The dimer-monomer equilibrium of SARS-CoV-2 main protease is affected by small molecule inhibitors

Abstract

The maturation of coronavirus SARS-CoV-2, which is the etiological agent at the origin of the COVID-19 pandemic, requires a main protease Mpro to cleave the virus-encoded polyproteins. Despite a wealth of experimental information already available, there is wide disagreement about the Mpro monomer-dimer equilibrium dissociation constant. Since the functional unit of Mpro is a homodimer, the detailed knowledge of the thermodynamics of this equilibrium is a key piece of information for possible therapeutic intervention, with small molecules interfering with dimerization being potential broad-spectrum antiviral drug leads. In the present study, we exploit Small Angle X-ray Scattering (SAXS) to investigate the structural features of SARS-CoV-2 Mpro in solution as a function of protein concentration and temperature. A detailed thermodynamic picture of the monomer-dimer equilibrium is derived, together with the temperature-dependent value of the dissociation constant. SAXS is also used to study how the Mpro dissociation process is affected by small inhibitors selected by virtual screening. We find that these inhibitors affect dimerization and enzymatic activity to a different extent and sometimes in an opposite way, likely due to the different molecular mechanisms underlying the two processes. The Mpro residues that emerge as key to optimize both dissociation and enzymatic activity inhibition are discussed.

Sub-Micromolar Inhibition of SARS-CoV-2 3CLpro by Natural Compounds

“…However, the presence of other high-order oligomers (e.g., tetramers and octamers) with considerable hydrolytic activity has been reported [ 9 , 23 ]. It is then possible that ligands binding to 3CLpro may modulate its conformational equilibrium, and even act as inhibitors by shifting it towards the inactive monomeric state [ 24 ]. Interestingly, we found that under our experimental conditions 3CLpro populated mainly dimers, with a minor fraction of larger oligomers, but the interaction with eugenol resulted in a considerable increase in high-order quaternary structures, as shown by electrophoresis ( Figure 4 ).…”

Support Vector Machine as a Supervised Learning for the Prioritization of Novel Potential SARS-CoV-2 Main Protease Inhibitors

“…This residue is found as conserved in other coronaviruses [ 24 ]. This is of special relevance, because it has been reported that Glu166 is important for the protomer dimerization and catalytic activity of the protease [ 29 , 30 , 31 ].…”

SARS-COV-2 Mpro conformational changes induced by covalently bound ligands

“…This indicates that the dimeric protein is more stable than monomer, at least when it comes to the S-III movement. It is interesting to note that the SARS-CoV-2 M pro is biologically active as a homodimer (Jin et al, 2020 ; Silvestrini et al, 2021 ; Suárez & Díaz, 2020 ).…”