An extended conformation of SARS-CoV-2 main protease reveals novel allosteric targets
The coronavirus main protease (Mpro) is required for viral replication, and has enzymatical activity only as a homodimer. In addition, it is highly conserved among the various coronaviruses. Thus, targeting its dimerization is an effective strategy for developing broad-spectrum allosteric inhibitors to suppress mutation escape. In this study, we first obtained the extended conformation of the native monomer of SARS-CoV-2 Mpro by trapping with nanobodies, and found that the catalytic domain and the helix domain dissociate, revealing novel allosteric targets. We also found another state, a compact conformation, similar to the dimeric form. The Mpro is in equilibrium among the monomeric extended conformation as the precursor of all other states, with the compact conformation as the intermediate state and the dimeric conformation as the active state. We designed an innovative NanoBiT-based high-throughput allosteric inhibitor assay based on the rearranged conformation. In addition, we also identified for the first time a set of allosteric inhibitory nanobodies against Mpro, one of which is also a competitive inhibitor for Mpro. Our results provide insight into the maturation of the coronavirus Mpro and a novel way to develop broad-spectrum anti-coronaviral drugs through targeting the maturation process to inhibit the auto-cleavage of the main protease.