Papain-like Protease Research Articles

Expression of recombinant SARS-CoV-2 papain-like protease (SARS-CoV-2 PLpro) in Escherichia coli RIPL

The SARS-CoV-2 virus that caused pandemic COVID-19 has spread rapidly to humans and causes a very serious health and social problem around the world. In order to develop vaccine and therapeutic approaches, a comprehensive study of viral structure and viral biological infection needs to be understood. In addition to four structural proteins; spike glycoprotein (S), envelope (E), membrane protein (M), and nucleocapsid protein (N), SARS-CoV-2 viral genome, consist of two open reading frame (ORF) which is located in N-terminus, ORF1a, and ORF1b. The ORF1a consist of Main protease (3CLpro) and together with papain-like protease (PLpro) play a role in the cleavage of polyproteins and translated from viral RNA to mature protein. Here we describe a method of production of 6xHis-tagged Papain-like protease fragment in E. coli RIPL, including gene expression, solubilization of inclusion bodies by different methods, and detection of gene product target by Western Blot Analyses.

Developing drugs against essential proteins of SARS-CoV-2 for the treatment of COVID-19

Infections caused by SARS-CoV-2 are causing catastrophic levels of morbidity in the world and new treatments are greatly needed. Essential proteins involved in the replication and infection of SARS-CoV-2 could be targeted to develop anti-viral drugs for the treatment of COVID-19. Two proteins, Papain-like protease (PLpro) and Non-structural protein 15 (Nsp15) from SARS-CoV-2, are essential for viral replication and evading the host immune response, respectively. PLpro is a crucial protease with deubiquitinating and deISGylating activity while Nsp15 is an uridylate-specific endonuclease which blocks the innate immune response pathways.

SARS-CoV-2 Inhibitors from Nigella Sativa.

The recently encountered severe acute respiratory syndrome coronavirus 2 creates huge predicaments among various countries. Lack of specific treatment of COVID-19 disease demands urgency in drug design against SARS-CoV-2 targets. Nigella sativa the miraculous herb native to South and Southwest Asia and belonging to the family Ranunculaceae, due to its beneficial bioactive properties, was used by us for performing in silico study to analyze the potential of its compounds so that they can target and inhibit SARS-COV-2 proteins including its main protease, the papain-like protease, its helicase, and also the RNA-dependent RNApolymerase, RNA-binding protein, Endoribonuclease, receptor-binding domain, and the RNA-binding domain of nucleocapsid phosphoprotein. The procedure of molecular docking was done with the help of AutoDock–Vina 1.1.2. and along with it the ADMET properties of the best suited ligands were found and Lipinski screening was performed. Among 58 ligands screened, various compounds showed binding energy less than the standard drug chloroquine. Three compounds alpha-hederin, rutin, and nigellamine A2 had the least binding energy with the specific SARS-Cov-2 proteins suggesting their best potential as SARS-CoV-2 inhibitor. Hence, in the future, studies including the in vitro and also the in vivo studies can be carried out for analyzing their true potential and encourage use of nutraceuticals like Nigella sativa to inhibit this virus.

Structure function characterization of SARS CoV2 proteases for COVID19 antiviral development

The COVID-19 pandemic has caused >4 million deaths and crippled the global economy. Effective control of the SARS-CoV2 virus requires development of antiviral therapies. The two proteases encoded in the CoV2 genome, Main protease (Mpro) and papain-like protease (PLpro), are important targets for antiviral development. Here we characterize structure-function relationships of these proteases and assess previously approved drugs as inhibitors of SARS-CoV2. We find different C and N terminal tags significantly affect homodimerization of the Mpro enzyme.

Catalytic function and substrate specificity of alpha and beta coronavirus papain-like proteases: targeting human NL63, SARS-CoV and SARS-CoV-2 (Covid-19) with designed inhibitors

Coronaviruses (CoVs) are single-stranded, positive-sense RNA viruses in the Coronaviridae family. Within thisfamily are four genera: Alpha-, Beta-, Gamma-, and Delta Coronavirus, with Alpha- and Beta- containing coronaviruses that infect humans and animals. NL63 is an Alpha-CoV causing 15-30% of severe upper and lower respiratory tract infections each year. Severe Acute Respiratory Syndrome (SARS) and SARS-CoV-2 are Beta-CoVs that have affected many people worldwide during their outbreaks in 2002-2003 (SARS-CoV) and now in 2019-2020 (SARS-CoV-2).

Inhibitory efficacy of 2, 4-diacetylphloroglucinol against SARS-COV-2 proteins: in silico study.

The pandemic of Severe Acute Respiratory Syndrome Coronavirus-2 has affected millions of people worldwide with common symptoms of fever, cough, and respiratory complications. The pandemic has posed a huge challenge to emergency health services due to unavailability of potent therapeutic drugs. The proteins associated with the viral pathogenesis has been identified as suitable targets for drug design and warrants effective drug discovery to abate COVID-19. The papain-like protease (PLpro), nucleocapsid (N), main protease (Mpro) and non-structural protein (nsp12) of SARS-CoV-2, key component of processing of viral polyproteins, transcription, assembly and replication. On this streak, present study evaluated the interaction of ligand 2,4-diacetylphloroglucinol (DAPG) with viral proteins using molecular docking with (i) AutoDock 4.2.6 and (ii) AutoDock Vina followed by molecular dynamic simulation studies of protein-ligand complex configuration. The analysis revealed that PLpro (3E9S) and N (4J3K) protein corresponds to the highest docking score and therefore, selected for molecular dynamics simulation study (100 ns). The study comprised analysis of parameters: (i) RMSD and RMSF, (ii) radius of gyration- which indicated interaction of protein entities with ligand supported steadiness of the complex, (iii) Coulombic and Lennard–Jones interactions, which played a significant role in complex stability. DAPG showed a good number of H-bonds with PLpro and MM-PBSA binding energy when compared to the N protein. This study showed DAPG as a potential bioactive molecule to act as an inhibitor for the PLpro thereby, DAPG can be used as potential inhibitor against SARS-CoV-2 and is potential drug candidate against COVID-19.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11756-021-00979-4.

Autoproteolysis of Procerain and Procerain B mediated by structural changes

Procerain (Pc) and Procerain B (PcB) are two latex proteases from Calotropis procera having potential applications in food and other industries. However, autolytic degradation of these proteases limits their potential use in industry. Nevertheless, basic mechanism underlying the autoproteolysis has not been detailed. In order to understand the same, we subjected the enzymes to various denaturing and activating conditions. The results showed that structural changes induced by different denaturing conditions trigger their autoproteolysis. We also observed differential response of Pc, PcB and other papain-like proteases towards autocatalysis in presence of reducing agent in-spite of sharing the same structural fold, including the number of disulfide bonds. The possible reason underlying this intriguing observation is also discussed. Further, present work establishes that structural changes in the proteases lead to autoproteolysis and the enzymes are stable unless they experience structural perturbation. These findings could thus be useful for their practical applications in industries.

Validation and Invalidation of SARS-CoV-2 Papain-like Protease Inhibitors.

SARS-CoV-2 encodes two viral cysteine proteases, the main protease (Mpro) and the papain-like protease (PLpro), both of which are validated antiviral drug targets. PLpro is involved in the cleavage of viral polyproteins as well as immune modulation by removing ubiquitin and interferon-stimulated gene product 15 (ISG15) from host proteins. Therefore, targeting PLpro might be a two-pronged approach. Several compounds including YM155, cryptotanshinone, tanshinone I, dihydrotanshinone I, tanshinone IIA, SJB2-043, 6-thioguanine, and 6-mercaptopurine were recently identified as SARS-CoV-2 PLpro inhibitors through high-throughput screenings. In this study, we aim to validate/invalidate the reported PLpro inhibitors using a combination of PLpro target-specific assays including enzymatic FRET assay, thermal shift binding assay (TSA), and cell-based FlipGFP assay. Collectively, our results showed that all compounds tested either did not show binding or led to denaturation of PLpro in the TSA binding assay, which might explain their weak enzymatic inhibition in the FRET assay. In addition, none of the compounds showed cellular PLpro inhibition as revealed by the FlipGFP assay. Therefore, more efforts are needed to search for potent and specific SARS-CoV-2 PLpro inhibitors.

SARS-COV-2 papain-like protease: Conformational changes in BL2' regions induced by PLpro inhibitors

SARS-COV-2 papain-like protease: Conformational changes in BL2' regions induced by PLpro inhibitors

Inhibitor induced conformational changes in SARS-COV-2 papain-like protease

Inhibitor induced conformational changes in SARS-COV-2 papain-like protease

Modelling the DFT structural and reactivity study of feverfew and evaluation of its potential antiviral activity against COVID-19 using molecular docking and MD simulations.

The unavailability of a proper drug against SARS-CoV-2 infections and the emergence of various variants created a global crisis. In the present work, we have studied the antiviral behavior of feverfew plant in treating COVID-19. We have reported a systematic in silico study with the antiviral effects of various phytoconstituents Borneol (C10H18O), Camphene (C10H16), Camphor (C10H16O), Alpha-thujene (C10H16), Eugenol (C10H14O), Carvacrol (C10H14O) and Parthenolide (C15H20O3) of feverfew on the viral protein of SARS-CoV-2. Parthenolide shows the best binding affinity with both main protease (Mpro) and papain-like protease (PLpro). The molecular electrostatic potential and Mulliken atomic charges of the Parthenolide molecule shows the high chemical reactivity of the molecule. The docking of Parthenolide with PLpro give score of −8.0 kcal/mol that validates the good binding of Parthenolide molecule with PLpro. This complex was further considered for molecular dynamics simulations. The binding energy of the complex seems to range in between −3.85 to −11.07 kcal/mol that is high enough to validate the stability of the complex. Free energy decomposition analysis have been also performed to understand the contribution of residues that reside into the binding site. Good binding affinity and reactivity response suggested that Parthenolide can be used as a promising drug against the COVID-19.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1007/s11696-022-02067-6.

A Dual-Color Fluorescent Probe Allows Simultaneous Imaging of Main and Papain-like Proteases of SARS-CoV-2-Infected Cells for Accurate Detection and Rapid Inhibitor Screening.

The main protease (Mpro) and papain‐like protease (PLpro) play critical roles in SARS‐CoV‐2 replication and are promising targets for antiviral inhibitors. The simultaneous visualization of Mpro and PLpro is extremely valuable for SARS‐CoV‐2 detection and rapid inhibitor screening. However, such a crucial investigation has remained challenging because of the lack of suitable probes. We have now developed a dual‐color probe (3MBP5) for the simultaneous detection of Mpro and PLpro by fluorescence (or Förster) resonance energy transfer (FRET). This probe produces fluorescence from both the Cy3 and Cy5 fluorophores that are cleaved by Mpro and PLpro. 3MBP5‐activatable specificity was demonstrated with recombinant proteins, inhibitors, plasmid‐transfected HEK 293T cells, and SARS‐CoV‐2‐infected TMPRSS2‐Vero cells. Results from the dual‐color probe first verified the simultaneous detection and intracellular distribution of SARS‐CoV‐2 Mpro and PLpro. This is a powerful tool for the simultaneous detection of different proteases with value for the rapid screening of inhibitors.

Drug Repurposing for the Identification of Compounds with Anti-SARS-CoV-2 Capability via Multiple Targets.

Since 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been rapidly spreading worldwide, causing hundreds of millions of infections. Despite the development of vaccines, insufficient protection remains a concern. Therefore, the screening of drugs for the treatment of coronavirus disease 2019 (COVID-19) is reasonable and necessary. This study utilized bioinformatics for the selection of compounds approved by the U.S. Food and Drug Administration with therapeutic potential in this setting. In addition, the inhibitory effect of these compounds on the enzyme activity of transmembrane protease serine 2 (TMPRSS2), papain-like protease (PLpro), and 3C-like protease (3CLpro) was evaluated. Furthermore, the capability of compounds to attach to the spike-receptor-binding domain (RBD) was considered an important factor in the present assessment. Finally, the antiviral potency of compounds was validated using a plaque reduction assay. Our funnel strategy revealed that tamoxifen possesses an anti-SARS-CoV-2 property owing to its inhibitory performance in multiple assays. The proposed time-saving and feasible strategy may accelerate drug screening for COVID-19 and other diseases.

Acriflavine, a clinically approved drug, inhibits SARS-CoV-2 and other betacoronaviruses

The COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort and available vaccines, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identify acriflavine (ACF) as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.

Liposome-Mediated Delivery of MERS Antigen Induces Potent Humoral and Cell-Mediated Immune Response in Mice.

The advancements in the field of nanotechnology have provided a great platform for the development of effective antiviral vaccines. Liposome-mediated delivery of antigens has been shown to induce the antigen-specific stimulation of the humoral and cell-mediated immune responses. Here, we prepared dried, reconstituted vesicles (DRVs) from DPPC liposomes and used them as the vaccine carrier system for the Middle East respiratory syndrome coronavirus papain-like protease (DRVs-MERS-CoV PLpro). MERS-CoV PLpro emulsified in the Incomplete Freund’s Adjuvant (IFA-MERS-CoV PLpro) was used as a control. Immunization of mice with DRVs-MERS-CoV PLpro did not induce any notable toxicity, as revealed by the levels of the serum alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN) and lactate dehydrogenase (LDH) in the blood of immunized mice. Immunization with DRVs-MERS-CoV PLpro induced greater antigen-specific antibody titer and switching of IgG1 isotyping to IgG2a as compared to immunization with IFA-MERS-CoV PLpro. Moreover, splenocytes from mice immunized with DRVs-MERS-CoV PLpro exhibited greater proliferation in response to antigen stimulation. Moreover, splenocytes from DRVs-MERS-CoV PLpro-immunized mice secreted significantly higher IFN-γ as compared to splenocytes from IFA-MERS-CoV PLpro mice. In summary, DRVs-MERS-CoV PLpro may prove to be an effective prophylactic formulation to prevent MERS-CoV infection.

Design and Evaluation of a Novel Peptide-Drug Conjugate Covalently Targeting SARS-CoV-2 Papain-like Protease.

Coronavirus disease 2019 (COVID-19) pandemic, a global health threat, was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 papain-like cysteine protease (PLpro) was recognized as a promising drug target because of multiple functions in virus maturation and antiviral immune responses. Inhibitor GRL0617 occupied the interferon-stimulated gene 15 (ISG15) C-terminus-binding pocket and showed an effective antiviral inhibition. Here, we described a novel peptide–drug conjugate (PDC), in which GRL0617 was linked to a sulfonium-tethered peptide derived from PLpro-specific substrate LRGG. The EM-C and EC-M PDCs showed a promising in vitro IC50 of 7.40 ± 0.37 and 8.63 ± 0.55 μM, respectively. EC-M could covalently label PLpro active site C111 and display anti-ISGylation activities in cellular assays. The results represent the first attempt to design PDCs composed of stabilized peptide inhibitors and GRL0617 to inhibit PLpro. These novel PDCs provide promising opportunities for antiviral drug design.

The Role of Proteases and Serpin Protease Inhibitors in β-Cell Biology and Diabetes.

Regulation of the equilibrium between proteases and their inhibitors is fundamental to health maintenance. Consequently, developing a means of targeting protease activity to promote tissue regeneration and inhibit inflammation may offer a new strategy in therapy development for diabetes and other diseases. Specifically, recent efforts have focused on serine protease inhibitors, known as serpins, as potential therapeutic targets. The serpin protein family comprises a broad range of protease inhibitors, which are categorized into 16 clades that are all extracellular, with the exception of Clade B, which controls mostly intracellular proteases, including both serine- and papain-like cysteine proteases. This review discusses the most salient, and sometimes opposing, views that either inhibition or augmentation of protease activity can bring about positive outcomes in pancreatic islet biology and inflammation. These potential discrepancies can be reconciled at the molecular level as specific proteases and serpins regulate distinct signaling pathways, thereby playing equally distinct roles in health and disease development.

<i>In Silico</i> Evaluation for the Inhibitory Action of Curcumin Derivatives on the SARS-CoV-2 Proteins

Purpose of the Study: COVID-19 is caused by the SARS-CoV-2 virus that had a global pandemic spread in the last two years. Symptoms of the disease include respiratory distress and, in severe cases may consequently lead to death. Blocking the viral proteins can aid in treating this disease and alleviating its symptoms. Two target proteins of the coronavirus that are hot spots in drug discovery are the papain-like protease PL-pro and the main protease M-pro. PL-pro is an enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread. M-pro is the major protease of SARS-CoV-2, which is a keystone in viral replication and transcription. Methods: In this study, we shed the light on the route of targeting viral proteins for disease alleviation, by targeting the two aforementioned enzymes, PL-pro and M-pro using in silico studies. Docking experiments, using AutoDock algorithms, were performed to predict the inhibitory effect of recently produced synthetic derivatives of curcumin on the viral proteins. Results: Most of the curcumin derivatives have shown variable levels of inhibition, e.g., S1 - S6, mainly on the papain-like protease, and to a lesser extent on the main protease. Conclusion: The results indicated that curcumin derivatives can be potent anti-viral drug of SARS-CoV-2, namely targeting the papain-like protease.

Pharmacoinformatic study of inhibitory potentials of selected flavonoids against papain-like protease and 3-chymotrypsin-like protease of SARS-CoV-2

BackgroundInhibition of papain-like protease (PLpro) and 3-chymotrypsin-like protease (3CLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is projected to terminate its replication. Hence, these proteases represent viable therapeutic targets.MethodsSixty-one flavonoids with reported activities against other RNA viruses were selected and docked in PLpro and 3CLpro. Flavonoids with better binding energies compared to reference inhibitors (lopinavir and ritonavir) in their interaction with PLpro and 3CLpro were selected for drug-likeness and ADMET analysis. The best representative flavonoid for each protease from the ADMET filtering analysis was subjected to molecular dynamics simulations (MDS) and clustering analysis of the trajectory files.ResultsLicorice, ugonin M, procyanidin, silymarin, and gallocatechin gallate had better binding energies (-11.8, -10.1, -9.8, -9.7 and -9.6 kcal/mol respectively) with PLpro compared to lopinavir and ritonavir (-9.1 and -8.5 kcal/mol respectively). Also, isonymphaeol B, baicalin, abyssinone II, tomentin A, and apigetrin had better binding energies (-8.7, -8.3, -8.2, -8.1, and -8.1 kcal/mol respectively) with 3CLpro compared to lopinavir and ritonavir (-7.3 and -7.1 kcal/mol respectively). These flavonoids interacted with the proteases via hydrogen and non-hydrogen bonding. Of these flavonoids, silymarin and isonymphaeol B demonstrated most favourable combination of attributes in terms of binding energies, compliance with Lipinski rule for drug-likeness and favourable pharmacokinetics in silico. These two flavonoids exhibited appreciable degree of structural stability, maintaining strong interaction with residues in the different representative clusters selected during the MDS run.ConclusionSilymarin and isonymphaeol B are proposed for further studies as compounds with potential activities against SARS-CoV-2.

Production of a functionally active recombinant SARS-CoV-2 (COVID-19) 3C-like protease and a soluble inactive 3C-like protease-RBD chimeric in a prokaryotic expression system.

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) intracellular life-cycle, two large polyproteins, pp1a and pp1ab, are produced. Processing of these by viral cysteine proteases, the papain-like protease (PLpro) and the chymotrypsin-like 3C-like protease (3CL-pro) release non-structural proteins necessary for the establishment of the viral replication and transcription complex (RTC), crucial for viral replication. Hence, these proteases are considered prime targets against which anti-coronavirus disease 2019 (COVID-19) drugs could be developed. Here, we describe the expression of a highly soluble and functionally active recombinant 3CL-pro using Escherichia coli BL21 cells. We show that the enzyme functions in a dimeric form and exhibits an unexpected inhibitory profile because its activity is potently blocked by serine rather than cysteine protease inhibitors. In addition, we assessed the ability of our 3CL-pro to function as a carrier for the receptor binding domain (RBD) of the Spike protein. The co-expressed chimeric protein, 3CLpro-RBD, did not exhibit 3CL-pro activity, but its enhanced solubility made purification easier and improved RBD antigenicity when tested against serum from vaccinated individuals in ELISAs. Chimeric proteins containing the 3CL-pro could represent an innovative approach to developing new COVID-19 vaccines.