Unraveling DPP4 Receptor Interactions with SARS-CoV-2 Variants and MERS-CoV: Insights into Pulmonary Disorders via Immunoinformatics and Molecular Dynamics.

Human coronaviruses like MERS CoV are known to utilize dipeptidyl peptidase 4 (DPP4), apart from angiotensin-converting enzyme 2(ACE2) as a potential co-receptor for viral cell entry. DPP4, the ubiquitous membrane-bound aminopeptidase, is closely associated with elevation of disease severity in comorbidities. In SARS-CoV-2, there is inadequate evidence for combination of spike protein variants with DPP4, and underlying adversity in COVID-19. To elucidate this mechanistic basis, we have investigated interaction of spike protein variants with DPP4 through molecular docking and simulation studies. The possible binding interactions between the receptor binding domain (RBD) of different spike variants of SARS-CoV-2 and DPP4 have been compared with interactions observed in the experimentally determined structure of the complex of MERS-CoV with DPP4. Comparative binding affinity confers that Delta-CoV-2: DPP4 shows close proximity with MERS-CoV:DPP4, as depicted from accessible surface area, radius of gyration and number of hydrogen bonding in the interface. Mutations in the delta variant, L452R and T478K directly participate in DPP4 interaction, enhancing DPP4 binding. E484K in alpha and gamma variants of spike protein is also found to interact with DPP4. Hence, DPP4 interaction with spike protein becomes more suitable due to mutation, especially due to L452R, T478K and E484K. Furthermore, perturbation in the nearby residues Y495, Q474 and Y489 is evident due to L452R, T478K and E484K, respectively. Virulent strains of spike protein are more susceptible to DPP4 interaction and are prone to be victimized in patients due to comorbidities. Our results will aid the rational optimization of DPP4 as a potential therapeutic target to manage COVID-19 disease severity.

The KDM6A-KMT2D-p300 axis regulates susceptibility to diverse coronaviruses by mediating viral receptor expression.

Identification of host determinants of coronavirus infection informs mechanisms of pathogenesis and may provide novel therapeutic targets. Here, we demonstrate that the histone demethylase KDM6A promotes infection of diverse coronaviruses, including SARS-CoV, SARS-CoV-2, MERS-CoV and mouse hepatitis virus (MHV) in a demethylase activity-independent manner. Mechanistic studies reveal that KDM6A promotes viral entry by regulating expression of multiple coronavirus receptors, including ACE2, DPP4 and Ceacam1. Importantly, the TPR domain of KDM6A is required for recruitment of the histone methyltransferase KMT2D and histone deacetylase p300. Together this KDM6A-KMT2D-p300 complex localizes to the proximal and distal enhancers of ACE2 and regulates receptor expression. Notably, small molecule inhibition of p300 catalytic activity abrogates ACE2 and DPP4 expression and confers resistance to all major SARS-CoV-2 variants and MERS-CoV in primary human airway and intestinal epithelial cells. These data highlight the role for KDM6A-KMT2D-p300 complex activities in conferring diverse coronaviruses susceptibility and reveal a potential pan-coronavirus therapeutic target to combat current and emerging coronaviruses. One Sentence Summary: The KDM6A/KMT2D/EP300 axis promotes expression of multiple viral receptors and represents a potential drug target for diverse coronaviruses.

Different electrostatic forces drive the binding kinetics of SARS-CoV, SARS-CoV-2 and MERS-CoV Envelope proteins with the PDZ2 domain of ZO1.

The Envelope protein (E) is a structural protein encoded by the genome of SARS-CoV, SARS-CoV-2 and MERS-CoV Coronaviruses. It is poorly present in the virus but highly expressed in the host cell, with prominent role in virus assembly and virulence. The E protein possesses a PDZ-binding motif (PBM) at its C terminus that allows it to interact with host PDZ domain containing proteins. ZO1 is a key protein in assembling the cytoplasmic plaque of epithelial and endothelial Tight Junctions (TJs) as well as in determining cell differentiation, proliferation and polarity. The PDZ2 domain of ZO1 is known to interact with the Coronaviruses Envelope proteins, however the molecular details of such interaction have not been established. In this paper we directly measured, through Fluorescence Resonance Energy Transfer and Stopped-Flow methodology, the binding kinetics of the PDZ2 domain of ZO1 with peptides mimicking the C-terminal portion of the Envelope protein from SARS-CoV, SARS-CoV-2 and MERS-CoV in different ionic strength conditions. Interestingly, the peptide mimicking the E protein from MERS-CoV display much higher microscopic association rate constant with PDZ2 compared to SARS-CoV and SARS-CoV-2 suggesting a stronger contribution of electrostatic forces in the early events of binding. A comparison of thermodynamic and kinetic data obtained at increasing ionic strengths put in evidence different contribution of electrostatics in the recognition and complex formation events for the three peptides. Our data are discussed under the light of available structural data of PDZ2 domain of ZO1 and of previous works about these protein systems.

A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry.

It is unknown whether pangolins, the most trafficked mammals, play a role in the zoonotic transmission of bat coronaviruses. We report the circulation of a novel MERS-like coronavirus in Malayan pangolins, named Manis javanica HKU4-related coronavirus (MjHKU4r-CoV). Among 86 animals, four tested positive by pan-CoV PCR, and seven tested seropositive (11 and 12.8%). Four nearly identical (99.9%) genome sequences were obtained, and one virus was isolated (MjHKU4r-CoV-1). This virus utilizes human dipeptidyl peptidase-4 (hDPP4) as a receptor and host proteases for cell infection, which is enhanced by a furin cleavage site that is absent in all known bat HKU4r-CoVs. The MjHKU4r-CoV-1 spike shows higher binding affinity for hDPP4, and MjHKU4r-CoV-1 has a wider host range than bat HKU4-CoV. MjHKU4r-CoV-1 is infectious and pathogenic in human airways and intestinal organs and in hDPP4-transgenic mice. Our study highlights the importance of pangolins as reservoir hosts of coronaviruses poised for human disease emergence.

Interactions of SARS-CoV-2 and MERS-CoV fusion peptides measured using single-molecule force methods.

We address the challenge of understanding how hydrophobic interactions are encoded by fusion peptide (FP) sequences within coronavirus (CoV) spike proteins. Within the FPs of severe acute respiratory syndrome CoV 2 and Middle East respiratory syndrome CoV (MERS-CoV), a largely conserved peptide sequence called FP1 (SFIEDLLFNK and SAIEDLLFDK in SARS-2 and MERS, respectively) has been proposed to play a key role in encoding hydrophobic interactions that drive viral-host cell membrane fusion. Although a non-polar triad (Leu-Leu-Phe (LLF)) is common to both FP1 sequences, and thought to dominate the encoding of hydrophobic interactions, FP1 from SARS-2 and MERS differ in two residues (Phe 2 versus Ala 2 and Asn 9 versus Asp 9, respectively). Here we explore whether single-molecule force measurements can quantify hydrophobic interactions encoded by FP1 sequences, and then ask whether sequence variations between FP1 from SARS-2 and MERS lead to significant differences in hydrophobic interactions. We find that both SARS-2 and MERS wild-type FP1 generate measurable hydrophobic interactions at the single-molecule level, but that SARS-2 FP1 encodes a substantially stronger hydrophobic interaction than its MERS counterpart (1.91 ± 0.03 nN versus 0.68 ± 0.03 nN, respectively). By performing force measurements with FP1 sequences with single amino acid substitutions, we determine that a single-residue mutation (Phe 2 versus Ala 2) causes the almost threefold difference in the hydrophobic interaction strength generated by the FP1 of SARS-2 versus MERS, despite the presence of LLF in both sequences. Infrared spectroscopy and circular dichroism measurements support the proposal that the outsized influence of Phe 2 versus Ala 2 on the hydrophobic interaction arises from variation in the secondary structure adopted by FP1. Overall, these insights reveal how single-residue diversity in viral FPs, including FP1 of SARS-CoV-2 and MERS-CoV, can lead to substantial changes in intermolecular interactions proposed to play a key role in viral fusion, and hint at strategies for regulating hydrophobic interactions of peptides in a range of contexts.

Close relatives of MERS-CoV in bats use ACE2 as their functional receptors.

Middle East respiratory syndrome coronavirus (MERS-CoV) and several bat coronaviruses use dipeptidyl peptidase-4 (DPP4) as an entry receptor1-4. However, the receptor for NeoCoV-the closest known MERS-CoV relative found in bats-remains unclear5. Here, using a pseudotype virus entry assay, we found that NeoCoV and its close relative, PDF-2180, can efficiently bind to and use specific bat angiotensin-converting enzyme 2 (ACE2) orthologues and, less favourably, human ACE2 as entry receptors through their receptor-binding domains (RBDs) on the spike (S) proteins. Cryo-electron microscopy analysis revealed an RBD-ACE2 binding interface involving protein-glycan interactions, distinct from those of other known ACE2-using coronaviruses. We identified residues 337-342 of human ACE2 as a molecular determinant restricting NeoCoV entry, whereas a NeoCoV S pseudotyped virus containing a T510F RBD mutation efficiently entered cells expressing human ACE2. Although polyclonal SARS-CoV-2 antibodies or MERS-CoV RBD-specific nanobodies did not cross-neutralize NeoCoV or PDF-2180, an ACE2-specific antibody and two broadly neutralizing betacoronavirus antibodies efficiently inhibited these two pseudotyped viruses. We describe MERS-CoV-related viruses that use ACE2 as an entry receptor, underscoring a promiscuity of receptor use and a potential zoonotic threat.

Exceptional diversity and selection pressure on coronavirus host receptors in bats compared to other mammals.

Pandemics originating from non-human animals highlight the need to understand how natural hosts have evolved in response to emerging human pathogens and which groups may be susceptible to infection and/or potential reservoirs to mitigate public health and conservation concerns. Multiple zoonotic coronaviruses, such as severe acute respiratory syndrome-associated coronavirus (SARS-CoV), SARS-CoV-2 and Middle Eastern respiratory syndrome-associated coronavirus (MERS-CoV), are hypothesized to have evolved in bats. We investigate angiotensin-converting enzyme 2 (ACE2), the host protein bound by SARS-CoV and SARS-CoV-2, and dipeptidyl-peptidase 4 (DPP4 or CD26), the host protein bound by MERS-CoV, in the largest bat datasets to date. Both the ACE2 and DPP4 genes are under strong selection pressure in bats, more so than in other mammals, and in residues that contact viruses. Additionally, mammalian groups vary in their similarity to humans in residues that contact SARS-CoV, SARS-CoV-2 and MERS-CoV, and increased similarity to humans in binding residues is broadly predictive of susceptibility to SARS-CoV-2. This work augments our understanding of the relationship between coronaviruses and mammals, particularly bats, provides taxonomically diverse data for studies of how host proteins are bound by coronaviruses and can inform surveillance, conservation and public health efforts.

Novel Engineered SARS-CoV-2 HR1 Trimer Exhibits Improved Potency and Broad-Spectrum Activity against SARS-CoV-2 and Its Variants.

The ongoing pandemic of COVID-19, caused by SARS-CoV-2, has substantially increased the risk to global public health. Multiple vaccines and neutralizing antibodies (nAbs) have been authorized for preventing and treating SARS-CoV-2 infection. However, the emergence and spread of the viral variants may limit the effectiveness of these vaccines and antibodies. Fusion inhibitors targeting the HR1 domain of the viral S protein have been shown to broadly inhibit SARS-CoV-2 and its variants. In theory, peptide inhibitors targeting the HR2 domain of the S protein should also be able to inhibit viral infection. However, previously reported HR1-derived peptide inhibitors targeting the HR2 domain exhibit poor inhibitory activities. Here, we engineered a novel HR1 trimer (HR1MFd) by conjugating the trimerization motif foldon to the C terminus of the HR1-derived peptide. HR1MFd showed significantly improved inhibitory activity against SARS-CoV-2, SARS-CoV-2 variants of concern (VOCs), SARS-CoV, and MERS-CoV. Mechanistically, HR1MFd possesses markedly increased α-helicity, thermostability, higher HR2 domain binding affinity, and better inhibition of S protein-mediated cell-cell fusion compared to the HR1 peptide. Therefore, HR1MFd lays the foundation to develop HR1-based fusion inhibitors against SARS-CoV-2. IMPORTANCE Peptides derived from the SARS-CoV-2 HR1 region are generally poor inhibitors. Here, we constructed a trimeric peptide HR1MFd by fusing the trimerization motif foldon to the C terminus of the HR1 peptide. HR1MFd was highly effective in blocking transductions by SARS-CoV-2, SARS-CoV-2 variants, SARS-CoV, and MERS-CoV pseudoviruses. In comparison with HR1M, HR1MFd adopted a much higher helical conformation, better thermostability, increased affinity to the viral HR2 domain, and better inhibition of S protein-mediated cell-cell fusion. Overall, HR1MFd provides the information to develop effective HR1-derived peptides as fusion inhibitors against SARS-CoV-2 and its variants.

Cellulosic copper nanoparticles and a hydrogen peroxide-based disinfectant trigger rapid inactivation of pseudoviral particles expressing the Spike protein of SARS-CoV-2, SARS-CoV, and MERS-CoV.

Severe acute respiratory syndrome (SARS) is a viral respiratory infection caused by human coronaviruses that include SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV). Although their primary mode of transmission is through contaminated respiratory droplets from infected carriers, the deposition of expelled virus particles onto surfaces and fomites could contribute to viral transmission. Here, we use replication-deficient murine leukemia virus (MLV) pseudoviral particles expressing SARS-CoV-2, SARS-CoV, or MERS-CoV Spike (S) protein on their surface. These surrogates of native coronavirus counterparts serve as a model to analyze the S-mediated entry into target cells. Carboxymethyl cellulose (CMC) nanofibers that are combined with copper (Cu) exhibit strong antimicrobial properties. S-pseudovirions that are exposed to CMC-Cu nanoparticles (30 s) display a dramatic reduction in their ability to infect target Vero E6 cells, with ∼97% less infectivity as compared to untreated pseudovirions. In contrast, addition of the Cu chelator tetrathiomolybdate protects S-pseudovirions from CMC-Cu-mediated inactivation. When S-pseudovirions were treated with a hydrogen peroxide-based disinfectant (denoted SaberTM) used at 1:250 dilution, their infectivity was dramatically reduced by ∼98%. However, the combined use of SaberTM and CMC-Cu is the most effective approach to restrict infectivity of SARS-CoV-2-S, SARS-CoV-S, and MERS-CoV-S pseudovirions in Vero E6 cell assays. Together, these results show that cellulosic Cu nanoparticles enhance the effectiveness of diluted SaberTM sanitizer, setting up an improved strategy to lower the risk of surface- and fomite-mediated transmission of enveloped respiratory viruses.

Inhibition of the IFN-α JAK/STAT Pathway by MERS-CoV and SARS-CoV-1 Proteins in Human Epithelial Cells.

Coronaviruses (CoVs) have caused several global outbreaks with relatively high mortality rates, including Middle East Respiratory Syndrome coronavirus (MERS)-CoV, which emerged in 2012, and Severe Acute Respiratory Syndrome (SARS)-CoV-1, which appeared in 2002. The recent emergence of SARS-CoV-2 highlights the need for immediate and greater understanding of the immune evasion mechanisms used by CoVs. Interferon (IFN)-α is the body's natural antiviral agent, but its Janus kinase/signal transducer and activators of transcription (JAK/STAT) signalling pathway is often antagonized by viruses, thereby preventing the upregulation of essential IFN stimulated genes (ISGs). Therapeutic IFN-α has disappointingly weak clinical responses in MERS-CoV and SARS-CoV-1 infected patients, indicating that these CoVs inhibit the IFN-α JAK/STAT pathway. Here we show that in lung alveolar A549 epithelial cells expression of MERS-CoV-nsp2 and SARS-CoV-1-nsp14, but not MERS-CoV-nsp5, increased basal levels of total and phosphorylated STAT1 & STAT2 protein, but reduced IFN-α-mediated phosphorylation of STAT1-3 and induction of MxA. While MERS-CoV-nsp2 and SARS-CoV-1-nsp14 similarly increased basal levels of STAT1 and STAT2 in bronchial BEAS-2B epithelial cells, unlike in A549 cells, they did not enhance basal pSTAT1 nor pSTAT2. However, both viral proteins reduced IFN-α-mediated induction of pSTAT1-3 and ISGs (MxA, ISG15 and PKR) in BEAS-2B cells. Furthermore, even though IFN-α-mediated induction of pSTAT1-3 was not affected by MERS-CoV-nsp5 expression in BEAS-2B cells, downstream ISG induction was reduced, revealing that MERS-CoV-nsp5 may use an alternative mechanism to reduce antiviral ISG induction in this cell line. Indeed, we subsequently discovered that all three viral proteins inhibited STAT1 nuclear translocation in BEAS-2B cells, unveiling another layer of inhibition by which these viral proteins suppress responses to Type 1 IFNs. While these observations highlight cell line-specific differences in the immune evasion effects of MERS-CoV and SARS-CoV-1 proteins, they also demonstrate the broad spectrum of immune evasion strategies these deadly coronaviruses use to stunt antiviral responses to Type IFN.

Host E3 ligase HUWE1 attenuates the proapoptotic activity of the MERS-CoV accessory protein ORF3 by promoting its ubiquitin-dependent degradation.

With the outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), coronaviruses have begun to attract great attention across the world. Of the known human coronaviruses, however, Middle East respiratory syndrome coronavirus (MERS-CoV) is the most lethal. Coronavirus proteins can be divided into three groups: nonstructural proteins, structural proteins, and accessory proteins. While the number of each of these proteins varies greatly among different coronaviruses, accessory proteins are most closely related to the pathogenicity of the virus. We found for the first time that the ORF3 accessory protein of MERS-CoV, which closely resembles the ORF3a proteins of severe acute respiratory syndrome coronavirus and SARS-CoV-2, has the ability to induce apoptosis in cells in a dose-dependent manner. Through bioinformatics analysis and validation, we revealed that ORF3 is an unstable protein and has a shorter half-life in cells compared to that of severe acute respiratory syndrome coronavirus and SARS-CoV-2 ORF3a proteins. After screening, we identified a host E3 ligase, HUWE1, that specifically induces MERS-CoV ORF3 protein ubiquitination and degradation through the ubiquitin-proteasome system. This results in the diminished ability of ORF3 to induce apoptosis, which might partially explain the lower spread of MERS-CoV compared to other coronaviruses. In summary, this study reveals a pathological function of MERS-CoV ORF3 protein and identifies a potential host antiviral protein, HUWE1, with an ability to antagonize MERS-CoV pathogenesis by inducing ORF3 degradation, thus enriching our knowledge of the pathogenesis of MERS-CoV and suggesting new targets and strategies for clinical development of drugs for MERS-CoV treatment.

Flavonol morin targets host ACE2, IMP-α, PARP-1 and viral proteins of SARS-CoV-2, SARS-CoV and MERS-CoV critical for infection and survival: a computational analysis.

A sudden outbreak of a novel coronavirus SARS-CoV-2 in 2019 has now emerged as a pandemic threatening to efface the existence of mankind. In absence of any valid and appropriate vaccines to combat this newly evolved agent, there is need of novel resource molecules for treatment and prophylaxis. To this effect, flavonol morin which is found in fruits, vegetables and various medicinal herbs has been evaluated for its antiviral potential in the present study. PASS analysis of morin versus reference antiviral drugs baricitinib, remdesivir and hydroxychloroquine revealed that morin displayed no violations of Lipinski's rule of five and other druglikeness filters. Morin also displayed no tumorigenic, reproductive or irritant effects and exhibited good absorption and permeation through GI (clogP <5). In principal component analysis, morin appeared closest to baricitinib in 3D space. Morin displayed potent binding to spike glycoprotein, main protease 3CLPro and papain-like protease PLPro of SARS-CoV-2, SARS-CoV and MERS-CoV using molecular docking and significant binding to three viral-specific host proteins viz. human ACE2, importin-α and poly (ADP-ribose) polymerase (PARP)-1, further lending support to its antiviral efficacy. Additionally, morin displayed potent binding to pro-inflammatory cytokines IL-6, 8 and 10 also supporting its anti-inflammatory activity. MD simulation of morin with SARS-CoV-2 3CLPro and PLPro displayed strong stability at 300 K. Both complexes exhibited constant RMSDs of protein side chains and Cα atoms throughout the simulation run time. In conclusion, morin might hold considerable therapeutic potential for the treatment and management of not only COVID-19, but also SARS and MERS if studied further. Communicated by Ramaswamy H. Sarma.

The lethal internal face of the coronaviruses: Kidney tropism of the SARS, MERS, and COVID19 viruses.

The kidney is one of the main targets attacked by viruses in patients with a coronavirus infection. Until now, SARS-CoV-2 has been identified as the seventh member of the coronavirus family capable of infecting humans. In the past two decades, humankind has experienced outbreaks triggered by two other extremely infective members of the coronavirus family; the MERS-CoV and the SARS-CoV. According to several investigations, SARS-CoV causes proteinuria and renal impairment or failure. The SARS-CoV was identified in the distal convoluted tubules of the kidney of infected patients. Also, renal dysfunction was observed in numerous cases of MERS-CoV infection. And recently, during the 2019-nCoV pandemic, it was found that the novel coronavirus not only induces acute respiratory distress syndrome (ARDS) but also can induce damages in various organs including the liver, heart, and kidney. The kidney tissue and its cells are targeted massively by the coronaviruses due to the abundant presence of ACE2 and Dpp4 receptors on kidney cells. These receptors are characterized as the main route of coronavirus entry to the victim cells. Renal failure due to massive viral invasion can lead to undesirable complications and enhanced mortality rate, thus more attention should be paid to the pathology of coronaviruses in the kidney. Here, we have provided the most recent knowledge on the coronaviruses (SARS, MERS, and COVID19) pathology and the mechanisms of their impact on the kidney tissue and functions.

The Interference between SARS-CoV-2 and Tyrosine Kinase Receptor Signaling in Cancer.

Cancer and viruses have a long history that has evolved over many decades. Much information about the interplay between viruses and cell proliferation and metabolism has come from the history of clinical cases of patients infected with virus-induced cancer. In addition, information from viruses used to treat some types of cancer is valuable. Now, since the global coronavirus pandemic erupted almost a year ago, the scientific community has invested countless time and resources to slow down the infection rate and diminish the number of casualties produced by this highly infectious pathogen. A large percentage of cancer cases diagnosed are strongly related to dysregulations of the tyrosine kinase receptor (TKR) family and its downstream signaling pathways. As such, many therapeutic agents have been developed to strategically target these structures in order to hinder certain mechanisms pertaining to the phenotypic characteristics of cancer cells such as division, invasion or metastatic potential. Interestingly, several authors have pointed out that a correlation between coronaviruses such as the SARS-CoV-1 and -2 or MERS viruses and dysregulations of signaling pathways activated by TKRs can be established. This information may help to accelerate the repurposing of clinically developed anti-TKR cancer drugs in COVID-19 management. Because the need for treatment is critical, drug repurposing may be an advantageous choice in the search for new and efficient therapeutic compounds. This approach would be advantageous from a financial point of view as well, given that the resources used for research and development would no longer be required and can be potentially redirected towards other key projects. This review aims to provide an overview of how SARS-CoV-2 interacts with different TKRs and their respective downstream signaling pathway and how several therapeutic agents targeted against these receptors can interfere with the viral infection. Additionally, this review aims to identify if SARS-CoV-2 can be repurposed to be a potential viral vector against different cancer types.

Computational designing of a peptide that potentially blocks the entry of SARS-CoV, SARS-CoV-2 and MERS-CoV.

Last decade has witnessed three major pandemics caused by SARS-CoV, SARS-CoV-2 and MERS-CoV that belong to Coronavirus family. Currently, there are no effective therapies available for corona virus infections. Since the three viruses belong to the same family and share many common features, we can theoretically design a drug that can be effective on all the three of them. In this study, using computational approach, we designed a peptide (Peptide 7) that can bind to the Receptor Binding Domain (RBD) of SARS-CoV, SARS-CoV-2 and MERS-CoV thereby preventing the entry of the viruses into the host cell. The peptide inhibitor was designed as a consensus peptide from three different peptides that might individually bind to the RBD of the three viruses. Docking studies and molecular dynamic simulations using Peptide 7 has shown that it binds with higher affinity than the native receptors of the RBD and forms a stable complex thereby preventing further viral-receptor interaction and inhibiting their cellular entry. This effective binding is observed for the three RBDs, despite the Peptide 7 interactions being slightly different. Hence; this peptide inhibitor can be used as a potential candidate for the development of peptide based anti-viral therapy against Corona viruses.

Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication.

SARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.

Rational design of multi epitope-based subunit vaccine by exploring MERS-COV proteome: Reverse vaccinology and molecular docking approach.

Middle East respiratory syndrome (MERS-COV), first identified in Saudi Arabia, was caused by a novel strain of coronavirus. Outbreaks were recorded from different regions of the world, especially South Korea and the Middle East, and were correlated with a 35% mortality rate. MERS-COV is a single-stranded, positive RNA virus that reaches the host by binding to the receptor of dipeptidyl-peptides. Because of the unavailability of the vaccine available for the protection from MERS-COV infection, the rapid case detection, isolation, infection prevention has been recommended to combat MERS-COV infection. So, vaccines for the treatment of MERS-COV infection need to be developed urgently. A possible antiviral mechanism for preventing MERS-CoV infection has been considered to be MERS-CoV vaccines that elicit unique T-cell responses. In the present study, we incorporated both molecular docking and immunoinformatic approach to introduce a multiepitope vaccine (MEP) against MERS-CoV by selecting 15 conserved epitopes from seven viral proteins such as three structural proteins (envelope, membrane, and nucleoprotein) and four non-structural proteins (ORF1a, ORF8, ORF3, ORF4a). The epitopes, which were examined for non-homologous to host and antigenicity, were selected on the basis of conservation between T-cell, B-cell, and IFN-γ epitopes. The selected epitopes were then connected to the adjuvant (ß-defensin) at the N-terminal through an AAY linker to increase the immunogenic potential. Structural modelling and physiochemical characteristic were applied to the vaccine construct developed. Afterwards the structure has been successfully docked with antigenic receptor, Toll-like receptor 3 (TLR-3) and in-silico cloning ensures that its expression efficiency is legitimate. Nonetheless the MEP presented needs tests to verify its safety and immunogenic profile.

Uncovering potential host proteins and pathways that may interact with eukaryotic short linear motifs in viral proteins of MERS, SARS and SARS2 coronaviruses that infect humans.

A coronavirus pandemic caused by a novel coronavirus (SARS-CoV-2) has spread rapidly worldwide since December 2019. Improved understanding and new strategies to cope with novel coronaviruses are urgently needed. Viruses (especially RNA viruses) encode a limited number and size (length of polypeptide chain) of viral proteins and must interact with the host cell components to control (hijack) the host cell machinery. To achieve this goal, the extensive mimicry of SLiMs in host proteins provides an effective strategy. However, little is known regarding SLiMs in coronavirus proteins and their potential targets in host cells. The objective of this study is to uncover SLiMs in coronavirus proteins that are present within host cells. These SLiMs have a high possibility of interacting with host intracellular proteins and hijacking the host cell machinery for virus replication and dissemination. In total, 1,479 SLiM hits were identified in the 16 proteins of 590 coronaviruses infecting humans. Overall, 106 host proteins were identified that may interact with SLiMs in 16 coronavirus proteins. These SLiM-interacting proteins are composed of many intracellular key regulators, such as receptors, transcription factors and kinases, and may have important contributions to virus replication, immune evasion and viral pathogenesis. A total of 209 pathways containing proteins that may interact with SLiMs in coronavirus proteins were identified. This study uncovers potential mechanisms by which coronaviruses hijack the host cell machinery. These results provide potential therapeutic targets for viral infections.

Elucidating the tunability of binding behavior for the MERS-CoV macro domain with NAD metabolites.

The macro domain is an ADP-ribose (ADPR) binding module, which is considered to act as a sensor to recognize nicotinamide adenine dinucleotide (NAD) metabolites, including poly ADPR (PAR) and other small molecules. The recognition of macro domains with various ligands is important for a variety of biological functions involved in NAD metabolism, including DNA repair, chromatin remodeling, maintenance of genomic stability, and response to viral infection. Nevertheless, how the macro domain binds to moieties with such structural obstacles using a simple cleft remains a puzzle. We systematically investigated the Middle East respiratory syndrome-coronavirus (MERS-CoV) macro domain for its ligand selectivity and binding properties by structural and biophysical approaches. Of interest, NAD, which is considered not to interact with macro domains, was co-crystallized with the MERS-CoV macro domain. Further studies at physiological temperature revealed that NAD has similar binding ability with ADPR because of the accommodation of the thermal-tunable binding pocket. This study provides the biochemical and structural bases of the detailed ligand-binding mode of the MERS-CoV macro domain. In addition, our observation of enhanced binding affinity of the MERS-CoV macro domain to NAD at physiological temperature highlights the need for further study to reveal the biological functions.

The roles of signaling pathways in SARS-CoV-2 infection; lessons learned from SARS-CoV and MERS-CoV.

The number of descriptions of emerging viruses has grown at an unprecedented rate since the beginning of the 21st century. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), is the third highly pathogenic coronavirus that has introduced itself into the human population in the current era, after SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Molecular and cellular studies of the pathogenesis of this novel coronavirus are still in the early stages of research; however, based on similarities of SARS-CoV-2 to other coronaviruses, it can be hypothesized that the NF-κB, cytokine regulation, ERK, and TNF-α signaling pathways are the likely causes of inflammation at the onset of COVID-19. Several drugs have been prescribed and used to alleviate the adverse effects of these inflammatory cellular signaling pathways, and these might be beneficial for developing novel therapeutic modalities against COVID-19. In this review, we briefly summarize alterations of cellular signaling pathways that are associated with coronavirus infection, particularly SARS-CoV and MERS-CoV, and tabulate the therapeutic agents that are currently approved for treating other human diseases.