Papain-like Protease Research Articles

In silico Molecular Docking of SARS-CoV-2 Surface Proteins with Benzimidazole Scaffolds: Strategy to Discover Lead Compounds

Background: The severe acute respiratory illness that was brought on because of the outbreak of COVID-19 caused by the SARS-CoV-2 infection has been designated as a public health emergency of worldwide concern. There is an immediate and pressing need to establish an effective therapeutic strategy to bring infections under control. COVID-19 viral spike glycoproteins and proteases both play important roles in the process of viral entrance as well as in the process of virus reproduction. Methods: Benzimidazole derivatives show antiviral activity against various RNA and DNA viruses and stop the early viral replication cycle. Based on this information, we designed eighteen new benzimidazole derivatives and screened them against the proteins S-glycoprotein 6VSB and papain-like protease 6W9C using molecular docking studies. Compounds that bind strongly to these proteins were evaluated again in an in vitro study. Results: When docked with SARS-CoV-2 spike glycoprotein, the binding affinity of R1 and R7 was –7.1 kcal/mol and -7.3 kcal/mol, respectively. This showed that they might be able to stop the SARS spike protein from binding to the ACE2 receptor on the human host, making it harder for the virus to get into the cells. The binding affinity of SARS-CoV-2 papain-like protease with R4, R14, and R15 was –6.7 kcal/mol, -6.5 kcal/mol, and –6.5 kcal/mol, respectively. COVID-19 could stop the protease from working by binding it. Conclusion: It was suggested, on the basis of the binding energy score, that these pharmacologically potent benzimidazole derivatives may be tested against SARS-CoV-2 and utilized in the production of efficient antiviral medicines

Novel SARS-COV2 poly epitope phage-based candidate vaccine and its immunogenicity

Background and purpose: The global emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prompted widespread concern. Bacteriophages have recently gained attention as a cost-effective and stable alternative for vaccine development due to their adjuvant properties. This study aimed to design and validate a poly epitope composed of viral proteins. Experimental approach: SARS-CoV-2 proteins (spike, nucleocapsid, membrane, envelope, papain-like protease, and RNA-dependent RNA polymerase) were selected for analysis. Immunoinformatic methods were employed to predict B and T cell epitopes, assessing their antigenicity, allergenicity, and toxicity. Epitopes meeting criteria for high antigenicity, non-allergenicity, and non-toxicity were linked to form poly epitopes. These sequences were synthesized and cloned into pHEN4 plasmids to generate Poly1 and Poly2 phagemid vectors. Recombinant Poly1 and Poly2 phages were produced by transforming M13ΔIII plasmids and phagemid vectors into E. coli TG1. Female Balb/c mice were immunized with a cocktail of Poly1 and Poly2 phages, and their serum was collected for ELISA testing. Interferon-gamma (IFN-γ) testing was performed on spleen-derived lymphocytes to evaluate immune system activation. Findings/Results: Recombinant Poly1 and Poly2 phages were produced, and their titer was determined as 1013 PFU/mL. Efficient humoral immune responses and cellular immunity activation in mice were achieved following phage administration. Conclusion and implication: Poly epitopes displayed on phages exhibit adjuvant properties, enhancing humoral and cellular immunity in mice. This suggests that phages could serve as adjuvants to bolster immunity against SARS-Cov-2. Recombinant phages could be applied as effective candidates for injectable and oral vaccine development strategies.

Discovery of SARS-CoV-2 papain-like protease (PLpro) inhibitors with efficacy in a murine infection model.

Vaccines and first-generation antiviral therapeutics have provided important protection against COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there remains a need for additional therapeutic options that provide enhanced efficacy and protection against potential viral resistance. The SARS-CoV-2 papain-like protease (PLpro) is one of the two essential cysteine proteases involved in viral replication. While inhibitors of the SARS-CoV-2 main protease have demonstrated clinical efficacy, known PLpro inhibitors have, to date, lacked the inhibitory potency and requisite pharmacokinetics to demonstrate that targeting PLpro translates to in vivo efficacy in a preclinical setting. Here, we report the machine learning-driven discovery of potent, selective, and orally available SARS-CoV-2 PLpro inhibitors, with lead compound PF-07957472 (4) providing robust efficacy in a mouse-adapted model of COVID-19 infection.

Genome-Wide Identification and Male Sterility-Related Expression Analysis of Papain-like Cysteine Protease Gene Family in Capsicum annuum

PLCPs (papain-like cysteine proteases) are one of the most abundant groups of cysteine proteases and play vital roles in multiple processes. The pepper (Capsicum annuum) is an important Solanaceae vegetable crop; its commercial hybrid seeds are widely used in production. Male sterility is a valuable trait for hybrid seed production. However, the function of PLCPs and the underlying mechanisms of male sterility in peppers remain unclear. In this study, we comprehensively identified the PLCP gene family in peppers, identifying 31 CaPLCPs. A phylogenetic analysis classified 31 members into eight clades. These CaPLCPs were unevenly distributed across eight chromosomes, and five segmental duplicated pairs were observed. The promoter cis-acting element analysis indicated that CaPLCP promoters contained abundant hormone-responsive and stress-responsive cis-elements, suggesting that CaPLCPs may play important roles in responding to abiotic stress, such as drought and low temperatures, as well as in plant immunity. The qRT-PCR analysis demonstrated that the expression levels of CaPLCP1, CaPLCP5, CaPLCP11, CaPLCP12, CaPLCP13, CaPLCP17, CaPLCP19, and CaPLCP21 were significantly reduced in the flowers of MS (male sterile pepper) at least at one stage, indicating their potential roles as regulatory factors in pepper male sterility. These findings provide important insights into the functional analysis of the PLCP gene family in peppers and other species, laying a crucial foundation for understanding the mechanisms of male sterility in peppers.

Discovery of potent SARS-CoV-2 nsp3 macrodomain inhibitors uncovers lack of translation to cellular antiviral response.

A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action. SARS-CoV-2 nsp3-mac1 is a viral macrodomain with ADP-ribosylhydrolase activity, which counteracts host immune response. Targeting the virus' immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV-2 compared to approved therapeutics, which target viral replication directly. Here we report a fragment-based lead generation campaign guided by computational approaches. We discover tool compounds which inhibit nsp3-mac1 activity at low nanomolar concentrations, and with responsive structure-activity relationships, high selectivity, and drug-like properties. Using our inhibitors, we show that inhibition of nsp3-mac1 increases ADP-ribosylation, but surprisingly does not translate to demonstrable antiviral activity in cell culture and iPSC-derived pneumocyte models. Further, no synergistic activity is observed in combination with interferon gamma, a main protease inhibitor, nor a papain-like protease inhibitor. Our results question the extent to which targeting modulation of innate immunity-driven ADP-ribosylation can influence SARS-CoV-2 replication. Moreover, these findings suggest that nsp3-mac1 might not be a suitable target for antiviral therapeutics development.

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.

Insights for Future Pharmacology: Exploring Phytochemicals as Potential Inhibitors Targeting SARS-CoV-2 Papain-like Protease

(1) Background: The SARS-CoV-2 papain-like protease (PLpro) remains an underexplored antiviral target so far. The reduced efficacy of approved treatments against novel variants highlights the importance of developing new agents. This review aims to provide a comprehensive understanding of phytochemicals as inhibitors of PLpro, identify gaps, and propose novel insights for future reference. (2) Methods: A thorough literature search was conducted using Google Scholar, ScienceDirect, and PubMed. Out of 150 articles reviewed, 57 met inclusion criteria, focusing on SARS-CoV-2 PLpro inhibitors, excluding studies on other coronaviruses or solely herbal extracts. Data were presented class-wise, and phytochemicals were grouped into virtual, weak, modest, and potential inhibitors. (3) Results: Approximately 100 phytochemicals are reported in the literature as PLpro inhibitors. We classified them as virtual inhibitors (70), weak inhibitors (13), modest inhibitors (11), and potential inhibitors (6). Flavonoids, terpenoids, and their glycosides predominated. Notably, six phytochemicals, including schaftoside, tanshinones, hypericin, and methyl 3,4-dihydroxybenzoate, emerged as potent PLpro inhibitors with favorable selectivity indices and disease-mitigation potential; (4) Conclusions: PLpro stands as a promising therapeutic target against SARS-CoV-2. The phytochemicals reported in the literature possess valuable drug potential; however, certain experimental and clinical gaps need to be filled to meet the therapeutic needs.

Non-Covalent Inhibitors of SARS-CoV-2 Papain-Like Protease (PLpro): In Vitro and In Vivo Antiviral Activity.

The SARS-CoV-2 papain-like protease (PLpro), essential for viral processing and immune response disruption, is a promising target for treating acute infection of SARS-CoV-2. To date, there have been no reports of PLpro inhibitors with both submicromolar potency and animal model efficacy. To address the challenge of PLpro's featureless active site, a noncovalent inhibitor library with over 50 new analogs was developed, targeting the PLpro active site by modulating the BL2-loop and engaging the BL2-groove. Notably, compounds 42 and 10 exhibited strong antiviral effects and were further analyzed pharmacokinetically. 10, in particular, showed a significant lung accumulation, up to 12.9-fold greater than plasma exposure, and was effective in a mouse model of SARS-CoV-2 infection, as well as against several SARS-CoV-2 variants. These findings highlight the potential of 10 as an in vivo chemical probe for studying PLpro inhibition in SARS-CoV-2 infection.

Disrupting protease and deubiquitinase activities of SARS-CoV-2 papain-like protease by natural and synthetic products discovered through multiple computational and biochemical approaches

The single-stranded RNA genome of SARS-CoV-2 encodes several structural and non-structural proteins, among which the papain-like protease (PLpro) is crucial for viral replication and immune evasion and has emerged as a promising therapeutic target. The current study aims to discover new inhibitors of PLpro that can simultaneously disrupt its protease and deubiquitinase activities. Using multiple computational approaches, six compounds (CP1-CP6) were selected from our in-house compounds database, with higher docking scores (−7.97 kcal/mol to −8.14 kcal/mol) and fitted well in the active pocket of PLpro. Furthermore, utilizing microscale molecular dynamics simulations (MD), the dynamic behavior of selected compounds was studied. Those molecules strongly binds at the PLpro active site and forms stable complexes. The dynamic motions suggest that the binding of CP1-CP6 brought the protein to a closed conformational state, thereby altering its normal function. In an in vitro evaluation, CP2 showed the most significant inhibitory potential for PLpro (protease activity = 2.71 ± 0.33 μM and deubiquitinase activity = 3.11 ± 0.75 μM), followed by CP1, CP5, CP4 and CP6. Additionally, CP1-CP6 showed no cytotoxicity at a concentration of 30 μM in the human BJ cell line.

SARS-CoV-2 PLpro Inhibition: Evaluating in Silico Repurposed Fidaxomicin's Antiviral Activity Through In Vitro Assessment.

The emergence of drug-resistant viruses and novel strains necessitates the rapid development of novel antiviral therapies. This need was particularly demanding during the COVID-19 pandemic. While de novo drug development is a time-consuming process, repurposing existing approved medications offers a more expedient approach. In our prior in silico screening of the DrugBank database, fidaxomicin emerged as a potential SARS-CoV-2 papain-like protease inhibitor. This study extends those findings by investigating fidaxomicin's antiviral properties in vitro. Our results support further exploration of fidaxomicin as a therapeutic candidate against SARS-CoV-2, given its promising in vitro antiviral activity and favorable safety profile.

Computational modeling and inhibition of SARS-COV-2 Papain-like protease enzyme: A potential therapeutic approach for COVID-19

This study aims to investigate the potential impact of inhibitors targeting the papain-like protease (PLpro) of SARS-CoV-2 on viral replication and the host immune response. A mathematical model was developed to simulate the interaction among susceptible cells, infected cells, PLpro, and immune cells, incorporating data on PLpro inhibition. Through numerical simulations using MATLAB, the model parameters were estimated based on available statistical data. The results indicate that strategically positioned inhibitors could impede the virus’s access to host cellular machinery, thereby enhancing the immune response and gradually reducing susceptible and infected cells over time. The dynamics of the viral enzyme PLpro showed reduced activity with the introduction of the inhibitor, leading to a decline in viral replication. Moreover, the immune cell population exhibited functional recovery as the inhibitor suppressed PLpro activity. These findings suggest that inhibitors targeting PLpro may serve as therapeutic interventions against SARS-CoV-2 by inhibiting viral replication and bolstering the immune response.

An ISG15-Based High-Throughput Screening Assay for Identification and Characterization of SARS-CoV-2 Inhibitors Targeting Papain-like Protease.

Emergence of newer variants of SARS-CoV-2 underscores the need for effective antivirals to complement the vaccination program in managing COVID-19. The multi-functional papain-like protease (PLpro) of SARS-CoV-2 is an essential viral protein that not only regulates the viral replication but also modulates the host immune system, making it a promising therapeutic target. To this end, we developed an in vitro interferon stimulating gene 15 (ISG15)-based Förster resonance energy transfer (FRET) assay and screened the National Cancer Institute (NCI) Diversity Set VI compound library, which comprises 1584 small molecules. Subsequently, we assessed the PLpro enzymatic activity in the presence of screened molecules. We identified three potential PLpro inhibitors, namely, NSC338106, 651084, and 679525, with IC50 values in the range from 3.3 to 6.0 µM. These molecules demonstrated in vitro inhibition of the enzyme activity and exhibited antiviral activity against SARS-CoV-2, with EC50 values ranging from 0.4 to 4.6 µM. The molecular docking of all three small molecules to PLpro suggested their specificity towards the enzyme's active site. Overall, our study contributes promising prospects for further developing potential antivirals to combat SARS-CoV-2 infection.

Noscapine shows antimalarial activity against Plasmodium falciparum 3D7, its clinical isolate Pf140/SS, and Plasmodium berghei ANKA

Objective: To evaluate the antimalarial activity of noscapine against Plasmodium falciparum 3D7 strain (Pf3D7), its clinical isolate (Pf140/SS), and Plasmodium berghei ANKA (PbA). Methods: Using ring-stage survival assay, phenotypic assessments, and SYBR-green-based fluorescence assay, the antimalarial activities of noscapine were assessed compared with dihydroartemisinin (DHA) in in vivo and in vitro studies. In addition, hemolysis and cytotoxicity tests were carried out to evaluate its safety. RT-PCR assay was also conducted to determine the effect of noscapine on papain-like cysteine protease Plasmodium falciparum falcipain-2 (PfFP-2). Results: The antimalarial efficacy of noscapine against Pf3D7 and Pf140/SS was comparable to DHA, with IC50 values of (7.68±0.88) and (5.57±0.74) nM/mL, respectively, and >95% inhibition of PbA infected rats. Noscapine also showed a safe profile, as evidenced by low hemolysis and cytotoxicity even at high concentrations. Moreover, PfFP-2 expression was significantly inhibited in both noscapine-treated Pf3D7 and Pf140/SS (P<0.01). Conclusions: Noscapine has antimalarial properties comparable to standard antimalarial DHA with better safety profiles, which may be further explored as a therapeutic candidate for the treatment of malaria.

On the dual role of (+)-catechin as primary antioxidant and inhibitor of viral proteases

Natural antioxidants have become the subject of many investigations due to the role that they play in the reduction of oxidative stress. Their main scavenging mechanisms concern the direct inactivation of free radicals and the coordination of metal ions involved in Fenton-like reactions. Recently, increasing attention has been paid to non-covalent inhibition of enzymes involved in different diseases by the antioxidants. Here, a computational investigation on the primary antioxidant power of (+)-catechin against the •OOH radical has been performed in both lipid-like and aqueous environments, taking into account the relevant species present in the simulated acid-base equilibria at the physiological pH. Hydrogen Atom Transfer (HAT), Single Electron Transfer (SET), and Radical Adduct Formation (RAF) mechanisms were studied, and relative rate constants were estimated. The potential inhibitory activity of the (+)-catechin towards the most important proteases from SARS-CoV-2, 3C-like (Mpro) and papain-like (PLpro) proteases was also investigated by MD simulations to provide deeper atomistic insights on the binding sites.Based on the antioxidant and antiviral properties also unravelled by comparison with other molecules having similar chemical scaffold, our results propose that (+)-CTc satisfies can explicate a dual action as antioxidant and antiviral in particular versus Mpro from SARS-CoV-2.

Thermal Inactivation, Denaturation and Aggregation Processes of Papain-Like Proteases.

The investigation into the behavior of ficin, bromelain, papain under thermal conditions holds both theoretical and practical significance. The production processes of medicines and cosmetics often involve exposure to high temperatures, particularly during the final product sterilization phase. Hence, it's crucial to identify the "critical" temperatures for each component within the mixture for effective technological regulation. In light of this, the objective of this study was to examine the thermal inactivation, aggregation, and denaturation processes of three papain-like proteases: ficin, bromelain, papain. To achieve this goal, the following experiments were conducted: (1) determination of the quantity of inactivated proteases using enzyme kinetics with BAPNA as a substrate; (2) differential scanning calorimetry (DSC); (3) assessment of protein aggregation using dynamic light scattering (DLS) and spectrophotometric analysis at 280 nm. Our findings suggest that the inactivation of ficin and papain exhibits single decay step which characterized by a rapid decline, then preservation of the same residual activity by enzyme stabilization. Only bromelain shows two steps with different kinetics. The molecular sizes of the active and inactive forms are similar across ficin, bromelain, and papain. Furthermore, the denaturation of these forms occurs at approximately the same rate and is accompanied by protein aggregation.

Fragment-Based Screen of SARS-CoV-2 Papain-like Protease (PLpro).

Coronaviruses have been responsible for numerous viral outbreaks in the past two decades due to the high transmission rate of this family of viruses. The deadliest outbreak is the recent Covid-19 pandemic, which resulted in over 7 million deaths worldwide. SARS-CoV-2 papain-like protease (PLPro) plays a key role in both viral replication and host immune suppression and is highly conserved across the coronavirus family, making it an ideal drug target. Herein we describe a fragment-based screen against PLPro using protein-observed NMR experiments, identifying 77 hit fragments. Analyses of NMR perturbation patterns and X-ray cocrystallized structures reveal fragments bind to two distinct regions of the protein. Importantly none of the fragments identified belong to the same chemical class as the few reported inhibitors, allowing for the discovery of a novel class of PLPro inhibitors.

Mutational profiling of SARS-CoV-2 papain-like protease reveals requirements for function, structure, and drug escape

Papain-like protease (PLpro) is an attractive drug target for SARS-CoV-2 because it is essential for viral replication, cleaving viral poly-proteins pp1a and pp1ab, and has de-ubiquitylation and de-ISGylation activities, affecting innate immune responses. We employ Deep Mutational Scanning to evaluate the mutational effects on PLpro enzymatic activity and protein stability in mammalian cells. We confirm features of the active site and identify mutations in neighboring residues that alter activity. We characterize residues responsible for substrate binding and demonstrate that although residues in the blocking loop are remarkably tolerant to mutation, blocking loop flexibility is important for function. We additionally find a connected network of mutations affecting activity that extends far from the active site. We leverage our library to identify drug-escape variants to a common PLpro inhibitor scaffold and predict that plasticity in both the S4 pocket and blocking loop sequence should be considered during the drug design process.

Modification of ionic liquid and lactoferrin-based small molecules as potential therapeutics against SARS-CoV-2: Molecular docking disclosed the predictable results

Ionic liquid MIE-NH2 displays a new role in the development of modification of glycoproteins of lactoferrin through a reductive amination mechanism to synthesize versatile pharmaceuticals. This work introduces a new strategy of modification of lactoferrin by using methyl imidazolium N-ethylamine MIE-NH2, this ionic liquid linked to N-glycans in lactoferrin derivatives in simple method. The modification of lactoferrin with MIE-NH2 as a novel small molecule (SM-IL-Lf) contains active amino groups is confirmed by UPLC/ESI-QTOF and MALDI-TOF mass spectrometry. Molecular docking disclosed the bioactivity of modifying glycoproteins - small IL-Lf-molecules by elucidating its interactions with the inspected targets. This providing of small molecules IL-Lf released a wide display with different functions as significantly important drug candidates inhibiting the targets of main protease (Mpro), RNA dependent RNA polymerase (RdRp), transmembrane protease serine 2 (TMPRSS2), and Papain-like protease (PLpro). The probability of the modeling N-glycans from lactoferrin modified and designed as small IL-Lf-molecules to inhibit any of these targets was investigated to find out its potency inhibitor of SARS-CoV-2.

The salivary gland transcriptome of Varroa destructor reveals suitable targets for RNAi-based mite control.

The mite Varroa destructorAnderson and Trueman (Mesostigmata: Varroidae) has a dramatic impact on beekeeping and is one of the main causes of honey bee colony losses. This ectoparasite feeds on honey bees' liquid tissues, through a wound created on the host integument, determining weight loss and a reduction of lifespan, as well as the transmission of viral pathogens. However, despite its importance, the mite feeding strategy and the host regulation role by the salivary secretions have been poorly explored. Here, we contribute to fill this gap by identifying the salivary components of V. destructor, to study their functional importance for mite feeding and survival. The differential expression analysis identified 30 salivary gland genes encoding putatively secreted proteins, among which only 15 were found to be functionally annotated. These latter include proteins with putative anti-bacterial, anti-fungal, cytolytic, digestive and immunosuppressive function. The three most highly transcribed genes, coding for a chitin-binding domain protein, a Kazal domain serine protease inhibitor and a papain-like cysteine protease were selected to study their functional importance by reverse genetics. Knockdown (90%-99%) by RNA interference (RNAi) of the transcript of a chitin-binding domain protein, likely interfering with the immune reaction to facilitate mite feeding, was associated with a 40%-50% decrease of mite survival. This work expands our knowledge of the host regulation and nutritional exploitation strategies adopted by ectoparasites of arthropods and allows the identification of potential targets for RNAi, paving the way towards the development of new strategies for Varroa mite control.

Myricetin inhibits transmissible gastroenteritis virus replication by targeting papain-like protease deubiquitinating enzyme activity.

Myricetin, a natural flavonoid found in various foods, was investigated for its antiviral effect against transmissible gastroenteritis virus (TGEV). This α-coronavirus causes significant economic losses in the global swine industry. The study focused on the papain-like protease (PLpro), which plays a crucial role in coronavirus immune evasion by mediating deubiquitination. Targeting PLpro could potentially disrupt viral replication and enhance antiviral responses. The results demonstrated that myricetin effectively inhibited TGEV-induced cytopathic effects in a dose-dependent manner, with an EC50 value of 31.19 μM. Myricetin significantly reduced TGEV viral load within 48 h after an 8-h co-incubation period. Further investigations revealed that myricetin at a concentration of 100 μM directly inactivated TGEV and suppressed its intracellular replication stage. Moreover, pretreatment with 100 μM myricetin conferred a protective effect on PK-15 cells against TGEV infection. Myricetin competitively inhibited PLpro with an IC50 value of 6.563 μM. Molecular docking experiments show that myricetin binds to the Cys102 residue of PLpro through conventional hydrogen bonds, Pi-sulfur, and Pi-alkyl interactions. This binding was confirmed through site-directed mutagenesis experiments, indicating myricetin as a potential candidate for preventing and treating TGEV infection.