A dual mechanism of action of AT-527 against SARS-CoV-2 polymerase

Figures

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  Fig. 1. Cryo-EM structure of the RTC with bound RNA and AT-9010 molecules and corresponding cryo-EM map. a Structure of the guanine analog phosphoramidate prodrug AT-527 (left) and its active triphosphate form AT-9010 (right) following activation by cellular kinases. b Ribbon and stick representation of the cryo-EM structure of nsp7-(nsp8)2-nsp12:AT-9010-terminated-RNA:(AT-9010)2 complex. RNA, AT-9010 and protein shown with the following colors: template RNA, green; RNA product, orange; AT-9010, magenta; nsp7, pink; nsp81 and nsp82, yellow and cyan respectively; and nsp12 in gray and blue for NiRAN and RdRp domains respectively. c Brown and black ovals are enlarged for RdRp domain and NiRAN domains, respectively, showing experimental cryo-EM map around AT-9010, In the RdRp, one AT-9010 monophosphate (AT-9010-MP) is incorporated at (+1) position, with an incoming AT-9010 occupying the (−1) position. In the NiRAN domain, AT-9010 is bound it its diphosphate form (AT-9010-DP). d Experimental cryo-EM map for RNA (stick representation), showing incorporated AT-9010-MP (+1) and incoming AT-9010 (−1). Bases of the template RNA involved in the interaction with AT-9010 are numbered as C24–C27. All…

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  Fig. 2. Structural basis for inhibition of the SARS-CoV-2 polymerase complex by AT-9010. a Nucplot molecular analysis with RNA, AT-9010 and nsp12. b Close up of RdRp catalytic site following AT-9010 incorporation. c Close up of RdRp catalytic site following remdesivir incorporation (PDB 7BV2). Both structures in panel b and c are in the same orientation, superimposed by least squares fit method (shown in circle). One AT-9010 5’-monophosphate (AT-9010-MP) is incorporated into the primer RNA strand, and terminates RNA elongation. The second AT-9010 molecule, coordinated by one ion, occupies the NTP-binding site in comparison, remdesivir is terminally incorporated, and untranslocated. Superimposition shows the incoming (−1) AT-9010 ribose group is shifted with the phosphates in a post-incorporation position. Ribbons are depicted as follows: nsp12, blue; nsp7, pink; nsp81 and nsp82, yellow and cyan, respectively; RNA, green sticks; AT-9010, magenta sticks. d Ligplot 2D analysis of the contacts of AT-9010 molecule with nsp12, and incorporated AT-9010-MP.

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  Fig. 3. Incorporation of AT-9010 and Sofosbuvir triphosphate (STP) by the SARS-CoV-2 RTC, and excision by the SARS-CoV-2 nsp14/10 exonuclease complex (ExoN). a Timecourse of AT-9010 incorporation (red dots) as a substitute for GTP (left panel, 50 µM each of ATP, UTP, and CTP), or in competition with GTP (right panel, 50 μM each NTP) at indicated concentrations of AT-9010 (0–250 µM). Fold-preference for GTP over AT-9010 incorporation is calculated by comparing the amount of AT-9010 insertion (red dots) relative to full-length product at two concentrations and at three time-points. b Timecourse of Sofosbuvir triphosphate (STP) incorporation (green dots) as a substitute for UTP (left panel, 50 µM each of ATP, GTP and CTP), or in competition with UTP (50 μM each NTP) at indicated concentrations (0 or 250 µM). For both a and b, reactions were run at least in duplicate at multiple nucleotide/analog concentrations for two different RNA substrates, with consistent results. c Incorporation of ATP+GTP control (black, left panel), ATP + AT-9010 (red, middle panel) and ATP + GTP + STP (green, right panel) by the polymerase complex (POL), followed by excision time-course with the nsp14 ExoN (EXO).…

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  Fig. 4. Structural basis for NiRAN inhibition of the SARS-CoV-2 by AT-9010. a Binding of AT-9010 5’-diphosphate (AT-9010-DP) in the NiRAN domain. Interacting residues are labeled and shown in stick representation. b Ligplot 2D representation of the detailed interactions of AT-9010-DP binding in the NiRAN cavity. c Surface representation of the NiRAN sliced orthogonally in Z showing AT-9010-DP engulfed in the cavity. d Surface representation of the NiRAN sliced orthogonally in Z showing GDP binding (PDB 7CYQ); Orientation is rotated by ~+90° compared to c. e Superimposition of GDP and AT-9010-DP showing the overlap of the α-phosphate of AT-9010 with the β-phosphate of GDP and Mg2+ in the two structures, same orientation as d. Color code is as follows: AT-9010, magenta sticks; GDP, yellow sticks; and Mg2+, light green.