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

The maturation of coronavirus SARS-CoV-2, which is the etiological agent at the origin of the COVID-19 pandemic, requires a main protease M to cleave the virus-encoded polyproteins. Despite a wealth of experimental information already available, there is wide disagreement about the M monomer-dimer equilibrium dissociation constant. Since the functional unit of M 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 M 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 M 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 M residues that emerge as key to optimize both dissociation and enzymatic activity inhibition are discussed.