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(10−12) PL pro is part of a membrane -anchored multidomain protein named nonstructural protein 3 (nsp-3), an essential component of the replicase–transcriptase complex. (13) While SARS-CoV PL pro prefers ubiquitinated substrates, SARS-CoV-2 PL pro prefers the ISGlyated proteins as substrates. (10−12) SARS-CoV-2 PL pro cleaves ISG15 and polyubiquitin modifications from cellular proteins, and inhibition of PL pro led to the accumulation of ISG15-conjugates and polyubiquitin conjugates. (9) PL pro has been shown to play additional roles in dysregulating the host immune response and impairing the host type I interferon antiviral effect through its deubiquitinating and deISG15ylating (interferon-induced gene 15) activities, respectively. PL pro cleaves at three sites with the recognition sequence “LXGG↓XX”. (7,8) M pro and PL pro are involved in the proteolytic digestion of the viral polyproteins pp1a and pp1ab, yielding individual functional viral proteins for the replication complex formation. Among the viral proteins that have been actively pursued as SARS-CoV-2 antiviral drug targets, the main protease (M pro) and papain-like protease (PL pro) are the most promising ones. Antivirals are not substitutes for vaccines but rather important complements that can be used for the treatment of infection from both wild-type (WT) and variant viruses. (6) Therefore, there is a dire need for additional antivirals with a novel mechanism of action. (3) Several variants have already emerged and have widely circulated among humans since the beginning of the pandemic. (5) Although the polymerase of SARS-CoV-2 has a proofreading function, it continues to mutate at a rate about 10 –6 per site per cycle. (4) For small molecule antivirals, remdesivir received FDA approval on October 22, 2020. Two mRNA vaccines developed by Pfizer/BioNtech and Moderna and one adenovirus-based vaccine by Johnson and Johnson have been approved by FDA in the United States. Overall, the PL pro inhibitors identified in this study represent promising candidates for further development as SARS-CoV-2 antivirals, and the FlipGFP-PL pro assay is a suitable surrogate for screening PL pro inhibitors in the BSL-2 setting. Molecular dynamics simulations showed that Jun9-72-2 and Jun9-75-4 engaged in more extensive interactions than GRL0617. X-ray crystal structure of PL pro in complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to a more closed conformation.
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Significantly, we developed a cell-based FlipGFP assay that can be applied to predict the cellular antiviral activity of PL pro inhibitors in the BSL-2 setting. Through a fluorescence resonance energy transfer-based high-throughput screening and subsequent lead optimization, we identified several PL pro inhibitors including Jun9-72-2 and Jun9-75-4 with improved enzymatic inhibition and antiviral activity compared to GRL0617, which was reported as a SARS-CoV PL pro inhibitor. The papain-like protease (PL pro) of SARS-CoV-2 is a validated antiviral drug target.