Suramin inhibits SARS-CoV-2 nucleocapsid phosphoprotein genome packaging function

Figures

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  Fig. 1. Suramin interaction with SARS-CoV2-N protein. (A) Schematic view of SARS-CoV-2 genome, virion, RNP protomer and structural organization of the N protein; the N gene is highlighted in lime green, NTD and CTD are depicted in blue bell and deep teal/wild willow green, RNA is depicted in pomegranate orange, respectively. (B) SDS-PAGE analysis of purified SARS-CoV-2 full-length N (left), NTD (middle) and CTD (right) (M, molecular weight marker). (C) Nano-DSF analysis of full-length SARS-CoV-2 N; thermal stability and conformational profile is shown by superimposed F330/350 ratio (lime green) and first derivative (deep-koamaru blue); conformational transitions corresponding to inflections in intrinsic fluorescence and related Tm1 (45.9 ± 0.1 °C) and Tm2 (64.4 ± 0.1 °C) are indicated as dashed and dotted lines, respectively. (D) Nano-DSF analysis of SARS-CoV-2 N in the presence of 100 μM Suramin; superimposed F330/350 ratio (lime green) and first derivative (pomegranate orange) are shown, with Tm1 (42.0 ± 0.1 °C; ∗∗∗∗ P < 0.0001) indicated as dashed line. Experimental points and Tm values are represented as averages and standard deviations from at least three independent experiments.…

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  Fig. 2. Suramin affinity to SARS-CoV-2 N NTD and CTD. (A) Nano-DSF thermal stability and conformational profile of SARS-CoV-2 N NTD; superimposed F330/350 ratio (blue bell) and first derivative (deep-koamaru blue), with conformational transition corresponding to intrinsic fluorescence inflection are shown; Tm (53.2 ± 0.1 °C) is indicated as dashed line. (B) SARS-CoV-2 N NTD Nano-DSF in the presence of 100 μM Suramin; superimposed F330/350 ratio (lime green) and first derivative (pomegranate orange) are shown, with shifted Tm (42.0 ± 0.1 °C; ΔT≈11∘C; ∗∗∗∗ P < 0.001) indicated as dashed line. (C) Nano-DSF thermal stability and conformational profile of SARS-CoV-2 N CTD; superimposed F330/350 ratio (wild willow green) and first derivative (deep-koamaru blue), with conformational transitions corresponding to intrinsic fluorescence inflections are shown; Tm1 (49.1 ± 0.1 °C) and Tm2 (53.7 ± 0.1 °C) are indicated as dashed and dotted lines, respectively. (D) SARS-CoV-2 N CTD Nano-DSF in the presence of 100 μM Suramin; superimposed F330/350 ratio (wild willow green) and first derivative (pomegranate orange) are shown, with shifted Tm1 (42.0 ± 0.1 °C; ΔT≈7∘C; ∗∗∗∗ P < 0.0001) and Tm2 (51.7 ± 0.1…

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  Fig. 3. Model and interaction map for Suramin binding to SARS-CoV-2 N NTD. Cartoon 3D representation of Suramin putative binding mode to SARS-CoV-2 N NTD, according to best pose by molecular docking; predicted 3D-intermolecular interactions at binding site are zoomed in the inset. (B) 2D-diagram of predicted Suramin-residue interactions within the N NTD binding pocket; H-bonds and hydrophobic contacts are shown as dashed lines with distances between atoms indicated in angstrom and as arcs with spokes, respectively.

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  Fig. 4. Model and interaction map for Suramin binding to SARS-CoV-2 N CTD. Cartoon 3D representation of Suramin putative binding mode to SARS-CoV-2 N CTD dimer interface, according to best pose by molecular docking for binding site 1. (B) 2D-diagram of predicted Suramin-residue interactions within the N CTD dimer interface. (C) Cartoon 3D representation of Suramin putative binding mode to SARS-CoV-2 N CTD dimer RNA binding groove, according to best pose by molecular docking for binding site 2. (D) 2D-diagram of predicted Suramin-residue interactions within the N CTD dimer RNA binding groove. 3D-intermolecular interactions at binding sites are zoomed in the inset; H-bonds and hydrophobic contacts are shown as dashed lines with distances between atoms indicated in angstrom and as arcs with spokes, respectively.

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  Fig. 5. Suramin inhibition of SARS-CoV-2 N NTD and CTD ssRNA binding. (A) MST analysis of SARS-CoV-2 N NTD binding and affinity to Cy5-ssRNA 7-mer showing low-micromolar apparent Kd (30.6 ± 4.9 μM). (B) MST analysis of SARS-CoV-2 N NTD interaction with Cy5-ssRNA 7-mer showing Suramin dose-dependent inhibition of ssRNA binding with low-micromolar IC50 (≈ 20 ± 2 μM). (C) MST analysis of SARS-CoV-2 N CTD binding and affinity to Cy5-ssRNA 7-mer showing low-micromolar apparent Kd (5.3 ± 0.3 μM). (D) MST analysis of SARS-CoV-2 N NTD interaction with Cy5-ssRNA 7-mer showing Suramin dose-dependent inhibition of ssRNA binding with low-micromolar IC50 (≈ 50 ± 6 μM). Dose-response curves were obtained by non-linear regression fitting (deep-koamaru blue and pomegranate orange dashed lines) on experimental points (blue bell and wild willow green circles). Experimental points, Kd and IC50 values are represented as averages and standard deviations (deep-koamaru blue and pomegranate orange bars) from at least three independent experiments.

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  Fig. 6. Suramin inhibition of SARS-CoV2 N genome packaging and virus replication. Representative micrographs from negative stain EM showing: (A) purified SARS-CoV-2 N in the absence of ssRNA; (B) in vitro reconstituted SARS-CoV-2 RNP complex-like intermediates formed upon incubation of SARS-CoV-2 N with ssRNA 56-mer; (C) inhibition of SARS-CoV-2 RNP complex-like intermediates formation in the presence of 50 μM Suramin. (D) Analysis of particle radius distribution between negative stain EM micrograph datasets of purified SARS-CoV-2 N in the absence of ssRNA (left box, lime green), RNP complex-like intermediates formed upon SARS-CoV-2 N interaction with ssRNA 56-mer in the absence (middle box, deep teal) or in the presence (right box, pomegranate orange) of 50 μM Suramin; median is shown as continuous line in the middle of each box, with top and bottom representing the 75th and 25th percentiles, respectively (**** P < 0.0001, unpaired two-tailed Student's t-test, N = 2). (E) Effect of Suramin on SARS-CoV-2 replication in Vero E6-GFP cells. (F) Effect of GC376 on SARS-CoV-2 replication in Vero E6-GFP cells. Results were expressed as percentage of virus-induced CPE over uninfected and…

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  Fig. 7. Suramin inhibition of SARS-CoV-2 N IFN-I antagonism. (A) Effect of Suramin on SARS-CoV-2 N inhibition of IFN-I promoter activation upon stimulation with viral RNA. (B) Effect of Suramin on SARS-CoV-2 N inhibition of ISRE promoter activation upon stimulation with IFN-α. Results were expressed as percentage of induction between Suramin untreated control (lime green bar) and Suramin treated samples (pomegranate orange bars) over unstimulated control transfected with the empty vector (black bar), and plotted as mean plus standard deviation of two independent experiments on triplicate (EV, empty vector). Statistical significance was evaluated by one-way analysis of variance (ANOVA, N = 3) followed by post-hoc Dunnett test (Supplementary Table 3).