A non-covalent inhibitor of 3C-like protease effectively inhibits SARS-CoV-2 replication


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In a recent study posted to the bioRxiv* preprint server, researchers assessed highly potent non-covalent inhibitors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3-chymotrypsin-like protease (3CLpro).

Study: Development of highly potent non-covalent inhibitors of SARS-CoV-2 3CLpro. Image Credit: Cristian Moga/Shutterstock
Study: Development of highly potent non-covalent inhibitors of SARS-CoV-2 3CLpro. Image Credit: Cristian Moga/Shutterstock


The widespread transmission of the causal virus of coronavirus disease 2019 (COVID-19), SARS-CoV-2, has necessitated the development of specific and potent antiviral agents against SARS-CoV-2. An enzyme called 3CLpro is essential for SARS-CoV-2 replication and was deemed a potential target for discovering effective drug therapies.    

About the study

In the present study, researchers explored the development of non-covalent and specific inhibitors of SARS-CoV-2 3CLpro.

The team screened deoxyribonucleic acid (DNA) encoded libraries (DELs) of over 49 million compounds with hexahistidine (His6)-tagged purified recombinant SARS-CoV-2 3CLpro, which was bound to nickel (Ni2+)-nitrilotriacetic acid (NTA) magnetic beads. Additionally, His6-tag was engineered into 3CLpro without impacting the protease activity. Furthermore, the 3CLpro-mHis was immobilized on the magnetic beads and incubated with DEL.

The bound DNA-encoded compounds were processed by quantitative polymerase chain reaction (qPCR) before performing DNA sequencing. DEL hits were further categorized according to chemotypes and ranked based on docking calculations. Compounds comprising a bromophenyl ring and an isoquinoline ring were recognized as potential candidates for SARS-CoV-2 3CLpro binders. The off-DNA version of five detected compounds was then synthesized, and their inhibitory activity against viral 3CLpro was assessed. 

The inhibitory ability of WU-04 against SARS-CoV-2 Omicron 3CLpro was assessed. Additionally, the activity of WU-02 and WU-04 to bind and inhibit 3CLpro was estimated by crystallizing 3CLpro with WU-02 and WU-04 and determining the structures via molecular replacement. The team also compared the binding modes associated with WU-04 and three non-covalent and six covalent inhibitors, including PF-00835231, of SARS-CoV-2 3CLpro.       


The study results showed that SARS-CoV-2 3CLpro crystal structures displayed Thr225 and Gly215 in the loop located at the far end of the dimer interface and catalytic site. A fluorescence-based assay revealed that the protease activity of the His6-tagged 3CLpro, called 3CLpro-mHis, was compared to that observed in the wild-type (WT) 3CLpro. Four of the potential 3CLpro binders inhibited 3CLpro with a half-maximal inhibitory concentration (IC50) of less than 1µM. The team noted that the most potent compounds were WU-02 with IC50 of 71 nM and WU-04 with IC50 of 72 nM. Furthermore, the isothermal titration calorimetry (ITC) data displayed a high binding affinity between 3CLpro and WU-04.

The team observed that the crystal structure of WU-04 showed significant binding to the 3CLpro catalytic pocket, which suggested that WU-04 blocked the 3CLpro substrates from interacting with the catalytic dyad. Hence, WU-04 was a competitive inhibitor of SARS-CoV-2 3CLpro. As per the interactions observed between 3CLpro and its corresponding peptide substrates, the 3CLpro catalytic pocket could be stratified into S1, S1’, S2, and S4. Additionally, the isoquinoline ring from WU-04 docked into the S1 site, while the 6-nitro and 4-bromo groups from the bromophenyl ring docked into the S2 and S4 sites, respectively. Notably, the amino-π interaction found between the Gln189 side chain and the phenyl ring improved WU-04 potency.

The covalent 3CLpro inhibitor called PF-00835231 had a γ-lactam moiety that interacted with the S1 site, similar to the glutamine sidechain at the P1 positions of the 3CLpro substrates. Furthermore, the γ-lactam moiety had carbonyl oxygen that formed a hydrogen bond with 3CLpro His163. This implied that a moiety with the ability to form a hydrogen bond with His163 was essential for 3CLpro inhibitory action.

Other reported structures had either a γ-lactam, a cyclobutyl, a pyridine, a benzotriazole, or a 1-methyl-1H-1,2,4- triazole moiety that docked into the 3CLpro S1 site. The team noted that the key distinction between the reported inhibitors and WU-04 was the presence of a bromophenyl ring in WU-04 that docked well into the S2 and S4 sites.

Furthermore, WU-04 displayed significant inhibition of SARS-CoV 3CLpro, while WU-06 exhibited no such activity. The binding affinity noted between SARS-CoV 3CLpro and WU-04 was approximately 65 nM. Moreover, the key residues found in the inhibitor-binding pocket associated with the SARS-CoV 3CLpro as well as the interactions between such residues and WU-04 were comparable to those noted between the SARS-CoV-2 3CLpro and WU-04 structures.

Notably, aligning the 3CLpro sequences of SARS-CoV-2 to those of SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), and four other human CoVs showed that the important residues that interacted directly with WU-04 were conserved. Moreover, other residues that were within 5 Å of the WU-04 structures in the SARS-CoV-2 3CLpro structure were also conserved, implying that WU-04 was an efficient pan-inhibitor of the CoV 3CLpro.


Overall, the study findings showed that the WU-04 was a potent and non-covalent SARS-CoV-2 3CLpro inhibitor. WU-04 also inhibited the enzyme activity of SARS-CoV and MERS-CoV with remarkable 3CLpro-binding affinity.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

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