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Authors: Gorshkov et al

Link to paper: https://www.biorxiv.org/content/10.1101/2020.05.16.091520v1

Journal/ Pre-Print: BioRxiv

Tags: Autophagy/Lysosomes, Therapeutics, Drug discovery/ Drug repurposing

Research Highlights

1. The autophagic flux inhibitors ROC-325 and clomipramine can inhibit the cytopathic effects of SARS-CoV-2 without causing severe toxicity.

2. The cytopathic effect inhibition correlates with LC3B and LysoTracker staining

Summary 

Hydroxycloroquine has been tested extensively as a potential drug for COVID-19 patients, but it can have toxic effects. The authors investigated the use of other autophagy modulators that can be considered for clinical use to avoid the toxicity of hydroxychloroquine. Several drugs were tested, with ROC-325 (EC50 = 3.28 μM) and clomipramine (EC50 = 13.6 μM) exhibiting highest inhibition of the cytopathic effect induced by SARS-CoV-2, without having toxic effects. The cytopathic effect inhibition correlated with LC3B and LysoTracker staining, showcasing the accumulation of autophagosomes and acidic endosomes/lysosomes. These results highlight the potential of autophagy inhibitors, other than hydroxychloroquine, to treat SARS-CoV-2 infection.

Impact for SARS-CoV2/COVID19 research efforts

Treatment of SARS-CoV2/COVID19 positive individuals: The authors explored several autophagy modulators for their inhibition of SARS-CoV-2 cytopathic effects, their cell toxicity, autophagy flux and lysosomal acidity markers.

Study Type

· In vitro study

Strengths and limitations of the paper

Novelty: This study is novel in bringing together multiple autophagic flux inhibitors to test their efficacy in blocking SARS-CoV-2 propagation. Clomipramine, one of the drugs used in this study, has previously been shown to inhibit SARS-CoV-2 (Weston et al, BioRxiv, 2020). 

Standing in the field: This study shows that autophagy inhibitors can help combat SARS-CoV-2 infection. On the other hand, autophagy inducers, such as spermidine, have been shown to hinder SARS-CoV-2 (Gassen, BioRxiv, 2020). Additionally, a different study revealed that autophagy pathway genes are both up- and down-regulated during SARS-CoV-2 infection (Singh et al, BioRxiv, 2020). These conflicting results suggest that SARS-CoV-2 virus might require specific steps of the autophagy pathway to replicate, but may downregulate other steps to escape degradation.

Appropriate statistics: Yes

Viral model used: Not mentioned

Translatability: The results are translatable, since autophagy modulators were examined for their ability to block cytopathic effects of SARS-CoV-2. However, we should keep in mind that (1) HCQ is not an effective drug for COVID-19 and (2) SARS-CoV-2 inhibits autophagy in cells, so these drugs could not easily be considered as an alternative strategy to treat COVID-19.

Main limitations:

1. Virus used not mentioned

2. It would be interesting to test these drugs in lung cell lines (such as Calu-3) and in vivo study.

3. Autophagy inhibition was very poorly addressed. A major limitation of the study is that the authors did not add a known lysosomal inhibitor to their drug assays such as bafilomyin A to establish that the increase of LC3B puncta is indeed due to autophagic flux inhibition rather than increased generation of autophagosomes.

4. LC3B and LysoTracker not shown in the viral setting.

5. Why should these modulators work if HCQ does not?.

6. Should show SARS-CoV-2 RNA release decreased with these modulators.

7. The mode of action of these drugs is different, they each have a different mechanism to inhibit autophagy, the impact of these different actions on SARS-CoV-2 was not experimentally explored, discussed or mentioned in the paper.

8. Hycanthone seems to be an autophagy inducer and not an inhibitor.