SARS‐CoV‐2 papain‐like protease (PLpro) is a key antiviral target as it plays a dual role in viral replication and modulation of innate immune responses by deubiquitinating or deISGylating host proteins. Therefore, therapeutically targeting PLpro may serve as a two‐pronged approach to mitigate SARS‐CoV‐2 infection. Interestingly, PLpro shares structural and functional similarities with cellular deubiquitinating enzymes (DUBs), and this fact was leveraged in our study to identify DUB inhibitors that target the ubiquitin/ISG15‐binding site and the known substrate‐binding pocket of PLpro. Among the identified compounds, flupenthixol, lithocholic acid, teneligliptin, and linagliptin markedly inhibited the proteolytic activity of purified PLpro and demonstrated potent antiviral effects against SARS‐CoV‐2 infection in a dose‐dependent manner. Treatment with lithocholic acid and linagliptin suppressed the expression of inflammatory mediators, thereby restoring immune responses. Here, crystal structures of SARS‐CoV‐2 PLpro in complex with linagliptin and with lithocholic acid were determined, revealing insights into the mechanism of inhibition and unique interactions within the ubiquitin/ISG15‐binding site (S2 site; Phe69, His73, Asn128, and His175) and the substrate‐binding cleft. Additionally, oral and intraperitoneal treatments with linagliptin increased survival, reduced lung viral load, and ameliorated histopathological damage in a mouse‐adapted model of SARS‐CoV‐2 infection. This study demonstrates for the first time that using DUB inhibitors that target the proteolytic activity of PLpro can simultaneously reinstate the host's immune response against SARS‐CoV‐2, highlighting the potential of this two‐pronged therapeutic approach.
Choudhary et al. (Mon,) studied this question.