Skip to main content
. 2022 Jul 27;23(15):8302. doi: 10.3390/ijms23158302

Figure 1.

Figure 1

Inhibitory effect of GS-443902 and cordycepin-TP on the SARS-CoV-2 RdRp complex. (A) Schematic of the polymerase reaction. A 40-mer RNA oligonucleotide was annealed with a 20-mer, radioactive label on its 5′ end. The green arrow indicates the incorporation position 1 of an adenosine or adenosine analog. The red arrow indicates position +3, after the first adenosine incorporation position. (B) Chemical structure of the used triphosphates GC-443902 and cordycepin-TP and their corresponding ProTide structures GS-5734 and NUC-7738. (C) Radiogram of the polymerase reaction. The drug concentrations are increasing from 500 nM, 5 μM, 50 μM to 500 μM. Lane 1: positive control reaction without inhibitor and 10 μM NTP concentration. Lanes 2–5: increasing concentrations of GS-443902, stalling position of the RdRp complex is indicated with a red arrow. Lane 6: positive control without inhibitor, indicating inefficient polymerization under ΔATP conditions, compared to the full NTP pool as shown as in lane 1. Lanes 7–10: ATP depleted reaction with increasing GS-443902 concentrations, stalling position is indicated with a red arrow. Lanes 7–9 show increasing amounts of full-length product (40-mer), due to the incorporation of GS-443902 that is used as an ATP substitute. Increasing the GS-443902 to 500 μM restores the inhibitory effect as shown in lane 10. Lanes 11–14: increasing concentrations of cordycepin-TP is able to inhibit the reaction at 50 μM (lane 13). A full inhibition is reached at 500 μM and indicated with a green arrow (lane 14). Lanes 15–18: NTP pool depleted of ATP, with increasing amounts of cordycepin-TP. A total of 500 nM of cordycepin-TP (lane 15) leads to an efficient inhibition without any full-length product, when compared to the control (lane 1). The intermediate products in lane 15 are diminished completely starting from lanes 16 to 18. The green arrow indicates the incorporation position 1 of an adenosine or adenosine analog. The red arrow indicates position +3, after the first adenosine incorporation position. For cordycepin-TP, the reaction stalls at position 1 (green arrow), while GC443902 stalls the reaction at position +3 (red arrow). This radiograph is a representative image of two independent experiments.