Stabilization of the Dimeric State of SARS-CoV-2 Main Protease by GC376 and Nirmatrelvir

Abstract

The main protease (Mpro or 3CLpro) is an enzyme that is evolutionarily conserved among different genera of coronaviruses. As it is essential for processing and maturing viral polyproteins, Mpro has been identified as a promising target for the development of broad-spectrum drugs against coronaviruses. Like SARS-CoV and MERS-CoV, the mature and active form of SARS-CoV-2 Mpro is a dimer composed of identical subunits, each with a single active site. Individual monomers, however, have very low or no catalytic activity. As such, inhibition of Mpro can be achieved by molecules that target the substrate binding pocket to block catalytic activity or target the dimerization process. In this study, we investigated GC376, a transition-state analog inhibitor of the main protease of feline infectious peritonitis coronavirus, and Nirmatrelvir (NMV), an oral, bioavailable SARS-CoV-2 Mpro inhibitor with pan-human coronavirus antiviral activity. Our results show that both GC376 and NMV are capable of strongly binding to SARS-CoV-2 Mpro and altering the monomer-dimer equilibrium by stabilizing the dimeric state. This behavior is proposed to be related to a structured hydrogen-bond network established at the Mpro active site, where hydrogen bonds between Ser1’ and Glu166/Phe140 are formed in addition to those achieved by the latter residues with GC376 or NMV.

High-Resolution Substrate Specificity Profiling of SARS-CoV-2 Mpro; Comparison to SARS-CoV Mpro

“…Although there is some uncertainty about the details because of differences seen among various structures, it is clear that Glu 166 also interacts with Ser 1 of the other monomer in the dimeric M pro structure. This interchain Glu 166–Ser 1 interaction likely influences the conformation of Glu 166 that forms the S1 pocket, helping to explain why dimerization facilitates M pro catalytic activity . Gln 189 makes significant VDW contact in a relatively deep S2 pocket that nicely accommodates the side chain of P2 Leu, although other similarly sized residues can likely fit as well.…”

“…This interchain Glu 166–Ser 1 interaction likely influences the conformation of Glu 166 that forms the S1 pocket, helping to explain why dimerization facilitates M pro catalytic activity. 18 Gln 189 makes significant VDW contact in a relatively deep S2 pocket that nicely accommodates the side chain of P2 Leu, although other similarly sized residues can likely fit as well. The P3 side chains for the substrates make contact in a hydrophobic region within M pro , and there is contact with P4 involving a hydrophobic pocket created by Met 165, Leu 167, and Gln 192.…”

The hope and hype of ellagic acid and urolithins as ligands of SARS-CoV-2 Nsp5 and inhibitors of viral replication

“…The active site is located between domains I and II, consisting of a catalytic dyad (Cys145 and His41) and a water molecule that forms a hydrogen bond with His41 thereby acting as a third component of the catalytic machinery 8 , 9 . Domain III is involved in the dimerisation process which is required for the catalytic activity of the enzyme 10 and affected by the presence of ligands 11 .…”