we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.
We have used affinity purification-mass spectrometry to identify 332 high-confidence SARS-CoV-2-human PPIs. We find the viral proteins connected to a wide array of biological processes, including protein trafficking, translation, transcription and ubiquitination regulation. Using a combination of a systematic chemoinformatic drug search with a pathway centric analysis, we uncovered close to 70 different drugs and compounds, including FDA approved drugs, compounds in clinical trials as well as preclinical compounds, targeting parts of the resulting network. We are currently testing these compounds for antiviral activity and encourage others to do the same as well as extract insights from the map that could have therapeutic value.
More generally, this proteomic/chemoinformatic analysis is not only identifying drug and clinical molecules that might perturb the viral-human interactome, it gives these potentially therapeutic perturbations a mechanistic context. Among those that may be infection relevant are the inhibition of lysosomal acidification and trafficking with Bafilomycin A1, via inhibition of V-ATPase71, and modulation of the ER stress and the protein unfolding response pathway by targeting the Sigma1 and Sigma2 receptor by drugs like haloperidol (Fig. 5a, Tables 1a,b). Indeed, several of the human proteins in the interactome are targeted by drugs that have emerged phenotypically as candidate therapeutics for treating Covid-19, such as chloroquine72,73. While we do not pretend to have identified the molecular basis of chloroquine’s putative activity, we do note that this drug targets the Sigma1 and Sigma2 receptors at mid-nM and low mM concentrations, respectively. Similarly, antibiotics like azithromycin have also been mooted as treatments for Covid-19. While this too remains to be demonstrated, we note that Azithromycin has off-target activity against human mitochondrial ribosomes, components of which interact with the SARS-CoV-2 Nsp8 protein (MRPS5, MRPS27, MRPS2, and MRPS25). Other antibiotics that also have an off-target effect on mitochondrial ribosomes, such as chloramphenicol, tigecycline, and Linezolid74,75 may also merit study for efficacy. Indeed, this logic may be extended. Many Covid-19 patients will be on the drugs identified here, treating pre-existing conditions. It may be useful to correlate clinical outcomes with the taking of these drugs, cross-referencing with the networks described here. In some senses, this is already occurring phenomenologically, leading to concerns about ACE inhibitors such as captopril and enalapril, and for NSAIDs. What this study provides is a systematic schema for making such clinical/drug associations going forward, giving them a mechanistic context that allows investigators to seek them directly.
Systematic genetic validation using genetic-based approaches76,77 will be key to determine the functional relevance of these interactions and if the human proteins are being used by the virus or are fighting off infection, information that would inform future pharmacological studies. It is important to note that pharmacological intervention with the agents we identified in this study could be either detrimental or beneficial for infection. For instance, the HDAC2 inhibitors may compound the potential action of the Nsp5 protease to hydrolyze this human protein. Future work will involve generation of protein-protein interaction maps in different human cell types, as well as bat cells, and the study of related coronaviruses including SARS-CoV, MERS-CoV and the less virulent OC435, data that will allow for valuable cross-species and viral evolution studies. Targeted biochemical and structural studies will also be crucial for a deeper understanding of the viral-host complexes, which will inform more targeted drug design.
Along with SARS-CoV-2, we have previously utilized global affinity purification-mass spectrometry (AP-MS) analysis to map the host-pathogen interfaces of a number of human pathogens including Ebola22, Dengue30, Zika30, Herpesvirus29, Hepatitis C28, Tuberculosis27, Chlamydia26, Enteroviruses25, HIV19, HPV24, and West Nile Fever23. Excitingly, we have uncovered both shared and unique mechanisms in which these pathogens co-opt the host machinery during the course of infection. Although host-directed therapy is often not explored for combatting pathogenic infections, it would be interesting to use this information to identify host factors that could serve as targets that would harbor pan-pathogenic activity so that when the next virus undergoes zoonosis, we will have treatment options available.
Reference & source Information:https://www.biorxiv.org/
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