TY - JOUR
T1 - A natural product compound inhibits coronaviral replication in vitro by binding to the conserved Nsp9 SARS-CoV-2 protein
AU - Littler, Dene R.
AU - Liu, Miaomiao
AU - McAuley, Julie L.
AU - Lowery, Shea A.
AU - Illing, Patricia T.
AU - Gully, Benjamin S.
AU - Purcell, Anthony W.
AU - Chandrashekaran, Indu R.
AU - Perlman, Stanley
AU - Purcell, Damian F.J.
AU - Quinn, Ronald J.
AU - Rossjohn, Jamie
N1 - Funding Information:
Acknowledgments—This research was undertaken in part using the MX beamlines at the Australian Synchrotron, part of ANSTO, and made use of the Australian Cancer Research Foundation (ACRF) detector. Additionally, we thank Dr Geoffrey Kong of the Monash Molecular Crystallisation Facility for his assistance with crystallographic screening and optimization. We thank A. Riboldi-Tunnicliffe and R. Williamson for assistance with data collection. We thank the Prof. E. Fodor from the William Dunn School of Pathology for supplying the SARS-CoV-2 Nsp12 pFastBac expression vector; Prof. D. Thal for help with baculoviral expression and Prof. J Baell for proofreading the manuscript. The University of Melbourne acknowledges the support of Melbourne Health, through its Victorian Infectious Diseases Reference Laboratory at the Doherty Institute, in providing our laboratory with isolated SARS-CoV-2 material. Computational resources were supported by the R@CMon/Monash Node of the NeCTAR Research Cloud, an initiative of the Australian Government’s Super Science Scheme and the Education Investment Fund.
Funding Information:
Funding and additional information—Funding for the work originated from the Australian Research Council Centre of Excellence for Advanced Molecular Imaging and the National Institutes of Health (USA) (PO1 AI060699, RO1 AI129269) (S. P.). A. W. P. is supported by a NHMRC PRF (APP1137739). P. T. I. was supported by a Monash University Faculty of Medicine, Nursing and Health Sciences Senior Postdoctoral Fellowship. J. R. is supported by an Australian Research Council Laureate Fellowship. D. F. J. P. and J. L. M. received antiviral program support from the DHHS COVID-19 Victorian Consortium. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2021 American Society for Biochemistry and Molecular Biology Inc.. All rights reserved.
PY - 2021/12
Y1 - 2021/12
N2 - The Nsp9 replicase is a conserved coronaviral protein that acts as an essential accessory component of the multi-subunit viral replication/transcription complex. Nsp9 is the predominant substrate for the essential nucleotidylation activity of Nsp12. Compounds specifically interfering with this viral activity would facilitate its study. Using a native massspectrometry- based approach to screen a natural product library for Nsp9 binders, we identified an ent-kaurane natural product, oridonin, capable of binding to purified SARS-CoV- 2 Nsp9 with micromolar affinities. By determining the crystal structure of the Nsp9-oridonin complex, we showed that oridonin binds through a conserved site near Nsp9's C-terminal GxxxG-helix. In enzymatic assays, oridonin's binding to Nsp9 reduces its potential to act as substrate for Nsp12's Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. We also showed using in vitro cellular assays oridonin, while cytotoxic at higher doses has broad antiviral activity, reducing viral titer following infection with either SARS-CoV-2 or, to a lesser extent, MERS-CoV. Accordingly, these preliminary findings suggest that the oridonin molecular scaffold may have the potential to be developed into an antiviral compound to inhibit the function of Nsp9 during coronaviral replication.
AB - The Nsp9 replicase is a conserved coronaviral protein that acts as an essential accessory component of the multi-subunit viral replication/transcription complex. Nsp9 is the predominant substrate for the essential nucleotidylation activity of Nsp12. Compounds specifically interfering with this viral activity would facilitate its study. Using a native massspectrometry- based approach to screen a natural product library for Nsp9 binders, we identified an ent-kaurane natural product, oridonin, capable of binding to purified SARS-CoV- 2 Nsp9 with micromolar affinities. By determining the crystal structure of the Nsp9-oridonin complex, we showed that oridonin binds through a conserved site near Nsp9's C-terminal GxxxG-helix. In enzymatic assays, oridonin's binding to Nsp9 reduces its potential to act as substrate for Nsp12's Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. We also showed using in vitro cellular assays oridonin, while cytotoxic at higher doses has broad antiviral activity, reducing viral titer following infection with either SARS-CoV-2 or, to a lesser extent, MERS-CoV. Accordingly, these preliminary findings suggest that the oridonin molecular scaffold may have the potential to be developed into an antiviral compound to inhibit the function of Nsp9 during coronaviral replication.
UR - http://www.scopus.com/inward/record.url?scp=85120046155&partnerID=8YFLogxK
U2 - 10.1016/j.jbc.2021.101362
DO - 10.1016/j.jbc.2021.101362
M3 - Article
C2 - 34756886
AN - SCOPUS:85120046155
SN - 0021-9258
VL - 297
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 6
M1 - 101362
ER -