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.

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.

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.

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.

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.

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.

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

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

Discovery of the Cryptic Sites of SARS-CoV-2 Papain-like Protease and Analysis of Its Druggability.

In late 2019, a new coronavirus (CoV) caused the outbreak of a deadly respiratory disease, resulting in the COVID-19 pandemic. In view of the ongoing pandemic, there is an immediate need to find drugs to treat patients. SARS-CoV-2 papain-like cysteine protease (PLpro) not only plays an important role in the pathogenesis of the virus but is also a target protein for the development of inhibitor drugs. Therefore, to develop targeted inhibitors, it is necessary to analyse and verify PLpro sites and explore whether there are other cryptic binding pockets with better activity. In this study, first, we detected the site of the whole PLpro protein by sitemap of Schrödinger (version 2018), the cavity of LigBuilder V3, and DeepSite, and roughly judged the possible activated binding site area. Then, we used the mixed solvent dynamics simulation (MixMD) of probe molecules to induce conformational changes in the protein to find the possible cryptic active sites. Finally, the TRAPP method was used to predict the druggability of cryptic pockets and analyse the changes in the physicochemical properties of residues around these sites. This work will help promote the research of SARS-CoV-2 PLpro inhibitors.

In Silico Study towards Repositioning of FDA-Approved Drug Candidates for Anticoronaviral Therapy: Molecular Docking, Molecular Dynamics and Binding Free Energy Calculations.

The SARS-CoV-2 targets were evaluated for a set of FDA-approved drugs using a combination of drug repositioning and rigorous computational modeling methodologies such as molecular docking and molecular dynamics (MD) simulations followed by binding free energy calculations. Six FDA-approved drugs including, Ouabain, Digitoxin, Digoxin, Proscillaridin, Salinomycin and Niclosamide with promising anti-SARS-CoV-2 activity were screened in silico against four SARS-CoV-2 proteins-papain-like protease (PLpro), RNA-dependent RNA polymerase (RdRp), SARS-CoV-2 main protease (Mpro), and adaptor-associated kinase 1 (AAK1)-in an attempt to define their promising targets. The applied computational techniques suggest that all the tested drugs exhibited excellent binding patterns with higher scores and stable complexes compared to the native protein cocrystallized inhibitors. Ouabain was suggested to act as a dual inhibitor for both PLpro and Mpro enzymes, while Digitoxin bonded perfectly to RdRp. In addition, Salinomycin targeted PLpro. Particularly, Niclosamide was found to target AAK1 with greater affinity compared to the reference drug. Our study provides comprehensive molecular-level insights for identifying or designing novel anti-COVID-19 drugs.

SARS-CoV-2 Papain-Like Protease: Structure, Function and Inhibition.

Emerging variants of SARS-CoV-2 and potential novel epidemic coronaviruses underline the importance of investigating various viral proteins as potential drug targets. The papain-like protease of coronaviruses has been less explored than other viral proteins; however, its substantive role in viral replication and impact on the host immune response make it a suitable target to study. This review article focuses on the structure and function of the papain-like protease (PLpro ) of SARS-CoV-2, including variants of concern, and compares it to those of other coronaviruses, such as SARS-CoV-1 and MERS-CoV. The protease's recognition motif is mirrored in ubiquitin and ISG15, which are involved in the antiviral immune response. Inhibitors, including GRL0617 derivatives, and their prospects as potential future antiviral agents are also discussed.

Antiviral activity of natural phenolic compounds in complex at an allosteric site of SARS-CoV-2 papain-like protease.

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, facilitating cleavage of the viral polypeptide chain, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to support coronaviruses in evading the host's innate immune responses. We identified three phenolic compounds bound to PLpro, preventing essential molecular interactions to ISG15 by screening a natural compound library. The compounds identified by X-ray screening and complexed to PLpro demonstrate clear inhibition of PLpro in a deISGylation activity assay. Two compounds exhibit distinct antiviral activity in Vero cell line assays and one inhibited a cytopathic effect in non-cytotoxic concentration ranges. In the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

A Review of In Silico Research, SARS-CoV-2, and Neurodegeneration: Focus on Papain-Like Protease.

Since the appearance of SARS-CoV-2 and the COVID-19 pandemic, the search for new approaches to treat this disease took place in the scientific community. The in silico approach has gained importance at this moment, once the methodologies used in this kind of study allow for the identification of specific protein-ligand interactions, which may serve as a filter step for molecules that can act as specific inhibitors. In addition, it is a low-cost and high-speed technology. Molecular docking has been widely used to find potential viral protein inhibitors for structural and non-structural proteins of the SARS-CoV-2, aiming to block the infection and the virus multiplication. The papain-like protease (PLpro) participates in the proteolytic processing of SARS-CoV-2 and composes one of the main targets studied for pharmacological intervention by in silico methodologies. Based on that, we performed a systematic review about PLpro inhibitors from the perspective of in silico research, including possible therapeutic molecules in relation to this viral protein. The neurological problems triggered by COVID-19 were also briefly discussed, especially relative to the similarities of neuroinflammation present in Alzheimer's disease. In this context, we focused on two molecules, curcumin and glycyrrhizinic acid, given their PLpro inhibitory actions and neuroprotective properties and potential therapeutic effects on COVID-19.

Genetically Encoded Fluorescent Sensors for SARS-CoV-2 Papain-like Protease PLpro.

In the SARS-CoV-2 lifecycle, papain-like protease PLpro cuts off the non-structural proteins nsp1, nsp2, and nsp3 from a large polyprotein. This is the earliest viral enzymatic activity, which is crucial for all downstream steps. Here, we designed two genetically encoded fluorescent sensors for the real-time detection of PLpro activity in live cells. The first sensor was based on the Förster resonance energy transfer (FRET) between the red fluorescent protein mScarlet as a donor and the biliverdin-binding near-infrared fluorescent protein miRFP670 as an acceptor. A linker with the PLpro recognition site LKGG in between made this FRET pair sensitive to PLpro cleavage. Upon the co-expression of mScarlet-LKGG-miRFP670 and PLpro in HeLa cells, we observed a gradual increase in the donor fluorescence intensity of about 1.5-fold. In the second sensor, both PLpro and its target-green mNeonGreen and red mScarletI fluorescent proteins separated by an LKGG-containing linker-were attached to the endoplasmic reticulum (ER) membrane. Upon cleavage by PLpro, mScarletI diffused from the ER throughout the cell. About a two-fold increase in the nucleus/cytoplasm ratio was observed as a result of the PLpro action. We believe that the new PLpro sensors can potentially be used to detect the earliest stages of SARS-CoV-2 propagation in live cells as well as for the screening of PLpro inhibitors.

Atomistic-Level Description of the Covalent Inhibition of SARS-CoV-2 Papain-like Protease.

Inhibition of the papain-like protease (PLpro) of SARS-CoV-2 has been demonstrated to be a successful target to prevent the spreading of the coronavirus in the infected body. In this regard, covalent inhibitors, such as the recently proposed VIR251 ligand, can irreversibly inactivate PLpro by forming a covalent bond with a specific residue of the catalytic site (Cys111), through a Michael addition reaction. An inhibition mechanism can therefore be proposed, including four steps: (i) ligand entry into the protease pocket; (ii) Cys111 deprotonation of the thiol group by a Brønsted-Lowry base; (iii) Cys111-S- addition to the ligand; and (iv) proton transfer from the protonated base to the covalently bound ligand. Evaluating the energetics and PLpro conformational changes at each of these steps could aid the design of more efficient and selective covalent inhibitors. For this aim, we have studied by means of MD simulations and QM/MM calculations the whole mechanism. Regarding the first step, we show that the inhibitor entry in the PLpro pocket is thermodynamically favorable only when considering the neutral Cys111, that is, prior to the Cys111 deprotonation. For the second step, MD simulations revealed that His272 would deprotonate Cys111 after overcoming an energy barrier of ca. 32 kcal/mol (at the QM/MM level), but implying a decrease of the inhibitor stability inside the protease pocket. This information points to a reversible Cys111 deprotonation, whose equilibrium is largely shifted toward the neutral Cys111 form. Although thermodynamically disfavored, if Cys111 is deprotonated in close proximity to the vinylic carbon of the ligand, then covalent binding takes place in an irreversible way (third step) to form the enolate intermediate. Finally, due to Cys111-S- negative charge redistribution over the bound ligand, proton transfer from the initially protonated His272 is favored, finally leading to an irreversibly modified Cys111 and a restored His272. These results elucidate the selectivity of Cys111 to enable formation of a covalent bond, even if a weak proton acceptor is available, as His272.

A computational essential dynamics approach to investigate structural influences of ligand binding on Papain like protease from SARS-CoV-2.

Papain like protease (PLpro) is a cysteine protease from the coronaviridae family of viruses. Coronaviruses possess a positive sense, single-strand RNA, leading to the translation of two viral polypeptides containing viral structural, non-structural and accessory proteins. PLpro is responsible for the cleavage of nsp1-3 from the viral polypeptide. PLpro also possesses deubiquitinating and deISGlyating activity, which sequesters the virus from the host's immune system. This indispensable attribute of PLpro makes it a protein of interest as a drug target. The present study aims to analyze the structural influences of ligand binding on PLpro. First, PLpro was screened against the ZINC-in-trials library, from which four lead compounds were identified based on estimated binding affinity and interaction patterns. Next, based on molecular docking results, ZINC000000596945, ZINC000064033452 and VIR251 (control molecule) were subjected to molecular dynamics simulation. The study evaluated global and essential dynamics analyses utilising principal component analyses, dynamic cross-correlation matrix, free energy landscape and time-dependant essential dynamics to predict the structural changes observed in PLpro upon ligand binding in a simulated environment. The MM/PBSA-based binding free energy calculations of the two selected molecules, ZINC000000596945 (-41.23 ± 3.70 kcal/mol) and ZINC000064033452 (-25.10 ± 2.65 kcal/mol), displayed significant values which delineate them as potential inhibitors of PLpro from SARS-CoV-2.

In Silico Screening of Semi-Synthesized Compounds as Potential Inhibitors for SARS-CoV-2 Papain-like Protease: Pharmacophoric Features, Molecular Docking, ADMET, Toxicity and DFT Studies.

Papain-like protease is an essential enzyme in the proteolytic processing required for the replication of SARS-CoV-2. Accordingly, such an enzyme is an important target for the development of anti-SARS-CoV-2 agents which may reduce the mortality associated with outbreaks of SARS-CoV-2. A set of 69 semi-synthesized molecules that exhibited the structural features of SARS-CoV-2 papain-like protease inhibitors (PLPI) were docked against the coronavirus papain-like protease (PLpro) enzyme (PDB ID: (4OW0). Docking studies showed that derivatives 34 and 58 were better than the co-crystallized ligand while derivatives 17, 28, 31, 40, 41, 43, 47, 54, and 65 exhibited good binding modes and binding free energies. The pharmacokinetic profiling study was conducted according to the four principles of the Lipinski rules and excluded derivative 31. Furthermore, ADMET and toxicity studies showed that derivatives 28, 34, and 47 have the potential to be drugs and have been demonstrated as safe when assessed via seven toxicity models. Finally, comparing the molecular orbital energies and the molecular electrostatic potential maps of 28, 34, and 47 against the co-crystallized ligand in a DFT study indicated that 28 is the most promising candidate to interact with the target receptor (PLpro).

A molecular sensor determines the ubiquitin substrate specificity of SARS-CoV-2 papain-like protease.

The SARS-CoV-2 papain-like protease (PLpro) is a target for antiviral drug development. It is essential for processing viral polyproteins for replication and functions in host immune evasion by cleaving ubiquitin (Ub) and ubiquitin-like protein (Ubl) conjugates. While highly conserved, SARS-CoV-2 and SARS-CoV PLpro have contrasting Ub/Ubl substrate preferences. Using a combination of structural analyses and functional assays, we identify a molecular sensor within the S1 Ub-binding site of PLpro that serves as a key determinant of substrate specificity. Variations within the S1 sensor specifically alter cleavage of Ub substrates but not of the Ubl interferon-stimulated gene 15 protein (ISG15). Significantly, a variant of concern associated with immune evasion carries a mutation in the S1 sensor that enhances PLpro activity on Ub substrates. Collectively, our data identify the S1 sensor region as a potential hotspot of variability that could alter host antiviral immune responses to newly emerging SARS-CoV-2 lineages.