which HeLa cells were either mock treated or treated with the hit compounds at 2 h before prior to (−2), at the same time (0), or 2 h after virus infection. Cells were fixed at 13 h PI and immunostained to detect S-Gn expression. As a control for viral cell entry, cells were treated with 20 µM NH₄Cl, a known inhibitor of endocytosis. (<b>D</b>) Time-of-addition assay in which HeLa cells were either mock treated or treated with the hit compounds at various times PI as indicated on the x-axis. Cells were fixed at 13 h PI and immunostained to detect S-Gn expression. (<b>E</b>) Relative changes in the RNA levels at cellular level or in the supernatants of compound treated cells at 12 h PI. Cells were mock treated or treated with the 4 hit compounds from 2 h prior to the start of infection through the entire duration of infection. In a duplicate experiment the percentage of cells expressing Gn and S-Gn was determined by HCI of the immunostained cells. The relative fold change was determined by normalizing data of compound treated cells with values of corresponding mock treated cells.</p