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. 2025 Apr 14;2025:10.17912/micropub.biology.001418. doi: 10.17912/micropub.biology.001418

Human Structural Homologues of SARS-CoV-2 PL pro as Anti-Targets: A Strategic Panel Analysis

Abdullah I Al-Homoudi 1, Joseph Engel 1, Michael D Muczynski 1, Joseph S Brunzelle 2, Navnath S Gavande 3,4, Ladislau C Kovari 1,§
Reviewed by: Anonymous
PMCID: PMC12035650  PMID: 40297819

Abstract

COVID-19 is caused by SARS-CoV-2, a highly transmissible and pathogenic RNA betacoronavirus. Developing small-molecule antiviral inhibitors of the SARS-CoV-2 papain-like protease (PL pro ) is advantageous due to the enzyme's role in processing viral polyproteins and disrupting host immune sensing. Given the structural and functional similarities between PL pro and human deubiquitinases (DUBs), small-molecule inhibitors are frequently counter-screened for off-target activity using a panel of human DUBs. Through X-ray crystallography, DALI structural comparisons, and in silico analysis, a high-quality crystal structure of SARS-CoV-2 PL pro enabled the identification of the closest structural human homologues of PL pro . Among the 27 human DUBs identified, USP46 and USP12 displayed the greatest structural similarity to PL pro , with alignment scores below 0.45 and RMSD values of 3.0 Å or less. Additionally, binding sites on ubiquitin-specific protease (USP46) and USP12, ancillary to the active site residues, share high sequence identity to the PL pro substrate binding sites that are often engaged by the most potent PL pro inhibitors. These findings offer a strong basis for choosing anti-targets and serve as a foundation for designing selective small-molecule PL pro inhibitors.

Figure 1. Identifying the closest structural human homologues of SARS-CoV-2 PL pro .

Figure 1. Identifying the closest structural human homologues of SARS-CoV-2 PL pro

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( A ) The structure of SARS-CoV-2 PL pro (9BF8.pdb) is represented by a green, pink, and red ribbon, alongside the binding poses of the most potent small-molecule inhibitors of SARS-CoV-2 PL pro . The inhibitors (thick tube representations) include GRL-0617 (orange, 7CJM.pdb), Compound 7 (blue, 8EUA.pdb), XR8-24 (black, 7LBS.pdb), and Jun12682 (purple, 8UOB.pdb), which interact with binding sites (pink) that are ancillary to active site residues Cys111, His272, and Asp286 (red). The relevant binding sites are known as the blocking loop (BL2) (Gly266-Gly-271), BL2 groove (Pro247, Pro248, Pro299, Gly266), and the Alpha-cleft (Asp164-Glu167) binding sites (pink). ( B ) Radar plot illustrating structural alignment results of related coronavirus (CoV) PL pro from SARS-CoV, MERS-CoV, HKU1-CoV, murine hepatitis virus (MHV), and the catalytic domains of 27 human deubiquitinases (DUBs). Alignment scores (green) lower than 0.6 indicate a good alignment, while scores greater than 0.6 indicate a failure of structural alignment calculation and insufficient structural similarity for a meaningful alignment. RMSD (Å) (purple) is the distance between superpositioned atoms, calculated based only on the residues that have been successfully aligned. The plot highlights the closest human structural homologues of SARS-CoV-2 PL pro , which are ubiquitin-specific protease 46 (USP46) and USP12 (red data blocks). ( C ) Binding site amino acid sequence alignment and Alignment scores of unrelated human structural protease homologues of SARS-CoV-2 PL pro (Created in BioRender. Alhomoudi, A. (2025) https://BioRender.com/gpsv9cp). ( D ) The figure illustrates the structural superposition of binding sites for USP46 (blue, 5L8H.pdb) and SARS-CoV-2 PL pro (green, 9BF8.pdb), emphasizing the BL2 groove, Alpha-cleft, and BL1, a secondary blocking loop present on USP46 in place of the BL2 groove.

Description

Despite the global availability of SARS-CoV-2 protease inhibitor regimens, there remains a critical need to enhance their pharmacological properties, particularly in terms of oral bioavailability, metabolic stability, and specificity (Eng et al., 2022; Jiang et al., 2023; Shimizu et al., 2023). The Papain-like protease (PL pro ) is essential for the replication of human coronaviruses (hCoVs) as it processes polyproteins translated from viral RNA into functional nonstructural proteins (NSPs). In addition to processing viral polyproteins, hCoV PL pro directly facilitates the evasion of host immune responses by cleaving ubiquitin and the ubiquitin-like interferon-stimulated gene 15 (ISG-15) from host protein conjugates (Thiel et al., 2003; Barretto et al., 2005; Linder et al., 2007; Linder et al., 2005; Shin et al., 2020). Therefore, inhibiting PL pro offers a promising approach to reducing viral replication and boosting antiviral immunity.

Deubiquitination and deISGylation are essential regulatory mechanisms in many biological processes, including NF-ĸB signaling following host innate immune system activation during viral infection (Hochstrasser, 2000; Song & Li, 2021). Seven human Deubiquitinase (DUB) subfamilies function to cleave ubiquitin and ubiquitin-like adducts from substrate proteins (Balakirev et al., 2003; Burnett et al., 2003; Evans et al., 2003; Gan-Erdene et al., 2003; Verma et al., 2002; Wilkinson, 1997; Yeh et al., 2000). The ubiquitin-specific protease (USP) subfamily has raised particular interest due to its presence in different eukaryotic organisms and the varied functions ascribed to this subfamily of diverse proteases in normal and pathological conditions (Quesada et al., 2004). In 2005, around the same time that Quesada and colleagues identified 22 novel human USPs, the first crystal structure of the SARS-CoV PL pro was elucidated (2FE8.pdb) (Ratia et al., 2006). Structural similarity screening through the entire Protein Data Bank (PDB) identified two significant human structural homologues of SARS-CoV PL pro : USP14 and HAUSP7 (USP7) (Ratia et al., 2006). Since then, tens of thousands of crystal structures have been deposited into the PDB, and the emergence of the deadly MERS-CoV in 2012 and SARS-CoV-2 in 2019 has accelerated the structure-based drug design (SBDD) of protease inhibitors.

The outbreak of SARS-CoV in 2002 facilitated the development of the naphthyl methyl amine core from GRL-0617 as an inhibitor for PL pro (Ghosh et al., 2009). Since then, GRL-0617 has been repurposed against SARS-CoV-2 PL pro , and several potent analogs have been optimized using SBDD, including Compound 19, Compound 7, XR8-24, and Jun12682 (Ratia et al., 2008; Sanders et al., 2023; Shan et al., 2021; Tan et al., 2023) ( Fig. 1A ). These potent antiviral compounds (Enzymatic IC 50 ≥ 0.09 μM, Antiviral EC 50 ≥ 0.42 μM) exhibit comparable binding poses, interacting with residues involved in ubiquitin and ISG-15 recognition, as evidenced by various hCoV PL pro X-ray co-crystal structures with ubiquitin (4MM3.pdb, 4RF1.pdb, 6XAA.pdb) and ISG-15 (6XA9.pdb, 6BI8.pdb, 5TL6.pdb). Notably, the flexible “blocking loop” (BL2) (Gly266-Gly271), the hydrophobic BL2 groove (Pro247, Pro248, Pro299, Gly266), and the rigidifying Alpha cleft (Asp164-Glu167) of PL pro , which are ancillary to the catalytic triad (Cys111, His272 & Asp286), improve inhibitor potency upon engagement. Although the efficacy of these compounds has been confirmed, the primary focus is now on enhancing pharmacological and drug-like attributes, including specificity and off-target activity.

Currently, the rationale for selecting specific USP panels for in vitro analysis to evaluate the specificity of PL pro inhibitors and their off-target activity remains unclear. In the development of GRL-0617, Ratia et al. counter-screened against USP7, USP18, UCH-L1, and UCH-L3 (Ratia et al., 2008). In the development of Compound 19, Shan et al. counter-screened against USP36, USP14, USP8, USP7, USP2, and related DUBs UCH-L1, SNEP1, OTUB-1, ATAXIN3, and AMSH (Shan et al., 2021). Sanders et al. screened Compound 7 against USP2c, USP4, USP7, USP8, USP15, USP30, and UCH-L1 (Sanders et al., 2023). Lastly, XR8-24 and Jun12682 were counter-screened against only USP7 and USP14 (Tan et al., 2023). SBDD efforts would greatly benefit from a clearly defined panel of human DUBs, facilitating a more rational counter-screening of lead PL pro inhibitors. To systematically address this concern, we identified a panel of the closest structural human homologues of SARS-CoV-2 PL pro to counter-screen potential drug candidates

We crystallized the SARS-CoV-2 PL pro enzyme (9BF8.pdb), and this high-quality crystal structure served as the query structure. The 9BF8 PDB Validation Report contains relevant data collection and refinement statistics. The diffraction data were collected by the Life Sciences Collaborative Access Team (LS-CAT) using the NSLS-II 17-1 AMX beamline at the Brookhaven National Laboratory, resulting in a 1.85 Å resolution data set. The resulting refined model had an overall B-factor of 34.1 Å 2 , allowing proper main and side chain modeling. The phased and refined structure resulted in a final R work of 17.75% ( R free = 20.00%), with zero Ramachandran and sidechain outliers.

A heuristic search of the PDB was performed using the DALI server (ekhidna2.biocenter.helsinki.fi/dali) to compare the coordinates of the query protein structure with all protein chains deposited in the PDB. The catalytic domains of 27 USPs were identified to have significant DALI Z-scores, where a higher score indicates statistical significance in similarity. DALI Z-scores for identified USPs ranged from 9.5 to 13.8, with most having similar scores ( Data Set Identifying Structural Homologues of SARS-CoV-2 PL pro ). SARS-CoV-2 PL pro sequence identity of the identified USPs ranged from 9% to 16%. Other hits include SARS-CoV PL pro and related hCoV PL pro from MERS-CoV, hCoV-HKU1, and the murine hepatitis virus (MHV), which had Z-scores ranging from 32 to 44 and SARS-CoV-2 PL pro sequence identities ranging from 28-83%.

Unlike the DALI server analysis, alignment metrics, such as the Alignment score in Schrödinger Maestro Suite release 2024-2 (version 14.0.134), provide a more comprehensive evaluation, offering a detailed and holistic view of protein similarity based on sequence and structure. In combination with the 27 USP catalytic cores identified by DALI, MERS-CoV PL pro , hCoV-HKU1 PLP2, and Murine Hepatitis Virus (MHV) PLP2 were processed using the Maestro Protein Preparation Workflow to prepare structures, primarily to fill in missing side chains, and then subjected to the Protein Structure Alignment tool (Sastry et al., 2013). As expected, related CoV PL pro had the best alignment scores to SARS-CoV-2 PL pro , ranging from 0.021 to 0.3, respectively ( Fig. 1B ). The catalytic domains of USP46 and USP12 exhibited the highest structural homology with SARS-CoV-2 PL pro , with alignment scores below 0.6 and RMSD values of ~3 Å. The alignment scores of USP46 (0.368) and USP12 (0.384) were similar to the alignment score of the MHV PL pro (0.361), highlighting the high similarity between the viral and human proteases ( Fig. 1C ). The catalytic domains of USP16, USP8, USP9X, USPL1, USP1, USP54, and USP7 are also significantly close structural homologues of PL pro with alignment scores of less than 0.459 and RMSD of less than 3.37 Å. Nonetheless, USP16 lacks essential catalytic residues and is categorized as a non-protease homologue in accordance with the MEROPS database classification ( Fig. 1C ). This designation rules USP16 out as a viable PL pro off-target. Furthermore, USPL1 and USP54 exhibit low PL pro sequence identity in the BL2 and Alpha-cleft regions. Lastly, USP7’s active site requires ubiquitin substrate binding for the catalytic Cys to align into an active conformation, and its BL2 is in an open state when HAUSP is unbound to ubiquitin (Hu et al., 2002), rendering it an irrational anti-PL pro off-target. Overall, USP46, USP12, and USP9X are the closest human structural homologues to PL pro ( Superposition of USP46, USP12, and USP9X to SARS-CoV-2 PL pro ) and serve as the most rational off-targets for testing the specificity of small-molecule PL pro inhibitors. Specifically, USP46 is notable for its superior alignment score, low RMSD value, and high sequence/structural identity with the ancillary binding sites of PL pro (e.g., BL2) ( Fig. 1D ).

The methodology described can identify anti-target panels for developing antiviral compounds. The findings presented herein provide a more transparent and economical array of human proteins suitable for counter-screening compounds. This advancement consequently facilitates the development of potent and selective antiviral small-molecule PL pro inhibitors.

Methods

PL pro Cloning, Expression, and Purification

The gene encoding the full-length SARS-CoV-2 PL pro (isolate Wuhan-Hu-1 NC_045512.2, pp1ab aa 1564–1878) with N-terminal His 6 -SUMO-cleavable tag was cloned into pMCSG7 (Midwest Center for Structural Genomics) vector following the Ligation Independent Cloning (LIC) procedure (Stols et al., 2002). Whole Plasmid Sequencing was performed by Plasmidsaurus using Oxford Nanopore Technology with custom analysis and annotation. The pHis 6 -SUMO vector containing the PL pro gene was transformed into Rosetta2 E. coli cells, plated onto Amp + agar plates, and incubated overnight at 37°C. Single colonies were cultured overnight in LB media with 100 µg/mL Amp at 37°C until OD 600 = 1.0. The temperature was then lowered to 20°C for 1 hour, followed by the induction of protein expression with 0.4 mM IPTG. The next day, cell pellets were harvested by centrifugation and frozen at -80°C. Frozen cell pellets were lysed by sonication in lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM DTT) containing 10 µg/mL lysozyme, 0.5 µg/mL Aprotinin, and 0.05 µg/mL Leupeptin. The lysate was clarified by centrifugation and loaded onto a 5 mL Ni-NTA resin equilibrated with Ni column wash buffer (25 mM Tris HCl, pH 7.5, 150 mM NaCl). Bound His 6 -SUMO-PL pro was eluted with elution buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 300 mM imidazole). Fractions containing His 6 -SUMO-PL pro were pooled and exchanged into SUMO dialysis buffer (20 mM Tris-HCl, pH 7.5, 250 mM NaCl, 1 mM DTT). A 1:100 molar ratio of ULPL1 (SUMO-specific protease) to PL pro was incubated at 4°C overnight to cleave the His 6 -tag. The reaction was loaded onto a Ni-NTA resin re-equilibrated with 25 mM Tris-HCl, pH 7.5, and 150 mM NaCl to remove the tag and the SUMO-specific protease. Cleaved PL pro eluted first, followed by gel filtration. PL pro fractions were pooled, concentrated, and frozen in liquid nitrogen for storage at -80°C in S75 buffer (20 mM Tris-HCl, pH 7.5, 250 mM NaCl, 1 mM DTT). A Bradford assay using BSA as a standard monitored protein yield at each step.

Crystallization and structure determination of SARS-CoV-2 PL pro

The crystals of SARS-CoV-2 PL pro were grown using the sitting drop vapor diffusion method against a 300 µL reservoir solution at 4°C. The PL pro crystals were formed by mixing 1-2 µL of 18.7 mg/mL protein with 1 µL of reservoir solution containing 0.1 M Tris buffer (pH 7.0), 1 M sodium phosphate monobasic monohydrate (NaH2PO4), 1 M potassium phosphate dibasic (K2HPO4), and 3% w/v sucrose. Crystals formed over 1-2 days, reaching dimensions of 0.5-0.8 mm with octahedral and dodecahedral external symmetries. Full-sized crystals were harvested after one week. They were carefully transferred into a cryoprotectant consisting of mother liquor and 25% (v/v) glycerol before being flash-frozen in liquid nitrogen. The samples were subsequently shipped to the NSLS-II 17-1 AMX beamline at Brookhaven National Laboratory for diffraction data collection. Reflections were integrated and scaled using XDS and AIMLESS via Xia2 (P. Evans, 2006; Kabsch, 2010; Winter, 2010). Phases were calculated through molecular replacement using PHASER within the PHENIX software package (Adams et al., 2010; Liebschner et al., 2019; McCoy et al., 2007) for molecular structure determination by employing a monomeric SARS-CoV-2 PL pro structure (8FWN.pdb) as the search model. Automated model building and multiple rounds of refinement were conducted using PHENIX Autobuild, WinCoot, and PHENIX Refine (Afonine et al., 2012; Afonine et al., 2018; Echols et al., 2012; Emsley, Lohkamp, Scott, & Cowtan, 2010; Terwilliger et al., 2008). The X-ray crystal structure of SARS-CoV-2 PL pro has been deposited in the PDB (9BF8.pdb), and the deposition validation file is also provided.

Structure Similarity Analysis

The query crystal structure PDB coordinates (9BF8.pdb) were uploaded into the DALI protein structure comparison server to perform a heuristic PDB search. Identified structures were prepared and aligned using the Protein Preparation Workflow and Protein Structure Alignment tool, employing the query crystal structure as reference residues using Schrödinger Maestro Suite release 2024-2 (version 14.0.134).

Reagents

Plasmid

Description

pHis 6 -SUMO-PL pro

Custom bacterial vector with an ampicillin resistance marker for regulated expression of SARS-CoV-2 PL pro with a cleavable His 6 -SUMO tag.

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Acknowledgments

This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817).

Funding Statement

Partial support for this research is provided by NIH (grant AI161570).

Extended Data

Description: Table with DALI-Z and Schrödinger Maestro Suite metrics used for analysis. . Resource Type: Dataset. DOI: https://doi.org/10.22002/cq58r-x9918

Description: Full wwPDB X-ray structure validation report and statistics for PDB code 9BF8 SARS-CoV-2 PLpro Untagged Crystal Structure.. Resource Type: Collection. DOI: https://doi.org/10.22002/ncxnv-byp09

Description: Structural superposition of USP46, USP12, and USP9X to SARS-CoV-2 PLpro highlighting relevant binding sites.. Resource Type: Image. DOI: https://doi.org/10.22002/7dxa1-p2863

References

  1. Adams Paul D., Afonine Pavel V., Bunkóczi Gábor, Chen Vincent B., Davis Ian W., Echols Nathaniel, Headd Jeffrey J., Hung Li-Wei, Kapral Gary J., Grosse-Kunstleve Ralf W., McCoy Airlie J., Moriarty Nigel W., Oeffner Robert, Read Randy J., Richardson David C., Richardson Jane S., Terwilliger Thomas C., Zwart Peter H. PHENIX : a comprehensive Python-based system for macromolecular structure solution . Acta Crystallographica Section D Biological Crystallography. 2010 Jan 22;66(2):213–221. doi: 10.1107/s0907444909052925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Afonine Pavel V., Grosse-Kunstleve Ralf W., Echols Nathaniel, Headd Jeffrey J., Moriarty Nigel W., Mustyakimov Marat, Terwilliger Thomas C., Urzhumtsev Alexandre, Zwart Peter H., Adams Paul D. Towards automated crystallographic structure refinement with phenix.refine . Acta Crystallographica Section D Biological Crystallography. 2012 Mar 16;68(4):352–367. doi: 10.1107/s0907444912001308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Afonine Pavel V., Poon Billy K., Read Randy J., Sobolev Oleg V., Terwilliger Thomas C., Urzhumtsev Alexandre, Adams Paul D. Real-space refinement in PHENIX for cryo-EM and crystallography . Acta Crystallographica Section D Structural Biology. 2018 May 30;74(6):531–544. doi: 10.1107/s2059798318006551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Balakirev Maxim Y, Tcherniuk Sergey O, Jaquinod Michel, Chroboczek Jadwiga. Otubains: a new family of cysteine proteases in the ubiquitin pathway. EMBO reports. 2003 Apr 18;4(5):517–522. doi: 10.1038/sj.embor.embor824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barretto N, Jukneliene D, Ratia K, Chen Z, Mesecar AD, Baker SC. The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity. J Virol. 2005 Dec 1;79(24):15189–15198. doi: 10.1128/JVI.79.24.15189-15198.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burnett B. The polyglutamine neurodegenerative protein ataxin-3 binds polyubiquitylated proteins and has ubiquitin protease activity. Human Molecular Genetics. 2003 Sep 30;12(23):3195–3205. doi: 10.1093/hmg/ddg344. [DOI] [PubMed] [Google Scholar]
  7. Echols Nathaniel, Grosse-Kunstleve Ralf W., Afonine Pavel V., Bunkóczi Gábor, Chen Vincent B., Headd Jeffrey J., McCoy Airlie J., Moriarty Nigel W., Read Randy J., Richardson David C., Richardson Jane S., Terwilliger Thomas C., Adams Paul D. Graphical tools for macromolecular crystallography in PHENIX . Journal of Applied Crystallography. 2012 May 16;45(3):581–586. doi: 10.1107/s0021889812017293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Emsley P., Lohkamp B., Scott W. G., Cowtan K. Features and development of Coot . Acta Crystallographica Section D Biological Crystallography. 2010 Mar 24;66(4):486–501. doi: 10.1107/s0907444910007493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eng Heather, Dantonio Alyssa L., Kadar Eugene P., Obach R. Scott, Di Li, Lin Jian, Patel Nandini C., Boras Britton, Walker Gregory S., Novak Jonathan J., Kimoto Emi, Singh Ravi Shankar P., Kalgutkar Amit S. Disposition of Nirmatrelvir, an Orally Bioavailable Inhibitor of SARS-CoV-2 3C-Like Protease, across Animals and Humans. Drug Metabolism and Disposition. 2022 May 1;50(5):576–590. doi: 10.1124/dmd.121.000801. [DOI] [PubMed] [Google Scholar]
  10. Evans Philip. Scaling and assessment of data quality. Acta Crystallographica Section D Biological Crystallography. 2005 Dec 14;62(1):72–82. doi: 10.1107/s0907444905036693. [DOI] [PubMed] [Google Scholar]
  11. Evans Paul C., Smith Trevor S., Lai Meng-Jiun, Williams Mark G., Burke David F., Heyninck Karen, Kreike Marja M., Beyaert Rudi, Blundell Tom L., Kilshaw Peter J. A Novel Type of Deubiquitinating Enzyme. Journal of Biological Chemistry. 2003 Jun 1;278(25):23180–23186. doi: 10.1074/jbc.m301863200. [DOI] [PubMed] [Google Scholar]
  12. Gan-Erdene Tudeviin, Nagamalleswari Kolli, Yin Luming, Wu Kenneth, Pan Zhen-Qiang, Wilkinson Keith D. Identification and Characterization of DEN1, a Deneddylase of the ULP Family. Journal of Biological Chemistry. 2003 Aug 1;278(31):28892–28900. doi: 10.1074/jbc.m302890200. [DOI] [PubMed] [Google Scholar]
  13. Ghosh Arun K., Takayama Jun, Aubin Yoann, Ratia Kiira, Chaudhuri Rima, Baez Yahira, Sleeman Katrina, Coughlin Melissa, Nichols Daniel B., Mulhearn Debbie C., Prabhakar Bellur S., Baker Susan C., Johnson Michael E., Mesecar Andrew D. Structure-Based Design, Synthesis, and Biological Evaluation of a Series of Novel and Reversible Inhibitors for the Severe Acute Respiratory Syndrome−Coronavirus Papain-Like Protease. Journal of Medicinal Chemistry. 2009 Jul 31;52(16):5228–5240. doi: 10.1021/jm900611t. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hochstrasser Mark. Evolution and function of ubiquitin-like protein-conjugation systems. Nature Cell Biology. 2000 Aug 1;2(8):E153–E157. doi: 10.1038/35019643. [DOI] [PubMed] [Google Scholar]
  15. Hu Min, Li Pingwei, Li Muyang, Li Wenyu, Yao Tingting, Wu Jia-Wei, Gu Wei, Cohen Robert E., Shi Yigong. Crystal Structure of a UBP-Family Deubiquitinating Enzyme in Isolation and in Complex with Ubiquitin Aldehyde. Cell. 2002 Dec 1;111(7):1041–1054. doi: 10.1016/s0092-8674(02)01199-6. [DOI] [PubMed] [Google Scholar]
  16. Jiang Xiangrui, Su Haixia, Shang Weijuan, Zhou Feng, Zhang Yan, Zhao Wenfeng, Zhang Qiumeng, Xie Hang, Jiang Lei, Nie Tianqing, Yang Feipu, Xiong Muya, Huang Xiaoxing, Li Minjun, Chen Ping, Peng Shaoping, Xiao Gengfu, Jiang Hualiang, Tang Renhong, Zhang Leike, Shen Jingshan, Xu Yechun. Structure-based development and preclinical evaluation of the SARS-CoV-2 3C-like protease inhibitor simnotrelvir. Nature Communications. 2023 Oct 13;14(1) doi: 10.1038/s41467-023-42102-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kabsch Wolfgang. XDS . Acta Crystallographica Section D Biological Crystallography. 2010 Jan 22;66(2):125–132. doi: 10.1107/s0907444909047337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Liebschner Dorothee, Afonine Pavel V., Baker Matthew L., Bunkóczi Gábor, Chen Vincent B., Croll Tristan I., Hintze Bradley, Hung Li-Wei, Jain Swati, McCoy Airlie J., Moriarty Nigel W., Oeffner Robert D., Poon Billy K., Prisant Michael G., Read Randy J., Richardson Jane S., Richardson David C., Sammito Massimo D., Sobolev Oleg V., Stockwell Duncan H., Terwilliger Thomas C., Urzhumtsev Alexandre G., Videau Lizbeth L., Williams Christopher J., Adams Paul D. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix . Acta Crystallographica Section D Structural Biology. 2019 Oct 1;75(10):861–877. doi: 10.1107/s2059798319011471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lindner HA, Lytvyn V, Qi H, Lachance P, Ziomek E, Ménard R. Selectivity in ISG15 and ubiquitin recognition by the SARS coronavirus papain-like protease. Arch Biochem Biophys. 2007 Jul 14;466(1):8–14. doi: 10.1016/j.abb.2007.07.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lindner HA, Fotouhi-Ardakani N, Lytvyn V, Lachance P, Sulea T, Ménard R. The papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme. J Virol. 2005 Dec 1;79(24):15199–15208. doi: 10.1128/JVI.79.24.15199-15208.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Madhavi Sastry G., Adzhigirey Matvey, Day Tyler, Annabhimoju Ramakrishna, Sherman Woody. Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. Journal of Computer-Aided Molecular Design. 2013 Mar 1;27(3):221–234. doi: 10.1007/s10822-013-9644-8. [DOI] [PubMed] [Google Scholar]
  22. McCoy Airlie J., Grosse-Kunstleve Ralf W., Adams Paul D., Winn Martyn D., Storoni Laurent C., Read Randy J. Phaser crystallographic software . Journal of Applied Crystallography. 2007 Jul 13;40(4):658–674. doi: 10.1107/s0021889807021206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Quesada Víctor, Díaz-Perales Araceli, Gutiérrez-Fernández Ana, Garabaya Cecilia, Cal Santiago, López-Otín Carlos. Cloning and enzymatic analysis of 22 novel human ubiquitin-specific proteases. Biochemical and Biophysical Research Communications. 2004 Jan 1;314(1):54–62. doi: 10.1016/j.bbrc.2003.12.050. [DOI] [PubMed] [Google Scholar]
  24. Ratia Kiira, Pegan Scott, Takayama Jun, Sleeman Katrina, Coughlin Melissa, Baliji Surendranath, Chaudhuri Rima, Fu Wentao, Prabhakar Bellur S., Johnson Michael E., Baker Susan C., Ghosh Arun K., Mesecar Andrew D. A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication. Proceedings of the National Academy of Sciences. 2008 Oct 21;105(42):16119–16124. doi: 10.1073/pnas.0805240105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ratia Kiira, Saikatendu Kumar Singh, Santarsiero Bernard D., Barretto Naina, Baker Susan C., Stevens Raymond C., Mesecar Andrew D. Severe acute respiratory syndrome coronavirus papain-like protease: Structure of a viral deubiquitinating enzyme. Proceedings of the National Academy of Sciences. 2006 Apr 11;103(15):5717–5722. doi: 10.1073/pnas.0510851103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sanders Brian C., Pokhrel Suman, Labbe Audrey D., Mathews Irimpan I., Cooper Connor J., Davidson Russell B., Phillips Gwyndalyn, Weiss Kevin L., Zhang Qiu, O’Neill Hugh, Kaur Manat, Schmidt Jurgen G., Reichard Walter, Surendranathan Surekha, Parvathareddy Jyothi, Phillips Lexi, Rainville Christopher, Sterner David E., Kumaran Desigan, Andi Babak, Babnigg Gyorgy, Moriarty Nigel W., Adams Paul D., Joachimiak Andrzej, Hurst Brett L., Kumar Suresh, Butt Tauseef R., Jonsson Colleen B., Ferrins Lori, Wakatsuki Soichi, Galanie Stephanie, Head Martha S., Parks Jerry M. Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2. Nature Communications. 2023 Mar 28;14(1) doi: 10.1038/s41467-023-37254-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shan Hengyue, Liu Jianping, Shen Jiali, Dai Jialin, Xu Gang, Lu Kuankuan, Han Chao, Wang Yaru, Xu Xiaolong, Tong Yilun, Xiang Huaijiang, Ai Zhiyuan, Zhuang Guanglei, Hu Junhao, Zhang Zheng, Li Ying, Pan Lifeng, Tan Li. Development of potent and selective inhibitors targeting the papain-like protease of SARS-CoV-2. Cell Chemical Biology. 2021 Jun 1;28(6):855–865.e9. doi: 10.1016/j.chembiol.2021.04.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shimizu Ryosuke, Sonoyama Takuhiro, Fukuhara Takahiro, Kuwata Aya, Matsuzaki Takanobu, Matsuo Yumiko, Kubota Ryuji. Evaluation of the Drug–Drug Interaction Potential of Ensitrelvir Fumaric Acid with Cytochrome P450 3A Substrates in Healthy Japanese Adults. Clinical Drug Investigation. 2023 May 1;43(5):335–346. doi: 10.1007/s40261-023-01265-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shin Donghyuk, Mukherjee Rukmini, Grewe Diana, Bojkova Denisa, Baek Kheewoong, Bhattacharya Anshu, Schulz Laura, Widera Marek, Mehdipour Ahmad Reza, Tascher Georg, Geurink Paul P., Wilhelm Alexander, van der Heden van Noort Gerbrand J., Ovaa Huib, Müller Stefan, Knobeloch Klaus-Peter, Rajalingam Krishnaraj, Schulman Brenda A., Cinatl Jindrich, Hummer Gerhard, Ciesek Sandra, Dikic Ivan. Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity. Nature. 2020 Jul 29;587(7835):657–662. doi: 10.1038/s41586-020-2601-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Song Kun, Li Shitao. The Role of Ubiquitination in NF-κB Signaling during Virus Infection. Viruses. 2021 Jan 20;13(2):145–145. doi: 10.3390/v13020145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stols Lucy, Gu Minyi, Dieckman Lynda, Raffen Rosemarie, Collart Frank R., Donnelly Mark I. A New Vector for High-Throughput, Ligation-Independent Cloning Encoding a Tobacco Etch Virus Protease Cleavage Site. Protein Expression and Purification. 2002 Jun 1;25(1):8–15. doi: 10.1006/prep.2001.1603. [DOI] [PubMed] [Google Scholar]
  32. Tan Bin, Zhang Xiaoming, Ansari Ahmadullah, Jadhav Prakash, Tan Haozhou, Li Kan, Chopra Ashima, Ford Alexandra, Chi Xiang, Ruiz Francesc Xavier, Arnold Eddy, Deng Xufang, Wang Jun. Design of SARS-CoV-2 papain-like protease inhibitor with antiviral efficacy in a mouse model. 2023 Dec 3; doi: 10.1101/2023.12.01.569653. [DOI] [PMC free article] [PubMed]
  33. Terwilliger Thomas C., Grosse-Kunstleve Ralf W., Afonine Pavel V., Moriarty Nigel W., Zwart Peter H., Hung Li-Wei, Read Randy J., Adams Paul D. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard . Acta Crystallographica Section D Biological Crystallography. 2007 Dec 4;64(1):61–69. doi: 10.1107/s090744490705024x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Thiel V, Ivanov KA, Putics Á, Hertzig T, Schelle B, Bayer S, Weißbrich B, Snijder EJ, Rabenau H, Doerr HW, Gorbalenya AE, Ziebuhr J. Mechanisms and enzymes involved in SARS coronavirus genome expression. J Gen Virol. 2003 Sep 1;84(Pt 9):2305–2315. doi: 10.1099/vir.0.19424-0. [DOI] [PubMed] [Google Scholar]
  35. Verma Rati, Aravind L., Oania Robert, McDonald W. Hayes, Yates John R., Koonin Eugene V., Deshaies Raymond J. Role of Rpn11 Metalloprotease in Deubiquitination and Degradation by the 26 S Proteasome . Science. 2002 Oct 18;298(5593):611–615. doi: 10.1126/science.1075898. [DOI] [PubMed] [Google Scholar]
  36. Wilkinson Keith D. Regulation of ubiquitin‐dependent processes by deubiquitinating enzymes. The FASEB Journal. 1997 Dec 1;11(14):1245–1256. doi: 10.1096/fasebj.11.14.9409543. [DOI] [PubMed] [Google Scholar]
  37. Winter G. xia2 : an expert system for macromolecular crystallography data reduction . Journal of Applied Crystallography. 2009 Dec 1;43(1):186–190. doi: 10.1107/s0021889809045701. [DOI] [Google Scholar]
  38. Yeh Edward T.H., Gong Limin, Kamitani Tetsu. Ubiquitin-like proteins: new wines in new bottles. Gene. 2000 May 1;248(1-2):1–14. doi: 10.1016/s0378-1119(00)00139-6. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Description: Table with DALI-Z and Schrödinger Maestro Suite metrics used for analysis. . Resource Type: Dataset. DOI: https://doi.org/10.22002/cq58r-x9918

Description: Full wwPDB X-ray structure validation report and statistics for PDB code 9BF8 SARS-CoV-2 PLpro Untagged Crystal Structure.. Resource Type: Collection. DOI: https://doi.org/10.22002/ncxnv-byp09

Description: Structural superposition of USP46, USP12, and USP9X to SARS-CoV-2 PLpro highlighting relevant binding sites.. Resource Type: Image. DOI: https://doi.org/10.22002/7dxa1-p2863

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