Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells
Adam Pickard, Ben C Calverley, Joan Chang, Richa Garva, Sara Gago, Yinhui Lu, Karl E Kadler
PLOS Pathogens, doi:10.1371/journal.ppat.1009840
COVID-19 vaccines based on the Spike protein of SARS-CoV-2 have been developed that appear to be largely successful in stopping infection. However, therapeutics that can help manage the disease are still required until immunity has been achieved globally. The identification of repurposed drugs that stop SARS-CoV-2 replication could have enormous utility in stemming the disease. Here, using a nano-luciferase tagged version of the virus (SARS-CoV-2-ΔOrf7a-NLuc) to quantitate viral load, we evaluated a range of human cell types for their ability to be infected and support replication of the virus, and performed a screen of 1971 FDA-approved drugs. Hepatocytes, kidney glomerulus, and proximal tubule cells were particularly effective in supporting SARS-CoV-2 replication, which is in-line with reported proteinuria and liver damage in patients with COVID-19. Using the nano-luciferase as a measure of virus replication we identified 35 drugs that reduced replication in Vero cells and human hepatocytes when treated prior to SARS-CoV-2 infection and found amodiaquine, atovaquone, bedaquiline, ebastine, LY2835219, manidipine, panobinostat, and vitamin D3 to be effective in slowing SARS-CoV-2 replication in human cells when used to treat infected cells. In conclusion, our study has identified strong candidates for drug repurposing, which could prove powerful additions to the treatment of COVID.
Generation of functional SARS-CoV-2 virus DNA encoding the genome of SARS-CoV-2 and SARS-CoV-2-ΔOrf7a-NLuc were purchased from Vectorbuilder Inc. (Chicago, US). Transfection of DNA encoding the viruses failed to generate replicative virus when electroporated into 293T cells. We therefore produced RNA molecules which encoded the virus in vitro. Briefly, virus encoding DNA (1 μg) was transcribed using T7 mMessenger mMachine (Thermo) with a GTP:Cap ratio of 2:1 used in a 20 μL reaction. In addition, RNA encoding the SARS-CoV-2 nucleocapsid was also generated by PCR using primers P1 and P2 (S5 Table ) . It has been reported that this aids the recovery of replicative virus [18] . Viral RNA genomes (10 μl) and 2.5 μL nucleocapsid RNA were electroporated into 293T cells within the BSL3 laboratory. Viral RNAs were electroporated (5,000,000 cells, 1100 V, 20 ms and 2 pulses) and grown in T75 cm 2 flasks and 24 well plates. Cells grown in 24 well plates for 24-120 hrs were used to monitor changes in NLuc activity.
Virus production, maintenance and assessment of titer Culture medium was collected from 293T cells 6 days after electroporation. This virus (P0) was used to infect cells of interest. As virus replication was slow in 293T cells, virus stocks were maintained by passage in Vero cells grown in DMEM supplemented with 2% FBS. Medium (1 mL) was used to infect Vero cells in order to generate P1 virus. Replication was assessed by measuring NLuc activity over 10 days. For..
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