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

Abstract

The guanosine analog AT-527 represents a promising candidate against Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2). AT-527 recently entered phase III clinical trials for the treatment of COVID-19. Once in cells, AT-527 is converted into its triphosphate form, AT-9010, that presumably targets the viral RNA-dependent RNA polymerase (RdRp, nsp12), for incorporation into viral RNA. Here we report a 2.98 Å cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-nsp8₂-RNA complex, showing AT-9010 bound at three sites of nsp12. In the RdRp active-site, one AT-9010 is incorporated at the 3' end of the RNA product strand. Its modified ribose group (2'-fluoro, 2'-methyl) prevents correct alignment of the incoming NTP, in this case a second AT-9010, causing immediate termination of RNA synthesis. The third AT-9010 is bound to the N-terminal domain of nsp12 - known as the NiRAN. In contrast to native NTPs, AT-9010 is in a flipped orientation in the active-site, with its guanine base unexpectedly occupying a previously unnoticed cavity. AT-9010 outcompetes all native nucleotides for NiRAN binding, inhibiting its nucleotidyltransferase activity. The dual mechanism of action of AT-527 at both RdRp and NiRAN active sites represents a promising research avenue against COVID-19.

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 cryo-EM maps are represented at 3.5σ representative of the general map of the entire complex at 2.98 Å resolution.

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.

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). For both AT-9010 and STP experiments, the next templated nucleotide for incorporation was additionally added. d Quantitation of remaining product after ExoN excision shown in c, a representative gel of experiments done in duplicate for two separate RNAs. The RNA marked at position 0 on each gel corresponds to the size of the fluorescently labeled primer shown above each gel, prior to elongation. Source data are provided as a Source Data file.

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.