Kite-Shaped Molecules Block SARS-CoV-2 Cell Entry at a Post-Attachment Step

Abstract

Anti-viral small molecules are currently lacking for treating coronavirus infection. The long development timescales for such drugs are a major problem, but could be shortened by repurposing existing drugs. We therefore screened a small library of FDA-approved compounds for potential severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antivirals using a pseudovirus system that allows a sensitive read-out of infectivity. A group of structurally-related compounds, showing moderate inhibitory activity with IC50 values in the 2–5 μM range, were identified. Further studies demonstrated that these “kite-shaped” molecules were surprisingly specific for SARS-CoV-1 and SARS-CoV-2 and that they acted early in the entry steps of the viral infectious cycle, but did not affect virus attachment to the cells. Moreover, the compounds were able to prevent infection in both kidney- and lung-derived human cell lines. The structural homology of the hits allowed the production of a well-defined pharmacophore that was found to be highly accurate in predicting the anti-viral activity of the compounds in the screen. We discuss the prospects of repurposing these existing drugs for treating current and future coronavirus outbreaks.

Antiviral efficacy of selective estrogen receptor modulators against SARS‐CoV‐2 infection in vitro and in vivo reveals bazedoxifene acetate as an entry inhibitor

“…The protocol used for the temperature shift assay has been previously reported 11,12 . Cells were seeded in 96‐well plates (4 × 10 4 cells/well).…”

“…The protocol used for the temperature shift assay has been previously reported. 11 , 12 Cells were seeded in 96‐well plates (4 × 10 4 cells/well). Cells were pre‐cooled at 4℃ for 1 h. For the attachment assay, compounds diluted in viruses co‐treated cells for 1 h at 4℃ (final concentration 10 μM, MOI = 2).…”

Fusion assays for screening of fusion inhibitors targeting SARS-CoV-2 entry and syncytia formation

“…293T/293T-ACE2/293T-ACE2-TMPRSS2 cells were transfected in either 100 mm dishes or 6-well plates at seeding densities of 4 × 10 6 cells/100 mm dish or 7.2 × 10 5 cells/well of a 6-well plate in 10% FCS/DMEM without antibiotics. Cells were transfected using calcium phosphate (125 mM CaCl 2 , 0.7 mM Na 2 HPO 4 , 70 mM NaCl, 25 mM (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid) (HEPES) pH 7.05, as described previously (Chan et al, 2021). Donor 293T cells were cotransfected with CAGT7 and either the pcDNA3.1, CMVSARS1S, CMVSARS2S or CAGVSVG in a 1:1 ratio (CAGVSVG was made up of 1/10th CAGVSVG and 9/10th pcDNA3.1).…”

“…Cells were lysed by the addition of 100 μl of passive lysis buffer (Promega/Biotium) to each well and shaken for >15min. Luciferase assay was carried out as described in (Chan and Egan, 2005;Chan and Egan, 2009;Egan et al, 2013;Chan et al, 2021) in a buffer containing 0.0165 M glycylglycine, 0.01 M MgSO 4 , 2.65 mM EGTA, 10.5 mM potassium phosphate, 1.4 mM adenosine 5′-triphosphate, 0.86 mM dithiothreitol (DTT), 0.175 mg/ml bovine serum albumin (BSA), and 0.035 mM luciferin (Promega) using 50 μl of the lysate and a luminometer (Berthold Technologies, Germany). A standard curve was constructed using recombinant luciferase (Promega).…”

“…Cell viability of the fusion co-cultures (293T + 293T-ACE2 or 293T + 293T-ACE2-TMPRSS2) at physiological pH was measured as described in (Chan et al, 2021) by addition of 50 μl of 1 mg/ml 2,3-Bis-(2-Methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt (XTT) (Biotium/Alfa Aezar) and 20 μM N-methyl dibenzopyrazine methyl sulfate (Cayman) in culture medium to each well for 2-4 h at 37 °C/5% CO 2 . Absorbance was read at 450 nm with a reference wavelength of 650 nm using a plate reader (Bio-Tek Synergy HT).…”