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. 2021 Feb 26;371(6532):926-931.
doi: 10.1126/science.abf4058. Epub 2021 Jan 25.

Plitidepsin has potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A

Kris M White #  1   2 , Romel Rosales #  3   2 , Soner Yildiz  3   2 , Thomas Kehrer  3   2 , Lisa Miorin  3   2 , Elena Moreno  3   2 , Sonia Jangra  3   2 , Melissa B Uccellini  3   2 , Raveen Rathnasinghe  3   2 , Lynda Coughlan  4 , Carles Martinez-Romero  3   2 , Jyoti Batra  5   6   7   8 , Ajda Rojc  5   6   7   8 , Mehdi Bouhaddou  5   6   7   8 , Jacqueline M Fabius  5   7 , Kirsten Obernier  5   6   7   8 , Marion Dejosez  9 , María José Guillén  10 , Alejandro Losada  10 , Pablo Avilés  10 , Michael Schotsaert  3   2 , Thomas Zwaka  9 , Marco Vignuzzi  11 , Kevan M Shokat  5   7   8   12 , Nevan J Krogan  1   5   6   7   8 , Adolfo García-Sastre  1   2   13   14
Affiliations

Plitidepsin has potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A

Kris M White et al. Science. .

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.

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Figures

Fig. 1
Fig. 1. Plitidepsin exhibits a strong antiviral activity in SARS-CoV-2 multiple cell lines.
(A to E) Vero E6 cells [(A) and (B)], hACE2-293T cells [(C) and (D)], or pneumocyte-like cells (E) were treated with indicated doses of remdesivir [(A) and (C)] or plitidepsin [(B), (D), and (E)]. IC50, IC90, 50% cytotoxic concentration (CC50), and CC10 values are indicated above the curves. All cells were pretreated for 2 hours and the drugs were maintained in the media throughout the experiment. SARS-CoV-2 infection and cell viability were measured at 48 hours. (F) The antiviral activities of plitidepsin and remdesivir were evaluated in pretreatment and post-infection time points in hACE2-293T cells. In all panels, data are means ± SD of three independent experiments performed in biological triplicate. DMSO, dimethyl sulfoxide.
Fig. 2
Fig. 2. Antiviral mechanism of action of plitidepsin is mediated through inhibition of eEF1A.
(A) Plitidepsin inhibition of SARS-CoV-2 replication in 293T cells transfected with eEF1A-WT or eEF1A-A399V expression vectors. Plitidepsin inhibition is reduced by expression of the A399V mutation, whereas virus replication in wild-type and eEF2A-transfected mutations remain susceptible to treatment with plitidepsin. (B and C) Plitidepsin (B) and remdesivir (C) inhibition of SARS-CoV-2 replication in a CRISPR 293T cell line carrying an A399V mutation in eEF1A. Viral replication in wild-type eEF1A preserves susceptibility to plitidepsin inhibition, whereas the presence of the eEF1A A399V mutation rendered the SARS-CoV-2 infection resistant to the eEF1A inhibitor. Remdesivir inhibition of SARS-CoV-2 viral replication was not affected by the A399V mutation. (D and E) Plitidepsin inhibition of cell proliferation, as measured by (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, is not affected by transfection of the A399V mutant (D) but is reduced by the 293T-A399V CRISPR cell line (E). (F) siRNA silencing of eEF1A greatly reduces N protein levels. In all panels, data are means ± SD of three independent experiments performed in biological triplicate. ****P < 0.0001.
Fig. 3
Fig. 3. Plitidepsin treatment causes a specific reduction in subgenomic RNA expression.
(A to D) Vero E6 cells were infected with SARS-CoV-2 at an MOI of 1 in the presence or absence of 3 nM plitidepsin or 5 μM remdesivir and samples were taken at the indicated time points. The levels of genomic RNA (A) and subgenomic N RNA (B) were analyzed with specific reverse transcription quantitative polymerase chain reactions (RT-qPCR). (C) Cell lysates were collected at the indicated times and subjected to Western blotting. (D) Each protein band was quantified by ImageJ and normalized to GAPDH levels. Data are means ± SD of three independent experiments performed in biological triplicate. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 4
Fig. 4. Plitidepsin treatment significantly reduces SARS-CoV-2 infection in BALB/c mice expressing human ACE2.
(A) Schematic of adenovirus expression of human ACE2 model of SARS-CoV-2 infection. BALB/c mice were transduced with human ACE2 expressing adenovirus. Mice were sensitized intranasally with 2.5 × 108 pfu. (B) Mice were intranasally infected with 104 pfu of SARS-CoV-2 and subcutaneously treated with either 0.3 mg/kg plitidepsin once daily for 3 days, a single dose of 1 mg/kg plitidepsin, or 50 mg/kg remdesivir once daily for 3 days. (C) SARS-CoV-2 lung titers in the plitidepsin-treated group relative to vehicle and remdesivir controls. Virus titers were determined in whole lung homogenates by median tissue culture infectious dose (TCID50) at day 3 after infection. The limit of detection for viral titers is indicated with a dotted line. Vehicle and remdesivir, N = 10; plitidepsin 1 mg/kg and 0.3 mg/kg, N = 8. ***P < 0.001, ****P < 0.0001.
Fig. 5
Fig. 5. Plitidepsin shows in vivo antiviral efficacy in the K18-hACE2 mouse model.
(A) Schematic of the K18-hACE2 model of SARS-CoV-2 infection. (B) Mice were intranasally infected with 104 pfu of SARS-CoV-2 and subcutaneously treated with 0.3 mg/kg plitidepsin once daily for 3 days or with 50 mg/kg remdesivir twice daily for 3 days. (C) SARS-CoV-2 lung titers in the plitidepsin-treated group relative to vehicle and remdesivir controls. Virus titers were determined in whole lung homogenates by TCID50 at day 3 after infection. Five mice were used in each group, except for the remdesivir control, which had 3. *P < 0.05, **P < 0.01. (D) Lungs were harvested on day 3 after infection, paraffin-embedded, and 5-μm sections stained for hematoxylin and eosin. Regions of the lung anatomy where inflammation was assessed are highlighted by black boxes, with the corresponding higher-magnification image indicated by matching letter. Regions where inflammation was detected are indicated by arrows. (E) Pathological severity scores in infected mice. To evaluate comprehensive histological changes, lung tissue sections were scored according to pathological changes outlined in the supplementary materials.
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