Deubiquitinase inhibitors: Targeting <scp>SARS</scp> ‐ <scp>CoV</scp> ‐2 papain‐like protease with antiviral efficacy in a murine model

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.