key: cord-0801230-sy2dpj6e authors: Dwivedy, Abhisek; Mariadasse, Richard; Ahmad, Mohammed; Chakraborty, Sayan; Kar, Deepsikha; Tiwari, Satish; Majumdar, Tanmay; Jeyakanthan, Jeyaraman; Biswal, Bichitra K. title: Characterization of the NiRAN domain from RNA-dependent RNA polymerase provides insights into a potential therapeutic target against SARS-CoV-2 date: 2021-02-03 journal: bioRxiv DOI: 10.1101/2021.02.03.429510 sha: c9819e8e0813dfd774390c9cc4466c9c1937f606 doc_id: 801230 cord_uid: sy2dpj6e Apart from the canonical fingers, palm and thumb domains, the RNA dependent RNA polymerases (RdRp) from the viral order Nidovirales possess two additional domains. Of these, the function of the Nidovirus RdRp associated nucleotidyl transferase domain (NiRAN) remains unanswered. The elucidation of the 3D structure of RdRp from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), provided the first ever insights into the domain organisation and possible functional characteristics of the NiRAN domain. Using in silico tools, we predict that the NiRAN domain assumes a kinase or phosphotransferase like fold and binds nucleoside triphosphates at its proposed active site. Additionally, using molecular docking we have predicted the binding of three widely used kinase inhibitors and five well characterized anti-microbial compounds at the NiRAN domain active site along with their drug-likeliness as well as DFT properties. For the first time ever, using basic biochemical tools, this study shows the presence of a kinase like activity exhibited by the SARS-CoV-2 RdRp. Interestingly, the proposed kinase inhibitors and a few of the predicted nucleotidyl transferase inhibitors significantly inhibited the aforementioned enzymatic activity. In line with the current global COVID-19 pandemic urgency and the emergence of newer strains with significantly higher infectivity, this study provides a new anti-SARS-CoV-2 drug target and potential lead compounds for drug repurposing against SARS-CoV-2. Using in silico tools, we predict that the NiRAN domain assumes a kinase or 23 phosphotransferase like fold and binds nucleoside triphosphates at its proposed active site. 24 Additionally, using molecular docking we have predicted the binding of three widely used Analysis of the NiRAN and Interface domains 90 As described in a recent study [6] , the NiRAN and the interface domains span over residues 91 1-365 of the SARS-CoV-2 RdRp polypeptide sequence. These two domains form an arrow 92 head like structure, which acts as a base for the RdRp region of protein (Fig. 1A ). Of these, 93 amino acids 4-28 and 69-249 have been designated as the NiRAN domain that comprises of 8 94 α-helices (H1-H8) and 5-β strands (S1-S5), respectively. Two independent β-strands (B1, B2) Table S4 . Table S5 . Table S6 . Table S7 . Lowering the energy gap between 280 HOMO-LUMO couples is known to improve the chemical reactivity of the inhibitors. The predicted molecules inhibit a kinase like activity exhibited by the NiRAN domain 282 14 In order to determine any putative kinase like activity being harboured by the CoV-2 RdRp, 283 the protein was overexpressed, purified ( Figure S7 A & B) and its identity was confirmed by 284 mass spectrometry ( Figure S7C & Table S8 ). The absence of any known accurately determining a kinase activity was verified using a known kinasehuman Akt2. The enzyme exhibited significant activity with a K m value of ~300 µM for ATP (Fig. 5A) , Interestingly, all the three kinases inhibitors significantly abrogated the kinase like activity of 306 CoV-2 RdRp (Fig. 5B) . For human Akt2, Sorafenib and SU6656 significantly inhibited its 307 15 kinase activity, while Sunitinib treatment demonstrated a mild inhibition (Fig. 5B) . This 308 provides a key insight into drug re-purposing for COVID-19, as all the three aforementioned 309 compounds are well approved drugs for human usage with high tolerance and without any CoV-2 RdRp kinase like activity at any of the two concentrations (Fig. 5C ). This suggests 319 that the three inhibitory compounds might be involved in blocking the binding of ATP to its 320 respective binding site, in a manner similar to the specific kinase inhibitors. The authors declare no conflicts of interests. Genome Sequence of a 2019 Novel Coronavirus (SARS-CoV-2) Strain Isolated in 559 The proximal origin of 562 SARS-CoV-2 Covid-19: New coronavirus variant is identified in UK Chemical Approaches to Inhibiting the 610 Hepatitis B Virus Ribonuclease H. 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