Fig. 4: 1664-TP inhibits SARS-CoV-2 and LASV RdRP activity by competing with ATP in a non-catalytic manner.

a, b Structural formulas of ATP (a) and 1664-TP (b). c 1664-TP inhibits SARS-CoV-2 RNA synthesis in a non-catalytic manner. Top: Schematic diagram of the minimal RNA substrate used for the SARS-CoV-2 incorporation assay. Bottom: Representative images showing the inhibitory effect of 1664-TP on SARS-CoV-2 RdRP replication. The amount of extended RNA at the longest time point tested (60 min) (lane 4) was used as the standard (set as 100) to quantify the extended RNA products. The reduction levels were calculated by comparing the lanes at the same time point tested (compare lane 2 with lanes 5 and 8 for 10 min, compare lane 3 with lanes 6 and 9 for 30 min, and compare lane 4 with lanes 7 and 10 for 60 min). Experiment was repeated three times independently with similar results. d 1664-TP inhibits LASV RNA synthesis in a non-catalytic manner. Top: The T30/P3 construct used for the assay. Bottom: Representative images showing the inhibition of LASV RdRP replication activity by 1664-TP. The amount of 4-mer in the marker lane (M, lane 1) was used as the standard (set as 100) to quantify the 4-mer products. Experiment was repeated twice independently with similar results. e Atomic model of 1664-TP binding with SARS-CoV-2 RdRP, viewed toward the edge of the 1664-TP purine base (left) and the 1664-TP purine plane (middle). Right: close contacts (4.1 Å or shorter in distance between nonhydrogen atoms) involving the 1664-TP base and ribose atoms are indicated by black dashed lines. Motifs A/B/F, each containing one invariant RdRP residue, are indicated by capital letters with a gray background. f Cryo-EM structure of a 1664-TP-bound LASV-RdRP-RNA complex. Top: Viewing toward the edge of the 1664-TP purine base; Bottom: Viewing toward the 1664-TP purine plane. Left: The overlay of the LASV-RNA-1664-TP structure with the EM density map (contoured at 6σ). Middle: The LASV-RNA-UMPNPP structure was used as a reference. Right: A superimposition of the 1664-TP- and UMPNPP-containing structures. The distances between the 1′-carbon atoms and the 4′-carbon atoms were used to estimate the positional difference between the NTP ribose moieties in these two structures. g Stereo pair images of the interaction details of 1664-TP with the LASV RdRP EC. The α-carbon atoms of the four invariant RdRP residues are shown as brown spheres. Close contacts (4.1 Å or shorter in distance between nonhydrogen atoms) involving the 1664-TP base and ribose atoms are indicated by purple dashed lines. Motifs A/B/C/F, each containing one invariant RdRP residue, are indicated by capital letters with a gray background.