Combined crystallographic fragment screening and deep mutational scanning enable discovery of Zika virus NS2B-NS3 protease inhibitors

Xiaomin Ni (), R. Blake Richardson, Andre Schutzer Godoy, Matteo P. Ferla, Caroline Kikawa, Jenke Scheen, William W. Hannon, Eda Capkin, Noa Lahav, Blake H. Balcomb, Peter G. Marples, Michael Fairhead, SiYi Wang, Eleanor P. Williams, Charles W. E. Tomlinson, Jasmin C. Aschenbrenner, Ryan M. Lithgo, Max Winokan, Charline Giroud, Isabela Dolci, Rafaela Sachetto Fernandes, Glaucius Oliva, Anu V. Chandran, Mary-Ann Xavier, Martin A. Walsh, Warren Thompson, Jesse D. Bloom, Nathaniel T. Kenton, Alpha A. Lee, Annette Delft, Haim Barr, Karla Kirkegaard, Lizbé Koekemoer, Daren Fearon, Matthew J. Evans () and Frank Delft ()
Additional contact information
Xiaomin Ni: University of Oxford, NDM research Building, Roosevelt Dr, Headington
R. Blake Richardson: Icahn School of Medicine at Mount Sinai
Andre Schutzer Godoy: Av. João Dagnone
Matteo P. Ferla: University of Oxford, NDM research Building, Roosevelt Dr, Headington
Caroline Kikawa: University of Washington
Jenke Scheen: Open Molecular Software Foundation
William W. Hannon: Fred Hutchinson Cancer Center
Eda Capkin: Harwell Science and Innovation Campus
Noa Lahav: Weizmann Institute of Science
Blake H. Balcomb: Harwell Science and Innovation Campus
Peter G. Marples: Harwell Science and Innovation Campus
Michael Fairhead: University of Oxford, NDM research Building, Roosevelt Dr, Headington
SiYi Wang: University of Oxford, NDM research Building, Roosevelt Dr, Headington
Eleanor P. Williams: University of Oxford, NDM research Building, Roosevelt Dr, Headington
Charles W. E. Tomlinson: Harwell Science and Innovation Campus
Jasmin C. Aschenbrenner: Harwell Science and Innovation Campus
Ryan M. Lithgo: Harwell Science and Innovation Campus
Max Winokan: Harwell Science and Innovation Campus
Charline Giroud: University of Oxford, NDM research Building, Roosevelt Dr, Headington
Isabela Dolci: Av. João Dagnone
Rafaela Sachetto Fernandes: Av. João Dagnone
Glaucius Oliva: Av. João Dagnone
Anu V. Chandran: Harwell Science and Innovation Campus
Mary-Ann Xavier: Harwell Science and Innovation Campus
Martin A. Walsh: Harwell Science and Innovation Campus
Warren Thompson: Harwell Science and Innovation Campus
Jesse D. Bloom: Fred Hutch Cancer Center
Nathaniel T. Kenton: PostEra Inc
Alpha A. Lee: PostEra Inc
Annette Delft: University of Oxford, NDM research Building, Roosevelt Dr, Headington
Haim Barr: Weizmann Institute of Science
Karla Kirkegaard: Stanford University
Lizbé Koekemoer: University of Oxford, NDM research Building, Roosevelt Dr, Headington
Daren Fearon: Harwell Science and Innovation Campus
Matthew J. Evans: Icahn School of Medicine at Mount Sinai
Frank Delft: University of Oxford, NDM research Building, Roosevelt Dr, Headington

Nature Communications, 2025, vol. 16, issue 1, 1-13

Abstract: Abstract The Zika viral protease NS2B-NS3 is essential for the cleavage of viral polyprotein precursor into individual structural and non-structural (NS) proteins and is therefore an attractive drug target. Generation of a robust crystal system of co-expressed NS2B-NS3 protease has enabled us to perform a crystallographic fragment screening campaign with 1076 fragments. 46 fragments with diverse scaffolds are identified to bind in the active site of the protease, with another 6 fragments observed in a potential allosteric site. To identify binding sites that are intolerant to mutation and thus suppress the outgrowth of viruses resistant to inhibitors developed from bound fragments, we perform deep mutational scanning of the NS2B-NS3 protease. Merging fragment hits yields an extensive set of ‘mergers’, defined as synthetically accessible compounds that recapitulate constellations of observed fragment-protein interactions. In addition, the highly sociable fragment hits enable rapid exploration of chemical space via algorithmic calculation and thus yield diverse possible starting points. In this work, we maximally explore the binding opportunities to NS2B-NS3 protease, facilitating its resistance-resilient antiviral development.

Date: 2025
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DOI: 10.1038/s41467-025-63602-z

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