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Comparative Study
. 2020 Mar:175:104706.
doi: 10.1016/j.antiviral.2020.104706. Epub 2020 Jan 10.

Comparison of broad-spectrum antiviral activities of the synthetic rocaglate CR-31-B (-) and the eIF4A-inhibitor Silvestrol

Christin Müller  1 , Wiebke Obermann  2 , Falk W Schulte  2 , Kerstin Lange-Grünweller  2 , Lisa Oestereich  3 , Fabian Elgner  4 , Mirco Glitscher  4 , Eberhard Hildt  4 , Kamini Singh  5 , Hans-Guido Wendel  5 , Roland K Hartmann  2 , John Ziebuhr  1 , Arnold Grünweller  6
Affiliations
Comparative Study

Comparison of broad-spectrum antiviral activities of the synthetic rocaglate CR-31-B (-) and the eIF4A-inhibitor Silvestrol

Christin Müller et al. Antiviral Res. 2020 Mar.

Abstract

Rocaglates, a class of natural compounds isolated from plants of the genus Aglaia, are potent inhibitors of translation initiation. They are proposed to form stacking interactions with polypurine sequences in the 5'-untranslated region (UTR) of selected mRNAs, thereby clamping the RNA substrate onto eIF4A and causing inhibition of the translation initiation complex. Since virus replication relies on the host translation machinery, it is not surprising that the rocaglate Silvestrol has broad-spectrum antiviral activity. Unfortunately, synthesis of Silvestrol is sophisticated and time-consuming, thus hampering the prospects for further antiviral drug development. Here, we present the less complex structured synthetic rocaglate CR-31-B (-) as a novel compound with potent broad-spectrum antiviral activity in primary cells and in an ex vivo bronchial epithelial cell system. CR-31-B (-) inhibited the replication of corona-, Zika-, Lassa-, Crimean Congo hemorrhagic fever viruses and, to a lesser extent, hepatitis E virus (HEV) at non-cytotoxic low nanomolar concentrations. Since HEV has a polypurine-free 5'-UTR that folds into a stable hairpin structure, we hypothesized that RNA clamping by Silvestrol and its derivatives may also occur in a polypurine-independent but structure-dependent manner. Interestingly, the HEV 5'-UTR conferred sensitivity towards Silvestrol but not to CR-31-B (-). However, if an exposed polypurine stretch was introduced into the HEV 5'-UTR, CR-31-B (-) became an active inhibitor comparable to Silvestrol. Moreover, thermodynamic destabilization of the HEV 5'-UTR led to reduced translational inhibition by Silvestrol, suggesting differences between rocaglates in their mode of action, most probably by engaging Silvestrol's additional dioxane moiety.

Keywords: Antiviral activity; CR-31-B; Rocaglates; Silvestrol; Translation initiation; eIF4A.

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Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Structures of the rocaglates Silvestrol, CR-31-B (−), CR-31-B (+) and RocA. The characteristic cyclopenta[b]benzofurane structure found in all rocaglates is indicated in red in the Silvestrol structure. The dioxane moiety that is only found in Silvestrol is shown on the left side in black. Blue ring: variable region in rocaglates.
Fig. 2
Fig. 2
Antiviral activities of the synthetic rocaglate CR-31-B (−) against HCoV-229E and MERS-CoV. (A) Western blot analysis of HCoV-229E N protein accumulation (top panel) in cells treated with different enantiomers of CR-31-B. β-Actin (lower panel) was used as a loading control. (B) Total (genomic and subgenomic) viral RNA produced in HCoV-229E-infected MRC-5 cells treated with the two enantiomers of CR-31-B. Relative changes in viral RNA levels were determined by RT-qPCR. The data were normalized to infected but untreated cells as well as GAPDH mRNA using the comparative ΔΔCt method. (C) HCoV-229E and MERS-CoV titers in supernatants collected from infected MRC-5 cells (MOI = 0,1) at 24 hpi. Cells were treated with CR-31-B (−/+) as indicated. Data from three independent experiments were used to calculate EC50 values (2.88 nM for HCoV-229E- and 1.87 nM for MERS-CoV).
Fig. 3
Fig. 3
Comparison of antiviral effects of CR-31-B (−) and Silvestrol using human bronchial epithelial cells infected with HCoV-229E. (A) Human bronchial epithelial cells were cultivated and differentiated at an air-liquid interface into different airway epithelial cell types (basal, ciliated, clara and goblet cells) und used to assess antiviral effects of the respective compounds. (B + C) HCoV-229E titers in cell culture supernatants collected at the indicated time points p.i. Cells obtained from two different donors were infected and treated with CR-31-B (−), Silvestrol (100/10 nM) or Cr-31-B (+) (100 nM), respectively.
Fig. 4
Fig. 4
CR-31-B (−) and Silvestrol inhibit LASV and CCHFV replication in primary murine hepatocytes with comparable efficiencies in a concentration range between 20 and 50 nM. (A) Potent antiviral activity of CR-31-B (−) against LASV and CCHFV without cytotoxicity in murine hepatocytes. (B) No antiviral effects of CR-31-B (+) up to a concentration of 5 μM. (C) Potent antiviral activity of Silvestrol against LASV and CCHFV without cytotoxicity in murine hepatocytes.
Fig. 5
Fig. 5
CR-31-B (−) and Silvestrol reduce the levels of extracellular HEV RNA at low nanomolar concentrations. qRT-PCR measurement of extracellular HEV RNA of CR-31-B (+), CR-31-B (−) and Silvestrol treated, persistently HEV-infected cells. All data are referred to the DMSO control.
Fig. 6
Fig. 6
Comparison of the inhibitory effects of CR-31-B (−) and Silvestrol on reporter gene expression constructs containing different viral 5′-UTRs. (A) Effects of 5 and 10 nM Silvestrol or CR-31-B (−) on reporter gene expression in the context of 5′-UTRs from coronaviruses HCoV-229E and MERS-CoV as well as EBOV VP30 and VP35. The VP35 5′-terminal hairpin and the VP35 hairpin with (AG)5 extensions were also analyzed. The predicted RNA secondary structures of the indicated 5′-UTRs are shown. Asterisks mark the positions of purines as part of the polypurine stretch in the VP35 hairpin. (B) The 5′-UTR of HEV and derivatives thereof were analyzed towards their sensitivity against 5 and 10 nM Silvestrol and CR-31-B (−) treatment in a dual luciferase assay. (AG)15 and (AC)15 sequences were used as positive and negative controls, respectively. Predicted RNA secondary structures of the HEV 5′-UTRs are shown. The reporter gene expression data were normalized to the transfection efficiencies and the corresponding DMSO controls. Blue circles indicate the mutated nucleotides in HEVgt3c-G4C and HEVgt3c-G4CC6A. Asterisks mark the positions of purines as part of the polypurine stretch in HEVgt3c-Purine. Standard errors of the mean of at least three independent experiments are shown. MFE = minimal free energy (kcal/mol).
Fig. 7
Fig. 7
Possible binding mode of Silvestrol and RNA clamping using structure-based comparative modeling of RocA or Silvestrol onto a 10-mer polyAG RNA bound to the surface of human eIF4A. (A) Surface of human eIF4A with a bound polyAG 10mer (adapted from Iwasaki et al., 2019). (B-E) Zoom in to show the binding region of RocA and Silvestrol. The dioxane moiety of Silvestrol is able to cross the RNA stretch to make additional contacts with proximal positioned arginine residues in eIF4A. Pymol was used for graphical illustration. eIF4A: grey; RNA: green; RocA: purple; Silvestrol; cyan.
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