key: cord-0856787-sb2bibh6
authors: Müller, Christin; Obermann, Wiebke; Karl, Nadja; Wendel, Hans-Guido; Taroncher-Oldenburg, Gaspar; Pleschka, Stephan; Hartmann, Roland K.; Grünweller, Arnold; Ziebuhr, John
title: The rocaglate CR-31-B (-) inhibits SARS-CoV-2 replication at non-cytotoxic, low nanomolar concentrations in vitro and ex vivo
date: 2020-11-24
journal: bioRxiv
DOI: 10.1101/2020.11.24.389627
sha: ddab54d70bea42a0b890915dff3e06c85640b349
doc_id: 856787
cord_uid: sb2bibh6

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a betacoronavirus in the subgenus Sarbecovirus causes a respiratory disease with varying symptoms referred to as coronavirus disease 2019 (COVID-19) and is responsible for a pandemic that started in early 2020. With no vaccines or effective antiviral treatments available, and infection and fatality numbers continuing to increase globally, the quest for novel therapeutic solutions remains an urgent priority. Rocaglates, a class of plant-derived cyclopenta[b]benzofurans, exhibit broad-spectrum antiviral activity against positive- and negative-sense RNA viruses. This compound class inhibits eukaryotic initiation factor 4A (eIF4A)-dependent mRNA translation initiation, resulting in strongly reduced viral RNA translation. The synthetic rocaglate CR-31-B (-) has previously been shown to inhibit the replication of human coronaviruses, such as HCoV-229E and MERS-CoV, as well as Zika-, Lassa-, Crimean Congo hemorrhagic fever virus in primary cells. Here, we assessed the antiviral activity of CR-31-B (-) against SARS-CoV-2 using both in vitro and ex vivo cell culture models. In African green monkey Vero E6 cells, CR-31-B (-) inhibited SARS-CoV-2 replication with an EC50 of ~1.8 nM. In line with this, viral protein accumulation and replication/transcription complex formation were found to be strongly reduced by this compound. In an ex vivo infection system using human airway epithelial cells, CR-31-B (-) was found to cause a massive reduction of SARS-CoV-2 titers by about 4 logs to nearly non-detectable levels. The data reveal a potent anti-SARS-CoV-2 activity by CR-31-B (-), corroborating previous results obtained for other coronaviruses and supporting the idea that rocaglates may be used in first-line antiviral intervention strategies against novel and emerging RNA virus outbreaks.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense, single-stranded RNA betacoronavirus of zoonotic origin first detected in Wuhan, China, at the end of 2019. The virus causes a potentially severe respiratory illness, coronavirus disease 2019 , with infection fatality rates ranging from 0.5% to 1% according to recent estimates (Rajgor et al., 2020) . Following its rapid spread first to Europe and then to the rest of the world, the World Health Organization declared SARS-CoV-2 a pandemic in March 2020 (WHO, 2020) . SARS-CoV-2 is genetically very closely related to SARS-CoV, the causative agent of the 2002-2003 SARS outbreak (Wu et al., 2020 , Zhou et al., 2020 , Gorbalenya et al., 2020 , and the two viruses have been assigned to the same virus species

Severe acute respiratory syndrome-related coronavirus (Gorbalenya et al., 2020) . The ongoing SARS-CoV-2 outbreak is the third documented case of a coronavirus outbreak of zoonotic origin to reach epidemic or pandemic scale since the beginning of the century (Gorbalenya et al., 2020) . The SARS-CoV outbreak in 2002-2003 was followed by the Middle East respiratory syndrome coronavirus (MERS-CoV) outbreak in 2012/2013 (which is still ongoing) and the current SARS-CoV-2 outbreak, all of which can lead to severe pneumonia and death in humans (Anderson and Baric, 2012) and have caused substantial social and economic disruptions. To date, no approved therapeutic is available against SARS-CoV, MERS-CoV or SARS-CoV-2, but a number of investigational antiviral compounds that target viral functions, e.g. remdesivir, ribavirin and favipiravir, which target viral RNA synthesis, as well as several protease inhibitors have entered clinical trials (Brown et al., 2019 , de Wit et al., 2020 , Sheahan et al., 2017 , de Wilde et al., 2014 , Choy et al., 2020 , Sanders et al., 2020 . Moreover, compounds that modulate the human immune system and/or have an antiinflammatory effect, including tocilizumab, interferon-beta and dexamethasone are also being tested for their potential to reduce the severity of COVID-19 progression (Oldenburg and Dohan, 2020 , Guaraldi et al., 2020 , Jalkanen et al., 2020 , Lam et al., 2020 . Finally, antivirals that target host mechanisms critical to viral replication and production of infectious virus progeny are also being developed, including inhibitors targeting host proteases required to activate the fusogenic activity of the SARS-CoV-2 spike protein (Bestle et al., 2020 , Hoffmann et al., 2020 .

Several groups, including ours, have focused on suppressing viral protein synthesis, another host function critical to viral proliferation. Incidentally, a recently published SARS-CoV-2 protein interaction map identified the host translational machinery as a top target for repurposing drugs to block SARS-CoV-2 (Gordon et al., 2020) . In particular, two eukaryotic factors involved in the initiation phase of translation, eukaryotic initiation factor-1A (eIF1A) and eukaryotic initiation factor-4A (eIF4A), have been singled out for further development.

eIF1A is a small protein that binds to the 40S ribosome subunit-mRNA complex (Gordon et al., 2020) , and eIF4A is a DEAD-box RNA helicase central to the activity of the eukaryotic translation initiation complex eIF4F (Chu and Pelletier, 2015) . As of this writing, the eIF1A inhibitor aplidin (PharmaMar) has entered a Phase 2 clinical trial for the treatment of COVID-19, and the eIF4A inhibitor zotatifin (eFFECTOR Therapeutics) is undergoing evaluation for potential COVID-19 clinical development (Harrison, 2020, Bronstrup and Sasse, 2020) .

We have recently shown the potential of rocaglates, a class of natural and synthetic compounds characterized by a common cyclopenta[b]benzofuran skeleton and originally extracted from plants in the genus Aglaia (Meliaceae) (Ebada et al., 2011) , to inhibit both the common cold human coronavirus HCoV-229E and MERS-CoV in vitro and ex vivo (Müller et al., 2018a , Müller et al., 2020 . Natural rocaglates such as rocaglamide A (RocA) or silvestrol, and synthetic rocaglates such as CR-31-B (-) and the above-mentioned zotatifin are specific nanomolar eIF4A inhibitors. Rocaglates form stacking interactions with polypurine sequences in the 5′-untranslated regions (UTRs) of capped mRNAs, clamping the mRNAs onto eIF4A and stalling mRNA unwinding, which results in depletion of the mRNA-eIF4A complex from eIF4F. Because eIF4A-mediated translation is key to the activation of a majority of oncogenes, several rocaglates are in advanced preclinical cancer studies and at least one compound, zotatifin, has been advanced into early stage clinical studies (Bordeleau et al., 2008 , Lucas et al., 2009 , Kogure et al., 2013 , Patton et al., 2015 , Ernst et al., 2020 .

Many viral RNAs contain structured 5'-UTRs analogous to eukaryotic capped mRNA UTRs and are dependent on eIF4A for translation (Madhugiri et al., 2016 , Schlereth et al., 2016 .

Silvestrol exhibits broad-spectrum antiviral activity against a range of positive-and negativesense RNA viruses such as Ebola virus, Zika virus, Chikungunya virus, Crimean Congo hemorrhagic fever virus, Lassa virus, hepatitis E and several coronaviruses (Biedenkopf et al., 2017 , Elgner et al., 2018 , Henss et al., 2018 , Müller et al., 2020 , Glitscher et al., 2018 , Todt et al., 2018 , Müller et al., 2018a . While silvestrol and other natural rocaglates represent promising therapeutic leads due to their low nanomolar activities and high selectivity indices (≥ 100), their natural availability is limited (Pannell, 1998) , and their chemical synthesis is not optimal due to their complex structures (Adams et al., 2009 , Pan et al., 2014 . As an alternative, a multiplicity of synthetic rocaglate analogs have been generated that exhibit similar or enhanced eIF4A-targeting characteristics and can be produced to high purity under GLP conditions and in large quantities.

Recently, we compared the antiviral activity of silvestrol with that of CR-31-B (-), which lacks the dioxane moiety of silvestrol and makes it structurally less complex and much more straightforward to synthesize (Wolfe et al., 2014) . We showed that CR-31-B (-) exhibits levels of viral replication inhibition analogous to those of silvestrol across a range of RNA viruses both in vitro and ex vivo (Müller et al., 2020) . So far, zotatifin has only been tested on one coronavirus, SARS-CoV-2, and only in the Vero E6 cell line, showing similar efficacy as silvestrol and CR-31-B (-) (Gordon et al., 2020) .

Here, we evaluated the potential activity of the synthetic rocaglate CR-31-B (-) against SARS-CoV-2 using both in vitro and human ex vivo cell culture systems.

Vero E6 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μ g/ml streptomycin at 37 °C in an atmosphere containing 5% CO 2 . HepG2 cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10 % fetal calf serum (FCS) at 37 °C and 5 % CO 2 .

Silvestrol was obtained from the Sarawak Biodiversity Centre (Kuching; North-Borneo, Malaysia; purity > 99%). A 6 mM stock solution was prepared in DMSO (sterile-filtered; Roth) and diluted in DMEM or IMDM. Control cells were treated with correspondingly concentrated DMSO dilutions lacking silvestrol. CR-31-B (−) and CR-31-B (+) (Wolfe et al., 2014) were dissolved in DMSO at a concentration of 10 mM and stored at -20 °C.

The dual luciferase assay was performed as described previously in at least three independent replicates (Müller et al., 2018a , Müller et al., 2020 . The total length of the 

Cell viability of Vero E6 cells in the presence of the respective compounds was determined by MTT assay as described previously (Müller et al., 2018a) .

Cryopreserved normal human bronchial epithelial (NHBE) cells were obtained from Lonza.

The undifferentiated cells were seeded on collagen IV-coated transwell plates (Corning Costar) and grown in a mixture of DMEM (Invitrogen) and BEGM (Lonza) supplemented with retinoic acid (75 nM). Every other day, fresh medium was added and, after reaching confluence, the cells were cultivated under air-liquid conditions for at least four additional weeks to differentiate into a pseudostratified human airway epithelium. During this period, medium from the basolateral compartment was renewed every 2-3 days and the apical surface was washed once a week with PBS (Invitrogen).

To determine the 50% effective concentration (EC 50 ) for CR-31-B ( For the infection of NHBE cells, the apical surface was washed 3 times with PBS and cells were infected with SARS-CoV-2 (MOI = 3). After 1 h, the inoculum was removed and the medium in the basal compartment was replaced with medium containing the indicated inhibitor concentration. At the indicated time points p.i., the apical surface of the cells was incubated with PBS for 15 min and virus titers in the supernatants were determined by plaque assay.

To analyze viral protein accumulation, Vero E6 cells were infected with SARS-CoV-2 at an MOI of 1. After inoculation, the supernatant was replaced with fresh medium supplemented with the indicated concentrations of the respective CR-31-B enantiomer. After 24 h, the medium was removed, the cells were washed with PBS and lysed using buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP40, and 1x protease inhibitor cocktail (P8340;

Sigma-Aldrich). The insoluble material was removed by centrifugation and the protein content in the supernatant was measured using a Qubit 3 fluorometer (Invitrogen) and equal amounts of proteins were separated in SDS-10 % polyacrylamide gels and blotted onto a nitrocellulose membrane (Amersham). Membranes were incubated with polyclonal rabbit anti-SARS nucleocapsid protein antibody (Rockland) and mouse-anti actin antibody (abcam), respectively, each diluted 1:500 in PBS containing 1 % bovine serum albumin (BSA). After 60 min, membranes were washed with PBS and incubated with appropriate secondary antibodies (IRDye-conjugated anti-mouse or anti-rabbit IgG mAb [Li-COR Biosciences]) diluted 1:10.000 in PBS containing 1% BSA. After 1 h, membranes were washed and analyzed using the LI-COR Odyssey imaging system.

Immunofluorescence was performed as described previously (Müller et al., 2018b) 

In a first set of experiments, we analyzed the inhibitory effect of CR-31-B (-) (Fig. 1A ) on 5'-UTRs of different coronaviruses including SARS-CoV-2 in a dual luciferase reporter assay ( Fig. 1B) to assess whether translation of mRNAs containing these viral 5'-UTRs depends on eIF4A. Beta-globin, as well as an unstructured (AC) 15 sequence served as negative controls, whereas the polypurine sequence (AG) 15 was used as a positive control since this sequence can be efficiently clamped by different rocaglates due to π -π stacking interactions on the surface of eIF4A-RNA complexes (Iwasaki et al., 2019 , Müller et al., 2020 . The data showed that the 5'-UTRs of SARS-CoV-2, HCoV-229E and MERS-CoV were similarly sensitive to translation inhibition by CR-31-B (-) in the dual luciferase reporter assay, when 10 nM CR-31-B (-) was used (Fig. 1C) . The observed eIF4A dependency indicated that CR-31-B (-) may have antiviral activity against the newly emerging SARS-CoV-2 in cell culture.

To analyze possible antiviral effects of CR-31-B (-) in cell culture, African green monkey Vero E6 cells were used because SARS-CoV-2 has previously been shown to grow to high titers in this cell line (Ogando et al., 2020) . Cytotoxicity of CR-31-B (-) was determined in an MTT assay after treating Vero E6 cells with increasing concentrations of CR-31-B (-) for 24 h ( Fig.   2A ). As shown in Fig 

The ongoing SARS-CoV-2 pandemic, a coronavirus outbreak of zoonotic origin reminiscent of the SARS and MERS outbreaks from a few years ago, has led to an increased awareness of the need for first-line, broad-spectrum 'pan-antivirals' that can be used as stopgaps until vaccines and specific therapies become available to prevent or treat infections caused by newly emerging viruses.

We and others have focused on the discovery and development of antivirals that target host mechanisms critical to viral proliferation. The rationale behind this approach is twofold: first, host mechanisms are not virus-specific, but are used by a broad range of viruses, and second, targeting a host mechanism preempts the risk for developing resistance typically associated with targeting viral structures or mechanisms.

Several host-targeting approaches against SARS-CoV-2 are currently under investigation.

Most of these represent efforts to repurpose approved drugs with known safety profiles.

Promising candidates are for example inhibitors of SARS-CoV-2 entry (e.g. compounds that block the viral spike protein activating cellular transmembrane protease serine 2 (TMPRSS2), (for review see Xiu et al., 2020) ), several immunomodulatory agents and also some kinase inhibitors that interfere with virus particle assembly (e.g. Janus kinase inhibitors) or with virus replication (imatinib). Angiotensin converting enzyme inhibitors or angiotensin receptor blockers, hydroxy methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors and several other compounds have also been tested as single usage or in combination approaches (Lam et al., 2020 , Al-Horani et al., 2020 , Sharma et al., 2020 , Santos et al., 2020 , thus illustrating an increasing relevance of host-targeting antiviral therapeutic strategies to combat COVID-19.

Here, we have further expanded the antiviral toolbox against SARS-CoV-2 by characterizing the antiviral activity of the synthetic rocaglate CR-31-B (-), a specific inhibitor of eIF4Adependent mRNA translation. We identified CR-31-B (-) as a potent and non-cytotoxic inhibitor of SARS-CoV-2 replication in vitro and ex vivo. The observed antiviral activities are directly comparable to those we have reported previously for other coronaviruses, namely

HCoV-229E and MERS-CoV, as well as a range of highly pathogenic positive-and negativesense single-stranded RNA viruses. This is to our knowledge the first report of a rocaglate inhibiting SARS-CoV-2 proliferation in a relevant ex vivo human bronchial cell system.

Against the backdrop of a number of reports we and others have published over the past three years documenting the broad-spectrum antiviral activity of CR-31-B (-) and related rocaglates, the data we report here strengthen the case for the potential use of rocaglates as first-line, broad-spectrum 'pan-antivirals'. The necessary next step is the in vivo evaluation of CR-31-B (-) and other rocaglates to confirm safety and determine appropriate dosing regimens. Extensive preclinical work with CR-31-B (-), zotatifin, and other rocaglates as cancer therapeutics is a priori highly encouraging for the potential antiviral application of rocaglates in humans. Minimal or no toxicities seen following long-term dosing regimens in animal models, favorable ADME-and bioavailability profiles (Wolfe et al., 2014 , Chan et al., 2019 , Rodrigo et al., 2012 , and the advancement of at least one rocaglate into the clinic for cancer, zotatifin, support the potential for this class of molecules to be used as antivirals in humans. At this point it is too early to speculate whether rocaglates could be applied prophylactically or therapeutically, but ongoing in vivo studies with CR-31-B (-) in relevant animal models of virus infection will provide answers to these questions.

In summary, the path to clinical implementation is still long and will most likely require further optimization of CR-31-B (-) or other lead rocaglates currently under investigation.

Nevertheless, the consistent antiviral efficacy and low toxicity of this class of compounds across viruses both in vitro and ex vivo and the more advanced parallel efforts to harness their activity as cancer therapeutics suggest that rocaglates represent a potentially powerful addition to the toolbox of interventions available to public health authorities to counter new and emerging RNA virus outbreaks globally.

We like to thank Ulrike Wend for technical assistance.

The authors declare no conflict of interest.

The work was supported by the LOEWE Center DRUID (projects A2 and B2 

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