Abstract: Journal Pre-proof
Identification of Cathepsin L as the molecular target of hydroxychloroquine with
chemical proteomics
Jin Shang, Bingjie Hu, Ka-Yan Karen Kung, Jiajun Jiang, Qi Zhang, Man-Kin Wong,
Hin Chu, Qian Zhao
PII:
S1535-9476(25)00413-X
DOI:
https://doi.org/10.1016/j.mcpro.2025.101314
Reference:
MCPRO 101314
To appear in:
Molecular & Cellular Proteomics
Received Date: 22 April 2025
Revised Date:
21 September 2025
Accepted Date: 15 October 2025
Please cite this article as: Shang J, Hu B, Kung KYK, Jiang J, Zhang Q, Wong MK, Chu H, Zhao Q,
Identification of Cathepsin L as the molecular target of hydroxychloroquine with chemical proteomics,
Molecular & Cellular Proteomics (2025), doi: https://doi.org/10.1016/j.mcpro.2025.101314.
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® 2025 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and
Molecular Biology.
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Identification of Cathepsin L as the molecular target of
hydroxychloroquine with chemical proteomics.
Jin Shang1, 2#, Bingjie Hu3#, Ka-Yan Karen Kung1, 3, Jiajun Jiang1, 4, Qi Zhang1, ManKin Wong1, 4*, Hin Chu3*, Qian Zhao1,2*
1
State Key Laboratory of Chemical Biology and Drug Discovery, Department of Applied Biological
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and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Hong Kong SAR,
China.
Department of Applied Biological and Chemical Technology, The Hong Kong Polytechnic
re
-p
r
2
University, Hung Hom, Hong Kong SAR, China.
3
State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, School of
Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong
4
lP
Kong SAR, China.
Department of Food Science and Nutrition, The Hong Kong Polytechnic University, Hung Hom,
Hong Kong SAR, China
The authors contributed equally
*
Corresponding Author: Qian ZHAO (q.zhao@polyu.edu.hk); Hin Chu (hinchu@hku.hk); Man-Kin
ur
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#
Wong (mankin.wong@polyu.edu.hk).
Jo
ABSTRACT: Hydroxychloroquine (HCQ) and chloroquine (CQ) have been utilized as antimalarial drugs for decades. Recently, these compounds were reported to inhibit various viruses
utilizing the endosomal entry pathway. However, their direct molecular targets in host cells
remain elusive. In this study, we developed a clickable photo-crosslinking probe in combination
with proteomic approaches to identified Cathepsin L (CTSL) as the binding target of HCQ.
Extensive biochemical and in silico analyses were conducted to validate the HCQ-CTSL
interactions. HCQ significantly inhibited the protease activity of CTSL and suppressed CTSLdependent coronavirus entry in cells that support endosomal entry pathway. These findings not
only reveal the underlying mechanism of how HCQ inhibits endosomal viral entry but also
guide the rational use of HCQ against other emerging infectious agents.
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