key: cord-0834960-zemtzfse authors: Mahmoudvand, Shahab; Shokri, Somayeh title: Interactions between SARS coronavirus 2 papain‐like protease and immune system: a potential drug target for the treatment of COVID‐19 date: 2021-04-19 journal: Scand J Immunol DOI: 10.1111/sji.13044 sha: dd330d3526b8949eb30d1ba8ca2d09d699c1e679 doc_id: 834960 cord_uid: zemtzfse Coronaviruses (CoVs) are a large family of respiratory viruses which can cause mild to moderate upper respiratory tract infections. Recently, new coronavirus named as Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has been identified which is a major threat to public health. Innate immune responses play a vital role in a host’s defense against viruses. Interestingly, CoVs have evolved elaborate strategies to evade the complex system of sensors and signaling molecules to suppress host immunity. SARS‐CoV‐2 papain‐like protease (PLpro), as an important coronavirus enzyme, regulates viral spread and innate immune responses. SCoV‐2 PLpro is multifunctional enzymes with deubiquitinating (DUB) and deISGylating activity. The PLpro can interact with key regulators in signaling pathways such as STING, NF‐κB, cytokine production, MAPK, and TGF‐β and hijack those to block the immune responses. Therefore, the PLpro can be as an important target for the treatment of COVID‐19. Until now, there are several drugs or compound have been identified that can inhibit PLpro activity. Here we discuss about the dysregulation effects of PLpro on immune system and drugs that have potential inhibitors for SCoV‐2 PLpro. The Many viruses encode proteins that antagonize both the innate and adapted arms of the immune response [5] . All CoVs encode at least one papain-like protease (PLpro) with deubiquitinating (DUB), deISGylating (deISG) and other activities that elicit the appropriate innate immune response [6] . Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) recently entered the human population at the end of 2019 from Hunnan seafood market in Wuhan, China [7] . The virus causes a pandemic infection in more than 119 million people with a case fatality ratio (CFR) of 1.4% with substantially higher ratios in older age groups, 0.32% in those aged <60 years, 6.4% in those aged ≥60 years and up to 13.4% in those aged 80 years or older [8, 9] . This raises an urgent need to develop an effective treatment based on identifying targets for viral factors which blocked or reduced innate immune responses. A majority of the newly reported studies showed that PLpro, which controls replication of the SCoV-2, has been identified as a potential drug target for the treatment. This article is protected by copyright. All rights reserved The CoVs belongs to the order Nidovirales, family of Coronaviridae, subfamily Orthocoronavirinae, and this subfamily including Alphacoronavirus (αCoV), Betacoronavirus (βCoV), Deltacoronavirus (δCoV), and Gammacoronavirus (γCoV) [10] . CoVs commonly infect humans and several other vertebrates and can cause acute respiratory distress syndrome (ARDS) with various symptoms such as weakness, fever, breathing difficulty, dry or hacking cough, headaches, pneumonia, enteric, hepatic, and neurologic diseases [11, 12] . has reported that there are more than 119 million confirmed cases globally with more than 2.5 million deaths [19] . SCoV-2 has structural similarity to the coronaviruses, more specifically SCoV-1, and contains a single stranded and positive sense RNA genome comprising approximately 29,903 nucleotides that has 5′ and 3′ terminal sequences containing 12 open reading frames (ORFs) (Figure 1 ) [20] . Like other coronaviruses, ORF1a/ORF1b is the biggest and encodes two large overlapping replicase polyproteins 1a (pp1a) and pp1ab. These precursors polyproteins are processed by two viral cysteine proteases (PLpro and 3CLpro) into 16 nonstructural proteins (NSP 1-16) (Table 1) [21] [22] [23] . SCoV-2 shares more than 79.5% of its genome and protein homology (95%-100%) with SCoV-1 A detailed knowledge of how pLpro interact with the host innate immune system is very important for understanding of the pathogenesis of SCoV-2. In this review, the interactions between PLpro of SCoV-2 and IFN response are described. A map of the pathways that will be discussed in the text is given in Figure 3 . [Insert Table 1] [Insert Figure 1] After the virion has entered the host cell, pp1a and pp1ab cleaved by two viral proteases, main protease (3CLpro, also called main protease) and PL pro [27] . 3CLpro is a dimer which utilizes a Cys/His catalytic dyad, whereas PLpro is a monomer with a Asp/His/Cys canonical catalytic triad Most coronaviruses encode two PLpro, termed PL1pro and PL2pro, whereas SCoV-1 and 2 encode a single PLpro (Table 1) [23, 29] . PLpro is the C-terminus of nsp3a and acts as a multifunctional cysteine protease along with phosphatase activity that processes the viral polyprotein and hosts cell proteins via the formation of an isopeptide bonds and hydrolyzing the peptide in viral and cellular substrates that participates in viral replication, regulates immune responses and antagonizes interferon This article is protected by copyright. All rights reserved (IFN) molecules [30, 31] . SCoV-2 PLpro is made up of an N-terminal ubiquitin-like domain (found in many ubiquitin-specific proteases or USPs) and a C-terminal domain containing thumb and palm, where the catalytic triad is situated, and the fingers, which include the zinc-finger motif [31] . SCoV-2 pLpro contains 945 nucleotides with 315 amino acid (residue 1564-1878, 35.6 kDa). The Amino acid sequence identity between the pLpro of SCoV-2 with SCoV-1 is 83%. As mentioned above, with this amino acid similarity there are differences in the biochemical characterization of SCoV-2 and SCoV- In addition to protease activity, PLpro is recognized to be involved in deubiquitination and deISG Analysis of purified CoV PLPs and the X-ray crystal structure of SCoV-1 PLpro reveal new information on viral DUB activity [44] . There is evidence for the structural relationship between This article is protected by copyright. All rights reserved SCoV-1 PLpro and ubiquitin C-terminal hydrolase (UCH-L1), ubiquitin-specific protease 14 (USP14) and herpes-associated ubiquitin-specific protease (HAUSP or USP7) [45, 46] . SCoV-1 PLpro with high affinity recognize poly Ub chains by reading units of a Lys48 and remove Lys48 from polyubiquitin chains [47, 48] .Recently studies showed that SCoV-2 PLpro have DUB activity [45, 49] . SCoV-2 PLpro cleaves K48-linked ubiquitin chains at a substantially slower rate than that of SCoV-1 PLpro. Like the SCoV-1, SCoV-2 PLpro shows no appreciable activity for K63 linked polyubiquitin chains [32]. (ISG15) conjugation in a process referred to as deISGylating [45, 50] . ISG15, an antiviral ubiquitinlike protein (Ubl) with two tandem ubiquitin-like folds, is expressed is secreted by human monocytes and lymphocytes and in response to IFN-α and β [51] . ISG15 conjugated with many targets (ISGylation), including Janus tyrosine kinase (JAK), signal transducer and activator of transcription (STAT) and IRF3 proteins and significantly leads to up-regulation following cellular stimulation by IFNs or viral infection [48, 51, 52] . Apart from the induction of immune responses, degradation or sequestration of viral proteins via ISGylation has also been found to play a role in host immunity [53] . In this context, researchers was found ISG15 significantly enriched in complexes with SCoV-2 PLpro Overall, SCoV-2 PLpro have deISGylating and DUB activities to promote the suppression of the innate immune response by effect on IFN and signaling pathways [57] . The PLpro as a drug candidate can a way to reduce cytokine storms associated with COVID-19. [Insert Figure 2] STING (stimulator of interferon genes, also known as MITA, ERIS and MPYS) is a key scaffolding protein which be essential for protecting the cell against a variety of pathogens and it could prevent the development of cancer by promoting anti tumour immune response [58] . STING, is an ERassociated membrane protein with four transmembrane domains in the N-terminal region [59] . Downstream effects of STING activation include Nuclear factor kappa B (NF-κB), IFN regulatory factor-3 (IRF3) and STAT6 activation, which leads to the production of type I IFNs [60] . Stimulation induces dimerization and phosphorylation of STING and triggers accumulation of STING complexed with TANK-binding kinase 1 (TBK1) from the ER to endosomal-lysosomal-perinuclear regions. Activated TBK1 leads to phosphorylation of IRF3 and NF-κB, which translocate to the nucleus and initiate innate immune gene transcription [61] . Actually, STING is a key scaffolding protein that links the cytosolic viral RNA sensors RIG-I and MDA5 to the mitochondrial antiviralsignaling protein (MAVS) [62] . The activation of STING facilitates the recruitment of IRF-3 and TBK-1 into a complex where IRF-3 is phosphorylated [63] . The NF-kB signaling pathway plays critical role in regulation of innate and adaptive immunity, inflammation, apoptosis, cancer, and tumor development [67] . NF-kB is a transcription factor, This article is protected by copyright. All rights reserved consists of five related proteins, p105/p50 (NF-κB1) and p100/p52 (NF-κB2), p65 (RelA), RelB and c-Rel (Rel), which in resting state remain in the cytoplasm as dimers associated with the IκB inhibitor [68] . There are eight IκB proteins, IκBα, IκBβ, IκBε, IκBζ, BCL-3, IκBns, and the precursor proteins NF-κB2 and NF-κB1, which are characterized by the presence of six to seven ankyrin repeat motifs (ANK) which have binding ability to NF-κB dimers [69, 70] . Therefore, in unstimulated cells, NF-κB dimers bind to IκB inhibitor proteins in the cytoplasm because all NF-κB proteins are characterized by the presence of a highly conserved Rel homology domain (RHD) in their N-terminus, which contains a nuclear localization signal (NLS) and is responsible interaction with IκBs [71] . Upon stimulation, IκB is phosphorylated in serine residues by the IκB kinase (IKK) complex, which consists of two catalytic subunits, IKKα (IKK1 or CHUK) and IKKβ (IKK2), and an NF-κB essential modifier (NEMO, also known as IKKγ, IKKAP1 or Fip-3) [72, 73] . Phosphorylated IκB creates a destruction motif recognized by the ubiquitin ligase complex and degraded by 26S proteasome, then NF-κB complexes translocate to the nucleus and regulates the expression of its target genes [38, 74] . Ubiquitination plays a crucial role in control of NF-κB pathway as a major regulator of the immune response [74] . USP15 inhibits the NF-κB pathway by removing K48-Ub from IκBα and consequently prevent degradation it. In this respect, Frieman et al. demonstrated that SARS PL pro stabilizes the IκBα and thereby blocks the activation of the NF-κB pathway [66] . In another study Ratia et al. indicated PLpro prevents this degradation of IκBα and leads to an increase in levels of IκBα [48] . Also TBK1 phosphorylates the IRF3, thereby no detectable level of IRF3 phosphorylation decrease of NF-κB p65 phosphorylation [75] . As mention above, SARS pLpro reduces the levels of ubiquitinated form of TBK1 [21] . This results conclude that SARS PLpro negatively regulates the NF-κB signal. The mitogen-activated protein kinase (MAPK), serine/threonine kinases, acts as an important factor in the intracellular signaling network [76] . MAPKs consist of four distinct groups: The extracellular signal-related kinases (ERKs), the c-jun N-terminal kinases (JNKs), the atypical MAPKs (ERK3, ERK5, and ERK8) and the p38 MAPKs [77] . ERK pathway plays a crucial role in the regulation of cellular processes. Activation of the ERK pathway includes three signal cascades, Raf, MEK1/2, and ERK1/2. Upon stimulation, Raf kinase is activated, which then activates the MEK1/2 and Subsequently the activated MEK1/2 phosphorylate and activate the ERK1/2. Finally, the activated This article is protected by copyright. All rights reserved ERK1/2 translocates from cytoplasm to nucleus and phosphorylates a large number of downstream substrates such as transcription factors regulating transcription for a large number of genes [78] . Therefore, the ERK signaling pathway involves a variety of cellular activities including cell growth, differentiation, survival or apoptosis [79] . Phosphorylation of STAT1 at serine 727 by ERK1/2 and p38 MAPK facilitates nuclear translocation of STAT1 for full expression of antiviral genes like protein kinase R (PKR), 29-59-oligoadenylate synthetase (OAS) and ISG15. Downregulation of ERK1 was identified with suppression of interaction between ERK1 and STAT1 as type I IFN antagonist function of SCoV PLpro [80] . The transforming growth factor-β (TGF-β) superfamily of signaling molecules controls a broad range of cellular processes, including cell differentiation, proliferation, embryonic development, and remodeling [81] . The effects of TGF-β are mediated by three known isoforms of TGF-β (TGF-β1, 2, 3) via TGF-β type I and II receptors and are transduced through Smad and non-Smad pathways. In patients with SCoV-2 virus infection, death has been caused by uncontrolled inflammatory responses, edema and fibrosis in the lungs [82] . Fibrosis is one of the most important consequences of TGF-β dysregulation [83]. SCoV-2 virus infection induces massive activation of the TGF-β in the lungs through neutrophil infiltration into the lungs, dysregulation of the coagulation and fibrinolytic pathways and apoptosis of bronchial epithelial cells, pneumocytes, and T lymphocytes [82] . It is interesting to note that SCoV-1 PLpro significantly increased TGF-β1 mRNA expression and protein production in cell-based assay and in mouse model [84, 85] and in early phase of SCoV-1 infection, TGF-β1 rises in plasma and lung tissues [85] . TGF-β stimulation can be activated under MAPK cascade, which represents an important mechanism for non-Smad pathways [86] . One study has shown that ERK1/2 and p38 MAPK inhibitors (U0126 and SB203580) dramatically decreased and expression of many TGF-β1-associated genes including HSP27, vimentin, protein disulfide isomerase A3 precursor, retinal dehydrogenase 2, glial fibrillary acidic protein, glutathione transferase omega-1 increased [84] . Vimentin is the major component of the type III intermediate filament protein which during virus entry it was observed a direct interaction between vimentin and SCoV spike protein [85] . PLpro up-regulates activating ubiquitin proteasome of UBE2K and proteasome subunit alpha type 5, and p38MAPK and ERK1/2 signaling via increase of This article is protected by copyright. All rights reserved HSP27 [84]. In li et al. study, SCoV-1 PLpro significantly triggered Egr-1 dependent activation of TGF-β1 promoter via ROS/p38 MAPK/STAT3 pathway [85] . Given the similarities between of SCoV-2 and SCoV-1, it could be predicted that PLpro can have positive role in the regulation of the cellular inflammatory and immune responses through TGF-β. [Insert Figure 3] Viral proteases are an attractive target for antiviral drug development [51] . In this context, a study published in Nature discusses about the role of SARS-CoV-1 and SARS-CoV-2 PLpro to host innate immune response and immune evasion [54] . Although the PLpro is a potential target for CoVs inhibitors, but no inhibitor approved drug by FDA. Table 2 shows sixteen FDA-approved drugs with good affinity for SARS-CoV-2 PLpro [31]. [Insert Table 2] Computational methods were used for the development of the inhibitors of SCoV-2 PLpro. Molecular docking indicated a series of drugs that exhibit a high binding affinity to SCoV-2 PLpro. Table 3 shows the binding energy scores along with interaction of compounds over the SCoV-2 PLpro. In inhibitors of the SCoV-2 Plpro. In this study dasatinib with the best docking score could efficiently bind to SCoV-2 PLpro [88] . Dasatinib was also shown to be active against SCoV-2 in a case report study [89] . It is interesting to know that curcumin, a polyphenol extracted from an East Indian plant Curcuma longa, can interact with a cysteine residue of Plpro [88] . Liu et al. reported that curcumin has a protective effect on the lung in case of severe pneumonia caused by SCoV-2, decreasing the expression of proinflammatory cytokines [90] . In addition, Terconazole and fluspirilene were shown to be active in cell-culture assays for SCoV-2 [89] . Another study have shown chloroquine might has anti-viral activity through its inhibition of This article is protected by copyright. All rights reserved SCoV-2 PLpro [91] . GRL-0617 with inhibition of SCoV-2 PLpro can reduced the virus cytopathogenic effect (CPE), viral replication and suppression of host innate immune responses in infected cells [32] . Recently, Fu et al. showed GRL-0617 (IC 50 = 2.1 μM) blocked the binding of the C-terminal tail of ISG15 with PLpro and can be a promising approach for combating COVID-19 [92] . One study showed 6-Thioguanine (6-TG) can be considered as a inhibiting PLpro de-ISGylation, polyprotein cleavage, and viral replication of SCoV-2 [93] . Disulfiram, an FDA-approved drug has also been identified to be a potential therapeutic target for SARS-CoV-2 infection. Tamburin et al showed that symptoms compatible with COVID-19 were significantly less common in patient under disulfiram treatment than control group (not taking disulfiram) [94] . Disulfiram is known to be a thiol-reactive compound that can covalently modify cysteine residues and may also act as a zinc ejector [95] . There are four Zn-sites in SARSCoV-2 PLpro that's why disulfiram (as Zn-ejector drug) can be used to disrupt Covid-19 protein structure/function [96] . It is noteworthy that disulfiram has now entered Phase 2 clinical trial. Primary outcome showed change in plasma inflammatory biomarker levels (e.g., IL-6, IL-1b) and viral load at days 5, 15, and 31 [97] . At the end of this topic, we comparison the activity of PLpro between SCoV-1, SCoV-2 for two Inhibitors which may contribute to speed up therapeutic development of COVID-19 (Table 4) [98]. [Insert Table 3] [Insert Table 4] This article is protected by copyright. All rights reserved Based on above mentions, the PLpro is responsible for suppression of host innate immune responses, so further characterization of the SARS-COV-2 PLpro may provide new targets for antiviral interventions. As an outcome of this study, more investigation regarding the ability of the some mentioned compounds with high binding affinity or energy in blocking the entrance of the PLpro active site and inhibiting PLpro enzyme activity is highly recommended. In this regard, in vivo and in vitro evaluations for candidate drugs and prepare for clinical trial applications is better to do. 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