YL1004 is a SARS-CoV-2 papain-like protease inhibitor with immunomodulatory and antiviral activity in mice.

The evolution of SARS-CoV-2, coupled with its immune evasion mechanisms, underscores the urgent need for antiviral strategies beyond vaccination. The papain-like protease (PLpro) is a dual-functional enzyme essential for viral replication and suppression of host innate immunity. Here, we present the rational design and characterization of YL1004, a tricyclic oral PLpro inhibitor demonstrating robust, cross-variant SARS-CoV-2 antiviral activity and favorable pharmacokinetic properties. YL1004 potently inhibits the enzymatic function of PLpro, disrupts deubiquitination and deISGylation processes, and restores antiviral immune signaling cascades. Notably, YL1004 suppresses the replication of SARS-CoV-2 wildtype, Delta and Omicron variants. Additionally, it is also effective against the Mpro E166V recombinant SARS-CoV-2 strain, which confers resistance to nirmatrelvir. In the lethal SARS-CoV-2 infection model using K18-hACE2 mice, YL1004 confers complete protection to animal survival, significantly reduces viral load in nasal turbinate and lung tissues, and alleviates virus-induced pathological tissue damages. Co-crystal structural analysis revealed a distinctive binding mode, enhancing target engagement through expanded hydrophobic interactive interface and additional hydrogen bonding interactions. Collectively, these findings establish YL1004 as a promising therapeutic candidate, harnessing dual antiviral and immunomodulatory mechanisms to combat SARS-CoV-2 and emerging variants.