TY - JOUR
T1 - Key allosteric and active site residues of SARS-CoV-2 3CLpro are promising drug targets
AU - Al Adem, Kenana
AU - Ferreira, Juliana C.
AU - Fadl, Samar
AU - Mustafa, Morad
AU - Rabeh, Wael M.
N1 - Publisher Copyright:
© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.
PY - 2023/6
Y1 - 2023/6
N2 - The main protease of SARS-CoV-2, 3-chymotrypsin-like protease (3CLpro), is a prominent target for antiviral development due to its essential role in the viral life cycle. Research has largely focused on competitive inhibitors of 3CLpro that target the active site. However, allosteric sites distal to the peptide substrate-binding region are also potential targets for the design of reversible noncompetitive inhibitors. Computational analyses have examined the importance of key contacts at allosteric sites of 3CLpro, but these contacts have not been validated experimentally. In this work, four druggable pockets spanning the surface of SARS-CoV-2 3CLpro were predicted: pocket 1 is the active site, whereas pockets 2, 3 and 4 are located away from the active site at the interface of domains II and III. Site-directed alanine mutagenesis of selected residues with important structural interactions revealed that 7 of 13 active site residues (N28, R40, Y54, S147, Y161, D187 and Q192) and 7 of 12 allosteric site residues (T111, R131, N133, D197, N203, D289 and D295) are essential for maintaining catalytically active and thermodynamically stable 3CLpro. Alanine substitution at these key amino acid residues inactivated or reduced the activity of 3CLpro. In addition, the thermodynamic stability of 3CLpro decreased in the presence of some of these mutations. This work provides experimental validation of essential contacts in the active and allosteric sites of 3CLpro that could be targeted with competitive and noncompetitive inhibitors as new therapeutics against COVID-19.
AB - The main protease of SARS-CoV-2, 3-chymotrypsin-like protease (3CLpro), is a prominent target for antiviral development due to its essential role in the viral life cycle. Research has largely focused on competitive inhibitors of 3CLpro that target the active site. However, allosteric sites distal to the peptide substrate-binding region are also potential targets for the design of reversible noncompetitive inhibitors. Computational analyses have examined the importance of key contacts at allosteric sites of 3CLpro, but these contacts have not been validated experimentally. In this work, four druggable pockets spanning the surface of SARS-CoV-2 3CLpro were predicted: pocket 1 is the active site, whereas pockets 2, 3 and 4 are located away from the active site at the interface of domains II and III. Site-directed alanine mutagenesis of selected residues with important structural interactions revealed that 7 of 13 active site residues (N28, R40, Y54, S147, Y161, D187 and Q192) and 7 of 12 allosteric site residues (T111, R131, N133, D197, N203, D289 and D295) are essential for maintaining catalytically active and thermodynamically stable 3CLpro. Alanine substitution at these key amino acid residues inactivated or reduced the activity of 3CLpro. In addition, the thermodynamic stability of 3CLpro decreased in the presence of some of these mutations. This work provides experimental validation of essential contacts in the active and allosteric sites of 3CLpro that could be targeted with competitive and noncompetitive inhibitors as new therapeutics against COVID-19.
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U2 - 10.1042/BCJ20230027
DO - 10.1042/BCJ20230027
M3 - Article
C2 - 37254750
AN - SCOPUS:85163809755
SN - 0264-6021
VL - 480
SP - 791
EP - 813
JO - Biochemical Journal
JF - Biochemical Journal
IS - 11
ER -