A molecular sensor determines the ubiquitin substrate specificity of SARS-CoV-2 papain-like protease.
Ontology highlight
ABSTRACT:
SUBMITTER: Patchett S
PROVIDER: S-EPMC8423903 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a papain-like protease (PLpro) with both deubiquitinating (DUB) and deISGylating activities that are proposed to counteract the post-translational modification of signaling molecules that activate the innate immune response. Here we examine the structural basis for PLpro's ubiquitin chain and interferon stimulated gene 15 (ISG15) specificity. We present the X-ray crystal structure of PLpro in complex with ubiquitin-aldehyde and model the interaction of PLpro with other ubiquitin-chain and ISG15 substrates. We show that PLpro greatly prefers K48- to K63-linked ubiquitin chains, and ISG15-based substrates to those that are mono-ubiquitinated. We propose that PLpro's higher affinity for K48-linked ubiquitin chains and ISG15 stems from a bivalent mechanism of binding, where two ubiquitin-like domains prefer to bind in the palm domain of PLpro with the most distal ubiquitin domain interacting with a "ridge" region of the thumb domain. Mutagenesis of residues within this ridge region revealed that these mutants retain viral protease activity and the ability to catalyze hydrolysis of mono-ubiquitin. However, a select number of these mutants have a significantly reduced ability to hydrolyze the substrate ISG15-AMC, or be inhibited by K48-linked diubuiquitin. For these latter residues, we found that PLpro antagonism of the nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) signaling pathway is abrogated. This identification of key and unique sites in PLpro required for recognition and processing of diubiquitin and ISG15 versus mono-ubiquitin and protease activity provides new insight into ubiquitin-chain and ISG15 recognition and highlights a role for PLpro DUB and deISGylase activity in antagonism of the innate immune response.
| S-EPMC4031219 | biostudies-literature
Project description:SARS-CoV-2 papain-like protease is considered as an important potential target for anti-SARS-CoV-2 drug discovery due to its crucial roles in viral spread and innate immunity. Here, we have utilized an in silico molecular docking approach to identify the possible inhibitors of the SARS-CoV-2 papain-like protease, by screening 21 antiviral, antifungal and anticancer compounds. Among them, Neobavaisoflavone has the highest binding energy for SARS-CoV-2 papain-like protease. These molecules could bind near the SARS-CoV-2 papain-like protease crucial catalytic triad, ubiquitination and ISGylation residues: Trp106, Asn109, Cys111, Met208, Lys232, Pro247, Tyr268, Gln269, His272, Asp286 and Thr301. Because blocking the papain-like protease is an important strategy in fighting against viruses, these compounds might be promising candidates for therapeutic intervention against COVID-19.
| S-EPMC7698687 | biostudies-literature
Project description:The Middle East Respiratory Syndrome coronavirus (MERS-CoV) papain-like protease (PLpro) blocking loop 2 (BL2) structure differs significantly from that of SARS-CoV PLpro, where it has been proven to play a crucial role in SARS-CoV PLpro inhibitor binding. Four SARS-CoV PLpro lead inhibitors were tested against MERS-CoV PLpro, none of which were effective against MERS-CoV PLpro. Structure and sequence alignments revealed that two residues, Y269 and Q270, responsible for inhibitor binding to SARS-CoV PLpro, were replaced by T274 and A275 in MERS-CoV PLpro, making critical binding interactions difficult to form for similar types of inhibitors. High-throughput screening (HTS) of 25 000 compounds against both PLpro enzymes identified a small fragment-like noncovalent dual inhibitor. Mode of inhibition studies by enzyme kinetics and competition surface plasmon resonance (SPR) analyses suggested that this compound acts as a competitive inhibitor with an IC50 of 6 μM against MERS-CoV PLpro, indicating that it binds to the active site, whereas it acts as an allosteric inhibitor against SARS-CoV PLpro with an IC50 of 11 μM. These results raised the possibility that inhibitor recognition specificity of MERS-CoV PLpro may differ from that of SARS-CoV PLpro. In addition, inhibitory activity of this compound was selective for SARS-CoV and MERS-CoV PLpro enzymes over two human homologues, the ubiquitin C-terminal hydrolases 1 and 3 (hUCH-L1 and hUCH-L3).
| S-EPMC4845099 | biostudies-literature
Project description:The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3727 unique approved drugs and clinical compounds against SARS2 PLpro, identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent selfprocessing of nsp3 in cells, and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.
| S-SCDT-EMBOJ-2020-106275 | biostudies-other
Project description:Covid-19 is caused by a novel form of coronavirus for which there are currently no vaccines or anti-viral drugs. This virus, termed SARS-CoV-2 (CoV2), contains Papain-like protease (PLpro) involved in viral replication and immune response evasion. Drugs targeting this protease therefore have great potential for inhibiting the virus, and have proven successful in older coronaviruses. Here, we introduce two effective inhibitors of SARS-CoV-1 (CoV1) and MERS-CoV to assess their potential for inhibiting CoV2 PLpro. We ran 1 ?s molecular dynamics (MD) simulations of CoV2, CoV1, and MERS-CoV ligand-free PLpro to characterize the dynamics of CoV2 PLpro, and made comparisons between the three to elucidate important similarities and differences relevant to drug design and ubiquitin-like protein binding for deubiquitinating and deISGylating activity of CoV2. Next, we simulated the inhibitors bound to CoV1 and CoV2 PLpro in various poses and at different known binding sites to analyze their binding modes. We found that the naphthalene-based ligand shows strong potential as an inhibitor of CoV2 PLpro by binding at the putative naphthalene inhibitor binding site in both computational predictions and experimental assays. Our modeling work suggested strategies to improve naphthalene-based compounds, and our results from molecular docking showed that the newly designed compounds exhibited improved binding affinity. The other ligand, chemotherapy drug 6-mercaptopurine (6MP), showed little to no stable intermolecular interaction with PLpro and quickly dissociated or remained highly mobile. We demonstrate multiple ways to improve the binding affinity of the naphthalene-based inhibitor scaffold by engaging new residues in the unused space of the binding site. Analysis of CoV2 PLpro also brings insights into recognition of ubiquitin-like proteins that may alter innate immune response.
| S-EPMC7417481 | biostudies-literature
Project description:The pandemic of coronavirus disease 2019 (COVID-19) is changing the world like never before. This crisis is unlikely contained in the absence of effective therapeutics or vaccine. The papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays essential roles in virus replication and immune evasion, presenting a charming drug target. Given the PLpro proteases of SARS-CoV-2 and SARS-CoV share significant homology, inhibitor developed for SARS-CoV PLpro is a promising starting point of therapeutic development. In this study, we sought to provide structural frameworks for PLpro inhibitor design. We determined the unliganded structure of SARS-CoV-2 PLpro mutant C111S, which shares many structural features of SARS-CoV PLpro. This crystal form has unique packing, high solvent content and reasonable resolution 2.5 Å, hence provides a good possibility for fragment-based screening using crystallographic approach. We characterized the protease activity of PLpro in cleaving synthetic peptide harboring nsp2/nsp3 juncture. We demonstrate that a potent SARS-CoV PLpro inhibitor GRL0617 is highly effective in inhibiting protease activity of SARS-CoV-2 with the IC50 of 2.2 ± 0.3 ?mol/L. We then determined the structure of SARS-CoV-2 PLpro complexed by GRL0617 to 2.6 Å, showing the inhibitor accommodates the S3-S4 pockets of the substrate binding cleft. The binding of GRL0617 induces closure of the BL2 loop and narrows the substrate binding cleft, whereas the binding of a tetrapeptide substrate enlarges the cleft. Hence, our results suggest a mechanism of GRL0617 inhibition, that GRL0617 not only occupies the substrate pockets, but also seals the entrance to the substrate binding cleft hence prevents the binding of the LXGG motif of the substrate.
| S-EPMC7467110 | biostudies-literature
Project description:As the pathogen of COVID-19, SARS-CoV-2 encodes two essential cysteine proteases that process the pathogen's two large polypeptide products pp1a and pp1ab in the human cell host to form 15 functionally important, mature nonstructural proteins. One of the two enzymes is papain-like protease or PLPro . It possesses deubiquitination and deISGylation activities that suppress host innate immune responses toward SARS-CoV-2 infection. To repurpose drugs for PLPro , we experimentally screened libraries of 33 deubiquitinase and 37 cysteine protease inhibitors on their inhibition of PLPro . Our results showed that 15 deubiquitinase and 1 cysteine protease inhibitors exhibit strong inhibition of PLPro at 200 μM. More comprehensive characterizations revealed seven inhibitors GRL0617, SJB2-043, TCID, DUB-IN-1, DUB-IN-3, PR-619, and S130 with an IC50 value below 40 μM and four inhibitors GRL0617, SJB2-043, TCID, and PR-619 with an IC50 value below 10 μM. Among four inhibitors with an IC50 value below 10 μM, SJB2-043 is the most unique in that it does not fully inhibit PLPro but has a noteworthy IC50 value of 0.56 μM. SJB2-043 likely binds to an allosteric site of PLPro to convene its inhibition effect, which needs to be further investigated. As a pilot study, the current work indicates that COVID-19 drug repurposing by targeting PLPro holds promise, but in-depth analysis of repurposed drugs is necessary to avoid omitting critical allosteric inhibitors.
| S-EPMC8653067 | biostudies-literature
Project description:Emerging variants of SARS-CoV-2 and potential novel epidemic coronaviruses underline the importance of investigating various viral proteins as potential drug targets. The papain-like protease of coronaviruses has been less explored than other viral proteins; however, its substantive role in viral replication and impact on the host immune response make it a suitable target to study. This review article focuses on the structure and function of the papain-like protease (PLpro ) of SARS-CoV-2, including variants of concern, and compares it to those of other coronaviruses, such as SARS-CoV-1 and MERS-CoV. The protease's recognition motif is mirrored in ubiquitin and ISG15, which are involved in the antiviral immune response. Inhibitors, including GRL0617 derivatives, and their prospects as potential future antiviral agents are also discussed.
| S-EPMC9538446 | biostudies-literature
Project description:SARS-CoV-2 encoded papain-like protease (PLpro) harbors a labile Zn site (Cys189-X-X-Cys192-X n -Cys224-X-Cys226) and a classic catalytic site (Cys111-His272-Asp286), which play key roles for viral replication and hence represent promising drug targets. In this Viewpoint, both sulfur-based drugs and peptides-based inhibitors may block Cys residues in the catalytic and/or Zn site of CoV-2-PLpro, leading to dysfunction of CoV-2-PLpro and thereby halting viral replication.
| S-EPMC7409920 | biostudies-literature
Project description:Papain-like protease (PLpro) of coronaviruses (CoVs) carries out proteolytic maturation of non-structural proteins that play a role in replication of the virus and performs deubiquitination of host cell factors to scuttle antiviral responses. Avian infectious bronchitis virus (IBV), the causative agent of bronchitis in chicken that results in huge economic losses every year in the poultry industry globally, encodes a PLpro. The substrate specificities of this PLpro are not clearly understood. Here, we show that IBV PLpro can degrade Lys(48)- and Lys(63)-linked polyubiquitin chains to monoubiquitin but not linear polyubiquitin. To explain the substrate specificities, we have solved the crystal structure of PLpro from IBV at 2.15-Å resolution. The overall structure is reminiscent of the structure of severe acute respiratory syndrome CoV PLpro. However, unlike the severe acute respiratory syndrome CoV PLpro that lacks blocking loop (BL) 1 of deubiquitinating enzymes, the IBV PLpro has a short BL1-like loop. Access to a conserved catalytic triad consisting of Cys(101), His(264), and Asp(275) is regulated by the flexible BL2. A model of ubiquitin-bound IBV CoV PLpro brings out key differences in substrate binding sites of PLpros. In particular, P3 and P4 subsites as well as residues interacting with the β-barrel of ubiquitin are different, suggesting different catalytic efficiencies and substrate specificities. We show that IBV PLpro cleaves peptide substrates KKAG-7-amino-4-methylcoumarin and LRGG-7-amino-4-methylcoumarin with different catalytic efficiencies. These results demonstrate that substrate specificities of IBV PLpro are different from other PLpros and that IBV PLpro might target different ubiquitinated host factors to aid the propagation of the virus.
| S-EPMC4358136 | biostudies-literature