Development and Evaluation of Peptidomimetic Compounds against SARS-CoV-2 Spike Protein: An in silico and in vitro Study.

BACKGROUND: Coronavirus disease 2019 (COVID-19) as global pandemic disease has been adversely affecting public health and social life with considerable loss of human life worldwide. Therefore, there is an urgent need for developing novel therapeutics to combat COVID-19. The causative agent of COVID-19 is SARS-CoV-2 which targets human angiotensin converting enzyme 2 (ACE2) as cellular receptor via its spike (S) protein. In this context, interfering with the binding of SARS-CoV-2 S protein to target molecules could provide a promising strategy to find novel therapeutic agents against SARS-CoV-2. The purpose of the current study was to identify potential peptidomimetics against S protein with a combination of structure-based virtual screening methods and in vitro assays. METHODS: The candidates were inspected in terms of ADME properties, drug-likeness, as well as toxicity profiles. Additionally, molecular docking and dynamics simulations were performed to predict binding of the studied ligands to spike protein. RESULTS: Biological evaluation of the compounds revealed that PM2 molecule exhibits some antiviral activity. CONCLUSION: In summary, this study highlights the importance of combining in silico and in vitro techniques in order to identify antiviral compound to tackle COVID-19 and presents a new scaffold that may be structurally optimized for improved antiviral activity.

Functional comparison of MERS-coronavirus lineages reveals increased replicative fitness of the recombinant lineage 5.

Middle East respiratory syndrome coronavirus (MERS-CoV) is enzootic in dromedary camels across the Middle East and Africa. Virus-induced pneumonia in humans results from animal contact, with a potential for limited onward transmission. Phenotypic changes have been suspected after a novel recombinant clade (lineage 5) caused large nosocomial outbreaks in Saudi Arabia and South Korea in 2016. However, there has been no functional assessment. Here we perform a comprehensive in vitro and ex vivo comparison of viruses from parental and recombinant virus lineages (lineage 3, n = 7; lineage 4, n = 8; lineage 5, n = 9 viruses) from Saudi Arabia, isolated immediately before and after the shift toward lineage 5. Replication of lineage 5 viruses is significantly increased. Transcriptional profiling finds reduced induction of immune genes IFNB1, CCL5, and IFNL1 in lung cells infected with lineage 5 strains. Phenotypic differences may be determined by IFN antagonism based on experiments using IFN receptor knock out and signaling inhibition. Additionally, lineage 5 is more resilient against IFN pre-treatment of Calu-3 cells (ca. 10-fold difference in replication). This phenotypic change associated with lineage 5 has remained undiscovered by viral sequence surveillance, but may be a relevant indicator of pandemic potential.

SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization.

Transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to farmed mink has been observed in Europe and the US. In the infected animals, viral variants arose that harbored mutations in the spike (S) protein, the target of neutralizing antibodies, and these variants were transmitted back to humans. This raised concerns that mink might become a constant source of human infection with SARS-CoV-2 variants associated with an increased threat to human health and resulted in mass culling of mink. Here, we report that mutations frequently found in the S proteins of SARS-CoV-2 from mink are mostly compatible with efficient entry into human cells and its inhibition by soluble angiotensin-converting enzyme 2 (ACE2). In contrast, mutation Y453F reduces neutralization by an antibody with emergency use authorization for coronavirus disease 2019 (COVID-19) therapy and sera/plasma from COVID-19 patients. These results suggest that antibody responses induced upon infection or certain antibodies used for treatment might offer insufficient protection against SARS-CoV-2 variants from mink.

SARS-CoV-2 and SARS-CoV Spike-Mediated Cell-Cell Fusion Differ in Their Requirements for Receptor Expression and Proteolytic Activation.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with angiotensin-converting enzyme 2 (ACE2) and activation by proteases, in particular transmembrane protease serine 2 (TMPRSS2). Viruses can also spread through fusion of infected with uninfected cells. We compared the requirements of ACE2 expression, proteolytic activation, and sensitivity to inhibitors for SARS2-S-mediated and SARS-CoV-S (SARS1-S)-mediated cell-cell fusion. SARS2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while SARS1-S-driven fusion was strongly increased by TMPRSS2 and less so by ACE2 expression. In contrast to that of SARS1-S, SARS2-S-mediated cell-cell fusion was efficiently activated by batimastat-sensitive metalloproteases. Mutation of the S1/S2 proteolytic cleavage site reduced effector cell-target cell fusion when ACE2 or TMPRSS2 was limiting and rendered SARS2-S-driven cell-cell fusion more dependent on TMPRSS2. When both ACE2 and TMPRSS2 were abundant, initial target cell-effector cell fusion was unaltered compared to that of wild-type (wt) SARS2-S, but syncytia remained smaller. Mutation of the S2 cleavage (S2') site specifically abrogated activation by TMPRSS2 for both cell-cell fusion and SARS2-S-driven pseudoparticle entry but still allowed for activation by metalloproteases for cell-cell fusion and by cathepsins for particle entry. Finally, we found that the TMPRSS2 inhibitor bromhexine, unlike the inhibitor camostat, was unable to reduce TMPRSS2-activated cell-cell fusion by SARS1-S and SARS2-S. Paradoxically, bromhexine enhanced cell-cell fusion in the presence of TMPRSS2, while its metabolite ambroxol exhibited inhibitory activity under some conditions. On Calu-3 lung cells, ambroxol weakly inhibited SARS2-S-driven lentiviral pseudoparticle entry, and both substances exhibited a dose-dependent trend toward weak inhibition of authentic SARS-CoV-2.IMPORTANCE Cell-cell fusion allows viruses to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2' cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those, greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion. Bromhexine, reportedly an inhibitor of TMPRSS2, is currently being tested in clinical trials against coronavirus disease 2019. Our results indicate that bromhexine enhances fusion under some conditions. We therefore caution against the use of bromhexine in high dosages until its effects on SARS-CoV-2 spike activation are better understood. The related compound ambroxol, which similarly to bromhexine is clinically used as an expectorant, did not exhibit activating effects on cell-cell fusion. Both compounds exhibited weak inhibitory activity against SARS-CoV-2 infection at high concentrations, which might be clinically attainable for ambroxol.

Low serum neutralizing anti-SARS-CoV-2 S antibody levels in mildly affected COVID-19 convalescent patients revealed by two different detection methods.

Neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into cells via surface-expressed angiotensin-converting enzyme 2 (ACE2). We used a surrogate virus neutralization test (sVNT) and SARS-CoV-2 S protein-pseudotyped vesicular stomatitis virus (VSV) vector-based neutralization assay (pVNT) to assess the degree to which serum antibodies from coronavirus disease 2019 (COVID-19) convalescent patients interfere with the binding of SARS-CoV-2 S to ACE2. Both tests revealed neutralizing anti-SARS-CoV-2 S antibodies in the sera of ~90% of mildly and 100% of severely affected COVID-19 convalescent patients. Importantly, sVNT and pVNT results correlated strongly with each other and to the levels of anti-SARS-CoV-2 S1 IgG and IgA antibodies. Moreover, levels of neutralizing antibodies correlated with the duration and severity of clinical symptoms but not with patient age. Compared to pVNT, sVNT is less sophisticated and does not require any biosafety labs. Since this assay is also much faster and cheaper, sVNT will not only be important for evaluating the prevalence of neutralizing antibodies in a population but also for identifying promising plasma donors for successful passive antibody therapy.

Chloroquine does not inhibit infection of human lung cells with SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been associated with more than 780,000 deaths worldwide (as of 20 August 2020). To develop antiviral interventions quickly, drugs used for the treatment of unrelated diseases are currently being repurposed to treat COVID-19. Chloroquine is an anti-malaria drug that is used for the treatment of COVID-19 as it inhibits the spread of SARS-CoV-2 in the African green monkey kidney-derived cell line Vero1-3. Here we show that engineered expression of TMPRSS2, a cellular protease that activates SARS-CoV-2 for entry into lung cells4, renders SARS-CoV-2 infection of Vero cells insensitive to chloroquine. Moreover, we report that chloroquine does not block infection with SARS-CoV-2 in the TMPRSS2-expressing human lung cell line Calu-3. These results indicate that chloroquine targets a pathway for viral activation that is not active in lung cells and is unlikely to protect against the spread of SARS-CoV-2 in and between patients.

LY6E impairs coronavirus fusion and confers immune control of viral disease.

Zoonotic coronaviruses (CoVs) are substantial threats to global health, as exemplified by the emergence of two severe acute respiratory syndrome CoVs (SARS-CoV and SARS-CoV-2) and Middle East respiratory syndrome CoV (MERS-CoV) within two decades1-3. Host immune responses to CoVs are complex and regulated in part through antiviral interferons. However, interferon-stimulated gene products that inhibit CoVs are not well characterized4. Here, we show that lymphocyte antigen 6 complex, locus E (LY6E) potently restricts infection by multiple CoVs, including SARS-CoV, SARS-CoV-2 and MERS-CoV. Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in immune cells were highly susceptible to a murine CoV-mouse hepatitis virus. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic immune cells, higher splenic viral burden and reduction in global antiviral gene pathways. Accordingly, we found that constitutive Ly6e directly protects primary B cells from murine CoV infection. Our results show that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo-knowledge that could help inform strategies to combat infection by emerging CoVs.

LY6E impairs coronavirus fusion and confers immune control of viral disease.

Zoonotic coronaviruses (CoVs) are significant threats to global health, as exemplified by the recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 1 . Host immune responses to CoV are complex and regulated in part through antiviral interferons. However, the interferon-stimulated gene products that inhibit CoV are not well characterized 2 . Here, we show that interferon-inducible lymphocyte antigen 6 complex, locus E (LY6E) potently restricts cellular infection by multiple CoVs, including SARS-CoV, SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in hematopoietic cells were highly susceptible to murine CoV infection. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic and splenic immune cells and reduction in global antiviral gene pathways. Accordingly, we found that Ly6e directly protects primary B cells and dendritic cells from murine CoV infection. Our results demonstrate that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo , knowledge that could help inform strategies to combat infection by emerging CoV.

A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells.

The pandemic coronavirus SARS-CoV-2 threatens public health worldwide. The viral spike protein mediates SARS-CoV-2 entry into host cells and harbors a S1/S2 cleavage site containing multiple arginine residues (multibasic) not found in closely related animal coronaviruses. However, the role of this multibasic cleavage site in SARS-CoV-2 infection is unknown. Here, we report that the cellular protease furin cleaves the spike protein at the S1/S2 site and that cleavage is essential for S-protein-mediated cell-cell fusion and entry into human lung cells. Moreover, optimizing the S1/S2 site increased cell-cell, but not virus-cell, fusion, suggesting that the corresponding viral variants might exhibit increased cell-cell spread and potentially altered virulence. Our results suggest that acquisition of a S1/S2 multibasic cleavage site was essential for SARS-CoV-2 infection of humans and identify furin as a potential target for therapeutic intervention.

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor.

The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.

Polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of Middle East respiratory syndrome coronavirus.

Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) causes a severe respiratory disease in humans. The MERS-CoV spike (S) glycoprotein mediates viral entry into target cells. For this, MERS-CoV S engages the host cell protein dipeptidyl peptidase 4 (DPP4, CD26) and the interface between MERS-CoV S and DPP4 has been resolved on the atomic level. Here, we asked whether naturally-occurring polymorphisms in DPP4, that alter amino acid residues required for MERS-CoV S binding, influence cellular entry of MERS-CoV. By screening of public databases, we identified fourteen such polymorphisms. Introduction of the respective mutations into DPP4 revealed that all except one (Δ346-348) were compatible with robust DPP4 expression. Four polymorphisms (K267E, K267N, A291P and Δ346-348) strongly reduced binding of MERS-CoV S to DPP4 and S protein-driven host cell entry, as determined using soluble S protein and S protein bearing rhabdoviral vectors, respectively. Two polymorphisms (K267E and A291P) were analyzed in the context of authentic MERS-CoV and were found to attenuate viral replication. Collectively, we identified naturally-occurring polymorphisms in DPP4 that negatively impact cellular entry of MERS-CoV and might thus modulate MERS development in infected patients.

Spike proteins of novel MERS-coronavirus isolates from North- and West-African dromedary camels mediate robust viral entry into human target cells.

The highly pathogenic Middle East respiratory syndrome (MERS)-related coronavirus (CoV) is transmitted from dromedary camels, the natural reservoir, to humans. For at present unclear reasons, MERS cases have so far only been observed in the Arabian Peninsula, although MERS-CoV also circulates in African dromedary camels. A recent study showed that MERS-CoV found in North/West- (Morocco) and West-African (Burkina Faso and Nigeria) dromedary camels are genetically distinct from Arabian viruses and have reduced replicative capacity in human cells, potentially due to amino acid changes in one or more viral proteins. Here, we show that the spike (S) proteins of the prototypic Arabian MERS-CoV strain, human betacoronavirus 2c EMC/2012, and the above stated African MERS-CoV variants do not appreciably differ in expression, DPP4 binding and ability to drive entry into target cells. Thus, virus-host-interactions at the entry stage may not limit spread of North- and West-African MERS-CoV in human cells.

Mutations in the Spike Protein of Middle East Respiratory Syndrome Coronavirus Transmitted in Korea Increase Resistance to Antibody-Mediated Neutralization.

Middle East respiratory syndrome coronavirus (MERS-CoV) poses a threat to public health. The virus is endemic in the Middle East but can be transmitted to other countries by travel activity. The introduction of MERS-CoV into the Republic of Korea by an infected traveler resulted in a hospital outbreak of MERS that entailed 186 cases and 38 deaths. The MERS-CoV spike (S) protein binds to the cellular protein DPP4 via its receptor binding domain (RBD) and mediates viral entry into target cells. During the MERS outbreak in Korea, emergence and spread of viral variants that harbored mutations in the RBD, D510G and I529T, was observed. Counterintuitively, these mutations were found to reduce DPP4 binding and viral entry into target cells. In this study, we investigated whether they also exerted proviral effects. We confirm that changes D510G and I529T reduce S protein binding to DPP4 but show that this reduction only translates into diminished viral entry when expression of DPP4 on target cells is low. Neither mutation modulated S protein binding to sialic acids, S protein activation by host cell proteases, or inhibition of S protein-driven entry by interferon-induced transmembrane proteins. In contrast, changes D510G and I529T increased resistance of S protein-driven entry to neutralization by monoclonal antibodies and sera from MERS patients. These findings indicate that MERS-CoV variants with reduced neutralization sensitivity were transmitted during the Korean outbreak and that the responsible mutations were compatible with robust infection of cells expressing high levels of DPP4.IMPORTANCE MERS-CoV has pandemic potential, and it is important to identify mutations in viral proteins that might augment viral spread. In the course of a large hospital outbreak of MERS in the Republic of Korea in 2015, the spread of a viral variant that contained mutations in the viral spike protein was observed. These mutations were found to reduce receptor binding and viral infectivity. However, it remained unclear whether they also exerted proviral effects. We demonstrate that these mutations reduce sensitivity to antibody-mediated neutralization and are compatible with robust infection of target cells expressing large amounts of the viral receptor DPP4.

Functional analysis of potential cleavage sites in the MERS-coronavirus spike protein.

The Middle East respiratory syndrome-related coronavirus (MERS-CoV) can cause severe disease and has pandemic potential. Therefore, development of antiviral strategies is an important task. The activation of the viral spike protein (S) by host cell proteases is essential for viral infectivity and the responsible enzymes are potential therapeutic targets. The cellular proteases furin, cathepsin L and TMPRSS2 can activate MERS-S and may cleave the S protein at two distinct sites, termed S1/S2 and S2'. Moreover, a potential cathepsin L cleavage site in MERS-S has been reported. However, the relative importance of these sites for MERS-S activation is incompletely understood. Here, we used mutagenic analysis and MERS-S-bearing vectors to study the contribution of specific cleavage sites to S protein-driven entry. We found that an intact S1/S2 site was only required for efficient entry into cells expressing endogenous TMPRSS2. In keeping with a previous study, pre-cleavage at the S1/S2 motif (RSVR) was important although not essential for subsequent MERS-S activation by TMPRSS2, and indirect evidence was obtained that this motif is processed by a protease depending on an intact RXXR motif, most likely furin. In contrast, the S2' site (RSAR) was required for robust viral entry into all cell lines tested and the integrity of one of the two arginines was sufficient for efficient entry. These findings suggest that cleavage at S2' is carried out by proteases recognizing a single arginine, most likely TMPRSS2 and cathepsin L. Finally, mutation of the proposed cathepsin L site did not impact viral entry and double mutation of S1/S2 and S2' site was compatible with cathepsin L- but not TMPRSS2-dependent host cell entry, indicating that cathepsin L can process the S protein at auxiliary sites. Collectively, our results indicate a rigid sequence requirement for S protein activation by TMPRSS2 but not cathepsin L.