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The SARS-coronavirus papain-like protease: Structure, function and inhibition by designed antiviral compounds

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... Papain like protease or PLpro is a cysteine protease specific to coronaviruses which comprises one part of the viral protease duo (the other part being the Main protease) responsible for the proteolysis of the viral polypeptide housing a myriad of proteins such as the structural, non-structural and the accessory proteins (Baez-Santos et al., 2015;Tripathi, 2021, 2022;Mishra and Tripathi, 2021). Coronaviruses from the coronaviridae family host a large genomic organisation of 26-32 kb, which hosts a distinctive replication-transcription complex component (Lu et al., 2020;Singh et al., 2021a). ...
... In addition, Nsp3 hosts Ubl proteins, a domain responsible for synthesising viral subgenomic RNAs, a domain that binds G-quadruplexes, a SARS unique domain (SUD), nucleic acid binding domain (NAB) with a chaperone function, marker domain and predicted transmembrane domains. This multifunctional attribute of nsp3 and its domains make it an attractive drug target for anti-viral therapeutics (Baez-Santos et al., 2015;Tripathi, 2021, 2022;Mishra and Tripathi, 2021). ...
... PLpro follows the same general catalytic mechanism as a cysteine protease, where the cysteine residue acts as a nucleophile with histidine as a general acid-base, and aspartic acid aids in stabilising the histidine residue (Klemm et al., 2020). The mechanism followed by PLpro (as proposed by Baez-Santos et al., 2015) begins with the deprotonation of the Cys111 by His273, which leads to a nucleophilic attack by the thiol group of Cys111 on the highly specific Glycine residue present in the substrate. This is followed by various intermediate states of the substrate and active site residue, which are stabilised by an oxyanion hole conferred by Trp106, which has been found to be critical for PLpro activity. ...
Article
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 utilizing 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.
... SARS-CoV-2 nsp3 is a large multidomain protein essential for viral replication [129]. PLpro, a~36 kDa protein, is one of the domains of nsp3 and exists as a monomer in biological settings [130]. It has a ubiquitin-like (UBL) domain at the N-terminus, and the rest of the protein has the architecture of the ubiquitin-specific protease (USP) fold ( Figure 9) [130,131]. ...
... PLpro, a~36 kDa protein, is one of the domains of nsp3 and exists as a monomer in biological settings [130]. It has a ubiquitin-like (UBL) domain at the N-terminus, and the rest of the protein has the architecture of the ubiquitin-specific protease (USP) fold ( Figure 9) [130,131]. The USP fold is topologically organized into three subdomains, thumb, palm and fingers, which together form a structure resembling a right hand [132]. ...
... The USP fold is topologically organized into three subdomains, thumb, palm and fingers, which together form a structure resembling a right hand [132]. The SARS-CoV-2 PLpro active site contains a canonical cysteine protease catalytic triad comprised of residues Cys111, His272, and Asp286 [130,131]. It cleaves at the N terminus of the LXGG motifs ( Figure 10) between nsp1, nsp2, nsp3 and nsp4, liberating the nsp1, nsp2, and nsp3 from the viral polyproteins [130,133]. ...
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The coronavirus disease 2019 (COVID-19) pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a recently emerged human coronavirus. COVID-19 vaccines have proven to be successful in protecting the vaccinated from infection, reducing the severity of disease, and deterring the transmission of infection. However, COVID-19 vaccination faces many challenges, such as the decline in vaccine-induced immunity over time, and the decrease in potency against some SARS-CoV-2 variants including the recently emerged Omicron variant, resulting in breakthrough infections. The challenges that COVID-19 vaccination is facing highlight the importance of the discovery of antivirals to serve as another means to tackle the pandemic. To date, neutralizing antibodies that block viral entry by targeting the viral spike protein make up the largest class of antivirals that has received US FDA emergency use authorization (EUA) for COVID-19 treatment. In addition to the spike protein, other key targets for the discovery of direct-acting antivirals include viral enzymes that are essential for SARS-CoV-2 replication, such as RNA-dependent RNA polymerase and proteases, as judged by US FDA approval for remdesivir, and EUA for Paxlovid (nirmatrelvir + ritonavir) for treating COVID-19 infections. This review presents an overview of the current status and future direction of antiviral drug discovery for treating SARS-CoV-2 infections, covering important antiviral targets such as the viral spike protein, non-structural protein (nsp) 3 papain-like protease, nsp5 main protease, and the nsp12/nsp7/nsp8 RNA-dependent RNA polymerase complex.
... The PLpro domain of about 250 amino acids is a part of the large (~2000 residues) nsp3 protein, which includes many domains [8]. Importantly, nsp3 has four transmembrane segments and locates to the endoplasmic reticulum (ER) membrane ( Figure 1). ...
... For the new PLpro sensor ("mScarlet-LKGG-miRFP670"), we selected brighter fluorescent proteins; namely, mScarlet [17] and miRFP670 [18]. The PLpro recognition site LKGG [8] was introduced into the linker between them. Similar to many previous FRET-based protease sensors, we expected the disappearance of the FRET after the linker cleavage and, hence, an increase in the fluorescence intensity of the mScarlet donor ( Figure 2A). ...
... Secondly, it was shown that in addition to performing its main function, viral polyprotein processing, PLpro was involved in the inhibition of the host immunity response. The protease dysregulates interferon antiviral signaling and reduces inflammation by removing ISG15 and ubiquitin from cellular proteins [8]. Therefore, PLpro inhibitors can potentially not only suppress viral replication, but also restore the regulation of impaired signaling cascades in infected cells [10]. ...
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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.
... Central components of this complex are NSP12 (RNAdependent RNA polymerase (RdRp)) as well as the cofactors NSP7 and NSP8. It has been shown previously that the Plpro of SARS-CoV-1 exhibits deubiquinating activity [30,40,41]. As Plpro of SARS-CoV-1 shares 82.9% sequence identity with the same protease of SARS-CoV-2 [42], it was legitimate to hypothesize that DIs might interfere with SARS-CoV-2 replication [43]. ...
... Next, we investigated the restriction capability of more specific DIs, namely, the USP7inhibitor HBX41108 and the USP14-inhibitor IU-1. Those DIs were purposely chosen as the tertiary structure of SARS-CoV-1 Plpro, which has DUB activity by itself and is known to be similar to the DUBs USP7 and 14 [41]. Interestingly, HBX41108 was able to inhibit the viral replication in SARS-CoV-2-infected Vero B4 cells in a similar way ( Figure 1B) as PR-619 ( Figure 1A), whereas the USP14-specific inhibitor IU-1 showed no effect ( Figure 1C). ...
... In one infection experiment, RDV was enrolled as positive control at 1 µM, which blocked the SARS-CoV-2 replication completely ( Figure 1C). and is known to be similar to the DUBs USP7 and 14 [41]. Interestingly, HBX41108 was able to inhibit the viral replication in SARS-CoV-2-infected Vero B4 cells in a similar way ( Figure 1B) as PR-619 ( Figure 1A), whereas the USP14-specific inhibitor IU-1 showed no effect ( Figure 1C). ...
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The ubiquitin proteasome system (UPS), particularly its deubiquitinating enzymes (DUBs), play a key role in the replication cycle of coronaviruses. The SARS-CoV-2 papain-like protease (Plpro) is known to process the viral polyproteins to form the replicase transcriptase complex and to counteract the host viral response. Recently, it was shown that this viral protease can also act as a deubiquitinating enzyme. In this study, we demonstrate that certain DUB-Inhibitors (DIs) interfere with SARS-CoV-2 replication. The DIs PR-619 and HBX41108 restrict SARS-CoV-2 in both Vero B4 and human Calu-3 lung cells where cells were infected with a Multiplicity of Infection (MOI) of 0.02. An in vitro protease assay using recombinant Plpro and Amido-4-methylcoumarin (AMC)-conjugated substrate revealed that PR-619 and HBX41108 are able to block the protease at concentrations where the interventions restricted virus replication. In contrast, DIs that do not inhibit Plpro had no influence on virus replication, which indicated that the protease might be at least one major target. Future vertical studies that would gain more insights into the mechanisms of how DUBs effect the replication of SARS-CoV-2 will further validate them as a potential therapeutic target.
... As mentioned, SARS-CoV-2, just as SARS-CoV-1, encodes four structural proteins involved in forming the viral envelope and in the packaging of the RNA genome and sixteen non-structural proteins (labelled as nsp1-16) [14,122] that take part in replication and transcription of the viral genome. The nsps are cleaved from PP1a and PP1ab by the 3Clike cysteine protease (3CLpro, also known as the main protease (Mpro) or non-structural protein 5 (nsp5)) and the papain-like protease (PLpro, the protease domain autocleaved from nsp3) [123][124][125][126]. ...
... PLpro is also essential for the RTC formation [127,128] and is involved in cleaving proteinaceous post-translational modifications on host proteins [129][130][131]. It contains a Cys 4 ZNF that tetrahedrally coordinates a zinc ion belonging to the "zinc ribbon" fold group [51,132] and it is essential for catalysis because it maintains the structural integrity of PLpro [122,[133][134][135][136][137]. ...
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An unprecedented effort to tackle the ongoing COVID-19 pandemic has characterized the activity of the global scientific community over the last two years. Hundreds of published studies have focused on the comprehension of the immune response to the virus and on the definition of the functional role of SARS-CoV-2 proteins. Proteins containing zinc fingers, both belonging to SARS-CoV-2 or to the host, play critical roles in COVID-19 participating in antiviral defenses and regulation of viral life cycle. Differentially expressed zinc finger proteins and their distinct activities could thus be important in determining the severity of the disease and represent important targets for drug development. Therefore, we here review the mechanisms of action of host and viral zinc finger proteins in COVID-19 as a contribution to the comprehension of the disease and also highlight strategies for therapeutic developments.
... Both enzymes are capable of recognizing and hydrolyzing fluorophores from the C-termini of RLRGG peptide, Ub, and ISG15 substrates, yet the kinetic parameters associated with these reactions are different (Baez-Santos et al. 2014). Despite that a number of SARS2-PLpro and SARS1-PLpro inhibitors have been reported, none of these reports have convincingly demonstrated the in vivo activity of the identified drug compounds (Ratia et al. 2008;Baez-Santos et al. 2014;Baez-Santos et al. 2015;Osipiuk et al. 2021;Shan et al. 2021;Zhao et al. 2021). Moreover, the reported coronavirus PLpro inhibitors are virus-specific and cannot serve as a ready-to-use treatment option for future coronavirus epidemics. ...
... Despite the considerable efforts lasting from SARS to MERS, and now COVID-19, none of the reported coronavirus PLpro inhibitors has been shown to be active in animal models. Pharmacokinetic characterization of F0123 is warranted to achieve enhanced metabolic stability in vivo, the major obstacle hindering anti-PLpro drug development (Ratia et al. 2008;Baez-Santos et al. 2014;Baez-Santos et al. 2015;Osipiuk et al. 2021;Shan et al. 2021;Zhao et al. 2021). Compared with GRL0617 and its analogue, a "model" compound that have been extensively analyzed in our and others' studies on SARS1 or SARS2-PLpro (Ratia et al. 2008;Shin et al. 2020;Fu et al. 2021;Gao et al. 2021), the versatile binding modes of F0213 may represent as a new generation of 'tool' compound to facilitate rational design of pan-coronavirus PLpro inhibitors. ...
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The emergence of SARS-CoV-2 variants of concern and repeated outbreaks of coronavirus epidemics in the past two decades emphasize the need for next-generation pan-coronaviral therapeutics. Drugging the multi-functional papain-like protease (PLpro) domain of the viral nsp3 holds promise. However, none of the known coronavirus PLpro inhibitors has been shown to be in vivo active. Herein, we screened a structurally diverse library of 50,080 compounds for potential coronavirus PLpro inhibitors and identified a noncovalent lead inhibitor F0213 that has broad-spectrum anti-coronaviral activity, including against the Sarbecoviruses (SARS-CoV-1 and SARS-CoV-2), Merbecovirus (MERS-CoV), as well as the Alphacoronavirus (hCoV-229E and hCoV-OC43). Importantly, F0213 confers protection in both SARS-CoV-2-infected hamsters and MERS-CoV-infected human DPP4-knockin mice. F0213 possesses a dual therapeutic functionality that suppresses coronavirus replication via blocking viral polyprotein cleavage, as well as promoting antiviral immunity by antagonizing the PLpro deubiquitinase activity. Despite the significant difference of substrate recognition, mode of inhibition studies suggest that F0213 is a competitive inhibitor against SARS2-PLpro via binding with the 157K amino acid residue, whereas an allosteric inhibitor of MERS-PLpro interacting with its 271E position. Our proof-of-concept findings demonstrated that PLpro is a valid target for the development of broad-spectrum anti-coronavirus agents. The orally administered F0213 may serve as a promising lead compound for combating the ongoing COVID-19 pandemic and future coronavirus outbreaks.
... In some cases, viruses encode proteins with E3 activity or even form a part of the E3 cell complex to degrade cellular proteins that limit the spread of the virus (99). The coronavirus genes include two open-reading frames (ORFs), ORFs 1a and 1b, which produce two viral polyproteins, pp1a and pp1ab, respectively, which are further cleaved by viral proteases to produce functional NSPs (100). Two coronavirus proteases, papain-like protease (PLpro; released from NSP3) and 3C protease (3CLpro), are especially critical for coronavirus replication (100). ...
... The coronavirus genes include two open-reading frames (ORFs), ORFs 1a and 1b, which produce two viral polyproteins, pp1a and pp1ab, respectively, which are further cleaved by viral proteases to produce functional NSPs (100). Two coronavirus proteases, papain-like protease (PLpro; released from NSP3) and 3C protease (3CLpro), are especially critical for coronavirus replication (100). The 3CLpro processes the C-terminus of the viral polyproteins pp1a and pp1ab, whereas PLpro processes its N-terminus. ...
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Melatonin, an endogenous indoleamine, is an antioxidant and anti-inflammatory molecule widely distributed in the body. It efficiently regulates pro-inflammatory and anti-inflammatory cytokines under various pathophysiological conditions. The melatonin rhythm, which is strongly associated with oxidative lesions and mitochondrial dysfunction, is also observed during the biological process of aging. Melatonin levels decline considerably with age and are related to numerous age-related illnesses. The signs of aging, including immune aging, increased basal inflammation, mitochondrial dysfunction, significant telomeric abrasion, and disrupted autophagy, contribute to the increased severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. These characteristics can worsen the pathophysiological response of the elderly to SARS-CoV-2 and pose an additional risk of accelerating biological aging even after recovery. This review explains that the death rate of coronavirus disease (COVID-19) increases with chronic diseases and age, and the decline in melatonin levels, which is closely related to the mitochondrial dysfunction in the patient, affects the virus-related death rate. Further, melatonin can enhance mitochondrial function and limit virus-related diseases. Hence, melatonin supplementation in older people may be beneficial for the treatment of COVID-19.
... The naphthalene-based inhibitors are potent, competitive inhibitors and bind within the active site of PLpro. Because of T-shaped π-π interaction with the naphthalene group of the inhibitor as well as other van der Waals interactions [84,99,100]. The peptidic inhibitors' drawbacks such as poor pharmacokinetic profiles and in vivo instability and cell membrane impermeability unappropriate PK profiles should be kept in mind when converting these to drug molecules [84]. ...
Article
The novel coronavirus that caused COVID-19 pandemic is SARS-CoV-2. Although various vaccines are currently being used to prevent the disease's severe consequences, there is still a need for medications for those who become infected. The SARS-CoV-2 has a variety of proteins that have been studied extensively since the virus's advent. In this review article, we looked at chemical to molecular aspects of the various structures studied that have pharmaceutical activity and attempted to find a link between drug activity and compound structure. For example, designing of the compounds which bind to the allosteric site and modify hydrogen bonds or the salt bridges can disrupt SARS-CoV2 RBD–ACE2 complex. It seems that quaternary ammonium moiety and quinolin-1-ium structure could act as a negative allosteric modulator to reduce the tendency between spike-ACE2. Pharmaceutical structures with amino heads and hydrophobic tails can block envelope protein to prevent making mature SARS-CoV-2. Also, structures based on naphthalene pharmacophores or isosteres can form a strong bond with the PLpro and form a π-π and the Mpro's active site can be occupied by octapeptide compounds or linear compounds with a similar fitting ability to octapeptide compounds. And for protein RdRp, it is critical to consider pH and pKa so that pKa regulation of compounds to comply with patients is very effective, thus, the presence of tetrazole, phenylpyrazole groups, and analogs of pyrophosphate in the designed drugs increase the likelihood of the RdRp active site inhibition. Finally, it can be deduced that designing hybrid drug molecules along with considering the aforementioned characteristics would be a suitable approach for developing medicines in order to accurate targeting and complete inhibition this virus.
... Likewise, flavonoids such as herbacetin, rhoifolin, and pectolinarin can effectively inhibit the enzymatic activities of SARS-CoV main protease [9]. By assessing fractionation-based anti-papain protease function of the methanolic extract of Paulownia tomentosa fruits, researchers have discovered that several geranylated flavonoid derivatives possess potent inhibitory impact on SARS-CoV papain protease [23]. ...
Article
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The current outbreak of coronavirus disease 2019 (COVID-19), which is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has negatively impacted the global health and economy. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shares many similarities with SARS-CoV and Middle East respiratory syndrome-related coronavirus. It has been identified that the 3C-like protease (3CLpro) enzyme in coronaviruses can be a major therapeutic target for combating these serious infections. Therefore, flavonoids are believed to hold high potential in eliminating the viruses and infections. Flavonoids are polyphenolic secondary metabolites found in plants and have been demonstrated for their notable benefits for health. The antiviral activity of flavonoids has been reported in recent studies. Flavonoids, such as apigenin, quercetin, luteolin, amentoflavone, epigallocatechin gallate, gallocatechin gallate, and kaempferol, are known to be able to fight against coronaviruses by reducing the 3CLpro activity, according to the docking studies. Besides, we also found that several flavonoids have the potential to suppress the inflammatory cytokines, which are generally expressed in the lungs of coronavirus-infected individuals. However, the studies utilizing 3CLpro using various scaffolds of flavonoids need to be performed for better understanding on the antiviral potential of flavonoid derivatives against 3CLpro.
... The second protease is the main protease or Mpro encoded by nsp5, which is responsible for cleaving the polyproteins at 11 further sites to release the remaining 12 nsps (Fan et al., 2004). Both activities are essential for viral replication and therefore, both PLpro and Mpro are prime drug targets in CoVs, including SARS-CoV-2 (Hilgenfeld, 2014;Báez-Santos et al., 2015;Lei et al., 2018). Indeed, in late 2021, an oral Mpro inhibitor termed Nirmatrelvir successfully completed clinical trials (clinical trial identifier NCT04960202, NCT05011513) , and became the third FDA approved small molecule drug against SARS-CoV-2, reaching 89% efficacy in clinical settings against severely ill patients (NCT04960202). ...
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The emergence of SARS-CoV-2 causing the COVID-19 pandemic, has highlighted how a combination of urgency, collaboration and building on existing research can enable rapid vaccine development to fight disease outbreaks. However, even countries with high vaccination rates still see surges in case numbers and high numbers of hospitalized patients. The development of antiviral treatments hence remains a top priority in preventing hospitalization and death of COVID-19 patients, and eventually bringing an end to the SARS-CoV-2 pandemic. The SARS-CoV-2 proteome contains several essential enzymatic activities embedded within its non-structural proteins (nsps). We here focus on nsp3, that harbours an essential papain-like protease (PLpro) domain responsible for cleaving the viral polyprotein as part of viral processing. Moreover, nsp3/PLpro also cleaves ubiquitin and ISG15 modifications within the host cell, derailing innate immune responses. Small molecule inhibition of the PLpro protease domain significantly reduces viral loads in SARS-CoV-2 infection models, suggesting that PLpro is an excellent drug target for next generation antivirals. In this review we discuss the conserved structure and function of PLpro and the ongoing efforts to design small molecule PLpro inhibitors that exploit this knowledge. We first discuss the many drug repurposing attempts, concluding that it is unlikely that PLpro-targeting drugs already exist. We next discuss the wealth of structural information on SARS-CoV-2 PLpro inhibition, for which there are now ∼30 distinct crystal structures with small molecule inhibitors bound in a surprising number of distinct crystallographic settings. We focus on optimisation of an existing compound class, based on SARS-CoV PLpro inhibitor GRL-0617, and recapitulate how new GRL-0617 derivatives exploit different features of PLpro, to overcome some compound liabilities.
... Nonstructural protein 2 (NSP2) binds two other host proteins, prohibitin 1 and prohibitin 2 (PHB1 and PHB2), disrupting the host cell environment (Cornillez-Ty et al., 2009). NSP3, the papain-like proteinase protein, has multiple functions and is considered the most important protease of the virus (Báez-Santos et al., 2015). ...
Article
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Coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2), is the largest pandemic in modern history with very high infection rates and considerable mortality. The disease, which emerged in China’s Wuhan province, had its first reported case on December 29, 2019, and spread rapidly worldwide. On March 11, 2020, the World Health Organization (WHO) declared the COVID-19 outbreak a pandemic and global health emergency. Since the outbreak, efforts to develop COVID-19 vaccines, engineer new drugs, and evaluate existing ones for drug repurposing have been intensively undertaken to find ways to control this pandemic. COVID-19 therapeutic strategies aim to impair molecular pathways involved in the virus entrance and replication or interfere in the patients’ overreaction and immunopathology. Moreover, nanotechnology could be an approach to boost the activity of new drugs. Several COVID-19 vaccine candidates have received emergency-use or full authorization in one or more countries, and others are being developed and tested. This review assesses the different strategies currently proposed to control COVID-19 and the issues or limitations imposed on some approaches by the human and viral genetic variability.
... In the publications carried out on virus binding and its other structures, "covalent bond" (B aez- Santos et al., 2015;Berry et al., 2015) was in the center, and most connections were among "acid," "metal-s" and "halogen." However, "hydrogen bond" and its connection to "Amphiphilin" (the active mechanism of soap influencing the effacement of the coronavirus) had many citations (de Haan et al., 1998;Kamata et al., 1994). ...
Article
Purpose Using science mapping analysis approach and co-word analysis, the present study explores and visualizes research fields and thematic evolution of the coronavirus. Based on this method, one can get a picture of the real content of the themes in the mentioned thematic area and identify the main minor and emerging themes. Design/methodology/approach This study was conducted based on co-word science mapping analysis under a longitudinal study (from 1988 to 2020). The collection of documents in this study was further divided into three subperiods: 1988–1998, 1999–2009 and 2010–2020. In order to perform science mapping analysis based on co-word bibliographic networks, SciMAT was utilized as a bibliometric tool. Moreover, WoS, PubMed and Scopus bibliographic databases were used to download all records. Findings In this study, strategic diagrams were demonstrated for the coronavirus research for a chronological period to assess the most relevant themes. Each diagram depended on the sum of documents linked to each research topic. In the first period (1988–1998), the most centralizations were on virology and evaluation of coronavirus structure and its structural and nonstructural proteins. In the second period (1999–2009), with due attention to high population density in eastern Asia and the increasing number of people affected with the new generation of coronavirus (named severe acute respiratory syndrome virus or SARS virus), publications have been concentrated on “antiviral activity.” In the third period (2010–2020), there was a tendency to investigate clinical syndromes, and most of the publications and citations were about hot topics like “severe acute respiratory syndrome,” “coronavirus” and “respiratory tract disease.” Scientometric analysis of the field of coronavirus can be regarded as a roadmap for future research and policymaking in this important area. Originality/value The originality of this research can be considered in two ways. First, the strategic diagrams of coronavirus are drawn in four thematic areas including motor cluster, basic and transversal cluster, highly developed cluster and emerging and declining cluster. Second, COVID-19 is mentioned as a hot topic of research. Keywords: Strategic diagram; COVID-19; Coronavirus; Thematic evolution of the coronavirus; Co-word science mapping analysis;
... Nonstructural protein 2 (NSP2) binds two other host proteins, prohibitin 1 and prohibitin 2 (PHB1 and PHB2), disrupting the host cell environment (Cornillez-Ty et al., 2009). NSP3, the papain-like proteinase protein, has multiple functions and is considered the most important protease of the virus (Báez-Santos et al., 2015). ...
Article
Full-text available
Coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2), is the largest pandemic in modern history with very high infection rates and considerable mortality. The disease, which emerged in China's Wuhan province, had its first reported case on December 29, 2019, and spread rapidly worldwide. On March 11, 2020, the World Health Organization (WHO) declared the COVID-19 outbreak a pandemic and global health emergency. Since the outbreak, efforts to develop COVID-19 vaccines, engineer new drugs, and evaluate existing ones for drug repurposing have been intensively undertaken to find ways to control this pandemic. COVID-19 therapeutic strategies aim to impair molecular pathways involved in the virus entrance and replication or interfere in the patients' overreaction and immunopathology. Moreover, nanotechnology could be an approach to boost the activity of new drugs. Several COVID-19 vaccine candidates have received emergency-use or full authorization in one or more countries, and others are being developed and tested. This review assesses the different strategies currently proposed to control COVID-19 and the issues or limitations imposed on some approaches by the human and viral genetic variability.
... Due to its importance in viral replication, conserved sequences, and 3D structures, M pro has become a potential target for drug discovery against the novel coronavirus [11]. In addition to the role in viral replication, PL pro also suppresses innate immunity through reversing the ubiquitination and ISGylation events where SARS-CoV-2 PLpro prefers the ISGylated proteins [12]. This dual functionality of PL pro makes it an attractive target for drug discovery against SARS-CoV-2. ...
Article
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Huge vaccination drives are underway around the world for the ongoing COVID-19 pandemic. However, the search for antiviral drugs is equally crucial. As new drug discovery is a time-consuming process, repurposing of existing drugs or developing drug candidates against SARS-CoV-2 will make the process faster. Considering this, 63 approved and developing antimalarial compounds were selected to screen against main protease (Mpro) and papain-like protease (PLpro) of SARS-CoV-2 using in silico methods to find out possible new drug candidate(s). Out of 63 compounds, epoxomicin showed the best binding affinity against the Mpro with CDocker energy of - 57.511 kcal/mol without any toxic effect. This compound was further taken for molecular dynamic simulation study, where the Mpro-epoxomicin complex was found to be stable with binding free energy - 79.315 kcal/mol. The possible inhibitory potential of the selected compound was determined by 3D-QSAR analysis and found to be 0.4447 µM against SARS-CoV-2 Mpro. Finally, the structure activity relationship of the compound was analyzed and two fragments responsible for overall good binding affinity of the compound at the active site of Mpro were identified. This study suggests a safe antimalarial drug, namely epoxomicin, as a probable inhibitor of SARS-CoV-2 Mpro which needs further validation by in vitro/in vivo studies before clinical use. Supplementary information: The online version contains supplementary material available at 10.1007/s11224-022-01916-0.
... To improve compound properties, a medicinal chemistry campaign was initiated, focusing on key aspects of the compound. We learned from previous published works that attempted to improve the piperidine scaffold for SARS-CoV PLpro (Báez-Santos et al., 2014;Báez-Santos et al., 2015;Ghosh et al., 2020), enabling us to explore novel chemical space. We also knew that 3k and 5c were metabolically labile (discussed below) and our designs also aimed to improve the ADME properties of compounds. ...
Article
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The COVID-19 pandemic continues unabated, emphasizing the need for additional antiviral treatment options to prevent hospitalization and death of patients infected with SARS-CoV-2. The papain-like protease (PLpro) domain is part of the SARS-CoV-2 non-structural protein (nsp)-3, and represents an essential protease and validated drug target for preventing viral replication. PLpro moonlights as a deubiquitinating (DUB) and deISGylating enzyme, enabling adaptation of a DUB high throughput (HTS) screen to identify PLpro inhibitors. Drug repurposing has been a major focus through the COVID-19 pandemic as it may provide a fast and efficient route for identifying clinic-ready, safe-in-human antivirals. We here report our effort to identify PLpro inhibitors by screening the ReFRAME library of 11,804 compounds, showing that none inhibit PLpro with any reasonable activity or specificity to justify further progression towards the clinic. We also report our latest efforts to improve piperidine-scaffold inhibitors, 5c and 3k, originally developed for SARS-CoV PLpro. We report molecular details of binding and selectivity, as well as in vitro absorption, distribution, metabolism and excretion (ADME) studies of this scaffold. A co-crystal structure of SARS-CoV-2 PLpro bound to inhibitor 3k guides medicinal chemistry efforts to improve binding and ADME characteristics. We arrive at compounds with improved and favorable solubility and stability characteristics that are tested for inhibiting viral replication. Whilst still requiring significant improvement, our optimized small molecule inhibitors of PLpro display decent antiviral activity in an in vitro SARS-CoV-2 infection model, justifying further optimization.
... PL pro is a 35 kDa domain withinNsp3, which is a 215 kDa multidomain protein that is a key component of the viral replication complex. 10 Compared to PL pro from SARS-CoV, SARS-CoV-2 PL pro displays decreased deubiquitination activity and enhanced deISGlyation activity. 9,11 In contrast to M pro , PL pro is a more challenging drug target mainly for two reasons. ...
Article
The global COVID-19 pandemic underscores the dire need for effective antivirals. Encouraging progress has been made in developing small-molecule inhibitors targeting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and main protease (Mpro). However, the development of papain-like protease (PLpro) inhibitors faces several obstacles. Nevertheless, PLpro represents a high-profile drug target given its multifaceted roles in viral replication. PLpro is involved in not only the cleavage of viral polyprotein but also the modulation of host immune response. In this study, we conducted a drug-repurposing screening of PLpro against the MedChemExpress bioactive compound library and identified three hits, EACC, KY-226, and tropifexor, as potent PLpro inhibitors with IC50 values ranging from 3.39 to 8.28 μM. The three hits showed dose-dependent binding to PLpro in the thermal shift assay. In addition, tropifexor inhibited the cellular PLpro activity in the FlipGFP assay with an IC50 of 10.6 μM. Gratifyingly, tropifexor showed antiviral activity against SARS-CoV-2 in Calu-3 cells at noncytotoxic concentrations. Overall, tropifexor represents a novel PLpro inhibitor that can be further developed as SARS-CoV-2 antivirals.
... Identification of specific active site inhibitors for PLpro, including approaches to analyse peptidic, non-peptidic and "dual target" inhibitors Zmudzinski et al., 2020;Shen et al., 2021), is particularly challenging due to a rather "featureless" active site and a high similarity to host deubiquitinases compared to proteases like Mpro. Access to the PLpro active site is regulated by a flexible blocking loop 2 (BL2) which is involved in substrate binding (Báez-Santos et al., 2015). PLpro binds ubiquitin and ubiquitinlike proteins at two distinct sites, S1 and S2, thereby providing specificity for K48-polyubiquitin (K48-Ub 2 ) and ISG15 ( Figures 1, 2) (Békés et al., 2016;Klemm et al., 2020). ...
Article
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The papain-like protease (PLpro) of SARS-CoV-2 is essential for viral propagation and, additionally, dysregulation of the host innate immune system. Using a library of 40 potential metal-chelating compounds we performed an X-ray crystallographic screening against PLpro. As outcome we identified six compounds binding to the target protein. Here we describe the interaction of one hydrazone (H1) and five thiosemicarbazone (T1-T5) compounds with the two distinct natural substrate binding sites of PLpro for ubiquitin and ISG15. H1 binds to a polar groove at the S1 binding site by forming several hydrogen bonds with PLpro. T1-T5 bind into a deep pocket close to the polyubiquitin and ISG15 binding site S2. Their interactions are mainly mediated by multiple hydrogen bonds and further hydrophobic interactions. In particular compound H1 interferes with natural substrate binding by sterical hindrance and induces conformational changes in protein residues involved in substrate binding, while compounds T1-T5 could have a more indirect effect. Fluorescence based enzyme activity assay and complementary thermal stability analysis reveal only weak inhibition properties in the high micromolar range thereby indicating the need for compound optimization. Nevertheless, the unique binding properties involving strong hydrogen bonding and the various options for structural optimization make the compounds ideal lead structures. In combination with the inexpensive and undemanding synthesis, the reported hydrazone and thiosemicarbazones represent an attractive scaffold for further structure-based development of novel PLpro inhibitors by interrupting protein-protein interactions at the S1 and S2 site.
... Because wild animals frequently come into contact with humans, zoonotic diseases are becoming more widespread. SARS-CoV and MERS-CoV are zoonotic diseases that cause severe respiratory disorders in humans (Báez-Santos et al., 2015;Venkataraman and Frieman 2017;Jackson et al., 2020;Singhal 2020;Zhao et al., 2020). ...
Article
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Coronavirus is an enclosed positive-sense RNA virus with club-like spikes protruding from its surface that causes acute respiratory infections in humans. Because it is considered a member of the complex pathogen group, it has been found to infect different host species and cause a variety of diseases. So far, it has been discovered that it may affect the immune, infection, and inflammatory systems, leading to the hypothesis that the immune and inflammatory systems (signaling pathways and components) fail to control infection, opening the door to look for potential targets primarily in these systems. The study’s main purpose is to identify highly overexpressed genes and their functional implications as a result of COVID-19 infection, as well as to investigate probable infections, inflammation, and immune systems to better understand the impact of coronavirus infection. We explored the genes and pathways mostly linked with infection, inflammation, and the immune systems using the datasets available for COVID-19 infection gene expression compendium. NFKBIA, FN1, FAP, KANK4, COMP, FAM101B, COL1A2, ANKRD1, TAGLN, SPARC, ADAM19, OLFM4, CXCL10/11, OASL, FOS, APOBEC3A, IFI44L, IFI27, IFIT1, RSAD2, NDUFS1, SRSF6, HECTD1, CBX3, and DDX17 are among the genes that may be impacted by infection, according to our findings. The functional changes are mainly associated with these pathways TNF, cytokine, NF—kB, TLR, TCR, BCR, Foxo, and TGF signaling pathways are among them and there are additional pathways such as hippo signaling, apoptosis, estrogen signaling, regulating pluropotency of stem cells, ErbB, Wnt, p53, cAMP, MAPK, PI3K—AKT, oxidative phosphorylation, protein processing in endoplasmic reticulum, prolactin signaling, adipocytokine, neurotrophine signaling, and longevity regulating pathways. Moreover, we have also explored the potential herbal drug (apigenin, quercetin, and resveratrol) targets for the top-rated genes based on the overall analysis where we observe that quercetin and resveratrol as most effective.
... A recent study reported the sensitivity of new coronavirus strains (SARS-CoV-2) to a copper surface (Van Doremalen et al. 2020). Other studies have revealed blockage of papain-like protease-2 by Cu 2+ , a protein required for replication by SARS-CoV-1; therefore, the need for high demand of copper supplementation is essential (Baez-Santos et al. 2015;Han et al. 2005). Due to the high competition in the absorption of Cu and Zn in the jejunum through metallothionein, high doses of zinc (greater than 150 mg per day) can lead to deficiency of Cu in healthy individuals. ...
Article
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The immune system protects human health from the effects of pathogenic organisms; however, its activity is affected when individuals become infected. These activities require a series of molecules, substrates, and energy sources that are derived from diets. The consumed nutrients from diets help to enhance the immunity of infected individuals as it relates to COVID-19 patients. This study aims to review and highlight requirement and role of macro- and micronutrients of COVID-19 patients in enhancing their immune systems. Series of studies were found to have demonstrated the enhancing potentials of macronutrients (carbohydrates, proteins, and fats) and micronutrients (vitamins, copper, zinc, iron, calcium, magnesium, and selenium) in supporting the immune system’s fight against respiratory infections. Each of these nutrients performs a vital role as an antiviral defense in COVID-19 patients. Appropriate consumption or intake of dietary sources that yield these nutrients will help provide the daily requirement to support the immune system in its fight against pathogenic viruses such as COVID-19.
... The two cysteine proteases, 3CL pro and PL pro, are conserved among major β-CoV human pathogens, SARS-CoV, MERS-CoV, and SARS-CoV-2 among the most critical drug targets for developing therapeutics [17,131,132]. The synthetic octapeptide, AVLQSGFR, forms strong hydrogen bonds with catalytic residues of SARS-CoV 3CL pro , actively inhibiting the replication of SARS-CoV in Vero cells [133]. ...
Article
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Betacoronaviruses (β-CoVs) have caused major viral outbreaks in the last two decades in the world. The mutation and recombination abilities in β-CoVs resulted in zoonotic diseases in humans. Proteins responsible for viral attachment and replication are highly conserved in β-CoVs. These conserved proteins have been extensively studied as targets for preventing infection and the spread of β-CoVs. Peptides are among the most promising candidates for developing vaccines and therapeutics against viral pathogens. The immunostimulatory and viral inhibitory potential of natural and synthetic peptides has been extensively studied since the SARS-CoV outbreak. Food-derived peptides demonstrating high antiviral activity can be used to develop effective therapeutics against β-CoVs. Specificity, tolerability, and customizability of peptides can be explored to develop potent drugs against β-CoVs. However, the proteolytic susceptibility and low bioavailability of peptides pose challenges for the development of therapeutics. This review illustrates the potential role of peptides in eliciting an adaptive immune response and inhibiting different stages of the β-CoV life cycle. Further, the challenges and future directions associated with developing peptide-based therapeutics and vaccines against existing and future β-CoV pathogens have been discussed.
... The viral proteases of SARS-CoV-2, including 3C-like protease (3CLpro), RNA-dependent RNA polymerase (RdRp), and papain-like protease (PLpro), play crucial roles in viral replication and have been proven to be important targets for the development of drugs against SARS-CoV-2 (Báez- Santos et al., 2015;Ullrich and Nitsche, 2020;Zhu et al., 2020). Some drugs targeting SARS-CoV-2 virus proteases have shown good clinical effects. ...
Article
In addition to severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2 has become the third deadly coronavirus that infects humans and causes the new coronavirus disease (COVID-19). COVID-19 has already caused more than six million deaths worldwide and it is likely the biggest pandemic of this century faced by mankind. Although many studies on SARS-CoV-2 have been conducted, a detailed understanding of SARS-CoV-2 and COVID-19 is still lacking. Animal models are indispensable for studying its pathogenesis and developing vaccines and antivirals. In this review, we analyze animal models of coronavirus infections and explore their applications on antivirals and vaccines.
... The selected target protein (PDB ID: 6LU7) for this study is 3CLpro a novel target for SARS-CoV-2 infections, significant research has been conducted on achieving the main protease inhibitors which could be potential drugs for SARS-CoV-2 and several pharmaceutical companies has developed antiviral drugs from different sources for SARS-CoV-2 and most of them are yet in clinical trials Tu et al., 2020). 3CLpro inhibitors for SARS-CoV-2 could block the entry of the virus into the host and stops their replication and translation activity (Báez-Santos et al., 2015;Ziebuhr et al., 2000). Molecular docking is performed to screen a potential 3CLpro inhibitor for SARS-CoV-2 by MOE software (Vilar et al., 2008) and resulted dock scores represent the best binding capacity of ligands in the substrate-binding active site of the target protein. ...
Article
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Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a more severe strain of coronavirus (CoV) that was first emerged in China in 2019. Available antiviral drugs could be repurposed and natural compounds with antiviral activity could be safer and cheaper source of medicine for SARS-CoV-2. 78 natural antiviral compounds database was identified from literature and virtual screening technique was applied to identify potential 3-chymotrypsin-like protease (3CLpro) inhibitors. Molecular docking studies were conducted to analyze the main protease (3CLpro) and inhibitors interactions with key residues of active site of target protein (PDB ID: 6LU7), active site constitute the part of active domain I and II of 3CLpro. 10 compounds with highest dock score were subjected to calculate ADMET parameters to figure out drug-likeness. Molecular dynamic (MD) simulation of the selected lead was performed by Amber simulation package to understand the conformational changes in docked complex. MD simulations analysis (RMSD, RMSF, Rg, BF, HBs, and SASA plots) of lead bounded with 3CLpro, hence revealed the important structural turns and twists during MD simulations from 0-100ns. MM-PBSA/GBSA methods has also been applied for the estimation binding free energy (BFE) of the selected lead-complex. The present study has identified lead compound “Forsythoside A” an active extract of Forsythia suspense as SARS-CoV-2 3CLpro inhibitor that can block the viral replication and translation. Structural analysis of target protein and lead compound performed in this study could contribute to the development of potential drug against SARS-CoV-2 infection.
... This latter mechanism is mediated by a (−1) ribosomal frameshift overreading the stop codon of ORF1a [72,73]. The polyproteins 1a and 1ab are processed into 16 mature proteins by two viral proteases, papain-like protease (PL pro ) and a 3C-like protease (3CL pro ) [74,75] ( Figure 1C). Both PL pro and 3CL pro are cysteine proteases, however, while PL pro adopts the papain-like fold [76,77], 3CL pro features the chymotrypsin-like fold [78,79]. ...
Article
As a frontline defense mechanism against viral infections, the innate immune system is the primary target of viral antagonism. A number of virulence factors encoded by viruses play roles in circumventing host defenses and augmenting viral replication. Among these factors are viral proteases, which are primarily responsible for maturation of viral proteins, but in addition cause proteolytic cleavage of cellular proteins involved in innate immune signaling. The study of these viral protease-mediated host cleavages has illuminated the intricacies of innate immune networks and yielded valuable insights into viral pathogenesis. In this review, we will provide a brief summary of how proteases of positive-strand RNA viruses, mainly from the Picornaviridae, Flaviviridae and Coronaviridae families, proteolytically process innate immune components and blunt their functions.
... The NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly inhibited cysteine proteases encoded by SARS-CoV-1 ORF1a and the membrane fusion of offspring virus S protein, decreasing viral replication by > 80% in VeroE6 cells [93][94][95][96][97]. Both SARS-CoV-2 and SARS-CoV exhibit a high degree of similarity in the receptor-binding domains of the spike proteins [98,99]. ...
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the cytokine release syndrome (CRS) and leads to multiorgan dysfunction. Mitochondrial dynamics are fundamental to protect against environmental insults, but they are highly susceptible to viral infections. Defective mitochondria are potential sources of reactive oxygen species (ROS). Infection with SARS-CoV-2 damages mitochondria, alters autophagy, reduces nitric oxide (NO), and increases both nicotinamide adenine dinucleotide phosphate oxidases (NOX) and ROS. Patients with coronavirus disease 2019 (COVID-19) exhibited activated toll-like receptors (TLRs) and the Nucleotide-binding and oligomerization domain (NOD-), leucine-rich repeat (LRR-), pyrin domain-containing protein 3 (NLRP3) inflammasome. The activation of TLRs and NLRP3 by SARS‐CoV‐2 induces interleukin 6 (IL-6), IL-1β, IL-18, and lactate dehydrogenase (LDH). Herein, we outline the inflammatory circuit of COVID-19 and what occurs behind the scene, the interplay of NOX/ROS and their role in hypoxia and thrombosis, and the important role of ROS scavengers to reduce COVID-19-related inflammation.
... Nonstructural proteins prevent the host immune response against the viral replication mediated by the nucleocapsid (N) protein 13,14,19 . Nonstructural proteins functions include inhibitory activities of the host immune response (nsp1) 20,21 ; perturbation of host intracellular signaling (nsp2) 22,23 ; polyprotein processing (nsp3) [24][25][26][27] , and (nsp5) 28,29 ; viral replication and transcription (nsp4, nsp12) [30][31][32][33] ; autophagosome (nsp6) 34,35 ; RNA primase function (nsp7, nsp8, nsp9, nsp10, nsp11) 11,33,[36][37][38] ; helicase core domain that binds ATP (nsp13) [39][40][41] ; mRNA capping (nsp14, nsp16) 11 ; evasion of host cell dsRNA sensors (nsp15) 9,42 . Protein network. ...
Article
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Several highly effective Covid-19 vaccines are in emergency use, although more-infectious coronavirus strains, could delay the end of the pandemic even further. Because of this, it is highly desirable to develop fast antiviral drug treatments to accelerate the lasting immunity against the virus. From a theoretical perspective, computational approaches are useful tools for antiviral drug development based on the data analysis of gene expression, chemical structure, molecular pathway, and protein interaction mapping. This work studies the structural stability of virus-host interactome networks based on the graphical representation of virus-host protein interactions as vertices or nodes connected by commonly shared proteins. These graphical network visualization methods are analogous to those use in the design of artificial neural networks in neuromorphic computing. In standard protein-node-based network representation, virus-host interaction merges with virus-protein and host-protein networks, introducing redundant links associated with the internal virus and host networks. On the contrary, our approach provides a direct geometrical representation of viral infection structure and allows the effective and fast detection of the structural robustness of the virus-host network through proteins removal. This method was validated by applying it to H1N1 and HIV viruses, in which we were able to pinpoint the changes in the Interactome Network produced by known vaccines. The application of this method to the SARS-CoV-2 virus-host protein interactome implies that nonstructural proteins nsp4, nsp12, nsp16, the nuclear pore membrane glycoprotein NUP210, and ubiquitin specific peptidase USP54 play a crucial role in the viral infection, and their removal may provide an efficient therapy. This method may be extended to any new mutations or other viruses for which the Interactome Network is experimentally determined. Since time is of the essence, because of the impact of more-infectious strains on controlling the spread of the virus, this method may be a useful tool for novel antiviral therapies. The current health emergency caused by the SARS-CoV-2 infection has prompted worldwide efforts to develop an antiviral treatment against Covid-19. The development of antiviral drugs requires an urgent, in-depth understanding of host-coronavirus protein-protein interactions. Many improvements in the SARS-CoV-2 interactome have been studied recently, but a disease treatment still remains elusive. Moreover, despite the genome sequences of the SARS-CoV-2 are quite similar to SARS-CoV-1 and MERS-CoV, and there are currently several vaccines in emergency use authorizations, there is no effective antiviral drug treatment yet. Current vaccines can be less effective against new variants of SARS-CoV-2 that could spread more quickly, develop more severe disease, or be capable of evading diagnostics. On the other hand, antiviral drugs can be easily administrated, possibly transported without a cold chain and at low cost. In fact, the US government invests $18.5 billion into vaccines but $8.2 billion in antiviral drugs development because it has not yet identified a highly effective drug to treat or prevent the Covid-19 infection (https:// www. nytim es. com/ 2021/ 01/ 30/ health/ covid-drugs-antiv irals. html). Based on available virus-host protein Interactome Network we developed a computational approach for the fast detection of crucial protein-protein interactions. Our approach is largely inspired on the graphical representation of multi-dimensional neural networks being used in various implementations of neuromorphic OPEN
... The main protease (Mpro), also known as 3-C like protease(3CLpro), has a crucial role in posttranslational processing of the replicase polyproteins [11]. Papain-like protease (PLpro) has the ability to disrupt the viral sequence and enhances viral load in host cell [12]. Spike protein, a transmembrane structural protein has two subunit S1 and S2; S1 with RBD region is responsible for the binding to the host cell whereas S2 for the viral cell membrane fusion [13,14]. ...
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Herein we have selected seventeen anti-lung cancer drugs to screen against Mpro, PLpro and spike glycoproteins of SARS-CoV-2to ascertain the potential therapeutic agent against COVID-19. ADMET profiling were employed to evaluate their pharmacokinetic properties. Molecular docking studies revealed that Capmatinib (CAP) showed highest binding affinity against the selected proteins of SARS-CoV-2. Molecular Dynamics (MD) simulation and the analysis of RMSD, RMSF, and binding energy confirmed the abrupt conformational changes of the proteins due to the presence of this drug. These findings provide an opportunity for doing advanced experimental research to evaluate the potential drug to combat COVID-19.
... Another identified potent antiviral drug target is SARS-CoV-2 PLpro (nsp3), which is essential for SARS-CoV-2 replication and is also suggested to play an important role in the innate immune response to viral infection analogous to SARS-CoV. This occurs via inhibition of the synthesis of cytokines and chemokines responsible for activation of the host's innate immune response against viral infection [13,118,119]. Inhibitors of this protease are potent anti-SARS-CoV-2 drug candidates (for review, see, e.g., [13]), but they still require further study. ...
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The appearance of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its spread all over the world is the cause of the coronavirus disease 2019 (COVID-19) pandemic, which has recently resulted in almost 400 million confirmed cases and 6 million deaths, not to mention unknown long-term or persistent side effects in convalescent individuals. In this short review, we discuss approaches to treat COVID-19 that are based on current knowledge of the mechanisms of viral cell receptor recognition, virus–host membrane fusion, and inhibition of viral RNA and viral assembly. Despite enormous progress in antiviral therapy and prevention, new effective therapies are still in great demand.
Preprint
The COVID-19 caused by novel strain of severe acute respiratory syndrome coronaviruses (SARS-CoV-2), has caused fatalities around the world since the end of 2019 due to its high virulence and quick multiplicity in the human body. Hence there has been an exigency to develop effective remedial measures to mitigate the mortalities. Scientists have been able to develop few corona vaccines to provide immunity, but there are no specific small molecule chemotherapeutics to combat the novel coronavirus which has spread to the whole world due to its contagiousness. In the viral genome exploration, it has been found that the main protease also known as chymotrypsin-like cysteine protease ([M pro ] or 3C-like protease [3CL pro ]) is responsible for the novel corona virus replication, transcription and host immunity destruction. Therefore, the main protease has been selected as one of the major targets for the design of new inhibitors. The protein crystallographic and molecular docking studies on SARS-CoV-2 Mpro inhibitors and some QSAR studies have been carried out on SARS-CoV main protease inhibitors to get some lead molecules for SARS-CoV-2 inhibition. But there is hardly any QSAR done on the diverse data of SARS-CoV-2 main protease inhibitors. In view of it, QSAR studies have been attempted on SARS-CoV-2 M pro inhibitors utilizing theoretical molecular descriptors solely computed from the structures of novel corona viral main protease inhibitors. The developed best QSAR model showing R ² , Q ² Loo and R ² pred values of 0.7389, 0.6666, and 0.6453 respectively has been further validated on external data set where a good correlation (r = 0.787) has been found. Therefore, this model may be useful for the design of new SARS-CoV-2 main protease inhibitors.
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Despite significant studies on the COVID-19 pandemic, scientists around the world are still battling to find a definitive therapy against the ongoing severe global health crisis. In this study, advanced computational approaches have been employed to identify bioactive food constituents as potential SARS-CoV-2 PLpro inhibitors-modulators. As a validated antiviral drug target, PLpro has gained tremendous attention for therapeutics developments. Therefore, targeting the multifunctional SARS-CoV-2 PLpro protein, ∼1039 bioactive dietary compounds have been screened extensively through novel techniques like negative image-based (NIB) screening and molecular docking approaches. In particular, the three different models of NIB screening have been generated and used to re-score the dietary compounds based on the negative image which is created by reversing the shape and electrostatics features of PLpro protein's ligand-binding cavity. Further, 100 ns molecular dynamics simulation has been performed and MM-GBSA based binding free energies have been estimated for the final proposed four dietary compounds (PC000550, PC000361, PC000558, and PC000573) as potential inhibitors/modulators of SARS-CoV-2 PLpro protein. Employed computational study outcome also has been compared with respect to the earlier experimentally investigated compound GRL0617 against SARS-CoV-2 PLpro protein, which suggests much greater interaction potential in terms of binding affinity and other energetic contributions for the proposed dietary compounds. Hence, the present study suggests that proposed dietary compounds can be suitable chemical entities for modulating the activity of PLpro protein or can be further utilized for optimizing or screening of novel chemical surrogates, however also needs experimental evaluation for entry in clinical studies for better assessment.
Chapter
Coronaviruses such as SARS and SARS-CoV-2 have established themselves as a global health concern after causing an epidemic and a pandemic in the last twenty years. Understanding the life cycle of such viruses is critical to reveal their pathogenic potential. As one of the essential viral enzymes, SARS proteases are indispensable for the processing of viral polypeptides and for the replication of the virus. SARS-CoV and SARS-CoV-2 encode for 2 viral proteases: the main protease (3CLpro) and the papain-like protease (PLPro), which are conserved among different coronaviruses and are absent in humans. This review summarizes the existing literature on the structure and function of these proteases; highlighting the similarity and differences between the enzymes of SARS and SARS-CoV-2. It also discusses the development of inhibitors to target viral proteases.
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The global spread of SARS-CoV-2 has proceeded at an unprecedented rate. Remarkably, characterization of the virus using modern tools in structural biology has also progressed at exceptional speed. Advances in electron-based imaging techniques, combined with decades of foundational studies on related viruses, have enabled the research community to rapidly investigate structural aspects of the novel coronavirus from the level of individual viral proteins to imaging the whole virus in a native context. Here, we provide a detailed review of the structural biology and pathobiology of SARS-CoV-2 as it relates to all facets of the viral life cycle, including cell entry, replication, and three-dimensional (3D) packaging based on insights obtained from X-ray crystallography, cryo-electron tomography, and single-particle cryo-electron microscopy. The structural comparison between SARS-CoV-2 and the related earlier viruses SARS-CoV and MERS-CoV is a common thread throughout this review. We conclude by highlighting some of the outstanding unanswered structural questions and underscore areas that are under rapid current development such as the design of effective therapeutics that block viral infection.
Preprint
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 (ISG15) from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we have designed 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 inhibited PLpro with k inact /K I = 10,000 M − 1 s − 1 , achieved sub-µM EC 50 values against three SARS-CoV-2 variants in mammalian cell lines, and did not inhibit a panel of human deubiquitinases at > 30 µM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validated our design strategy and established 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.
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SARS‐CoV‐2 has been responsible for causing 6,218,308 deaths globally till date and has garnered worldwide attention. The lack of effective preventive and therapeutic drugs against SARS‐CoV‐2 has further worsened the scenario and has bolstered research in the area. The N‐terminal and C‐terminal RNA binding domains (NTD and CTD) of SARS‐CoV‐2 nucleocapsid protein represent attractive therapeutic drug targets. Naturally occurring compounds are an excellent source of novel drug candidates due to their structural diversity and safety. Ten major bioactive compounds were identified in ethanolic extract (s) of Cinnamomum zeylanicum, Cinnamomum tamala, Origanum vulgare, and Petroselinum crispum using HPLC and their cytotoxic potential was determined against cancer and normal cell lines by MTT assay to ascertain their biological activity in vitro. To evaluate their antiviral potential, the binding efficacy to NTD and CTD of SARS‐CoV‐2 nucleocapsid protein was determined using in silico biology tools. In silico assessment of the phytocomponents revealed that most of the phytoconstituents displayed a druglike character with no predicted toxicity. Binding affinities were in the order apigenin > catechin > apiin toward SARS‐CoV‐2 nucleocapsid NTD. Toward nucleocapsid CTD, the affinity decreased as apigenin > cinnamic acid > catechin. Remdesivir displayed lesser affinity with NTD and CTD of SARS‐CoV‐2 nucleocapsid proteins than any of the studied phytoconstituents. Molecular dynamics (MD) simulation results revealed that throughout the 100 ns simulation, SARS‐CoV‐2 nucleocapsid protein NTD‐apigenin complex displayed greater stability than SARS‐CoV‐2 nucleocapsid protein NTD‐cinnamic acid complex. Hence, apigenin, catechin, apiin and cinnamic acid might prove as effective prophylactic and therapeutic candidates against SARS‐CoV‐2, if examined further in vitro and in vivo. Practical applications Ten major bioactive compounds were identified in the extract(s) of four medicinally important plants viz. Cinnamomum zeylanicum, Cinnamomum tamala, Origanum vulgare and Petroselinum crispum using HPLC and their biological activity was also evaluated against cancer and normal cell lines. Interestingly, while all extract(s) wielded significant cytotoxicity against cancer cells, no significant toxicity was found against normal cells. The outcome of the results prompted evaluation of the antiviral potential of the ten bioactive compounds using in silico biology tools. The present study emphasizes on the application of computational approaches to understand the binding interaction and efficacy of the ten bioactive compounds from the above plants with SARS‐CoV‐2 nucleocapsid protein N‐terminal and C‐terminal RNA binding domains in preventing and/or treating COVID‐19 using in silico tools. Druglikeness and toxicity profiles of the compounds were carried out to check the therapeutic application of the components. Additionally, molecular dynamics (MD) simulation was performed to check the stability of ligand‐protein complexes. The results provided useful insights into the structural binding interaction(s) that can be exploited for the further development of potential antiviral agents targeting SARS‐CoV‐2 especially since no specific therapy is still available to combat the rapidly evolving virus and the existing treatment is more or less symptomatic which makes search for novel antiviral agents all the more necessary and crucial.
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The world has been suffering from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, and millions of people have been infected through human-to-human transmission and lost their lives within months. Although multidisciplinary scientific approaches have been employed to fight against this deadly pandemic, various mutations and diverse environments keep producing constraints in treating SARS-CoV-2. Indeed, the efficacy of the developed vaccines has been limited, and inoculation with the vaccines does not guarantee complete protection even though multiple doses are required, which is a frustrating process. Historically, coinage metals (Cu, Ag, and Au) have been well-known for their effectiveness in antiviral action as well as good biocompatibility, binding receptor inhibition, reactive oxygen species, and phototherapy properties. Thus, this review highlights the diagnostic and therapeutic mechanisms of SARS-CoV-2 using the antivirus ability and mode of action of coinage metals such as viral entry mechanisms into host cells and the NP-inhibition process, which are explained in detail. This article also draws attention to coinage metal nanomaterial-based approaches to treat other contagious viruses. In addition, coinage metal-based biosensors and an overview of some other biocompatible metal-based nanomaterials to fight against SARS-CoV-2 variants are discussed. Finally, the advantages, perspectives and challenges of coinage metal nanoparticles are given to fight against viral infections in the future.
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COVID-19 is still a global pandemic that has not been stopped. Many traditional medicines have been demonstrated to be incredibly helpful for treating COVID-19 patients while fighting the disease worldwide. We introduced 10 bioactive compounds derived from traditional medicinal plants and assessed their potential for inhibiting viral spike protein (S-protein), Papain-like protease (PLpro), and RNA dependent RNA polymerase (RdRp) using molecular docking protocols where we simulate the inhibitors bound to target proteins in various poses and at different known binding sites using Autodock version 4.0 and Chimera 1.8.1 software. Results found that the chicoric acid, quinine, and withaferin A ligand strongly inhibited CoV-2 S -protein with a binding energy of −8.63, −7.85, and −7.85 kcal/mol, respectively. Our modeling work also suggested that curcumin, quinine, and demothoxycurcumin exhibited high binding affinity toward RdRp with a binding energy of −7.80, −7.80, and −7.64 kcal/mol, respectively. The other ligands, namely chicoric acid, demothoxycurcumin, and curcumin express high binding energy than the other tested ligands docked to PLpro with −7.62, −6.81, and −6.70 kcal/mol, respectively. Prediction of drug-likeness properties revealed that all tested ligands have no violations to Lipinski’s Rule of Five except cepharanthine, chicoric acid, and theaflavin. Regarding the pharmacokinetic behavior, all ligand predicted to have high GI-absorption except chicoric acid and theaflavin. At the same way chicoric acid, withaferin A, and withanolide D predicted to be substrate for multidrug resistance protein (P-gp substrate). Caffeic acid, cepharanthine, chicoric acid, withaferin A, and withanolide D also have no inhibitory effect on any cytochrome P450 enzymes. Promisingly, chicoric acid, quinine, curcumin, and demothoxycurcumin exhibited high binding affinity on SARS-CoV-2 target proteins and expressed good drug-likeness and pharmacokinetic properties. Further research is required to investigate the potential uses of these compounds in the treatment of SARS-CoV-2. View Full-Text
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The Middle-East Respiratory Syndrome coronavirus (MERS-CoV) causes severe acute pneumonia and renal failure. The MERS-CoV papain-like protease (PLpro) is a potential target for the development of antiviral drugs. To facilitate these efforts, we determined the three-dimensional structure of the enzyme by X-ray crystallography. The molecule consists of a ubiquitin-like domain and a catalytic core domain. The catalytic domain displays an extended right-hand fold with a zinc ribbon and embraces a solvent-exposed substrate-binding region. The overall structure of the MERS-CoV PLpro is similar to that of the corresponding SARS-CoV enzyme, but the architecture of the oxyanion hole and of the S3 as well as the S5 specificity sites differ from the latter. These differences are the likely reason for reduced in-vitro peptide hydrolysis and deubiquitinating activities of the MERS-CoV PLpro, compared to the homologous enzyme from the SARS coronavirus. Introduction of a side-chain capable of oxyanion stabilization through the Leu106Trp mutation greatly enhances the in-vitro catalytic activity of the MERS-CoV PLpro. The unique features observed in the crystal structure of the MERS-CoV PLpro should allow the design of antivirals that would not interfere with host ubiquitin-specific proteases.
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Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a papain-like protease (PLpro) with both deubiquitinating (DUB) and deISGylating activities that are proposed to counteract the post-translational modification of signaling molecules that activate the innate immune response. Here we examine the structural basis for PLpro's ubiquitin chain and interferon stimulated gene 15 (ISG15) specificity. We present the X-ray crystal structure of PLpro in complex with ubiquitin-aldehyde and model the interaction of PLpro with other ubiquitin-chain and ISG15 substrates. We show that PLpro greatly prefers K48- to K63-linked ubiquitin chains, and ISG15-based substrates to those that are mono-ubiquitinated. We propose that PLpro's higher affinity for K48-linked ubiquitin chains and ISG15 stems from a bivalent mechanism of binding, where two ubiquitin-like domains prefer to bind in the palm domain of PLpro with the most distal ubiquitin domain interacting with a "ridge" region of the thumb domain. Mutagenesis of residues within this ridge region revealed that these mutants retain viral protease activity and the ability to catalyze hydrolysis of mono-ubiquitin. However, a select number of these mutants have a significantly reduced ability to hydrolyze the substrate ISG15-AMC, or be inhibited by K48-linked diubuiquitin. For these latter residues, we found that PLpro antagonism of the nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) signaling pathway is abrogated. This identification of key and unique sites in PLpro required for recognition and processing of diubiquitin and ISG15 versus mono-ubiquitin and protease activity provides new insight into ubiquitin-chain and ISG15 recognition and highlights a role for PLpro DUB and deISGylase activity in antagonism of the innate immune response.
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Through the combined action of ubiquitinating and deubiquitinating enzymes, conjugation of ubiquitin to a target protein acts as a reversible post-translational modification functionally similar to phosphorylation. Indeed, ubiquitination is more and more recognized as a central process for the fine regulation of many cellular pathways. Due to their nature as obligate intracellular parasites, viruses rely on the most conserved host cell machineries for their own replication. Thus, it is not surprising that members from almost every viral family are challenged by ubiquitin mediated mechanisms in different steps of their life cycle and have evolved in order to by-pass or exploit the cellular ubiquitin conjugating system to maximize their chance to establish a successful infection. In this review we will present several examples of the complex interplay that links viruses and the ubiquitin conjugation machinery, with a special focus on the mechanisms evolved by the human immunodeficiency virus to escape from cellular restriction factors and to exit from infected cells.
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Papain-like protease (PL(pro)) is one of two cysteine proteases involved in the proteolytic processing of the polyproteins of Severe acute respiratory syndrome coronavirus (SARS-CoV). PL(pro) also shows significant in vitro deubiquitinating and de-ISGylating activities, although the detailed mechanism is still unclear. Here, the crystal structure of SARS-CoV PL(pro) C112S mutant in complex with ubiquitin (Ub) is reported at 1.4 Å resolution. The Ub core makes mostly hydrophilic interactions with PL(pro), while the Leu-Arg-Gly-Gly C-terminus of Ub is located in the catalytic cleft of PL(pro), mimicking the P4-P1 residues and providing the first atomic insights into its catalysis. One of the O atoms of the C-terminal Gly residue of Ub is located in the oxyanion hole consisting of the main-chain amides of residues 112 and 113. Mutations of residues in the PL(pro)-Ub interface lead to reduced catalytic activity, confirming their importance for Ub binding and/or catalysis. The structure also revealed an N-cyclohexyl-2-aminethanesulfonic acid molecule near the catalytic triad, and kinetic studies suggest that this binding site is also used by other PL(pro) inhibitors. Overall, the structure provides a foundation for understanding the molecular basis of coronaviral PL(pro) catalysis.
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Unlabelled: Coronaviruses (CoV), like other positive-stranded RNA viruses, redirect and rearrange host cell membranes for use as part of the viral genome replication and transcription machinery. Specifically, coronaviruses induce the formation of double-membrane vesicles in infected cells. Although these double-membrane vesicles have been well characterized, the mechanism behind their formation remains unclear, including which viral proteins are responsible. Here, we use transfection of plasmid constructs encoding full-length versions of the three transmembrane-containing nonstructural proteins (nsps) of the severe acute respiratory syndrome (SARS) coronavirus to examine the ability of each to induce double-membrane vesicles in tissue culture. nsp3 has membrane disordering and proliferation ability, both in its full-length form and in a C-terminal-truncated form. nsp3 and nsp4 working together have the ability to pair membranes. nsp6 has membrane proliferation ability as well, inducing perinuclear vesicles localized around the microtubule organizing center. Together, nsp3, nsp4, and nsp6 have the ability to induce double-membrane vesicles that are similar to those observed in SARS coronavirus-infected cells. This activity appears to require the full-length form of nsp3 for action, as double-membrane vesicles were not seen in cells coexpressing the C-terminal truncation nsp3 with nsp4 and nsp6. Importance: Although the majority of infections caused by coronaviruses in humans are relatively mild, the SARS outbreak of 2002 to 2003 and the emergence of the human coronavirus Middle Eastern respiratory syndrome (MERS-CoV) in 2012 highlight the ability of these viruses to cause severe pathology and fatality. Insight into the molecular biology of how coronaviruses take over the host cell is critical for a full understanding of any known and possible future outbreaks caused by these viruses. Additionally, since membrane rearrangement is a tactic used by all known positive-sense single-stranded RNA viruses, this work adds to that body of knowledge and may prove beneficial in the development of future therapies not only for human coronavirus infections but for other pathogens as well.
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Viruses have evolved elaborate mechanisms to evade or inactivate the complex system of sensors and signaling molecules that make up the host innate immune response. Here we show that human coronavirus (HCoV) NL63 and severe acute respiratory syndrome (SARS) CoV papain-like proteases (PLP) antagonize innate immune signaling mediated by STING (stimulator of interferon genes, also known as MITA/ERIS/MYPS). STING resides in the endoplasmic reticulum and upon activation, forms dimers which assemble with MAVS, TBK-1 and IKKε, leading to IRF-3 activation and subsequent induction of interferon (IFN). We found that expression of the membrane anchored PLP domain from human HCoV-NL63 (PLP2-TM) or SARS-CoV (PLpro-TM) inhibits STING-mediated activation of IRF-3 nuclear translocation and induction of IRF-3 dependent promoters. Both catalytically active and inactive forms of CoV PLPs co-immunoprecipitated with STING, and viral replicase proteins co-localize with STING in HCoV-NL63-infected cells. Ectopic expression of catalytically active PLP2-TM blocks STING dimer formation and negatively regulates assembly of STING-MAVS-TBK1/IKKε complexes required for activation of IRF-3. STING dimerization was also substantially reduced in cells infected with SARS-CoV. Furthermore, the level of ubiquitinated forms of STING, RIG-I, TBK1 and IRF-3 are reduced in cells expressing wild type or catalytic mutants of PLP2-TM, likely contributing to disruption of signaling required for IFN induction. These results describe a new mechanism used by CoVs in which CoV PLPs negatively regulate antiviral defenses by disrupting the STING-mediated IFN induction.
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Severe acute respiratory coronavirus (SARS-CoV) emerged in 2002, resulting in roughly 8000 cases worldwide and 10% mortality. The animal reservoirs for SARS-CoV precursors still exist and the likelihood of future outbreaks in the human population is high. The SARS-CoV papain-like protease (PLP) is an attractive target for pharmaceutical development because it is essential for virus replication and is conserved among human coronaviruses. A yeast-based assay was established for PLP activity that relies on the ability of PLP to induce a pronounced slow-growth phenotype when expressed in S. cerevisiae. Induction of the slow-growth phenotype was shown to take place over a 60-hour time course, providing the basis for conducting a screen for small molecules that restore growth by inhibiting the function of PLP. Five chemical suppressors of the slow-growth phenotype were identified from the 2000 member NIH Diversity Set library. One of these, NSC158362, potently inhibited SARS-CoV replication in cell culture without toxic effects on cells, and it specifically inhibited SARS-CoV replication but not influenza virus replication. The effect of NSC158362 on PLP protease, deubiquitinase and anti-interferon activities was investigated but the compound did not alter these activities. Another suppressor, NSC158011, demonstrated the ability to inhibit PLP protease activity in a cell-based assay. The identification of these inhibitors demonstrated a strong functional connection between the PLP-based yeast assay, the inhibitory compounds, and SARS-CoV biology. Furthermore the data with NSC158362 suggest a novel mechanism for inhibition of SARS-CoV replication that may involve an unknown activity of PLP, or alternatively a direct effect on a cellular target that modifies or bypasses PLP function in yeast and mammalian cells.
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Coronaviruses encode multifunctional proteins that are critical for viral replication and for blocking the innate immune response to viral infection. One such multifunctional domain is the coronavirus papain-like protease (PLP), which processes the viral replicase polyprotein, has deubiquitinating (DUB) activity, and antagonizes the induction of type I interferon (IFN). Here we characterized the DUB and IFN antagonism activities of the PLP domains of human coronavirus NL63 and severe acute respiratory syndrome (SARS) coronavirus to determine if DUB activity mediates interferon antagonism. We found that NL63 PLP2 deconjugated ubiquitin (Ub) and the Ub-line molecule ISG15 from cellular substrates and processed both lysine-48- and lysine-63- linked polyubiquitin chains. This PLP2 DUB activity was dependent on an intact catalytic cysteine residue. We demonstrated that in contrast to PLP2 DUB activity, PLP2-mediated interferon antagonism did not require enzymatic activity. Furthermore, addition of an inhibitor that blocks coronavirus protease/DUB activity did not abrogate interferon antagonism. These results indicated that a component of coronavirus PLP-mediated interferon antagonism was independent of protease and DUB activity. Overall, these results demonstrate the multifunctional nature of the coronavirus PLP domain as a viral protease, DUB, and IFN antagonist and suggest that these independent activities may provide multiple targets for antiviral therapies.
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Coronaviruses induce in infected cells the formation of double-membrane vesicles (DMVs) in which the replication-transcription complexes (RTCs) are anchored. To study the dynamics of these coronavirus replicative structures, we generated recombinant murine hepatitis coronaviruses that express tagged versions of the nonstructural protein nsp2. We demonstrated by using immunofluorescence assays and electron microscopy that this protein is recruited to the DMV-anchored RTCs, for which its C terminus is essential. Live-cell imaging of infected cells demonstrated that small nsp2-positive structures move through the cytoplasm in a microtubule-dependent manner. In contrast, large fluorescent structures are rather immobile. Microtubule-mediated transport of DMVs, however, is not required for efficient replication. Biochemical analyses indicated that the nsp2 protein is associated with the cytoplasmic side of the DMVs. Yet, no recovery of fluorescence was observed when (part of) the nsp2-positive foci were bleached. This result was confirmed by the observation that preexisting RTCs did not exchange fluorescence after fusion of cells expressing either a green or a red fluorescent nsp2. Apparently, nsp2, once recruited to the RTCs, is not exchanged with nsp2 present in the cytoplasm or at other DMVs. Our data show a remarkable resemblance to results obtained recently by others with hepatitis C virus. The observations point to intriguing and as yet unrecognized similarities between the RTC dynamics of different plus-strand RNA viruses.
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The nuclear magnetic resonance (NMR) structure of a globular domain of residues 1071 to 1178 within the previously annotated nucleic acid-binding region (NAB) of severe acute respiratory syndrome coronavirus nonstructural protein 3 (nsp3) has been determined, and N- and C-terminally adjoining polypeptide segments of 37 and 25 residues, respectively, have been shown to form flexibly extended linkers to the preceding globular domain and to the following, as yet uncharacterized domain. This extension of the structural coverage of nsp3 was obtained from NMR studies with an nsp3 construct comprising residues 1066 to 1181 [nsp3(1066-1181)] and the constructs nsp3(1066-1203) and nsp3(1035-1181). A search of the protein structure database indicates that the globular domain of the NAB represents a new fold, with a parallel four-strand β-sheet holding two α-helices of three and four turns that are oriented antiparallel to the β-strands. Two antiparallel two-strand β-sheets and two 310-helices are anchored against the surface of this barrel-like molecular core. Chemical shift changes upon the addition of single-stranded RNAs (ssRNAs) identified a group of residues that form a positively charged patch on the protein surface as the binding site responsible for the previously reported affinity for nucleic acids. This binding site is similar to the ssRNA-binding site of the sterile alpha motif domain of the Saccharomyces cerevisiae Vts1p protein, although the two proteins do not share a common globular fold.
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Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV), the non-structural proteins (Nsps), have been determined. However, within the large Nsp3 (1922 amino-acid residues), the structure and function of the so-called SARS-unique domain (SUD) have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389-652 ("SUD(core)") of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 A resolution, respectively) revealed that SUD(core) forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUD(core) as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5-6 nucleotides, but more extended G-stretches are found in the 3'-nontranslated regions of mRNAs coding for certain host-cell proteins involved in apoptosis or signal transduction, and have been shown to bind to SUD in vitro. Therefore, SUD may be involved in controlling the host cell's response to the viral infection. Possible interference with poly(ADP-ribose) polymerase-like domains is also discussed.
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Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nsp1, nsp2, and nsp3 and describe trans-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione S-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nsp1 (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a approximately 300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro trans-cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nsp1/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro can cleave in trans at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site.