A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry (preprint)

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.

Activation of ACE2 and interferon-stimulated transcriptomes in human airway epithelium is curbed by Janus Kinase inhibitors (preprint)

SARS-CoV-2 infection of human airway epithelium activates genetic programs that lead to progressive hyperinflammation in COVID-19 patients. Here we report on the transcriptomic response of airway epithelium to interferons and its suppression by the JAK inhibitors Baricitinib and Ruxolitinib. There is a debate on the regulation of the conventional versus the novel intronic promoter inducing the short ACE2 isoform. Through RNA-seq and ChIP-seq analyses for activating chromatin marks and Polymerase II, we define the interferon-activated intronic regulatory region. Our results also support that the conventional ACE2 promoter is controlled by interferon.

Proteolytic activation of the SARS-CoV-2 spike S1/S2 site: a re-evaluation of furin cleavage (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its spike (S) protein to mediate viral entry into host cells. Cleavage of the S protein at the S1/S2 and/or S2 site(s) is associated with viral entry, which can occur at either the cell plasma membrane (early pathway) or the endosomal membrane (late pathway), depending on the cell type. Previous studies show that SARS-CoV-2 has a unique insert at the S1/S2 site that can be cleaved by furin, which appears to expand viral tropism to cells with suitable protease and receptor expression. Here, we utilize viral pseudoparticles and protease inhibitors to study the impact of the S1/S2 cleavage on infectivity. Our results demonstrate that S1/S2 pre-cleavage is essential for early pathway entry into Calu-3 cells, a model lung epithelial cell line, but not for late pathway entry into Vero E6 cells, a model cell line. The S1/S2 cleavage was found to be processed by other proteases beyond furin. Using bioinformatic tools, we also analyze the presence of a furin S1/S2 site in related CoVs and offer thoughts on the origin of the insertion of the furin-like cleavage site in SARS-CoV-2.

FDA approved calcium channel blockers inhibit SARS CoV 2 infectivity in epithelial lung cells (preprint)

Covid-19 has infected more than 14 million people worldwide causing over 600.000 deaths1. The disease is caused by the severe acute respiratory syndrome coronavirus (CoV) 2 (SARS-CoV-2), which shares a high sequence similarity to SARS CoV2. Currently there are no vaccinations available to provide protection and the only antiviral therapy in active use in patients is remdesivir, which currently provides only limited benefit3. Hence, there is an urgent need for antiviral therapies against SARS CoV 2. SARS CoV requires Ca2+ ions for host cell entry4 and based on the similarity between SARS CoV and SARS CoV 25 it is highly likely that the same requirements exist for the two viruses. Here, we tested whether FDA-approved calcium channel blocker (CCB) drugs are efficacious to inhibit the spread of SARS CoV 2 in cell culture. Our data shows that amlodipine, felodipine and nifedipine limit the growth of SARS CoV 2 in epithelial kidney (Vero E6) and epithelial lung (Calu-3) cells. We observed some differences in the inhibition efficacy of the drugs in the two different cell lines, but with felodopine and nifedipine having the greatest effect. Overall, our data suggest that CCBs have a high potential to treat SARS CoV 2 infections and their current FDA approval would allow for a fast repurposing of these drugs.

Identifying SARS-CoV-2 entry inhibitors through drug repurposing screens of SARS- S and MERS-S pseudotyped particles (preprint)

While vaccine development will hopefully quell the global pandemic of COVID-19 caused by SARS-CoV-2, small molecule drugs that can effectively control SARS-CoV-2 infection are urgently needed. Here inhibitors of two coronavirus spike proteins (S) were identified by screening a library of approved drugs with SARS-S and MERS-S pseudotyped particle entry assays. Using high-throughput screening technology, we discovered three compounds (cepharanthine, abemaciclib and trimipramine) to be broad spectrum inhibitors for spike-mediated entry. This work should contribute to the development of effective treatments against the initial stage of viral infection, thus reducing viral burden in COVID-19 patients.