We developed a rational protocol with a minimal number of mutated residues to create highly potent and selective protein-based inhibitors. Guided by an interaction and dihedral correlation network of ubiquitin (Ub) and MERS coronaviral papain-like protease (PLpro) complex, our designed ubiquitin variant (UbV) with 3 mutated residues (A46F, K48E, and E64Y) resulted in a ~3,500-fold increase in functional inhibition as compared with the wild-type Ub (wtUb). Further optimization with C-terminal R74N and G75S mutations led to a KD of 1.5 nM and IC50 of 9.7 nM and 27,000-fold and 5,500-fold increases in affinity and potency and selectivity, respectively, without destabilizing the UbV structure. This approach effectively designs tight binding inhibitors, which assists the development of therapeutics for COVID-19 and other coronaviruses.
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