Novel SARS-CoV-2 allosteric inhibitors that destabilize the Main Protease Mpro dimer

SARS-CoV-2 Main protease (Mpro) is the most explored coronavirus antiviral target, being most antivirals approved or under development protease inhibitors. Mpro is active as a dimer and the molecular details of its maturation are poorly understood. Some compounds that crystallize at the dimerization interface rather than at the catalytic pocket have been proposed as allosteric inhibitors. Here, we characterize a series of novel compounds starting from a scaffold identified by an in silico screening for Mpro catalytic pocket. Several compounds showed anti-SARS-CoV-2 activity in infected cells, but they did not inhibit Mpro in vitro. Time-of-addition studies pointed to a stage compatible with Mpro targeting. Molecular modelling studies suggested that compounds 1 and 11 bind Mpro similarly to the allosteric inhibitor AT7519. Small-angle X-ray scattering studies revealed that 1 and 11 strongly shift Mpro equilibrium to the monomeric form, while the allosteric inhibitor pelitinib and the catalytic inhibitors nirmatrelvir and GC376 stabilize the dimer. Compounds 1 and 11 inhibited Mpro proteolytic activity in SARS-CoV-2 infected cells acting as allosteric inhibitors that stabilize the monomeric form. In conclusion, we validated an allosteric site in Mpro that could be exploited for the development of effective anti-SARS-CoV-2 antivirals targeting Mpro with a novel mechanism.