So far, the only FDA (US Food and Drug Administration) approved drug for the treatment of Covid-19 is Remdesivir, which acts by inhibiting the protein responsible for the viral genome replication (the RNA dependent RNA polymerase, RdRP). However, the search of other effective compounds is still ongoing. For instance, a recent study has identified, based on the screening of 12.000 compounds, a number of small molecules inhibiting virus replication and infection.
Drugs work by interfering with key mechanisms of viral infection, and SARS-CoV2 relies on several host factors to infect the cells and replicate. For instance, ACE2 receptor –expressed on the surface of human cells− is needed for virus binding to human cells and infection. Moreover, after the viral protein Spike binding to ACE2, Spike protein needs to be cleaved by host proteins (eg TMPRSS2, cathepsin L), and the cleavage is necessary for Spike activation, the fusion of the virus with the host membrane and the release of the viral genome in the host cell. Then, the virus uses both its own and the host factors to replicate.
Main Finding. In a paper recently published in Cell, authors identify a number of host genes which are required for virus infection, whose inhibition reduces SARS-CoV2 infection. These genes may thus represent potential pharmacological targets for the treatment of SARS-CoV2 infection.
Experimental details. The authors performed a CRISPR1-based loss of function screen (meaning CRISPR-mediated deletion of specific genes in order to identify those that conferred to the cells the capability to survive SARS-CoV2 infection) in human lung cell line (A549) expressing ACE2 receptor.
Identification of key host genes in viral infection. The screen led to the identification of a number of genes involved in key aspects of viral entry and replication, such as ACE2; genes involved in spike protein cleavage and fusion of the virus membrane with the human cell membrane; genes of the Endoplasmic Reticulum/Golgi trafficking (movement of proteins between the two cellular compartments); endosome recycling (process in which portions of the plasma membrane internalized are processed back to the cell surface for reuse); modulators of gene expression. Except for ACE2, the genes identified were broadly expressed in nearly all tissues, suggesting that the underlying mechanisms may be the same independently from the tissue or cell type. Moreover, some of the genes/gene products identified have been previously reported to directly interact with different viral proteins.
Interestingly, this screening approach has been used in the past to identify genes required for ZIKA virus and H1N1 virus infection, and the authors found that several genes identified as critical in ZIKA virus infection were also found critical in SARS-CoV2 infection.
Validation. Thirty genes among those identified by the screening were chosen for a further validation analysis. Deletion (CRISPR-mediated loss of function) of all these 30 genes strongly reduced SARS-CoV2 infection (by 10 times). The greatest protection from infection was given by the loss of genes such as the known ACE2, cathepsin L, and genes of the vesicle trafficking (vesicle movement inside the cell).
Mechanism of action. What is the role in SARS-CoV2 infection of the genes identified in the screening? The authors observed that the loss of one of these genes (RAB7A, a protein regulating vesicular transport and membrane trafficking) reduces cell surface expression of ACE2, which accumulates instead in vesicles in the cytoplasm.
Moreover, the loss of 11 of the 30 host genes identified by the screening alters the gene expression profile, and 6 of these genes lead to similar gene expression changes, which includes the upregulation of pathways of lipid and cholesterol homeostasis (cholesterol is a component of animal cell membranes strongly affecting the its physical properties). A previous work had shown that SARS-CoV-2 infection negatively modulates the cholesterol synthesis pathway and pharmacological induction of this pathway effectively prevents infection2. Even though future studies are warranted to unveil the mechanistic link between cholesterol synthesis pathway and resistance to SARS-CoV2 infection, it is worth to mention that changes in lipid composition of the cell have been previously shown to affect Hepatitis C and Influenza A virus maturation and ability to infect.
Clinical translation of the findings. What is the relevance of these findings from a clinical point of view? Interestingly, by comparing the whole list of genes identified by the screen with the list of genes of the Drug Gene Interaction database (DGIdb) (namely genes or gene products that are known or predicted to interact with drugs), authors identified 69 genes for which potential targeting drugs are available. In particular, among these 69 genes, 9 resulted to be potential targets of 26 inhibitors. ACE2-expressing human cells were thus pre-treated with the inhibitors and then infected with SARS-CoV2: seven of the 26 inhibitors performed better (PIK-III, Compound-19, SAR405, autophinib, ALLN, tamoxifen and ilomastat), significantly reducing the viral load. Among these 7 best performing molecules, 4 (Compound-19, PIK-III, autophinib, SAR405) targeted the same gene –PIK3C3– and 2 (autophinib and ALLN) reduced the viral load more than 1.000-fold. Importantly, the combination of these drugs showed additive effect, with enhanced protection against SARS-CoV2 infection.
Conclusions. The authors identified a number of human genes playing a key role in SARS-CoV2 infection (whose loss is able to confer resistance to infection). Importantly, their results highlight the clinical relevance of these findings, showing that available drugs targeting some of these genes are able to reduce the viral load of SARS-CoV2-infected cells. It is worth to mention that among the drugs identified, ALLN is currently in clinical trial (for the treatment of different diseases), and tamoxifen is approved for breast cancer treatment.
Reference. Identification of required host factors for SARS-CoV-2 infection in human cells. Zharko Daniloski, Tristan X. Jordan, […], Benjamin R. tenOever, Neville E. Sanjana. Cell 2020.
1CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats): gene editing tool that cuts DNA in a precise, directed manner.
2Modulating the transcriptional landscape of SARS-CoV-2 as an effective method for developing antiviral compounds. Hoagland, D.A., Clarke, D.J.B., Møller, R., Han, Y., Yang, L., Wojciechowicz, M.L., Lachmann, A., Oguntuyo, K.Y., Stevens, C., Lee, B., et al. (2020). BioRxiv 2020.07.12.199687.