Fig 1.
The CoV-2-RdRp NiRAN domain presents a kinase/phosphotransferase like structural organisation.
(A) The NiRAN and the interface domains of the CoV-2-RdRp present an arrow head like structure (helices in royal blue, strands in peach and loops in green). (B) The overall topology of the NiRAN (brown background, helices as marked as H and strands are marked as S) and interface domain (grey background, helices as marked as h and strands are marked as s) along with β-hairpin structure (brick red background, strands are marked as B). (C) The NiRAN domain possess a topology (left panel) that borrows elements from the canonical kinase fold (centre panel) as well as the non-canonical kinase fold of TgBPK1 (right panel). (D-I) The structural superimpositions of the secondary structural elements of CoV-2-RdRp NiRAN with known kinases reveal significant alignment in the antiparallel β-sheet and in the helices that follow. (D) Lim 2 kinase domain. (E) Syk kinase domain (F) O-mannosyl kinase domain. (G) IRAK4 kinase domain. (H) FGFR2 kinase domain. (I) Insulin receptor kinase domain (The aforementioned kinases’ structural elements are shown in cyan, CoV-2-RdRp NiRAN structural elements shown in yellow).
Fig 2.
The active site of CoV-2-RdRp NiRAN domain binds ATP, GTP and UTP and exhibits kinase like motifs.
ATP, GTP and UTP bind at the probable active site of the NiRAN domain with notably low free binding energies. (A) ATP within the active site pocket. (B) GTP within the active site pocket. (C) UTP within the active site pocket (Blue indicates positively charged regions, red indicates negatively charged regions and green indicates neutral regions, grey indicates regions beyond GTP-binding pocket). (A’) ATP forms salt bridge with K73 and H-bonding with D208. (B’) GTP binding reveals salt bridge interaction with K73 and H-bonding with Asp 116. (C’) UTP binding exhibits salt bridge interaction with K73.
Fig 3.
The active site of CoV-2-RdRp NiRAN domain binds kinase inhibitors.
(A, C and E) Broad specificity kinase inhibitors bind within the predicted NiRAN active site with significantly low free binding energies. (Blue indicates positively charged regions, red indicates negatively charged regions and green indicates neutral regions, grey indicates regions beyond GTP-binding pocket): (A) Sunitinib within the active site pocket; (B) Sorafenib within the active site pocket; and (C) SU6656 within the active site pocket. The kinases inhibitors demonstrate H-bond interactions between with the enzymatically critical aspartate and lysine residues lining the active site- (A’) Sunitinib; (B’) Sorafenib; and (C’) SU6656.
Fig 4.
The binding and molecular interactions of the best five predicted nucleotidyl transferase inhibitors at the active site of CoV-2-RdRp NiRAN.
The computationally directed binding of- (A) 65482; (B) 122108; (C) 135659024; (D) 4534; and (E) 23673624; within the active site pocket. (Blue indicates positively charged regions, red indicates negatively charged regions and green indicates neutral regions, grey indicates regions beyond GTP-binding pocket). The molecular interactions between the inhibitors and the active site pocket reveal H-bonds, salt bridges and pi-pi interactions- (A’) 65482; (B’) 122108; (C’) 135659024; (D’) 4534; (E’) 23673624. Of note, the best predicted inhibitor- 65482 presents all the aforementioned molecular interactions.
Fig 5.
SARS-CoV-2 RdRp exhibits a kinase/phosphotransferase like activity.
(A) The CoV-2 RdRp exhibits a kinase like activity akin to that of purified human Akt2. While mutations in the probable NiRAN active site (K73A, D218A) cause decline in the kinase like catalytic activity of the SARS-CoV-2 RdRp, mutating the RdRp replication active site (D760A+D761A) has negligible effect on the same. The ATP binding protein Bovine Serum albumin serves as a negative control (Data points show mean and standard error. The connecting curves represent non-linear regressions). (B) MS-MS spectra profile for peptide KSLVSKGTLVQTK from Histone H1 treated with SARS-CoV-2 RdRp in presence of ATP and Mg2+ shows the phosphorylation at two serine residues. (C) MS-MS spectra profile for the same peptide from Histone H1 treated with human Akt2 in presence of ATP and Mg2+ shows the phosphorylation at a single threonine residue. (D) MS-MS spectra profile for the aforementioned peptide from Histone H1 treated with SARS-CoV-2 RdRp in the presence of only Mg2+ shows no phosphorylation, confirming the phosphotransferase activity of SARS-CoV-2 RdRp NiRAN domain (Further details are presented in S7 and S8 Figs and S8 Table).
Fig 6.
Impeding the activity of the SARS-CoV-2 RdRp NiRAN domain reduces viral load in infected cells.
(A) While treatment with all the kinase inhibitors abrogate the kinase like activity of CoV-2 RdRp, the activity human Akt2 is majorly inhibited by Sorafenib and SU6656 with Sunitinib only exhibiting mild inhibition. (B) Treatment of CoV2-RdRp with nucleotidyl transferase inhibitors 135659024, 122108 and 65482 exhibit conspicuous inhibition of the kinase activity in micromolar concentrations. The compound 4534 however fails to exhibit any inhibitory potential. (C) 5 μM Sorafenib effectively reduces viral replication in SARS-CoV-2 infected Vero E6 cells. In addition, a combination of Sorafenib with 1 μM Remdesivir completely neutralizes SARS-CoV-2 as evident by a Ct value similar to that of uninfected cells (The bars represent mean and standard error. The symbols “*” and “**” represent significance for p-value less than 0.05 and 0.005, respectively. The symbol “ns” represents non-significance).