The SARS-CoV-2 papain-like protease suppresses type I interferon responses by deubiquitinating STING.

The SARS-CoV-2 papain-like protease (PLpro), which has deubiquitinating activity, suppresses the type I interferon (IFN-I) antiviral response. We investigated the mechanism by which PLpro antagonizes cellular antiviral responses. In HEK392T cells, PLpro removed K63-linked polyubiquitin chains from Lys289 of the stimulator of interferon genes (STING). PLpro-mediated deubiquitination of STING disrupted the STING-IKKε-IRF3 complex that induces the production of IFN-ß and IFN-stimulated cytokines and chemokines. In human airway cells infected with SARS-CoV-2, the combined treatment with the STING agonist diABZi and the PLpro inhibitor GRL0617 resulted in the synergistic inhibition of SARS-CoV-2 replication and increased IFN-I responses. The PLpros of seven human coronaviruses (SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-HKU1, HCoV-OC43, and HCoV-NL63) and four SARS-CoV-2 variants of concern (α, ß, γ, and δ) all bound to STING and suppressed STING-stimulated IFN-I responses in HEK293T cells. These findings reveal how SARS-CoV-2 PLpro inhibits IFN-I signaling through STING deubiquitination and a general mechanism used by seven human coronaviral PLpros to dysregulate STING and to facilitate viral innate immune evasion. We also identified simultaneous pharmacological STING activation and PLpro inhibition as a potentially effective strategy for antiviral therapy against SARS-CoV-2.

The substrate selectivity of papain-like proteases from human-infecting coronaviruses correlates with innate immune suppression.

Coronaviruses that can infect humans can cause either common colds (HCoV-NL63, HCoV-229E, HCoV-HKU1, and HCoV-OC43) or severe respiratory symptoms (SARS-CoV-2, SARS-CoV, and MERS-CoV). The papain-like proteases (PLPs) of SARS-CoV, SARS-CoV-2, MERS-CoV, and HCoV-NL63 function in viral innate immune evasion and have deubiquitinating (DUB) and deISGylating activities. We identified the PLPs of HCoV-229E, HCoV-HKU1, and HCoV-OC43 and found that their enzymatic properties correlated with their ability to suppress innate immune responses. A conserved noncatalytic aspartic acid residue was critical for both DUB and deISGylating activities, but the PLPs had differing ubiquitin (Ub) chain cleavage selectivities and binding affinities for Ub, K48-linked diUb, and interferon-stimulated gene 15 (ISG15) substrates. The crystal structure of HKU1-PLP2 in complex with Ub revealed binding interfaces that accounted for the unusually high binding affinity between this PLP and Ub. In cellular assays, the PLPs from the severe disease-causing coronaviruses strongly suppressed innate immune IFN-I and NF-κB signaling and stimulated autophagy, whereas the PLPs from the mild disease-causing coronaviruses generally showed weaker effects on immune suppression and autophagy induction. In addition, a PLP from a SARS-CoV-2 variant of concern showed increased suppression of innate immune signaling pathways. Overall, these results demonstrated that the DUB and deISGylating activities and substrate selectivities of these PLPs differentially contribute to viral innate immune evasion and may affect viral pathogenicity.

Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin.

The Papain-like protease (PLpro) is a domain of a multi-functional, non-structural protein 3 of coronaviruses. PLpro cleaves viral polyproteins and posttranslational conjugates with poly-ubiquitin and protective ISG15, composed of two ubiquitin-like (UBL) domains. Across coronaviruses, PLpro showed divergent selectivity for recognition and cleavage of posttranslational conjugates despite sequence conservation. We show that SARS-CoV-2 PLpro binds human ISG15 and K48-linked di-ubiquitin (K48-Ub2) with nanomolar affinity and detect alternate weaker-binding modes. Crystal structures of untethered PLpro complexes with ISG15 and K48-Ub2 combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub2 are differently utilized in interactions with PLpro. Analysis of protein interface energetics predicted differential binding stabilities of the two UBL/Ub domains that were validated experimentally. We emphasize how substrate recognition can be tuned to cleave specifically ISG15 or K48-Ub2 modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function.

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.

Discovery of novel papain-like protease inhibitors for potential treatment of COVID-19.

The recent emergence of different SARS-CoV-2 variants creates an urgent need to develop more effective therapeutic agents to prevent COVID-19 outbreaks. Among SARS-CoV-2 essential proteases is papain-like protease (SARS-CoV-2 PLpro), which plays multiple roles in regulating SARS-CoV-2 viral spread and innate immunity such as deubiquitinating and deISG15ylating (interferon-induced gene 15) activities. Many studies are currently focused on targeting this protease to tackle SARS-CoV-2 infection. In this context, we performed a phenotypic screening using an in-house pilot compounds collection possessing a diverse skeleta against SARS-CoV-2 PLpro. This screen identified SIMR3030 as a potent inhibitor of SARS-CoV-2. SIMR3030 has been shown to exhibit deubiquitinating activity and inhibition of SARS-CoV-2 specific gene expression (ORF1b and Spike) in infected host cells and possessing virucidal activity. Moreover, SIMR3030 was demonstrated to inhibit the expression of inflammatory markers, including IFN-α, IL-6, and OAS1, which are reported to mediate the development of cytokine storms and aggressive immune responses. In vitro absorption, distribution, metabolism, and excretion (ADME) assessment of the drug-likeness properties of SIMR3030 demonstrated good microsomal stability in liver microsomes. Furthermore, SIMR3030 demonstrated very low potency as an inhibitor of CYP450, CYP3A4, CYP2D6 and CYP2C9 which rules out any potential drug-drug interactions. In addition, SIMR3030 showed moderate permeability in Caco2-cells. Critically, SIMR3030 has maintained a high in vivo safety profile at different concentrations. Molecular modeling studies of SIMR3030 in the active sites of SARS-CoV-2 and MERS-CoV PLpro were performed to shed light on the binding modes of this inhibitor. This study demonstrates that SIMR3030 is a potent inhibitor of SARS-CoV-2 PLpro that forms the foundation for developing new drugs to tackle the COVID-19 pandemic and may pave the way for the development of novel therapeutics for a possible future outbreak of new SARS-CoV-2 variants or other Coronavirus species.

Identifying Drug Candidates for COVID-19 with Large-Scale Drug Screening.

Papain-like protease (PLpro) is critical to COVID-19 infection. Therefore, it is a significant target protein for drug development. We virtually screened a 26,193 compound library against the PLpro of SARS-CoV-2 and identified several drug candidates with convincing binding affinities. The three best compounds all had better estimated binding energy than those of the drug candidates proposed in previous studies. By analyzing the docking results for the drug candidates identified in this and previous studies, we demonstrate that the critical interactions between the compounds and PLpro proposed by the computational approaches are consistent with those proposed by the biological experiments. In addition, the predicted binding energies of the compounds in the dataset showed a similar trend as their IC50 values. The predicted ADME and drug-likeness properties also suggested that these identified compounds can be used for COVID-19 treatment.

Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2.

Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we design a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibits PLpro with kinact/KI = 9,600 M-1 s-1, achieves sub-µM EC50 values against three SARS-CoV-2 variants in mammalian cell lines, and does not inhibit a panel of human deubiquitinases (DUBs) at >30 µM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validates our design strategy and establishes the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.

Autophagy is induced by swine acute diarrhea syndrome coronavirus through the cellular IRE1-JNK-Beclin 1 signaling pathway after an interaction of viral membrane-associated papain-like protease and GRP78.

Autophagy plays an important role in the infectious processes of diverse pathogens. For instance, cellular autophagy could be harnessed by viruses to facilitate replication. However, it is still uncertain about the interplay of autophagy and swine acute diarrhea syndrome coronavirus (SADS-CoV) in cells. In this study, we reported that SADS-CoV infection could induce a complete autophagy process both in vitro and in vivo, and an inhibition of autophagy significantly decreased SADS-CoV production, thus suggesting that autophagy facilitated the replication of SADS-CoV. We found that ER stress and its downstream IRE1 pathway were indispensable in the processes of SADS-CoV-induced autophagy. We also demonstrated that IRE1-JNK-Beclin 1 signaling pathway, neither PERK-EIF2S1 nor ATF6 pathways, was essential during SADS-CoV-induced autophagy. Importantly, our work provided the first evidence that expression of SADS-CoV PLP2-TM protein induced autophagy through the IRE1-JNK-Beclin 1 signaling pathway. Furthermore, the interaction of viral PLP2-TMF451-L490 domain and substrate-binding domain of GRP78 was identified to activate the IRE1-JNK-Beclin 1 signaling pathway, and thus resulting in autophagy, and in turn, enhancing SADS-CoV replication. Collectively, these results not only showed that autophagy promoted SADS-CoV replication in cultured cells, but also revealed that the molecular mechanism underlying SADS-CoV-induced autophagy in cells.

Engineering potent live attenuated coronavirus vaccines by targeted inactivation of the immune evasive viral deubiquitinase.

Coronaviruses express a papain-like protease (PLpro) that is required for replicase polyprotein maturation and also serves as a deubiquitinating enzyme (DUB). In this study, using a Middle East respiratory syndrome virus (MERS-CoV) PLpro modified virus in which the DUB is selectively inactivated, we show that the PLpro DUB is an important MERS-CoV interferon antagonist and virulence factor. Although the DUB-negative rMERS-CoVMA replicates robustly in the lungs of human dipeptidyl peptidase 4 knock-in (hDPP4 KI) mice, it does not cause clinical symptoms. Interestingly, a single intranasal vaccination with DUB-negative rMERS-CoVMA induces strong and sustained neutralizing antibody responses and sterilizing immunity after a lethal wt virus challenge. The survival of naïve animals also significantly increases when sera from animals vaccinated with the DUB-negative rMERS-CoVMA are passively transferred, prior to receiving a lethal virus dose. These data demonstrate that DUB-negative coronaviruses could be the basis of effective modified live attenuated vaccines.

Diphenyl Diselenide and SARS-CoV-2: in silico Exploration of the Mechanisms of Inhibition of Main Protease (Mpro) and Papain-like Protease (PLpro).

The SARS-CoV-2 pandemic has prompted global efforts to develop therapeutics. The main protease of SARS-CoV-2 (Mpro) and the papain-like protease (PLpro) are essential for viral replication and are key targets for therapeutic development. In this work, we investigate the mechanisms of SARS-CoV-2 inhibition by diphenyl diselenide (PhSe)2 which is an archetypal model of diselenides and a renowned potential therapeutic agent. The in vitro inhibitory concentration of (PhSe)2 against SARS-CoV-2 in Vero E6 cells falls in the low micromolar range. Molecular dynamics (MD) simulations and density functional theory (DFT) calculations [level of theory: SMD-B3LYP-D3(BJ)/6-311G(d,p), cc-pVTZ] are used to inspect non-covalent inhibition modes of both proteases via π-stacking and the mechanism of covalent (PhSe)2 + Mpro product formation involving the catalytic residue C145, respectively. The in vitro CC50 (24.61 µM) and EC50 (2.39 µM) data indicate that (PhSe)2 is a good inhibitor of the SARS-CoV-2 virus replication in a cell culture model. The in silico findings indicate potential mechanisms of proteases' inhibition by (PhSe)2; in particular, the results of the covalent inhibition here discussed for Mpro, whose thermodynamics is approximatively isoergonic, prompt further investigation in the design of antiviral organodiselenides.

Parthenolide reveals an allosteric mode to inhibit the deISGylation activity of SARS-CoV­2 papain-like protease.

The coronavirus papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for viral polypeptide cleavage and the deISGylation of interferon-stimulated gene 15 (ISG15), which enable it to participate in virus replication and host innate immune pathways. Therefore, PLpro is considered an attractive antiviral drug target. Here, we show that parthenolide, a germacrane sesquiterpene lactone, has SARS-CoV-2 PLpro inhibitory activity. Parthenolide covalently binds to Cys-191 or Cys-194 of the PLpro protein, but not the Cys-111 at the PLpro catalytic site. Mutation of Cys-191 or Cys-194 reduces the activity of PLpro. Molecular docking studies show that parthenolide may also form hydrogen bonds with Lys-192, Thr-193, and Gln-231. Furthermore, parthenolide inhibits the deISGylation but not the deubiquitinating activity of PLpro in vitro. These results reveal that parthenolide inhibits PLpro activity by allosteric regulation.

Identification and characterization of aurintricarboxylic acid as a potential inhibitor of SARS-CoV-2 PLpro.

As the global health crisis due to evolution of mutations in SARS-CoV-2 continues, it is important to develop several effective antivirals to control the disease. Targeting papain-like protease (PLpro) of SARS-CoV-2 for drug development is a promising strategy due to its dual role in promoting viral replication and dysregulating host immune responses. Here, we screened a library of compounds to find potential inhibitors of PLpro. We find aurintricarboxylic acid (ATA) inhibits PLpro with Ki and IC50 values of 16 µM and 30 µM, respectively. The binding of ATA to PLpro was further characterized using isothermal titration calorimetry, differential scanning fluorimetry, dynamic light scattering and circular dichroism spectrometry. In vitro assays showed the antiviral potential of ATA with IC50 of 50 µM. In vivo efficacy was studied in Syrian hamsters and the results are being discussed.

Ubiquitin variants potently inhibit SARS-CoV-2 PLpro and viral replication via a novel site distal to the protease active site.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has made it clear that combating coronavirus outbreaks benefits from a combination of vaccines and therapeutics. A promising drug target common to all coronaviruses-including SARS-CoV, MERS-CoV, and SARS-CoV-2-is the papain-like protease (PLpro). PLpro cleaves part of the viral replicase polyproteins into non-structural protein subunits, which are essential to the viral replication cycle. Additionally, PLpro can cleave both ubiquitin and the ubiquitin-like protein ISG15 from host cell substrates as a mechanism to evade innate immune responses during infection. These roles make PLpro an attractive antiviral drug target. Here we demonstrate that ubiquitin variants (UbVs) can be selected from a phage-displayed library and used to specifically and potently block SARS-CoV-2 PLpro activity. A crystal structure of SARS-CoV-2 PLpro in complex with a representative UbV reveals a dimeric UbV bound to PLpro at a site distal to the catalytic site. Yet, the UbV inhibits the essential cleavage activities of the protease in vitro and in cells, and it reduces viral replication in cell culture by almost five orders of magnitude.

Biobran/MGN-3, an Arabinoxylan Rice Bran, Protects against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): An In Vitro and In Silico Study.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), poses a serious global public health threat for which there is currently no satisfactory treatment. This study examines the efficacy of Biobran/MGN-3 against SARS-CoV-2. Biobran is an arabinoxylan rice bran that has been shown to significantly inhibit the related influenza virus in geriatric subjects. Here, Biobran's anti-SARS-CoV-2 activity was assessed using MTT and plaque reduction assays, RT-PCR, ELISA techniques, and measurements of SARS-CoV-2-related gene expression and protein levels. For Vero E6 cells infected with SARS-CoV-2, Biobran reduced the viral load by 91.9% at a dose of 100 µg/mL, it reduced viral counts (PFU/mL) by 90.6% at 50 µg/mL, and it exhibited a significant selectivity index (EC50/IC50) of 22.5. In addition, Biobran at 10 µg/mL inhibited papain-like proteinase (PLpro) by 87% and ACE2 SARS-CoV-2 S-protein RBD by 90.5%, and it significantly suppressed SARS-CoV-2 gene expression, down-regulating E-gene and RdRp gene expression by 93% each at a dose of 50 µg/mL and inhibiting the E-protein by 91.3%. An in silico docking study was also performed to examine the protein-protein interaction (PPI) between SARS-CoV-2 RBD and DC-SIGN as well as between serine carboxypeptidase and papain-like protease PLpro. Serine carboxypeptidase, an active ingredient in Biobran, was found to interfere with the binding of SARS-CoV-2 to its receptor DC-SIGN on Vero cells, thus preventing the cell entry of SARS-CoV-2. In addition, it impairs the viral replication cycle by binding to PLpro. We conclude that Biobran possesses potent antiviral activity against SARS-CoV-2 in vitro and suggest that Biobran may be able to prevent SARS-CoV-2 infection. This warrants further investigation in clinical trials.

Structure-Based Design of a Dual-Targeted Covalent Inhibitor Against Papain-like and Main Proteases of SARS-CoV-2.

The two proteases, PLpro and Mpro, of SARS-CoV-2 are essential for replication of the virus. Using a structure-based co-pharmacophore screening approach, we developed a novel dual-targeted inhibitor that is equally potent in inhibiting PLpro and Mpro of SARS-CoV-2. The inhibitor contains a novel warhead, which can form a covalent bond with the catalytic cysteine residue of either enzyme. The maximum rate of the covalent inactivation is comparable to that of the most potent inhibitors reported for the viral proteases and covalent inhibitor drugs currently in clinical use. The covalent inhibition appears to be very specific for the viral proteases. The inhibitor has a potent antiviral activity against SARS-CoV-2 and is also well tolerated by mice and rats in toxicity studies. These results suggest that the inhibitor is a promising lead for development of drugs for treatment of COVID-19.

Amino acid variants of SARS-CoV-2 papain-like protease have impact on drug binding.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused both a health and economic crisis around the world. Its papain-like protease (PLpro) is one of the protein targets utilized in designing new drugs that would aid vaccines in the fight against the virus. Although there are already several potential candidates for a good inhibitor of this protein, the degree of variability of the protein itself is not taken into account. As an RNA virus, SARS-CoV-2 can mutate to a high degree, but PLpro variability has not been studied to date. Based on sequence data available in databases, we analyzed the mutational potential of this protein. We focused on the effect of observed mutations on inhibitors' binding mode and their efficacy as well as protein's activity. Our analysis identifies five mutations that should be monitored and included in the drug design process: P247S, E263D-Y264H and T265A-Y268C.

Insights into the Dynamics and Binding of Two Polyprotein Substrate Cleavage Points in the Context of the SARS-CoV-2 Main and Papain-like Proteases.

It is well known that vital enzymes in the replication process of the coronavirus are the SARS-CoV-2 PLpro and SARS-CoV-2 3CLpro, both of which are important targets in the search for anti-coronavirus agents. These two enzymes are responsible for cleavage at various polyprotein sites in the SARS-CoV-2 lifecycle. Herein, the dynamics of the polyprotein cleavage sequences for the boundary between non-structural proteins Nsp1 and Nsp2 (CS1) and between Nsp2 and Nsp3 (CS2) in complex with both the papain-like protein PLpro and the main protease 3CLpro were explored using computational methods. The post dynamics analysis reveals that CS1 and CS2 both have greater stability when complexed with PLpro. Of these two, greater stability is observed for the CS1-PLpro complex, while destabilization resulting in loss of CS2 from the PLpro active site is observed for CS2-PLpro, suggesting the rate of exchange by the papain-like protease is faster for CS2 compared to CS1. On the other hand, the 3CLpro main protease also reveals stability for CS1 suggesting that the main protease could also play a potential role in the cleavage at point CS1. However, destabilization occurs early in the simulation for the complex CLpro-CS2 suggesting a poor interaction and non-plausible protease cleavage of the polyprotein at CS2 by the main protease. These findings could be used as a guide in the development and design of potent COVID-19 antiviral inhibitors that mimic the CS1 cleavage site.

Characterization of the Ubiquitin and ISG15 Deconjugase Activity of SARS-CoV-1 and SARS-CoV-2 Papain-Like Protease.

Both severe acute respiratory syndrome coronavirus 1 and 2 (SARS-CoV-1 and SARS-CoV-2) encode a papain-like protease (PLpro), which plays a vital role in viral propagation. PLpro accomplishes this function by processing the viral polyproteins essential for viral replication and removing the small proteins, ubiquitin and ISG15 from the host's key immune signaling proteins, thereby preventing the host's innate immune response. Although PLpro from both SARS-CoV-1 and SARS-CoV-2 are structurally highly similar (83% sequence identity), they exhibit functional variability. Hence, to further elucidate the mechanism and aid in drug discovery efforts, the biochemical and kinetic characterization of PLpro is needed. This chapter describes step-by-step experimental procedures for evaluating PLpro activity in vitro using activity-based probes (ABPs) along with fluorescence-based substrates. Herein we describe a step-by-step experimental procedure to assess the activity of PLpro in vitro using a suite of activity-based probes (ABPs) and fluorescent substrates and how they can be applied as fast and yet sensitive methods to calculate kinetic parameters.

Identification of Doxorubicin as Repurposing Inhibitory Drug for MERS-CoV PLpro.

Middle East respiratory syndrome coronavirus (MERS-CoV), belonging to the betacoronavirus genus can cause severe respiratory illnesses, accompanied by pneumonia, multiorgan failure, and ultimately death. CoVs have the ability to transgress species barriers and spread swiftly into new host species, with human-to-human transmission causing epidemic diseases. Despite the severe public health threat of MERS-CoV, there are currently no vaccines or drugs available for its treatment. MERS-CoV papain-like protease (PLpro) is a key enzyme that plays an important role in its replication. In the present study, we evaluated the inhibitory activities of doxorubicin (DOX) against the recombinant MERS-CoV PLpro by employing protease inhibition assays. Hydrolysis of fluorogenic peptide from the Z-RLRGG-AMC-peptide bond in the presence of DOX showed an IC50 value of 1.67 µM at 30 min. Subsequently, we confirmed the interaction between DOX and MERS-CoV PLpro by thermal shift assay (TSA), and DOX increased ΔTm by ~20 °C, clearly indicating a coherent interaction between the MERS-CoV PL protease and DOX. The binding site of DOX on MERS-CoV PLpro was assessed using docking techniques and molecular dynamic (MD) simulations. DOX bound to the thumb region of the catalytic domain of the MERS-CoV PLpro. MD simulation results showed flexible BL2 loops, as well as other potential residues, such as R231, R233, and G276 of MERS-CoV PLpro. Development of drug repurposing is a remarkable opportunity to quickly examine the efficacy of different aspects of treating various diseases. Protease inhibitors have been found to be effective against MERS-CoV to date, and numerous candidates are currently undergoing clinical trials to prove this. Our effort follows a in similar direction.