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

## TL;DR

This study investigates how small molecule inhibitors affect the dimer-monomer balance of the SARS-CoV-2 main protease, which could lead to new antiviral drugs.

## Contribution

The study provides a detailed thermodynamic analysis of Mpro dimerization and how inhibitors affect both dimerization and enzymatic activity.

## Key findings

- SAXS reveals temperature-dependent dissociation constants for Mpro monomer-dimer equilibrium.
- Inhibitors affect dimerization and enzymatic activity through different molecular mechanisms.
- Key Mpro residues for optimizing both dimerization and activity inhibition are identified.

## 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.