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

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.

Authors

Institutions

Publication Details

Journal
Nature Communications
Published
2025-10-08
DOI
https://doi.org/10.1038/s41467-025-63602-z
Citations
6
Primary Topic
Mosquito-borne diseases and control
Type
article
Field-Weighted Citation Impact
11.27

Funders

Controls
|||
ALL TIME
JAN
FEB
MAR
APR
MAY
JUN
JUL
AUG
SEP
OCT
article

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

Eleanor Williams, Charles W.E. Tomlinson, Eda Çapkın, R. Blake Richardson et al.
6 citations
Nature Communications
Mosquito-borne diseases and control
11.27
article

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

Eleanor Williams, Charles W.E. Tomlinson, Eda Çapkın, R. Blake Richardson, Caroline Kikawa, M. Fairhead, Blake Balcomb, Glaucius Oliva, Warren Thompson, Mary-Ann Xavier, Matthew J. Evans, Charline Giroud, Haim Michael Barr, Si-Yi Wang, Anu V. Chandran, Noa Lahav, André S. Godoy, Xiaomin Ni, L. Koekemoer, F. von Delft, Ryan Lithgo, Jenke Scheen, J.C. Aschenbrenner, R.S. Fernandes, Alpha A. Lee, Karla Kirkegaard, William W. Hannon, Isabela Dolci, D. Fearon, Max Winokan, Annette von Delft, Martin Walsh, Matteo P. Ferla, Jesse D. Bloom, Peter Marples, Nathaniel T. Kenton
article en
6 citations

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.

Nature CommunicationsVol. 16(1)
Broad Institute (US), Cape Town HVTN Immunology Laboratory / Hutchinson Centre Research Institute of South Africa (ZA), Howard Hughes Medical Institute (US), University of Washington (US), University of Johannesburg (ZA), Diamond Light Source (GB), University of Oxford (GB), Open Geospatial Consortium (US), Institute of Physics (PL), Fred Hutch Cancer Center (US), Research Complex at Harwell (GB), Oxford Centre for Diabetes, Endocrinology and Metabolism (GB), Oxford BioMedica (United Kingdom) (GB), Weizmann Institute of Science (IL), Stanford University (US), Icahn School of Medicine at Mount Sinai (US)
Diamond Light Source, Fundação de Amparo à Pesquisa do Estado de São Paulo, National Institute of Allergy and Infectious Diseases
Openalex Percentile: Top 1%
Mosquito-borne diseases and control
11.27
DOIOpen Access PDFOpenAlex
AI Navigator

Ask Laika to Summarize, Analyze, and Connect papers live on the map.

Summarize Papers & Methodologies

Extract key findings, datasets, and comparative methods across publications.

Benchmark Rankings & Visual Analytics

Rank top research institutions, authors, funders, topics, and journals by Field-Weighted Citation Impact (FWCI) and paper volume with instant charts.

Connect Distant Disciplines

Bridge topological clusters on the map to find hidden collaborative intersections.