Review 1: "Elucidation of remdesivir cytotoxicity pathways through genome-wide CRISPR-Cas9 screening and transcriptomics"

RR:C19 Evidence Scale rating by reviewer:

• Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review:

The manuscript by Akinci et. al investigates the molecular basis of the cytotoxicity of two antiviral drugs: remdesivir and hydroxychloroquine (HCQ). The active remdesivir metabolite is known to inhibit viral RNA-dependent RNA polymerase (RdRp) enzymes while HCQ inhibits the endosome and lysosome trafficking. As previously observed, HCQ treatment upregulates in RNA-seq the cholesterol metabolism and the autophagy and starvation pathways while it downregulates the rRNA and tRNA production. This was confirmed experimentally by showing that HCQ impairs cellular LDL uptake, which results in increased LDL retention in endosomes. The CRISPR/Cas9 screen performed with HCQ found that the knockout of genes involved in endocytosis and cholesterol metabolism increased HCQ lethality. In contrast, enriched genes in the CRISPR/Cas9 screen after HCQ treatment are involved in plasmalogen synthesis in the peroxisome and peroxisome maintenance. Interestingly, genes part of the vesicle trafficking pathway were found to be enriched in both over-represented (higher survival) and under-represented (higher lethality) groups.   

In RNA-seq experiments, remdesivir treatment resulted in a specific decrease in expression of genes involved in the mitochondrial respiratory complexes, which was confirmed experimentally by a decrease in ATP production in cells treated with remdesivir. A CRISPR/Cas9 screening was used to find genes involved in its metabolism and its cytotoxicity. The authors found that the remdesivir prodrug is metabolized by Cathepsin A in the lysosome and then phosphorylated in its active form by AK2 in the mitochondrial intermembrane space. SLC29A3 is the potential protein modulating the entry of remdesivir in the lysosome and the mitochondrial intermembrane space. Active remdesivir competes with ATP which promotes inhibition of the mitochondrial RNA polymerase, leading to cytotoxicity.  The authors then investigated by knocking out SLC29A3 or AK2 whether cytotoxicity could be decreased without affecting the potency of remdesivir against SARS-CoV-2. They found that SLC29A3 is a good candidate with an 8-fold decrease in cytotoxicity and no major decrease in antiviral potency.

This is overall an important and well-performed study that provides insights into the molecular pathways leading to remdesivir cytotoxicity, which is due to the inhibition of the mitochondrial RNA polymerase. However, there is currently no inhibitor specific for SLC29A3 which limits the potential therapeutic approach through this protein to reduce the toxicity of remdesivir or other nucleoside drugs. Nevertheless, this study provides significant information and opens new research avenues to decrease unwanted side effects of remdesivir.