3C-like protease

The 3C-like protease (3CLpro) or main protease (Mpro), formally known as C30 endopeptidase or 3-chymotrypsin-like protease,[2] is the main protease found in coronaviruses. It cleaves the coronavirus polyprotein at eleven conserved sites. It is a cysteine protease and a member of the PA clan of proteases. It has a cysteine-histidine catalytic dyad at its active site and cleaves a Gln–(Ser/Ala/Gly) peptide bond.

See also: COVID-19 drug development
SARS-CoV-2 main proteinase dimer with the catalytic dyad (H41; C145) in complex with a covalent peptidomimetic protease inhibitor ("11a", magenta). From PDB: 6LZE​.[1]
Identifiers
EC no.3.4.22.69
Databases
IntEnzIntEnz view
BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum
Search
PMCarticles
PubMedarticles
NCBIproteins
Peptidase C30, Coronavirus endopeptidase
Identifiers
SymbolPeptidase_C30
PfamPF05409
InterProIPR008740
PROSITEPS51442
MEROPSC30
SCOP2d1q2wb1 / SCOPe / SUPFAM
Available protein structures:
Pfam  structures / ECOD  
PDBRCSB PDB; PDBe; PDBj
PDBsumstructure summary

The Enzyme Commission refers to this family as SARS coronavirus main proteinase (Mpro; EC 3.4.22.69). The 3CL protease corresponds to coronavirus nonstructural protein 5 (nsp5). The "3C" in the common name refers to the 3C protease (3Cpro) which is a homologous protease found in picornaviruses.

Function

The 3C-like protease is able to catalytically cleave a peptide bond between a glutamine at position P1 and a small amino acid (serine, alanine, or glycine) at position P1'. The SARS coronavirus 3CLpro can for instance self-cleave the following peptides:[3][4][5]

TSAVLQ-SGFRK-NH2 and SGVTFQ-GKFKK are the two peptides corresponding to the two self-cleavage sites of the SARS 3C-like proteinase

The protease is important in the processing of the coronavirus replicase polyprotein (P0C6U8). It is the main protease in coronaviruses and corresponds to nonstructural protein 5 (nsp5).[6] It cleaves the coronavirus polyprotein at 11 conserved sites. The 3CL protease has a cysteine-histidine catalytic dyad at its active site.[4] The sulfur of the cysteine acts as a nucleophile and the imidazole ring of the histidine as a general base.[7]

Substrate preferences for 3CL proteases (from table 2)[8]
PositionSubstrate preference
P5No strong preference
P4Small hydrophobic residues
P3Positively charged residue
P2High hydrophobicity and absence of beta-branch
P1Glutamine
P1'Small residues
P2'Small residues
P3'No strong preference

Nomenclature

Alternative names provided by the EC include 3CLpro, 3C-like protease, coronavirus 3C-like protease, Mpro, SARS 3C-like protease, SARS coronavirus 3CL protease, SARS coronavirus main peptidase, SARS coronavirus main protease, SARS-CoV 3CLpro enzyme, SARS-CoV main protease, SARS-CoV Mpro and severe acute respiratory syndrome coronavirus main protease.

As a treatment target
Nirmatrelvir bound to 3CL PDB: 7RFW​
Nirmatrelvir, a 3CLpro inhibitor developed by Pfizer in phase II/III clinical trials as a combination drug with ritonavir.[9][10]

The protease 3CLpro is used as a drug target for coronavirus infections due to its essential role in processing the polyproteins that are translated from the viral RNA.[11][12] The X-ray structures of the unliganded SARS-CoV-2 protease 3CLpro and its complex with an α-ketoamide inhibitor provides a basis for design of α-ketoamide inhibitors[13] for a treatment of SARS-CoV-2 infection.[14][15][16][17][18]

A number of protease inhibitors being developed targeting 3CLpro and homologous 3Cpro, including CLpro-1, GC376, rupintrivir, lufotrelvir, PF-07321332, and AG7404.[19][20][21][22][1] The intravenous administered prodrug PF-07304814 (lufotrelvir) entered clinical trials in September 2020.[23]

After clinical trials, in December 2021, the oral medication nirmatrelvir (formerly PF-07321332) became commercially available under emergency regulatory authorizations, as part of the nirmatrelvir/ritonavir combination therapy (brand name Paxlovid).

In 2022, an ultralarge virtual screening campaign of 235 million molecules was able to identify a novel broad-spectrum inhibitor targeting the main protease of several coronaviruses. It is unusually not a peptidomimetic.[24]

A ligand-binding diagram showing the amino acid residues in contact with a covalently bound peptidomimetic protease inhibitor. The small red spheres are water molecules.[1]

Other 3C(-like) proteases

3C-like proteases (3C(L)pro) are widely found in (+)ssRNA viruses. All of them are cysteine proteases with a chymotrypsin-like fold (PA clan), using a catalytic dyad or triad. They share some general similarities on substrate specificity and inhibitor effectiveness. They are divided into subfamilies by sequence similarity, corresponding to the family of viruses they are found in:[25]

Additional members are known from Potyviridae and non-Coronaviridae Nidovirales.[26]

See also

References
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Further reading
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