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

X Xiaomin Ni (Institute of Biomedical and Health Engineering, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences) R R. Blake Richardson A Andre Schutzer Godoy M Matteo P. Ferla C Caroline Kikawa (Human Biology Division, Fred Hutchinson Cancer Center) J Jenke Scheen W William W. Hannon E Eda Capkin N Noa Lahav B Blake H. Balcomb P Peter G. Marples M Michael Fairhead S Siyi Wang (State Key Laboratory of Advanced Fiber Materials, Key Laboratory of Science and Technology of Eco-Textile, Ministry of Education, College of Chemistry and Chemical Engineering) E Eleanor P. Williams C Charles W. E. Tomlinson (Department of Biology, University of York) J Jasmin C. Aschenbrenner R Ryan M. Lithgo M Max Winokan C Charline Giroud I Isabela Dolci R Rafaela Sachetto Fernandes G Glaucius Oliva A Anu V. Chandran M Mary-Ann Xavier M Martin A. Walsh W Warren Thompson J Jesse D. Bloom N Nathaniel T. Kenton A Alpha A. Lee A Annette von Delft H Haim Barr K Karla Kirkegaard (Violetta L. Horton Professor, Departments of Genetics and of Microbiology and Immunology, Stanford University School of Medicine) L Lizbé Koekemoer D Daren Fearon M Matthew J. Evans (School of Chemistry, Monash University, P.O. Box 23, Melbourne, Victoria 3800, Australia) F Frank von Delft

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.

Article Details

Volume / Issue Vol. 16, Issue 1
Published October 08, 2025
ISSN 2041-1723
Publisher Nature Portfolio

Journal Info

Nature Communications

Nature Portfolio

ISSN: 2041-1723 Open Access Life Sciences

Authors (36)

X

Xiaomin Ni

Institute of Biomedical and Health Engineering, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences

R

R. Blake Richardson

A

Andre Schutzer Godoy

M

Matteo P. Ferla

C

Caroline Kikawa

Human Biology Division, Fred Hutchinson Cancer Center

J

Jenke Scheen

W

William W. Hannon

E

Eda Capkin

N

Noa Lahav

B

Blake H. Balcomb

P

Peter G. Marples

M

Michael Fairhead

S

Siyi Wang

State Key Laboratory of Advanced Fiber Materials, Key Laboratory of Science and Technology of Eco-Textile, Ministry of Education, College of Chemistry and Chemical Engineering

E

Eleanor P. Williams

C

Charles W. E. Tomlinson

Department of Biology, University of York

J

Jasmin C. Aschenbrenner

R

Ryan M. Lithgo

M

Max Winokan

C

Charline Giroud

I

Isabela Dolci

R

Rafaela Sachetto Fernandes

G

Glaucius Oliva

A

Anu V. Chandran

M

Mary-Ann Xavier

M

Martin A. Walsh

W

Warren Thompson

J

Jesse D. Bloom

N

Nathaniel T. Kenton

A

Alpha A. Lee

A

Annette von Delft

H

Haim Barr

K

Karla Kirkegaard

Violetta L. Horton Professor, Departments of Genetics and of Microbiology and Immunology, Stanford University School of Medicine

L

Lizbé Koekemoer

D

Daren Fearon

M

Matthew J. Evans

School of Chemistry, Monash University, P.O. Box 23, Melbourne, Victoria 3800, Australia

F

Frank von Delft