The NSP14/NSP10 RNA repair complex as a Pan-coronavirus therapeutic target

Figures

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  Fig. 1. In vitro NSP14/10 activity assay and inhibitor screening. a Schematics showing dsRNA FRET-based NSP14 exonuclease activity assay. NSP14/10 recognizes the terminal mismatch and removes bases from the 3ʹ end of the substrate strand until the two RNA strands separate and the fluorescence signal increases. b Activity curves of SARS-CoV-2, SARS-CoV and MERS-CoV NSP14/10 complex using Oligo D. RNase A was used as a positive control and NSP10 alone was used as a negative control. For all downstream assays SARS-CoV-2 NSP14/10 was used in combination with Oligo D unless otherwise stated. Data shown from one independent measurement using technical duplicates. Error bars represent SD. c Bar graph shows exonuclease activity of the NSP14:NSP10 complex at different molar ratios. Relative activity was normalized to NSP14 alone. Fluorescence was read after 1 h of reaction time. d Specificity of the NSP14/10 complex over a variety of purified proteins (8-Oxoguanine DNA Glycosylase [OGG1]; BTB Domain and CNC Homolog 1 [Bach1]; Ubiquitin conjugating enzyme UbcH3; bovine serum albumin [BSA]). As proposed by the literature, NSP14 alone barely shows activity. Relative activity was normalized to a…

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  Fig. 2. Characterization of novel NSP14/10 inhibitors. a Chemical structures of the three compounds (#79, #96 and #102) that performed the best in the viral infection assays. Compound #38, which does not inhibit NSP14, was included in downstream assays as a negative control. b Dose-response curves of NSP14/10 activity in the presence of compounds #38 (negative control), #79, #96 and #102. Average IC50 values were calculated from three independent measurements using technical duplicates. Error bars represent SD. c A gel-based assay was used to verify inhibitory potential of compounds #79, #96, #102, and #38. Inhibition of the NSP14/10 exonuclease activity results in the reduction of the full-length dsRNA oligo and increase in faster migrating bands. Compounds were used at the indicated concentrations. d Differential scanning fluorimetry performed in the presence or absence of the indicated compounds at 50 μM concentrations. Graph shows calculated mean melting temperatures of the NSP14/10 complex in the presence of the indicated compounds. Error bars represent SD. e Docking poses of compounds, predicted to stack either against the last nucleobase (#79, #96) or under the RNA (#102) while…

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  Fig. 3. Synergistic effect of select compounds with remdesivir using HCoV-OC43 viral infection assay. a Schematics showing the viral infection assay workflow. Drug treated cells were infected with HCoV-OC43 or SARS-CoV-2, fixed, stained, and imaged at indicated times post infection. Cytotoxicity was measured in similarly drug-treated uninfected cells using either the CellTiter-Glo or the alamarBlue assay. b Representative graphs are shown of the antiviral activity (full symbols) and cytotoxicity (empty symbols) of compounds #79, #96 and #102 in HCM3 cells infected with HCoV-OC43. Compounds were applied at the following concentrations: #79: 60 μM, #96: 15 μM, #102: 25 μM. Remdesivir concentrations are indicated on the X-axis in μM. Error bars represents SEM. IF images of representative wells show anti-N staining (red) and DAPI signal (blue) at indicated drug concentrations. Graph shows the EC50 values from three independent experiments using technical triplicates. Error bars represents SEM.

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  Fig. 4. Synergistic effect of select compounds with remdesivir using SARS-CoV-2 viral infection assay. Representative graphs are shown of the antiviral activity (full symbols) and cytotoxicity (empty symbols) of compounds #79, #96 and #102 in A549+ACE2 cells infected with SARS-CoV-2. Compounds were applied at the following concentrations: #79: 60 μM, #96: 15 μM, #102: 25 μM. Remdesivir concentrations are indicated on the X-axis in μM. Error bars represents SEM. IF images of representative wells show anti-N staining (red) and DAPI signal (blue) at indicated drug concentrations. Graph shows the EC50 values from three independent experiments using technical triplicates. Error bars represents SEM.

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  Fig. 5. NSP14/10 inhibition as a therapeutic approach. Schematics showing our therapeutic approach where inhibiting NSP14 potentiates the inhibition of NSP12 (with remdesivir, a ribonucleoside analog).