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PLoS Pathogens Mar 2024The coronavirus papain-like protease (PLpro) is crucial for viral replicase polyprotein processing. Additionally, PLpro can subvert host defense mechanisms by its...
The coronavirus papain-like protease (PLpro) is crucial for viral replicase polyprotein processing. Additionally, PLpro can subvert host defense mechanisms by its deubiquitinating (DUB) and deISGylating activities. To elucidate the role of these activities during SARS-CoV-2 infection, we introduced mutations that disrupt binding of PLpro to ubiquitin or ISG15. We identified several mutations that strongly reduced DUB activity of PLpro, without affecting viral polyprotein processing. In contrast, mutations that abrogated deISGylating activity also hampered viral polyprotein processing and when introduced into the virus these mutants were not viable. SARS-CoV-2 mutants exhibiting reduced DUB activity elicited a stronger interferon response in human lung cells. In a mouse model of severe disease, disruption of PLpro DUB activity did not affect lethality, virus replication, or innate immune responses in the lungs. This suggests that the DUB activity of SARS-CoV-2 PLpro is dispensable for virus replication and does not affect innate immune responses in vivo. Interestingly, the DUB mutant of SARS-CoV replicated to slightly lower titers in mice and elicited a diminished immune response early in infection, although lethality was unaffected. We previously showed that a MERS-CoV mutant deficient in DUB and deISGylating activity was strongly attenuated in mice. Here, we demonstrate that the role of PLpro DUB activity during infection can vary considerably between highly pathogenic coronaviruses. Therefore, careful considerations should be taken when developing pan-coronavirus antiviral strategies targeting PLpro.
Topics: Humans; Animals; Mice; Coronavirus Papain-Like Proteases; COVID-19; SARS-CoV-2; Immunity, Innate; Papain; Peptide Hydrolases; Virus Replication; Polyproteins
PubMed: 38527094
DOI: 10.1371/journal.ppat.1012100 -
Biomedicine & Pharmacotherapy =... Apr 2024Several investigational nitric oxide donors were originally created to correct vascular endothelial dysfunction in cardiovascular diseases. These 48 compounds contain an...
Several investigational nitric oxide donors were originally created to correct vascular endothelial dysfunction in cardiovascular diseases. These 48 compounds contain an urea-like moiety attached to the well-known NO donors isosorbide 2- and 5-mononitrate. CR-0305 and CR-0202 were synthesized and found to be nontoxic in the cell lines HMEC-1, A549/hACE2 and VeroE6. CR-0305 induced vasodilation in human coronary arteries ex vivo. Since NO can also have antiviral properties, a study of drug-protein interactions with SARS-CoV-2 was undertaken using in silico modeling. CR-0305 experimentally outperformed the other compounds, including CR-0202, in binding the catalytic site of SARS-CoV-2 papain-like protease (PL). PL is a primary target for therapeutic inhibition of SARS-CoV-2 as it mediates viral replication and modulates host innate immune responses. CR-0305 is predicted to sit firmly in the PL catalytic pocket as confirmed by molecular dynamics simulations, wherein stability of binding to the catalytic site of PL induces a conformational change in the BL2 loop to a more closed conformation as observed previously with GRL0617. Surface plasmon resonance was performed with CR-0305 and CR-0202 to characterize binding affinity to purified SARS-CoV-2 PL protein. CR-0305 and CR-0202 also inhibited SARS-CoV-2 infection compared to vehicle as measured by virus N protein staining with a specific antibody in A549-ACE2 and VeroE6 cells at 20 µM. CR-0305 is a coronary vasodilator that appears to bind to the catalytic site of the PL of SARS-CoV-2 while targeting delivery of antiviral NO to cells infected by SARS-CoV-2, suggesting multiple indications for future development.
Topics: Humans; Peptide Hydrolases; Papain; SARS-CoV-2; Nitric Oxide Donors; COVID-19; Vasodilator Agents; Antiviral Agents; Protease Inhibitors; Molecular Docking Simulation
PubMed: 38492437
DOI: 10.1016/j.biopha.2024.116378 -
Saudi Pharmaceutical Journal : SPJ :... Apr 2024Concerns 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...
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 IC values of 68.58 μM (of SA). The TSA showed SA raised temperature of melting to 54.61 °C near IC and at approximately 2X IC 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.
PubMed: 38487020
DOI: 10.1016/j.jsps.2024.101986 -
PLoS Pathogens Mar 2024Papain-like cysteine proteases (PLCPs) play pivotal roles in plant defense against pathogen invasions. While pathogens can secrete effectors to target and inhibit PLCP...
Papain-like cysteine proteases (PLCPs) play pivotal roles in plant defense against pathogen invasions. While pathogens can secrete effectors to target and inhibit PLCP activities, the roles of PLCPs in plant-virus interactions and the mechanisms through which viruses neutralize PLCP activities remain largely uncharted. Here, we demonstrate that the expression and activity of a maize PLCP CCP1 (Corn Cysteine Protease), is upregulated following sugarcane mosaic virus (SCMV) infection. Transient silencing of CCP1 led to a reduction in PLCP activities, thereby promoting SCMV infection in maize. Furthermore, the knockdown of CCP1 resulted in diminished salicylic acid (SA) levels and suppressed expression of SA-responsive pathogenesis-related genes. This suggests that CCP1 plays a role in modulating the SA signaling pathway. Interestingly, NIa-Pro, the primary protease of SCMV, was found to interact with CCP1, subsequently inhibiting its protease activity. A specific motif within NIa-Pro termed the inhibitor motif was identified as essential for its interaction with CCP1 and the suppression of its activity. We have also discovered that the key amino acids responsible for the interaction between NIa-Pro and CCP1 are crucial for the virulence of SCMV. In conclusion, our findings offer compelling evidence that SCMV undermines maize defense mechanisms through the interaction of NIa-Pro with CCP1. Together, these findings shed a new light on the mechanism(s) controlling the arms races between virus and plant.
Topics: Zea mays; Cysteine Proteases; Salicylic Acid; Potyvirus; Mosaic Viruses; Plant Diseases
PubMed: 38484013
DOI: 10.1371/journal.ppat.1012086 -
Journal of Virology Apr 2024As for all single-stranded, positive-sense RNA (+RNA) viruses, intracellular RNA synthesis relies on extensive remodeling of host cell membranes that leads to the...
UNLABELLED
As for all single-stranded, positive-sense RNA (+RNA) viruses, intracellular RNA synthesis relies on extensive remodeling of host cell membranes that leads to the formation of specialized structures. In the case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus causing COVID-19, endoplasmic reticulum membranes are modified, resulting in the formation of double-membrane vesicles (DMVs), which contain the viral dsRNA intermediate and constitute membrane-bound replication organelles. The non-structural and transmembrane protein nsp3 is a key player in the biogenesis of DMVs and, therefore, represents an interesting antiviral target. However, as an integral transmembrane protein, it is challenging to express for structural biology. The C-terminus of nsp3 encompasses all the membrane-spanning, -interacting, and -remodeling elements. By using a cell-free expression system, we successfully produced the C-terminal region of nsp3 (nsp3C) and reconstituted purified nsp3C into phospholipid nanodiscs, opening the way for structural studies. Negative-stain transmission electron microscopy revealed the presence of nsp3C oligomers very similar to the region abutting and spanning the membrane on the cytosolic side of DMVs in a recent subtomogram average of the SARS-CoV-2 nsp3-4 pore (1). AlphaFold-predicted structural models fit particularly well with our experimental data and support a pore-forming hexameric assembly. Altogether, our data give unprecedented clues to understand the structural organization of nsp3, the principal component that shapes the molecular pore that spans the DMVs and is required for the export of RNA .
IMPORTANCE
Membrane remodeling is at the heart of intracellular replication for single-stranded, positive-sense RNA viruses. In the case of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this leads to the formation of a network of double-membrane vesicles (DMVs). Targeting DMV biogenesis offers promising prospects for antiviral therapies. This requires a better understanding of the molecular mechanisms and proteins involved. Three non-structural proteins (nsp3, nsp4, and nsp6) direct the intracellular membrane rearrangements upon SARS-CoV-2 infection. All of them contain transmembrane helices. The nsp3 component, the largest and multi-functional protein of the virus, plays an essential role in this process. Aiming to understand its structural organization, we used a cell-free protein synthesis assay to produce and reconstitute the C-terminal part of nsp3 (nsp3C) including transmembrane domains into phospholipid nanodiscs. Our work reveals the oligomeric organization of one key player in the biogenesis of SARS-CoV-2 DMVs, providing basis for the design of future antiviral strategies.
Topics: Humans; Coronavirus Papain-Like Proteases; COVID-19; Endoplasmic Reticulum; Phospholipids; RNA, Viral; SARS-CoV-2; Viral Nonstructural Proteins; Virus Replication
PubMed: 38483167
DOI: 10.1128/jvi.01575-23 -
Molecules (Basel, Switzerland) Feb 2024We provide promising computational (in silico) data on phytochemicals (compounds -) from Arabian Peninsula medicinal plants as strong binders, targeting...
Identification of Phytochemicals from Arabian Peninsula Medicinal Plants as Strong Binders to SARS-CoV-2 Proteases (3CL and PL) by Molecular Docking and Dynamic Simulation Studies.
We provide promising computational (in silico) data on phytochemicals (compounds -) from Arabian Peninsula medicinal plants as strong binders, targeting 3-chymotrypsin-like protease (3CL) and papain-like proteases (PL) of SARS-CoV-2. Compounds - followed the Lipinski rules of five (RO5) and ADMET analysis, exhibiting drug-like characters. Non-covalent (reversible) docking of compounds - demonstrated their binding with the catalytic dyad (CYS145 and HIS41) of 3CL and catalytic triad (CYS111, HIS272, and ASP286) of PL. Moreover, the implementation of the covalent (irreversible) docking protocol revealed that only compounds , , and possess covalent warheads, which allowed the formation of the covalent bond with the catalytic dyad (CYS145) in 3CL and the catalytic triad (CYS111) in PL. Root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and radius of gyration (Rg) analysis from molecular dynamic (MD) simulations revealed that complexation between ligands (compounds , , and ) and 3CL and PL was stable, and there was less deviation of ligands. Overall, the in silico data on the inherent properties of the above phytochemicals unravel the fact that they can act as reversible inhibitors for 3CL and PL. Moreover, compounds , , and also showed their novel properties to inhibit dual targets by irreversible inhibition, indicating their effectiveness for possibly developing future drugs against SARS-CoV-2. Nonetheless, to confirm the theoretical findings here, the effectiveness of the above compounds as inhibitors of 3CL and PL warrants future investigations using suitable in vitro and in vivo tests.
Topics: Peptide Hydrolases; Molecular Docking Simulation; SARS-CoV-2; Plants, Medicinal; COVID-19; Papain; Molecular Dynamics Simulation; Phytochemicals; Antiviral Agents; Protease Inhibitors
PubMed: 38474509
DOI: 10.3390/molecules29050998 -
International Journal of Molecular... Mar 2024Since the appearance of SARS-CoV-2 in 2019, the ensuing COVID-19 (Corona Virus Disease 2019) pandemic has posed a significant threat to the global public health system,... (Review)
Review
Since the appearance of SARS-CoV-2 in 2019, the ensuing COVID-19 (Corona Virus Disease 2019) pandemic has posed a significant threat to the global public health system, human health, life, and economic well-being. Researchers worldwide have devoted considerable efforts to curb its spread and development. The latest studies have identified five viral proteins, spike protein (Spike), viral main protease (3CLpro), papain-like protease (PLpro), RNA-dependent RNA polymerase (RdRp), and viral helicase (Helicase), which play crucial roles in the invasion of SARS-CoV-2 into the human body and its lifecycle. The development of novel anti-SARS-CoV-2 drugs targeting these five viral proteins holds immense promise. Therefore, the development of efficient, high-throughput screening methodologies specifically designed for these viral proteins is of utmost importance. Currently, a plethora of screening techniques exists, with fluorescence-based assays emerging as predominant contenders. In this review, we elucidate the foundational principles and methodologies underpinning fluorescence-based screening approaches directed at these pivotal viral targets, hoping to guide researchers in the judicious selection and refinement of screening strategies, thereby facilitating the discovery and development of lead compounds for anti-SARS-CoV-2 pharmaceuticals.
Topics: Humans; COVID-19; SARS-CoV-2; Viral Proteins; Antiviral Agents; Peptide Hydrolases
PubMed: 38474097
DOI: 10.3390/ijms25052850 -
Foods (Basel, Switzerland) Mar 2024Papain-like cysteine proteases are widespread and can be detected in all domains of life. They share structural and enzymatic properties with the group's namesake... (Review)
Review
Papain-like cysteine proteases are widespread and can be detected in all domains of life. They share structural and enzymatic properties with the group's namesake member, papain. They show a broad range of protein substrates and are involved in several biological processes. These proteases are widely exploited for food, pharmaceutical, chemical and cosmetic biotechnological applications. However, some of them are known to cause allergic reactions. In this context, the objective of this review is to report an overview of some general properties of papain-like cysteine proteases and to highlight their contributions to allergy reactions observed in humans. For instance, the literature shows that their proteolytic activity can cause an increase in tissue permeability, which favours the crossing of allergens through the skin, intestinal and respiratory barriers. The observation that allergy to PLCPs is mostly detected for inhaled proteins is in line with the reports describing mite homologs, such as Der p 1 and Der f 1, as major allergens showing a frequent correlation between sensitisation and clinical allergic reactions. In contrast, the plant food homologs are often digested in the gastrointestinal tract. Therefore, they only rarely can cause allergic reactions in humans. Accordingly, they are reported mainly as a cause of occupational diseases.
PubMed: 38472904
DOI: 10.3390/foods13050790 -
Dental and Medical Problems 2024The dentin substrate can be modified by proteolytic agents, which may affect the bonding strength of adhesive systems to the treated dentin surface. Papain, a cysteine... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
The dentin substrate can be modified by proteolytic agents, which may affect the bonding strength of adhesive systems to the treated dentin surface. Papain, a cysteine protease enzyme with antibacterial and anti-inflammatory properties, can be used for deproteinization of dentin. An alternative deproteinizing enzyme is bromelain.
OBJECTIVES
This study aimed to evaluate the impact of deproteinization on the shear bond strength (SBS) of composite resin to deep dentin using different concentrations of bromelain and papain.
MATERIAL AND METHODS
Sixty upper premolars were extracted and randomly divided into 5 groups (n = 12 per group). In all groups, the dentin surface was etched with 37% phosphoric acid. Group 1 did not receive any enzyme treatment, group 2 was treated with a 10% papain solution, group 3 was treated with a 15% papain solution, group 4 was treated with a 6% bromelain solution, and group 5 was treated with a 10% bromelain solution. After applying an etch-and-rinse adhesive system, the specimens were restored with composite resin and the SBS was measured.
RESULTS
Statistically significant differences were found between groups 2 and 3 (10% papain and 15% papain, p = 0.004), groups 2 and 4 (10% papain and 6% bromelain, p = 0.017), groups 4 and 5 (6% bromelain and 10% bromelain, p = 0.021), and groups 3 and 5 (15% papain and 10% bromelain, p = 0.005).
CONCLUSIONS
Deproteinization with papain and bromelain at different concentrations after acid etching did not affect the SBS of composite resin to deep dentin when using an etch-and-rinse adhesive system. However, the group deproteinized with 15% papain demonstrated a higher SBS than the group deproteinized with 10% papain, and the group deproteinized with 6% bromelain showed a higher SBS compared to the group deproteinized with 10% bromelain.
Topics: Humans; Anti-Bacterial Agents; Bromelains; Composite Resins; Dentin; Papain
PubMed: 38441350
DOI: 10.17219/dmp/133404 -
PeerJ 2024(HI), commonly known as the black soldier fly, has been recognized for its prowess in resource utilization and environmental protection because of its ability to...
BACKGROUND
(HI), commonly known as the black soldier fly, has been recognized for its prowess in resource utilization and environmental protection because of its ability to transform organic waste into animal feed for livestock, poultry, and aquaculture. However, the potential of the black soldier fly's high protein content for more than cheap feedstock is still largely unexplored.
METHODS
This study innovatively explores the potential of larvae (HIL) protein as a peptone substitute for microbial culture media. Four commercial proteases (alkaline protease, trypsin, trypsase, and papain) were explored to hydrolyze the defatted HIL, and the experimental conditions were optimized response surface methodology experimental design. The hydrolysate of the defatted HIL was subsequently vacuum freeze-dried and deployed as a growth medium for three bacterial strains (, , and ) to determine the growth kinetics between the HIL peptone and commercial peptone.
RESULTS
The optimal conditions were 1.70% w/w complex enzyme (alkaline protease: trypsin at 1:1 ratio) at pH 7.0 and 54 °C for a duration of 4 h. Under these conditions, the hydrolysis of defatted HIL yielded 19.25% ±0.49%. A growth kinetic analysis showed no significant difference in growth parameters (, , and ) between the HIL peptone and commercial peptone, demonstrating that the HIL hydrolysate could serve as an effective, low-cost alternative to commercial peptone. This study introduces an innovative approach to HIL protein resource utilization, broadening its application beyond its current use in animal feed.
Topics: Animals; Peptones; Trypsin; Hydrolysis; Kinetics; Diptera; Larva; Culture Media
PubMed: 38426145
DOI: 10.7717/peerj.16995