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Chem Biol Interact
. 2025 Jun 24:111620.
doi: 10.1016/j.cbi.2025.111620. Online ahead of print. Thiazole-fused thiazoliniums with ketone warheads as covalent inhibitors targeting Cys44 of SARS-CoV-2 3CLpro
Qizhen Cao 1 , Zhaoqin Zhang 2 , Guanghao Zhu 2 , Sanfeng Dong 3 , Xue Sun 2 , Yuqing Song 2 , Ya Zhang 2 , Tingting Cai 3 , Xinben Zhang 3 , Yong Zhang 3 , Zhijian Xu 4 , Ruisheng Xiong 5 , Weiliang Zhu 6 , Guangbo Ge 7 , Bo Li 8
Affiliations
The 3-chymotrypsin-like protease (3CLpro) 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 3CLpro. We designed and synthesized 60 derivatives to systematically study their structure-activity relationships (SAR). Of these, compound 46 demonstrated the most potent inhibition against 3CLpro (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 3CLpro. The inactivation kinetics indicated that compound 46 reduced the 3CLpro activity in a time- and dose-dependent manner, with an inactivation efficiency constant (kinact/Ki) 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.
. 2025 Jun 24:111620.
doi: 10.1016/j.cbi.2025.111620. Online ahead of print. Thiazole-fused thiazoliniums with ketone warheads as covalent inhibitors targeting Cys44 of SARS-CoV-2 3CLpro
Qizhen Cao 1 , Zhaoqin Zhang 2 , Guanghao Zhu 2 , Sanfeng Dong 3 , Xue Sun 2 , Yuqing Song 2 , Ya Zhang 2 , Tingting Cai 3 , Xinben Zhang 3 , Yong Zhang 3 , Zhijian Xu 4 , Ruisheng Xiong 5 , Weiliang Zhu 6 , Guangbo Ge 7 , Bo Li 8
Affiliations
- PMID: 40571175
- DOI: 10.1016/j.cbi.2025.111620
The 3-chymotrypsin-like protease (3CLpro) 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 3CLpro. We designed and synthesized 60 derivatives to systematically study their structure-activity relationships (SAR). Of these, compound 46 demonstrated the most potent inhibition against 3CLpro (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 3CLpro. The inactivation kinetics indicated that compound 46 reduced the 3CLpro activity in a time- and dose-dependent manner, with an inactivation efficiency constant (kinact/Ki) 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.