Journal: bioRxiv

Article Title: An Oral Combination therapy against SARS-CoV-2 based on Synergistic Action of Auranofin and Remdesivir

doi: 10.64898/2025.12.19.695342

Figure Lengend Snippet: (A) Schematic of pseudotype virus entry. HEK293T ACE2 cells pretreated with 1 μM Auranofin were infected with SARS-CoV-2 spike pseudotyped particles and 3 hours post-infection (hpi) luciferase activity was measured. (B) Graph showing firefly luciferase activity for infection control and Auranofin-treated conditions. (C) Schematic of time-of-addition (ToA) assay. HEK293T ACE2 cells pretreated for 3 h with 1 μM Auranofin were infected with 10 MOI SARS-CoV-2 in the presence of drug (T-3). After 3 h, cells were harvested for western blot analysis. (D) Western blot showing expression of SARS-CoV-2 spike upon Auranofin treatment, compared to infected control (VC) and (E) quantification of band intensity. (F) Schematic of SARS-CoV-2 FlipGFP Mpro assay. HEK293T cells were co-transfected with FlipGFP Mpro reporter construct and SARS-CoV-2 NSP5 WT plasmid, 1 μM Auranofin was added after 3 h and 36 h later cells were fixed and GFP expression was analyzed under a fluorescence microscope. Treatment with Nirmatrelvir served as a positive control. (G) Representative fluorescence images showing differences in GFP fluorescence intensity in 3 different conditions as indicated. (H) Total GFP and mCherry signals from each field were quantified using ImageJ/Fiji. The GFP signals were normalized to the mCherry signal, and then the GFP values for each treatment condition were normalized to the no-drug control. All data are from 3 independent biological replicates. ∗∗p < 0.01, ∗∗∗p < 0.001; ns, non-significant using two-tailed unpaired t-test or Welch ANOVA with Dunnett’s T3 multiple comparison tests, where applicable. Error bars represent mean ± SD.

Article Snippet: For optimizing the reporter system, cells were cotransfected with 125 ng of FlipGFP Mpro plasmid (Addgene, 163078) and 375 ng of NSP5 WT or NSP5 C145A plasmids (kind gift from Prof. Nevan Krogan - University of California San Francisco) using Lipofectamine 2000 transfection reagent (Invitrogen, 11668019).

Techniques: Virus, Infection, Luciferase, Activity Assay, Control, Western Blot, Expressing, Transfection, Construct, Plasmid Preparation, Fluorescence, Microscopy, Positive Control, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: An Oral Combination therapy against SARS-CoV-2 based on Synergistic Action of Auranofin and Remdesivir

doi: 10.64898/2025.12.19.695342

Figure Lengend Snippet: Binding free energy and docking of Auranofin against SARS-CoV-2 Mpro were performed using Autodock. (A-B) Docked structures of SARS-CoV-2 Mpro to Auranofin and corresponding Hydrogen bonding interactions with key amino acids are shown in (A) and (B) respectively. (C) Shows binding free energy for Auranofin and Nirmatrelvir. (D-F) ITC Sensogram and isothermodynamics for binding interactions between Mpro and auranofin and nirmatrelvir are shown in (D) and (E) respectively. (F) shows similar data set for interactions between the catalytic mutant of Mpro C145A and auranofin.

Article Snippet: For optimizing the reporter system, cells were cotransfected with 125 ng of FlipGFP Mpro plasmid (Addgene, 163078) and 375 ng of NSP5 WT or NSP5 C145A plasmids (kind gift from Prof. Nevan Krogan - University of California San Francisco) using Lipofectamine 2000 transfection reagent (Invitrogen, 11668019).

Techniques: Binding Assay, Mutagenesis