tetano
Editor, Senior Moderator
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 3CL[SUP]pro[/SUP]
Qizhen Cao[SUP] 1 [/SUP], Zhaoqin Zhang[SUP] 2 [/SUP], Guanghao Zhu[SUP] 2 [/SUP], Sanfeng Dong[SUP] 3 [/SUP], Xue Sun[SUP] 2 [/SUP], Yuqing Song[SUP] 2 [/SUP], Ya Zhang[SUP] 2 [/SUP], Tingting Cai[SUP] 3 [/SUP], Xinben Zhang[SUP] 3 [/SUP], Yong Zhang[SUP] 3 [/SUP], Zhijian Xu[SUP] 4 [/SUP], Ruisheng Xiong[SUP] 5 [/SUP], Weiliang Zhu[SUP] 6 [/SUP], Guangbo Ge[SUP] 7 [/SUP], Bo Li[SUP] 8 [/SUP]
Affiliations
The 3-chymotrypsin-like protease (3CL[SUP]pro[/SUP]) 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[SUP]pro[/SUP]. 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[SUP]pro[/SUP] (IC[SUB]50[/SUB] = 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[SUP]pro[/SUP]. The inactivation kinetics indicated that compound 46 reduced the 3CL[SUP]pro[/SUP] activity in a time- and dose-dependent manner, with an inactivation efficiency constant (k[SUB]inact[/SUB]/K[SUB]i[/SUB]) of 0.011 min[SUP]-1[/SUP] μM[SUP]-1[/SUP]. 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 3CL[SUP]pro[/SUP]
Qizhen Cao[SUP] 1 [/SUP], Zhaoqin Zhang[SUP] 2 [/SUP], Guanghao Zhu[SUP] 2 [/SUP], Sanfeng Dong[SUP] 3 [/SUP], Xue Sun[SUP] 2 [/SUP], Yuqing Song[SUP] 2 [/SUP], Ya Zhang[SUP] 2 [/SUP], Tingting Cai[SUP] 3 [/SUP], Xinben Zhang[SUP] 3 [/SUP], Yong Zhang[SUP] 3 [/SUP], Zhijian Xu[SUP] 4 [/SUP], Ruisheng Xiong[SUP] 5 [/SUP], Weiliang Zhu[SUP] 6 [/SUP], Guangbo Ge[SUP] 7 [/SUP], Bo Li[SUP] 8 [/SUP]
Affiliations
- PMID: 40571175
- DOI: 10.1016/j.cbi.2025.111620
The 3-chymotrypsin-like protease (3CL[SUP]pro[/SUP]) 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[SUP]pro[/SUP]. 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[SUP]pro[/SUP] (IC[SUB]50[/SUB] = 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[SUP]pro[/SUP]. The inactivation kinetics indicated that compound 46 reduced the 3CL[SUP]pro[/SUP] activity in a time- and dose-dependent manner, with an inactivation efficiency constant (k[SUB]inact[/SUB]/K[SUB]i[/SUB]) of 0.011 min[SUP]-1[/SUP] μM[SUP]-1[/SUP]. 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.