SARS-CoV-2 papain-like protease inhibits ISGylation of the viral nucleocapsid protein to evade host anti-viral immunity.

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes mild-to-severe respiratory symptoms, including acute respiratory distress. Despite remarkable efforts to investigate the virological and pathological impacts of SARS-CoV-2, many of the characteristics of SARS-CoV-2 infection still remain unknown. The interferon-inducible ubiquitin-like protein ISG15 is covalently conjugated to several viral proteins to suppress their functions. It was reported that SARS-CoV-2 utilizes its papain-like protease (PLpro) to impede ISG15 conjugation, ISGylation. However, the role of ISGylation in SARS-CoV-2 infection remains unclear. We aimed to elucidate the role of ISGylation in SARS-CoV-2 replication. We observed that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation in cultured cells. Site-directed mutagenesis reveals that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation, alongside conserved lysine residue in MERS-CoV (K372) and SARS-CoV (K375). We also observed that the nucleocapsid-ISGylation results in the disruption of nucleocapsid oligomerization, thereby inhibiting viral replication. Knockdown of ISG15 mRNA enhanced SARS-CoV-2 replication in the SARS-CoV-2 reporter replicon cells, while exogenous expression of ISGylation components partially hampered SARS-CoV-2 replication. Taken together, these results suggest that SARS-CoV-2 PLpro inhibits ISGylation of the nucleocapsid protein to promote viral replication by evading ISGylation-mediated disruption of the nucleocapsid oligomerization.IMPORTANCEISG15 is an interferon-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation in many viruses. However, the role of ISGylation in SARS-CoV-2 infection remains largely unclear. Here, we demonstrated that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation. We also found that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation. We obtained evidence suggesting that nucleocapsid-ISGylation results in the disruption of nucleocapsid-oligomerization, thereby suppressing SARS-CoV-2 replication. We discovered that SARS-CoV-2 papain-like protease inhibits ISG15 conjugation of nucleocapsid protein via its de-conjugating enzyme activity. The present study may contribute to gaining new insight into the roles of ISGylation-mediated anti-viral function in SARS-CoV-2 infection and may lead to the development of more potent and selective inhibitors targeted to SARS-CoV-2 nucleocapsid protein.

Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2.

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 3,727 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 self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.

The Papain-Like Protease of Porcine Reproductive and Respiratory Syndrome Virus Impedes STING Translocation from the Endoplasmic Reticulum to the Golgi Apparatus by Deubiquitinating STIM1.

Stimulator of interferon (IFN) genes (STING) was recently pinpointed as an antiviral innate immune factor during the infection of RNA viruses. Porcine reproductive and respiratory syndrome virus (PRRSV), the swine arterivirus, is an enveloped RNA virus which has evolved many strategies to evade innate immunity. To date, the interactive network between PRRSV and STING remains to be fully established. Herein, we report that STING suppresses PRRSV replication through type I interferon signaling. However, PRRSV impedes STING trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus, leading to the decreased phosphorylation of TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). Furthermore, PRRSV nonstructural protein 2 (Nsp2) colocalizes with STING, blocks STING translocation, and disrupts the STING-TBK1-IRF3 complex. Mechanistically, PRRSV Nsp2 retains STING at the ER by increasing the level of Ca2+ sensor stromal interaction molecule 1 (STIM1) protein. Functional analysis reveals that PRRSV Nsp2 deubiquitinates STIM1 by virtue of its papain-like protease 2 (PLP2) deubiquitinating (DUB) activity. Finally, we demonstrate that loss of STIM1 is associated with an elevated IFN response and restricts PRRSV replication. This work delineates the relationship between PRRSV infection and STING signaling and the importance of papain-like proteases (PLPs) in interfering in this axis. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the family Arteriviridae, is responsible for reproductive disorders in pregnant sows and respiratory problems in piglets, resulting in huge losses in the swine industry worldwide. Of note, PRRSV infection causes immunosuppression, of which the mechanism is not completely understood. Here, we demonstrate for the first time that STING, a protein typically associated with the antiviral response in DNA viruses, plays a critical role in controlling PRRSV infection. However, PRRSV utilizes its encoded protein Nsp2 to inhibit STING activity by blocking its translocation from the ER to the Golgi apparatus. In particular, Nsp2 retains STING at the ER by interacting with and further deubiquitinating STIM1. For this process, the activity of the viral PLP2 DUB enzyme is indispensable. The study describes a novel mechanism by which PLP2 plays a critical role in suppressing the innate immune response against arteriviruses and potentially other viruses that encode similar proteases.

Ciclesonide Inhibits SARS-CoV-2 Papain-Like Protease in Vitro.

The emergence of coronavirus disease 2019 (COVID-19), a novel identified pneumonia resulting from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, has significantly impacted and posed significant challenges to human society. The papain-like protease (PLpro) found in the nonstructural protein 3 of SARS-CoV-2 plays a vital role in viral replication. Moreover, PLpro disrupts the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 from host proteins. Consequently, PLpro has emerged as a promising drug target against SARS-CoV-2 infection. Computational studies have reported that ciclesonide can bind to SARS-CoV-2 PLpro. However, the inhibitory effects of ciclenoside on the PLpro have not been experimentally evaluated. Here, we evaluated the inhibitory effects of synthetic glucocorticoids (sGCs), including ciclesonide, on SARS-CoV-2 PLpro in vitro assay. Ciclesonide significantly inhibited the enzymatic activity of PLpro, compared with other sGCs and its IC50 was 18.4 ± 1.89 µM. These findings provide insights into the development of PLpro inhibitors.

Entropy driven cooperativity effect in multi-site drug optimization targeting SARS-CoV-2 papain-like protease.

Papain-like protease (PLpro), a non-structural protein encoded by SARS-CoV-2, is an important therapeutic target. Regions 1 and 5 of an existing drug, GRL0617, can be optimized to produce cooperativity with PLpro binding, resulting in stronger binding affinity. This work investigated the origin of the cooperativity using molecular dynamics simulations combined with the interaction entropy (IE) method. The regions' improvement exhibits cooperativity by calculating the binding free energies between the complex of PLpro-inhibitor. The thermodynamic integration method further verified the cooperativity generated in the drug improvement. To further determine the specific source of cooperativity, enthalpy and entropy in the complexes were calculated using molecular mechanics/generalized Born surface area and IE. The results show that the entropic change is an important contributor to the cooperativity. Our study also identified residues P248, Q269, and T301 that play a significant role in cooperativity. The optimization of the inhibitor stabilizes these residues and minimizes the entropic loss, and the cooperativity observed in the binding free energy can be attributed to the change in the entropic contribution of these residues. Based on our research, the application of cooperativity can facilitate drug optimization, and provide theoretical ideas for drug development that leverage cooperativity by reducing the contribution of entropy through multi-locus binding.

In-cell bioluminescence resonance energy transfer (BRET)-based assay uncovers ceritinib and CA-074 as SARS-CoV-2 papain-like protease inhibitors.

Papain-like protease (PLpro) is an attractive anti-coronavirus target. The development of PLpro inhibitors, however, is hampered by the limitations of the existing PLpro assay and the scarcity of validated active compounds. We developed a novel in-cell PLpro assay based on BRET and used it to evaluate and discover SARS-CoV-2 PLpro inhibitors. The developed assay demonstrated remarkable sensitivity for detecting the reduction of intracellular PLpro activity while presenting high reliability and performance for inhibitor evaluation and high-throughput screening. Using this assay, three protease inhibitors were identified as novel PLpro inhibitors that are structurally disparate from those previously known. Subsequent enzymatic assays and ligand-protein interaction analysis based on molecular docking revealed that ceritinib directly inhibited PLpro, showing high geometric complementarity with the substrate-binding pocket in PLpro, whereas CA-074 methyl ester underwent intracellular hydrolysis, exposing a free carboxyhydroxyl group essential for hydrogen bonding with G266 in the BL2 groove, resulting in PLpro inhibition.

Utilizing sinapic acid as an inhibitory antiviral agent against MERS-CoV PLpro.

Concerns about the social and economic collapse, high mortality rates, and stress on the healthcare system are developing due to the coronavirus onslaught in the form of various species and their variants. In the recent past, infections brought on by coronaviruses severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) as well as middle east respiratory syndrome coronavirus (MERS-CoV) have been reported. There is a severe lack of medications to treat various coronavirus types including MERS-CoV which is hazard to public health due to its ability for pandemic spread by human-to-human transmission. Here, we utilized sinapic acid (SA) against papain-like protease (PLpro), a crucial enzyme involved in MERS-CoV replication, because phytomedicine derived from nature has less well-known negative effects. The thermal shift assay (TSA) was used in the current study to determine whether the drug interact with the recombinant MERS-CoV PLpro. Also, inhibition assay was conducted as the hydrolysis of fluorogenic peptide from the Z-RLRGG-AMC-peptide bond in the presence of SA to determine the level of inhibition of the MERS-CoV PLpro. To study the structural binding efficiency Autodock Vina was used to dock SA to the MERS-CoV PLpro and results were analyzed using PyMOL and Maestro Schrödinger programs. Our results show a convincing interaction between SA and the MERS protease, as SA reduced MERS-CoV PLpro in a dose-dependent way IC50 values of 68.58 µM (of SA). The TSA showed SA raised temperature of melting to 54.61 °C near IC50 and at approximately 2X IC50 concentration (111.5 µM) the Tm for SA + MERS-CoV PLpro was 59.72 °C. SA was docked to MERS-CoV PLpro to identify the binding site. SA bound to the blocking loop (BL2) region of MERS-CoV PLpro interacts with F268, E272, V275, and P249 residues of MERS-CoV PLpro. The effectiveness of protease inhibitors against MERS-CoV has been established and SA is already known for broad range biological activity including antiviral properties; it can be a suitable candidate for anti-MERS-CoV treatment.

Structural and Biochemical Characterization of Porcine Epidemic Diarrhea Virus Papain-Like Protease 2.

Coronaviral papain-like proteases (PLpros) are essential enzymes that mediate not only the proteolytic processes of viral polyproteins during virus replication but also the deubiquitination and deISGylation of cellular proteins that attenuate host innate immune responses. Therefore, PLpros are attractive targets for antiviral drug development. Here, we report the crystal structure of papain-like protease 2 (PLP2) of porcine epidemic diarrhea virus (PEDV) in complex with ubiquitin (Ub). The X-ray structural analyses reveal that PEDV PLP2 interacts with the Ub substrate mainly through the Ub core region and C-terminal tail. Mutations of Ub-interacting residues resulted in a moderately or completely abolished deubiquitinylating function of PEDV PLP2. In addition, our analyses also indicate that 2-residue-extended blocking loop 2 at the S4 subsite contributes to the substrate selectivity and binding affinity of PEDV PLP2. Furthermore, the PEDV PLP2 Glu99 residue, conserved in alphacoronavirus PLpros, was found to govern the preference of a positively charged P4 residue of peptidyl substrates. Collectively, our data provided structure-based information for the substrate binding and selectivity of PEDV PLP2. These findings may help us gain insights into the deubiquitinating (DUB) and proteolytic functions of PEDV PLP2 from a structural perspective. IMPORTANCE Current challenges in coronaviruses (CoVs) include a comprehensive understanding of the mechanistic effects of associated enzymes, including the 3C-like and papain-like proteases. We have previously reported that the PEDV PLP2 exhibits a broader substrate preference, superior DUB function, and inferior peptidase activity. However, the structural basis for these functions remains largely unclear. Here, we show the high-resolution X-ray crystal structure of PEDV PLP2 in complex with Ub. Integrated structural and biochemical analyses revealed that (i) three Ub core-interacting residues are essential for DUB function, (ii) 2-residue-elongated blocking loop 2 regulates substrate selectivity, and (iii) a conserved glutamate residue governs the substrate specificity of PEDV PLP2. Collectively, our findings provide not only structural insights into the catalytic mechanism of PEDV PLP2 but also a model for developing antiviral strategies.

Preparation and characterization of nanobodies targeting SARS-CoV-2 papain-like protease.

Coronavirus Papain-like protease (PLpro) mediates the cleavage of viral polyproteins and assists the virus escaping from innate immune response. Thus, PLpro is an attractive target for the development of broad-spectrum drugs as it has a conserved structure across different coronaviruses. In this study, we purified SARS-CoV-2 PLpro as an immune antigen, constructed a nanobody phage display library, and identified a set of nanobodies with high affinity for SARS-CoV-2. In addition, enzyme activity experiments demonstrated that two nanobodies had a significant inhibitory effect on the PLpro. These nanobodies should therefore be investigated as candidates for the treatment of coronaviruses.

Mass spectrometric assays monitoring the deubiquitinase activity of the SARS-CoV-2 papain-like protease inform on the basis of substrate selectivity and have utility for substrate identification.

The SARS-CoV-2 papain-like protease (PLpro) and main protease (Mpro) are nucleophilic cysteine enzymes that catalyze hydrolysis of the viral polyproteins pp1a/1ab. By contrast with Mpro, PLpro is also a deubiquitinase (DUB) that accepts post-translationally modified human proteins as substrates. Here we report studies on the DUB activity of PLpro using synthetic Nε-lysine-branched oligopeptides as substrates that mimic post-translational protein modifications by ubiquitin (Ub) or Ub-like modifiers (UBLs), such as interferon stimulated gene 15 (ISG15). Mass spectrometry (MS)-based assays confirm the DUB activity of isolated recombinant PLpro. They reveal that the sequence of both the peptide fragment derived from the post-translationally modified protein and that derived from the UBL affects PLpro catalysis; the nature of substrate binding in the S sites appears to be more important for catalytic efficiency than binding in the S' sites. Importantly, the results reflect the reported cellular substrate selectivity of PLpro, i.e. human proteins conjugated to ISG15 are better substrates than those conjugated to Ub or other UBLs. The combined experimental and modelling results imply that PLpro catalysis is affected not only by the identity of the substrate residues binding in the S and S' sites, but also by the substrate fold and the conformational dynamics of the blocking loop 2 of the PLpro:substrate complex. Nε-Lysine-branched oligopeptides thus have potential to help the identification of PLpro substrates. More generally, the results imply that MS-based assays with Nε-lysine-branched oligopeptides have potential to monitor catalysis by human DUBs and hence to inform on their substrate preferences.

A snake venom-analog peptide that inhibits SARS-CoV-2 and papain-like protease displays antithrombotic activity in mice arterial thrombosis model, without interfering with bleeding time.

BACKGROUND: (p-BthTX-I)2 K, a dimeric analog peptide derived from the C-terminal region of phospholipase A2-like bothropstoxin-I (p-BthTX-I), is resistant to plasma proteolysis and inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains with weak cytotoxic effects. Complications of SARS-CoV-2 infection include vascular problems and increased risk of thrombosis; therefore, studies to identify new drugs for treating SARS-CoV-2 infections that also inhibit thrombosis and minimize the risk of bleeding are required. OBJECTIVES: To determine whether (p-BthTX-I)2 K affects the hemostatic system. METHODS: Platelet aggregation was induced by collagen, arachidonic acid, and adenosine diphosphate (ADP) in the Chronolog Lumi-aggregometer. The coagulation activity was evaluated by determining activated partial thromboplastin clotting time (aPTT) and prothrombin time (PT) with (p-BthTX-I)2 K (5.0-434.5 µg) or 0.9% NaCl. Arterial thrombosis was induced with a 540 nm laser and 3.5-20 mg kg- 1 Rose Bengal in the carotid artery of male C57BL/6J mice using (p-BthTX-I)2 K. Bleeding time was determined in mouse tails immersed in saline at 37 °C after (p-BthTX-I)2 K (4.0 mg/kg and 8.0 mg/kg) or saline administration. RESULTS: (p-BthTX-I)2 K prolonged the aPTT and PT by blocking kallikrein and FXa-like activities. Moreover, (p-BthTX-I)2 K inhibited ADP-, collagen-, and arachidonic acid-induced platelet aggregation in a dose-dependent manner. Further, low concentrations of (p-BthTX-I)2 K extended the time to artery occlusion by the formed thrombus. However, (p-BthTX-I)2 K did not prolong the bleeding time in the mouse model of arterial thrombosis. CONCLUSION: These results demonstrate the antithrombotic activity of the peptide (p-BthTX-I)2 K possibly by kallikrein inhibition, suggesting its strong biotechnological potential.

Discovery of new non-covalent and covalent inhibitors targeting SARS-CoV-2 papain-like protease and main protease.

Global coronavirus disease 2019 (COVID-19) pandemic still threatens human health and public safety, and the development of effective antiviral agent is urgently needed. The SARS-CoV-2 main protease (Mpro) and papain-like protease (PLpro) are vital proteins in viral replication and promising therapeutic targets. Additionally, PLpro also modulates host immune response by cleaving ubiquitin and interferon-stimulated gene product 15 (ISG15) from ISGylated host proteins. In this report, we identified [1,2]selenazolo[5,4-c]pyridin-3(2H)-one and benzo[d]isothiazol-3(2H)-one as attractive scaffolds of PLpro and Mpro inhibitors. The representative compounds 6c and 7e exhibited excellent PLpro inhibition with percent inhibition of 42.9% and 44.9% at 50 nM, respectively. The preliminary enzyme kinetics experiment and fluorescent labelling experiment results determined that 6c was identified as a covalent PLpro inhibitor, while 7e was a non-covalent inhibitor. Molecular docking and dynamics simulations revealed that 6c and 7e bound to Zn-finger domain of PLpro. Compounds 6c and 7e were also identified to potent Mpro inhibitors, and they exhibited potent antiviral activities in SARS-CoV-2 infected Vero E6 cells, with EC50 value of 3.9 µM and 7.4 µM, respectively. In addition, the rat liver homogenate half-life of 6c and 7e exceeded 24 h. These findings suggest that 6c and 7e are promising led compounds for further development of PLpro/Mpro dual-target antiviral drugs.

Green and efficient one-pot three-component synthesis of novel drug-like furo[2,3-d]pyrimidines as potential active site inhibitors and putative allosteric hotspots modulators of both SARS-CoV-2 MPro and PLPro.

In this paper, an environmentally benign, convenient, and efficient one-pot three-component reaction has been developed for the regioselective synthesis of novel 5-aroyl(or heteroaroyl)-6-(alkylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-diones (4a‒n) through the sequential condensation of aryl(or heteroaryl)glyoxal monohydrates (1a‒g), 1,3-dimethylbarbituric acid (2), and alkyl(viz. cyclohexyl or tert-butyl)isocyanides (3a or 3b) catalyzed by ultra-low loading ZrOCl2•8H2O (just 2 mol%) in water at 50 ˚C. After synthesis and characterization of the mentioned furo[2,3-d]pyrimidines (4a‒n), their multi-targeting inhibitory properties were investigated against the active site and putative allosteric hotspots of both SARS-CoV-2 main protease (MPro) and papain-like protease (PLPro) based on molecular docking studies and compare the attained results with various medicinal compounds which approximately in three past years were used, introduced, and or repurposed to fight against COVID-19. Furthermore, drug-likeness properties of the mentioned small heterocyclic frameworks (4a‒n) have been explored using in silico ADMET analyses. Interestingly, the molecular docking studies and ADMET-related data revealed that the novel series of furo[2,3-d]pyrimidines (4a‒n), especially 5-(3,4-methylendioxybenzoyl)-6-(cyclohexylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4g) as hit one is potential COVID-19 drug candidate, can subject to further in vitro and in vivo studies. It is worthwhile to note that the protein-ligand-type molecular docking studies on the human body temperature-dependent MPro protein that surprisingly contains zincII (ZnII) ion between His41/Cys145 catalytic dyad in the active site, which undoubtedly can make new plans for designing novel SARS-CoV-2 MPro inhibitors, is performed for the first time in this paper, to the best of our knowledge.

The role of IBV PL1pro in virus replication and suppression of host innate immune responses.

BACKGROUND: Coronavirus papain-like proteases (PLpros) play a crucial role in virus replication and the evasion of the host immune response. Infectious bronchitis virus (IBV) encodes a proteolytically defective remnant of PL1pro and an active PL2pro. However, the function of PL1pro in IBV remains largely unknown. This study aims to explore the effect of PL1pro on virus replication and underlying mechanisms. RESULTS: The recombinant viruses rIBV-ΔPL1pro and rIBV-ΔPL1pro-N were obtained using reverse genetic techniques through the deletion of the IBV PL1pro domain and the N-terminal conserved sequence of PL1pro (PL1pro-N). We observed significantly lower replication of rIBV-ΔPL1pro and rIBV-ΔPL1pro-N than wild-type IBV. Further investigation revealed that the lack of PL1pro-N in IBV decreased virus resistance to interferon (IFN) while also inducing host immune response by enhancing the production of IFN-ß and activating the downstream STAT1 signaling pathway of IFNs. In addition, the overexpression of PL1pro-N significantly suppressed type I IFN response by down-regulating the expressions of genes in the IFN pathway. CONCLUSIONS: Our data demonstrated that IBV PL1pro plays a crucial role in IBV replication and the suppression of host innate immune responses, suggesting that IBV PL1pro could serve as a promising molecular target for antiviral therapy.

Therapeutic potential of metal ions for COVID-19: insights from the papain-like protease of SARS-CoV-2.

Coronaviruses have been responsible for multiple challenging global pandemics, including coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Papain-like protease (PLpro), one of two cysteine proteases responsible for the maturation and infectivity of SARS-CoV-2, processes and liberates functional proteins from the viral polyproteins and cleaves ubiquitin and ISG15 modifications to inhibit innate immune sensing. Consequently, PLpro is an attractive target for developing COVID-19 therapies. PLpro contains a zinc-finger domain important for substrate binding and structural stability. However, the impact of metal ions on the activity and biophysical properties of SARS-CoV-2 PLpro has not been comprehensively studied. Here, we assessed the impacts of metal ions on the catalytic activity of PLpro. Zinc had the largest inhibitory effect on PLpro, followed by manganese. Calcium, magnesium, and iron had smaller or no effects on PLpro activity. EDTA at a concentration of 0.5 mM was essential for PLpro activity, likely by chelating trace metals that inhibit PLpro. IC50 values for ZnCl2, ZnSO4, and MnCl2 of 0.42 ± 0.02 mM, 0.35 ± 0.01 mM, and 2.6 ± 0.3 mM were obtained in the presence of 0.5 mM EDTA; in the absence of EDTA, the estimated IC50 of ZnCl2 was 14 µM. Tryptophan intrinsic fluorescence analysis confirmed the binding of zinc and manganese to PLpro, and differential scanning calorimetry revealed that zinc but not manganese reduced ΔHcal of PLpro. The results of this study provide a reference for further work targeting PLpro to prevent and treat COVID-19.

In Silico Identification of Inhibitory Compounds for SARS-Cov-2 Papain-Like Protease.

Virtual screening with high-performance computers is a powerful and cost-effective technique in drug discovery. A chemical database is searched to find candidate compounds firmly bound to a target protein, judging from the binding poses and/or binding scores. The severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) infectious disease has spread worldwide for the last three years, causing severe slumps in economic and social activities. SARS-Cov-2 has two viral proteases: 3-chymotrypsin-like (3CL) and papain-like (PL) protease. While approved drugs have already been released for the 3CL protease, no approved agent is available for PL protease. In this work, we carried out in silico screening for the PL protease inhibitors, combining docking simulation and molecular mechanics calculation. Docking simulations were applied to 8,820 molecules in a chemical database of approved and investigational compounds. Based on the binding poses generated by the docking simulations, molecular mechanics calculations were performed to optimize the binding structures and to obtain the binding scores. Based on the binding scores, 57 compounds were selected for in vitro assay of the inhibitory activity. Five inhibitory compounds were identified from the in vitro measurement. The predicted binding structures of the identified five compounds were examined, and the significant interaction between the individual compound and the protease catalytic site was clarified. This work demonstrates that computational virtual screening by combining docking simulation with molecular mechanics calculation is effective for searching candidate compounds in drug discovery.

Synthesis and structure-activity relationships of USP48 deubiquitinylase inhibitors.

Ubiquitin-specific proteases represent a family of enzymes that catalyze the cleavage of ubiquitin from specific substrate proteins to regulate their activity. USP48 is a rarely studied USP, which has recently been linked to inflammatory signaling via regulation of the transcription factor nuclear factor kappa B. Nonetheless, a crystal structure of USP48 has not yet been resolved and potent inhibitors are not known. We screened a set of 14 commercially available USP inhibitors for their activity against USP48 and identified the USP2 inhibitor "ML364" as a candidate for further optimization. Using a ligand-based approach, we derived and synthesized a series of ML364 analogs. The IC50 concentrations of the new compounds to inhibit USP48 were determined in a deubiquitinylase activity assay by measuring the fluorescence intensity using tetra-ubiquitin rhodamine110 as substrate. A compound containing a carboxylic acid functionalization (17e) inhibited USP48 activity toward tetra-ubiquitin rhodamine110 with an IC50 of 12.6 µM. Further structure-based refinements are required to improve the inhibition activity and specificity.

Glycyrrhizin as a promising kryptonite against SARS-CoV-2: Clinical, experimental, and theoretical evidences.

COVID-19 is the most devastating disease in recent times affecting most people globally. The higher rate of transmissibility and mutations of SARS-CoV-2 along with the lack of potential therapeutics has made it a global crisis. Potential molecules from natural sources could be a fruitful remedy to combat COVID-19. This systematic review highlights the detailed therapeutic implication of naturally occurring glycyrrhizin and its related derivatives against COVID-19. Glycyrrhizin has already been established for blocking different biomolecular targets related to the SARS-CoV-2 replication cycle. In this article, several experimental and theoretical evidences of glycyrrhizin and related derivatives have been discussed in detail to evaluate their potential as a promising therapeutic strategy against COVID-19. Moreover, the implication of glycyrrhizin in traditional Chinese medicines for alleviating the symptoms of COVID-19 has been reviewed. The potential role of glycyrrhizin and related compounds in affecting various stages of the SARS-CoV-2 life cycle has also been discussed in detail. Derivatization of glycyrrhizin for designing potential lead compounds along with combination therapy with other anti-SARS-CoV-2 agents followed by extensive evaluation may assist in the formulation of novel anti-coronaviral therapy for better treatment to combat COVID-19.

Screening, Synthesis and Biochemical Characterization of SARS-CoV-2 Protease Inhibitors.

The severe acute respiratory syndrome-causing coronavirus 2 (SARS-CoV-2) papain-like protease (PLpro) and main protease (Mpro) play an important role in viral replication events and are important targets for anti-coronavirus drug discovery. In search of these protease inhibitors, we screened a library of 1300 compounds using a fluorescence thermal shift assay (FTSA) and identified 53 hits that thermally stabilized or destabilized PLpro. The hit compounds structurally belonged to two classes of small molecules: thiazole derivatives and symmetrical disulfide compounds. Compound dissociation constants (Kd) were determined using an enzymatic inhibition method. Seven aromatic disulfide compounds were identified as efficient PLpro inhibitors with Kd values in the micromolar range. Two disulfides displayed six-fold higher potency for PLpro (Kd = 0.5 µM) than for Mpro. The disulfide derivatives bound covalently to both proteases, as confirmed through mass spectrometry. The identified compounds can serve as lead compounds for further chemical optimization toward anti-COVID-19 drugs.

Structure-Based Design of Potent Peptidomimetic Inhibitors Covalently Targeting SARS-CoV-2 Papain-like Protease.

The papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a critical role in the proteolytic processing of viral polyproteins and the dysregulation of the host immune response, providing a promising therapeutic target. Here, we report the structure-guide design of novel peptidomimetic inhibitors covalently targeting SARS-CoV-2 PLpro. The resulting inhibitors demonstrate submicromolar potency in the enzymatic assay (IC50 = 0.23 µM) and significant inhibition of SARS-CoV-2 PLpro in the HEK293T cells using a cell-based protease assay (EC50 = 3.61 µM). Moreover, an X-ray crystal structure of SARS-CoV-2 PLpro in complex with compound 2 confirms the covalent binding of the inhibitor to the catalytic residue cysteine 111 (C111) and emphasizes the importance of interactions with tyrosine 268 (Y268). Together, our findings reveal a new scaffold of SARS-CoV-2 PLpro inhibitors and provide an attractive starting point for further optimization.