Nucleoside Analogs That Inhibit SARS-CoV-2 Replication by Blocking Interaction of Virus Polymerase with RNA

Figure 1. Directions of 1-(4’-hydroxy-2’-cyclopenten-1-yl)-6-(4-pentylphenyl)-3H-pyrrolo [2,3-d]-pyrimidine-2-one (compound 1) modification.

Figure 2. Directions of 1-(4’-hydroxy-2’-cyclopenten-1-yl)-6-(4-tert-butylphenyl)-3H-pyrrolo [2,3-d]-pyrimidine-2-one (compound 2) modification.

Scheme 1. Reaction conditions: (a) HMDS, Py, (NH4)2SO4; (b) Bz3AcRib or Ac4Rib, TMSOTf; (c) NH4OH/EtOH.

Scheme 2. Reaction conditions: (a) HMDS, Py, (NH4)2SO4; (b) Ac4Rib, TMSOTf; (c) CuI, 10%Pd/C, 1-ethynyl-4-pentylbenzene, NEt3, CH3CN; (d) NH3/MeOH.

Scheme 3. Reaction conditions: (a) HMDS, Py, (NH4)2SO4; (b) AcO(CH2)2OCH2OAc, TMSOTf; (c) CuI, 10%Pd/C, 1-ethynyl-4-pentylbenzene or 1-ethynyl-4-tert-butylbenzene, NEt3, CH3CN; (d) NH3/MeOH.

Figure 3. Inhibition of RdRp activity by the tested compounds. The primer–template RNA substrate (P32-labeled at the 5′-end of the primer strand) is shown on the top. The assays were performed with different orders of the addition of the reaction components. (A) RdRp was first pre-incubated with indicated inhibitors and then the RNA substrate was added. (B) RdRp was mixed with the RNA substrate and inhibitors were added. (C) RNA was first pre-incubated with inhibitors and then RdRp was added. The final concentrations of RdRp, RNA, and inhibitors in all assays were 1 μM, 25 nM, and 1 mM, respectively. DMSO was added in control reactions in the absence of inhibitors. ATP was added to 100 μM and the reactions were performed for 1 min at 30 °C. Positions of the starting 20 nt RNA primer and the extended full-length 30 nt RNA product are indicated. (D) Quantification of the inhibition efficiencies (averages from two independent measurements).

Figure 4. Inhibition of RNA binding by RdRp by the tested inhibitors. (A) Analysis of RNA-RdRp binding by EMSA. (Left) RdRp was first pre-incubated with indicated inhibitors and then the primer–template RNA substrate (labeled at the 5′-end of the primer strand) was added. (Right) RdRp was first pre-incubated with the RNA substrate and then inhibitors were added. The samples were separated by non-denaturing PAGE. Positions of free RNA and the RdRp/RNA complex are indicated. (B) Quantification of data from panel A (averages from two independent measurements). In each case, RNA binding was normalized by the binding in the absence of inhibitors.

Figure 5. Titration of RdRp inhibitors. RdRp was pre-incubated with increasing concentrations of individual inhibitors, and its activity was measured after adding the RNA substrate and ATP. (A) Analysis of the RNA products at different concentrations of 1. Positions of the starting 20 nt RNA primer and the extended full-length 30 nt RNA product are indicated. (B) Quantification of the inhibition of RdRp activity by 1, 5, and 14 (means and standard deviations from three independent measurements). The data were fitted to a hyperbolic equation (see Section 4 for details).

Figure 6. Docking of compound 1 on the RdRp structure. (A) The structure of SARS-CoV-2 RdRp in complex with RNA (template strand, black; primer strand, orange) (PDB: 6YYT) [33]. Only the catalytic subunit nsp12 is shown, cofactor subunits nsp7 and nsp8 are omitted for clarity. (B) The structure of SARS-CoV-2 nsp12 (PDB: 6M71) [34] was used for molecular docking of compound 1 using the SwissDock web service [35], resulting in 33 binding site clusters; one representative molecule of compound 1 with the highest binding energy was selected for each cluster. (C) Predicted binding sites of compound 1 in the RNA-binding channel of RdRp. Representative binding sites were manually selected based on their overlap with the RNA primer–template in the elongation complex (panel A). The nsp12 domains are colored as follows: palm, green; fingers, gray; thumb, violet; NiRAN and interface, blue. The catalytic residues D760 and D761 in the active site of nsp12 are shown in light green as stick models.