Thiazole-fused thiazoliniums with ketone warheads as covalent inhibitors targeting Cys44 of SARS-CoV-2 3CLpro

Qizhen Cao; Zhaoqin Zhang; Guanghao Zhu; Sanfeng Dong; Xue Sun; Yuqing Song; Ya Zhang; Tingting Cai; Xinben Zhang; Yong Zhang; Zhijian Xu; Ruisheng Xiong; Weiliang Zhu; Guangbo Ge; Bo Li

doi:10.1016/j.cbi.2025.111620

Thiazole-fused thiazoliniums with ketone warheads as covalent inhibitors targeting Cys44 of SARS-CoV-2 3CL^pro

Chem Biol Interact. 2025 Jun 24:111620. doi: 10.1016/j.cbi.2025.111620. Online ahead of print.

Authors

Qizhen Cao¹, Zhaoqin Zhang², Guanghao Zhu², Sanfeng Dong³, Xue Sun², Yuqing Song², Ya Zhang², Tingting Cai³, Xinben Zhang³, Yong Zhang³, Zhijian Xu⁴, Ruisheng Xiong⁵, Weiliang Zhu⁶, Guangbo Ge⁷, Bo Li⁸

Affiliations

¹ School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210046, China; State Key Laboratory of Drug Research, Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
² Shanghai Frontiers Science Center of TCM Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.
³ State Key Laboratory of Drug Research, Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
⁴ State Key Laboratory of Drug Research, Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China.
⁵ Department of Chemistry - BMC, Science for Life Laboratory, Uppsala University, Uppsala 75123, Sweden.
⁶ School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210046, China; State Key Laboratory of Drug Research, Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China. Electronic address: wlzhu@simm.ac.cn.
⁷ Shanghai Frontiers Science Center of TCM Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. Electronic address: geguangbo@dicp.ac.cn.
⁸ Shanghai Frontiers Science Center of TCM Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; State Key Laboratory of Drug Research, Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China; Department of Chemistry - BMC, Science for Life Laboratory, Uppsala University, Uppsala 75123, Sweden. Electronic address: boli@simm.ac.cn.

PMID: 40571175
DOI: 10.1016/j.cbi.2025.111620

Abstract

The 3-chymotrypsin-like protease (3CL^pro) is a crucial enzyme for the replication of coronaviruses, notable for its high conservation across viral species and the lack of human analogs. These characteristics make it a prime target for the development of broad-spectrum antiviral medications. In this work, we incorporated the zolinium as a hydrophilic group and a ketone as the covalent warhead to develop novel agents targeting SARS-CoV-2 3CL^pro. We designed and synthesized 60 derivatives to systematically study their structure-activity relationships (SAR). Of these, compound 46 demonstrated the most potent inhibition against 3CL^pro (IC₅₀ = 1.75 ± 0.039 μM) and good selectivity against other five enzymes, with reasonable chemical stability and rapid reactivity with cysteine. Mass spectrometry-based peptide mapping revealed that the ketone group of compound 46 covalently modified Cys44 of SARS-CoV-2 3CL^pro. The inactivation kinetics indicated that compound 46 reduced the 3CL^pro activity in a time- and dose-dependent manner, with an inactivation efficiency constant (k_inact/K_i) of 0.011 min^-1 μM^-1. Further covalent docking and molecular dynamics simulations elucidated the binding mechanism involving the disruption of protein's dimer interface and stability, which was partially validated by Native-PAGE analysis. Moreover, compound 46 exhibited negligible cytotoxicity and good metabolic stability in liver microsome assays, positioning it as a promising covalent lead for the advancement of broad-spectrum anti-coronavirus therapies.

Keywords: Covalent inhibitor; Cys44; Ketone; SARS-CoV-2 3CL(pro); Thiazole-fused thiazolinium.