The nsp15 Nuclease as a Good Target to Combat SARS-CoV-2: Mechanism of Action and Its Inactivation with FDA-Approved Drugs

Figure 1. (A) Orthogonal views of the hexameric form of nsp15, which is formed by dimers of trimers. Monomers are colored in cyan, pink, violet, blue, green and yellow. Active site residues are shown as sticks. (B) nsp15 domain organization. ND, amino acids (aa) 1–63; MD, aa 64–181; NendoU, aa 206–346. The location of the conserved residues is represented. (C) Localization of the nsp15 domains in the monomer (PDB code 6W01). Mutated residues are shown as sticks. (D) Sequence alignment of nsp15 from SARS-CoV-2 (UniProt ID: P0DTD1), SARS-CoV (UniProt ID: P0C6X7) and MERS-CoV (YP_009047225). Residues are colored according to their conservation: in black, residues that are identical; in grey, residues that are not identical but have similar properties; in white, residues that are different. Residues mutated in this work are highlighted with a red box.

Figure 2. Characterization of SARS-CoV-2 nsp15. (A) Left panel: chromatogram obtained during nsp15 size exclusion chromatography; right panels: SDS-PAGE gel with the purified fractions collected during the SEC and the respective Western blot. M, monomer; T, trimer; H, hexamer; L, Protein ladder. (B) In vitro activity of the purified fractions H, T and M using 50 nM of R16.4 RNA substrate and 500 nM of protein. (C) In vitro activity of nsp15 in the hexamer conformation (500 nM) in the presence of a single-stranded RNA (16comp) and a double-stranded RNA (16–30 ds), which are schematically represented in the bottom. Reactions were analyzed on 7-M urea/20% polyacrylamide gels. C, control reactions; time points are indicated in the top of each panel. All the experiments were performed at least in triplicate.

Figure 3. Effect of divalent ions in SARS-CoV-2 nsp15. (A) Five hundred nM of nsp15 were incubated with 50 nM of R16.4 RNA substrate in the presence of 10 mM of different divalent ions. (B) Five hundred nM of nsp15 were incubated with 50 nM of R16.4 RNA substrate in the presence of different MnCl2 concentrations (1–20 mM). Reactions were analyzed on 7 M urea/20% polyacrylamide gels. C, control reactions; time points are indicated in the top of each panel. All the experiments were performed at least in triplicate. (C) Quantification of nsp15 protein activity (% of RNA degradation product generated) in the presence of different divalent ions (on the top) and in a growing range of different MnCl2 concentrations (on the bottom).

Figure 4. The influence of different buffer compositions in SARS-CoV-2 nsp15 activity. On the top, 500 nM of nsp15 were incubated with 50 nM of R16.4 RNA substrate in the presence of buffers with different compositions, as indicated on the top of each panel. Reactions were analyzed on 7 M urea/20% polyacrylamide gels. C, control reactions; time points are indicated in the top of each panel. All the experiments were performed at least in triplicate. On the bottom, quantification of nsp15 protein activity (% of RNA degradation product generated) in the presence of buffers with different compositions.

Figure 5. Effect of nsp15 mutations on its endonucleolytic activity. (A) On the top, chromatograms obtained during nsp15 wt, N164A and K290A size exclusion chromatographies; M, monomer; T, trimer; H, hexamer; on the bottom, a SDS-PAGE gel with all hexameric fractions of the purified proteins. (B) On the top, 800 nM of either nsp15 wt or nsp15 mutant versions were incubated with 50 nM of R16.4 RNA substrate; on the bottom, quantification of nsp15 wt and mutants activity in the presence of R16.4 RNA (% of RNA substrate degraded). The activity of nsp15 wt was considered as 100%. (C) 800 nM of nsp15 wt or each mutant version were incubated with 50 nM of 16comp RNA substrate. Reactions were analyzed on 7 M urea/20% polyacrylamide gels. C, control reactions; time points are indicated in the top of each panel. All the experiments were performed at least in triplicate.

Figure 6. Docking solutions for the six selected drugs (A) Dutasteride, (B) Etoposide, (C) Golvatinib, (D) Irinotecan, (E) Meprednisone and (F) Tasosartan are shown with the carbon atoms in yellow. Key residues of the nsp15 active site are depicted with the carbon atoms in black. The active site and the phosphate ion (PO4) are covered by Dutasteride, Irinotecan, Golvatinib and Tasosartan. Meprednisone and Etoposide bind only to the top portion of the nsp15 active site.

Figure 7. Effect of different drugs in SARS-CoV-2 nsp15 activity. (A) 500 nM of nsp15 were incubated with 50 nM of R16.4 RNA substrate in the presence of 1 mM of each drug indicated on the top of the respective panel. Reactions were analyzed on 7-M urea/20% polyacrylamide gels. C, control reactions; time points are indicated in the top of each panel. All the experiments were performed at least in triplicate. (B) Quantification of nsp15 activity in the presence of different concentrations of Dutasteride, Meprednisone and Tasosartan. The activity of nsp15 in the presence of DMSO was considered 100%.