S2 SUBUNIT FUSION AT 2019-NCOV

S2 SUBUNIT FUSION AT 2019-NCOV

S2 SUBUNIT FUSION

SARS-CoV spike (S) protein S2 subunit plays a key role in mediating virus fusion with and entry into the host cell, in which the heptad repeat 1 (HR1) and heptad repeat 2 (HR2) can interact to form six-helical bundle (6-HB), thereby bringing viral and cellular membranes in close proximity for fusion. Using S-HR1 as a target, we have previously designed and developed several potent fusion inhibitors against SARS-CoV (e.g., SARS-HR2P)4 and Middle East respiratory syndrome (MERS)-CoV (e.g., MERS-HR2P). However, it is unclear whether 2019-nCoV also possesses a similar fusion and entry mechanism as that of SARS-CoV and MERS-CoV, and if so, whether a 2019-nCoV S-HR1 can also serve as an important target for the development of 2019-nCoV fusion/entry inhibitors. In the post-fusion hairpin conformation of the SARS-CoV or MERS-CoV S protein, the HR2 domain forms both rigid helix and flexible loop to interact with HR1 domain. ). According to the sequence alignment, the 2019-nCoV and SARS-CoV S2 subunits are highly conserved, with 92.6% and 100% overall identity in HR1 and HR2 domains, respectively. However, inside the HR1core region, 8 of the 21 residues show mutation (~38% difference). This is significantly different from the HR1core region of previously identified SARS-like viruses, such as WIV1, Rs3367, and RsSHC014, which are 100% identical to that of SARS-CoV (Fig. 1b). These novel point mutations in 2019-nCoV S2 subunit may change the interaction pattern between HR1 and HR2 domains in the post-fusion core, thus affecting the 6-HB formation. These results confirm, for the first time, that 2019-nCoV HR1 and HR2 regions are able to interact with each other to form 6HB and suggest that 2019-nCoV-HR2P may inhibit 2019-nCoV fusion with and entry into the target cell, as we showed before with SARS-CoV, MERS-CoV, and other human CoVs.

HR1 region in various coronaviruses is a conserved target site, and based on that evidence, we designed a pan-coronavirus fusion inhibitor, denoted as EK1.8 Compared with 2019-nCoV-HR2P, EK1 shows significant sequence variation, but interestingly, EK1 could also bind 2019-nCoV-HR1P in native-PAGE in a manner similar to that of 2019-nCoV-HR2P. these results suggest that the 2019-nCoV S-HR1 region is also a promising conserved target for developing effective CoV fusion/entry inhibitors. Both 2019-nCoVHR2P and EK1 peptides could significantly inhibit 2019-nCoV pseudovirus infection in a dose-dependent manner with an IC50 values of 0.98 and 2.38µM, respectively. . Notably, both 2019-nCoVHR2P and EK1, the pan-CoV fusion inhibitor, exhibited potent inhibitory activity against S-mediated cell–cell fusion and 2019-nCoV pseudovirus infection, suggesting potential development of either 2019-nCoV-HR2P or EK1 peptide in nasal spray and inhalation formulations, respectively, to prevent and treat 2019nCoV infection(1). (4)

     ACE2-Spikeprotein RBD complex interaction with salt bridge(Glu329 and Arg426). May be future design for antibodies.

NEUTRALIZING ANTIBODY

SARS-CoV infection has been thoroughly reviewed elsewhere. Entry of SARS-CoV into the host cell is mediated by the attachment of S protein and ACE-2 receptor. The S protein is the major inducer of NAbs. Most of NAbs have been identified to recognize RBD region. Interestingly, some NAbs still showed to recognize epitopes on S2 unit, suggesting that other mechanisms could be involved in the neutralization. . Currently, several strategies are used in the clinic or under development, such as viral-targeting therapeutics and host-targeting agents (such as interferons, glucocorticoids) for the treatment of COVID-19. As compared with these therapeutic strategies, NAbs appear to be more specific for virions. Understanding of action mechanisms of NAbs may provide valuable implications for the rapid development of antibody therapy and vaccine for SARS-CoV-2. However, the development of NAb-based therapeutics is a time- consuming and laborious process. To date, no NAb agents for either SARS-CoV or (Middle East Respiratory Syndrome Coronavirus) MERS-CoV are available in the market. Meanwhile, a note of caution is that the effect of antibody immune response in protecting against pulmonary pathogenesis of SARS-CoV is controversial. Some patients who died of SARS showed the strong NAb responses and pulmonary proinflammatory accumulation, suggesting NAbs could be associated with fatal acute lung injury. Therefore, it is important to take insight into humoral and cellular responses of SARS-CoV-2 when antiviral immunotherapy is developed(2). 

HIV-1 PROTEASE INHIBITOR

The coronavirus genome encodes four structural proteins: spike glycoprotein (S), small envelope protein (E), matrix glycoprotein (M) and nucleocapsid protein (N). In addition to the above four structural genes, the 3CLpro, a main protease required for the maturation of coronaviruses, is vital for the viral life cycle, making it an attractive target of anti-coronavirus drug development. By sequence alignment, it is found that SARS-CoV-2 and SARS-CoV 3CLpro share remarkable 96% sequence identity. The crytal structure of SARS-CoV-2 3CLpro (PDB ID: 6LU7) is highly similar to its SARS sister. Both of SARS-CoV-2 and SARS-CoV 3CLpro protomers contain nine α-helices and 13 β-strands that make up three distinct domains, i.e. domain I, domain II and domain III. Similar to other CoV proteases, Domains I (residues 8–101) and II (residues 102–184) contain one antiparallel β-barrel, which resemble the trypsin-like serine proteases structure. Domain III (residues 201–306) consists of 5 α-helices (α5-α9), which are connected by a long loop (residues185–200) with domain II. In contrast to the common Ser–His–Asp catalytic triad of serine proteases, the SARS-CoV-2 and SARS-CoV 3CLpro has a catalytic dyad, which are composed of the conserved residues H41 and C145. The main substrate-binding site of the 3CLpro is formed by a cleft between domains I and II. Although the SARS-CoV-2 3CLpro 3D structure provides deep insight into viral life cycle and facilitate for screening anti-COVID-19 drugs, no approved drugs have been found to effectively inhibit the virus so far. Since the emergency of this outbreak and it has been reported that the HIV-1 protease inhibitors can be used as anti-SARS drugs by tegarting SARS-CoV 3CLpro [11-13], we choose six public anti-HIV-1 drugs to evaluate their potential to become clinical drugs for COVID-19 by means of molecular docking. Two of the six drug-3CLpro complexes (indinavir and darunavir) showing high docking scores were futher subjected to molecular dynamics (MD) simulation and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations. The molecular interactions between these two HIV-1 proteinase inhibitors and the 3CLpro were detailed analyzed, and the reason for the difference of binding ability between SARS-CoV-2 and SARS-CoV 3CLpro and these inhibitors was also discussed. It has been reported that HIV-1 inhibitors can be used as anti-SARS clinical treatment drugs by targeting the SARS-CoV 3CLpro. . Our results show that darunavir has the best binding affinity with SARS-CoV-2 and SARS-CoV 3CLpro among all inhibitors, indicating it has the potential to become an anti-COVID-19 clinical drug(3).

 

InhibitorBinding energy (kJ/mol)
 SARS-CoV-2SARS-CoVHIV-1 proteinase
lopinavir-5.49-2.12-5.78
ritonavir-2.34-1.42-5.17
indinavir-10.02-7.49-11.56
saquinavir-8.26-5.3-11.82
darunavir-10.24-7.5-10.85
tipranavir-5.8-4.4-11.07

Chemical structures of HIV-1 protease inhibitors used in this study. (A) lopinavir (C37H48N4O5). (B) ritonavir (C37H48N6O5S2). (C) indinavir (C36H47N5O4). (D) saquinavir (C38H50N6O5). (E) darunavir (C27H37N3O7S). (F) tipranavir (C31H33F3N2O5S).

 

TMPRSS2

The spike (S) protein of coronaviruses facilitates viral entry in to target cells. Entry depends on binding of the surface unit, S1, of the S protein to a cellular receptor, which facilitates viral attachment to the surface of target cells. In addition, entry requires S protein priming by cellular proteases, which entails S protein cleavage at the S1/S2 and the S2’ site and allows fusion of viraland cellular membranes, a process driven by the S2 subunit. SARS-S engages angiotensin-converting enzyme 2(ACE2) as the entry receptor and employs the cellular serine protease TMPRSS2 for S protein priming. The SARS-S/ACE2 interface has been elucidated at the atomic level, and the efficiency of ACE2 usage was found to be a key determinant of SARS-CoV transmissibility. SARS-S und SARS-2-S share76% amino acid identity. However, it is unknown whether SARS-2-S like SARS-S employs ACE2 and TMPRSS2 for host cell entry. SARS-CoV can use the endosomal cysteine proteases cathepsin B and L (CatB/L) and the serine protease TMPRSS2 for S protein priming in cell lines, and inhibition of both proteases is required for robust blockade of viral entry. However, only TMPRSS2 activity is essential for viral spread and pathogenesis in the infected host whereas CatB/L activity is dispensable. The present study provides evidence that host cell entry of SARS-CoV-2 depends on the SARS-CoV receptor ACE2 and can be blocked by a clinically proven inhibitor of the cellular serine protease TMPRSS2, which is employed by SARS-CoV-2 for S protein priming. Moreover, it suggests that antibody responses raised against SARS-CoV could at least partially protect against SARS-CoV-2 infection. The S proteins of SARS-CoV can use the endosomal cysteine proteases CatB/L for S protein priming in TMPRSS2 cells. However, S protein priming by TMPRSS2 but not CatB/L is essential for viral entry into primary target cells and for viral spread in the infected host. The present study indicates that SARS-CoV-2 spread also depends on TMPRSS2 activity, although we note that SARS-CoV-2 infection of Calu-3 cells was inhibited but not abrogated by camostat mesylate, likely reflecting residual S protein priming by CatB/L. One can speculate that furin-mediated precleavage at the S1/S2 site in infected cells might promote subsequent TMPRSS2-dependent entry into target cells, as reported for MERS-CoV. Collectively, our present findings and previous work highlight TMPRSS2 as a host cell factor that is critical for spread of several clinically relevant viruses, including influenza A viruses and coronaviruses. In contrast, TMPRSS2 is dispensable for development and homeostasis and thus constitutes an attractive drug target. In this context, it is noteworthy that the serine protease inhibitor camostat mesylate, which blocks TMPRSS2 activity, has been approved in Japan for human use, but for an unrelated indication. This compound or related ones with potentially increased antiviral activity could thus be considered for off-label treatment of SARS-CoV-2-infected patients(5).

ANNTI SARS COV-2 NEUTRALIZING ANTIBODIES

Coronavirus neutralizing antibodies primarily target the trimeric spike (S) glycoproteins on the viral surface that mediate entry into host cells. The S protein has two functional subunits that mediate cell attachment (the S1 subunit, existing of four core domains S1A through S1D) and fusion of the viral and cellular membrane (the S2 subunit). Potent neutralizing antibodies often target the receptor interaction site in S1, disabling receptor interactions. In order to identify SARS-CoV-2 neutralizing antibodies, ELISA-(cross)reactivity was assessed of antibody-containing supernatants of a collection of 51 SARS-S hybridoma’s derived from immunized transgenic H2L2 mice that encode chimeric immunoglobulins with human variable heavy and light chains and constant regions of rat origin. The human 47D11 antibody binds to cells expressing the full-length spike proteins of SARS-CoV and SARS-CoV-2. The 47D11 antibody was found to potently inhibit infection of VeroE6 cells with SARS-S and SARS2-S pseudotyped VSV with IC50 values of 0.06 and 0.08 μg/ml respectively.  47D11 binds a conserved epitope on the spike receptor binding domain explaining its ability to cross-neutralize SARS-CoV and SARS-CoV-2, using a mechanism that is independent of receptor binding inhibition. This antibody will be useful for development of antigen detection tests and serological assays targeting SARS-CoV-2. Neutralizing antibodies can alter the course of infection in the infected host supporting virus clearance or protect an uninfected host that is exposed to the virus. Hence, this antibody offers the potential to prevent and/or treat COVID-19, and possibly also other future emerging diseases in humans caused by viruses from the Sarbecovirus subgenus(6).

REFERENCES

  1. Fusion mechanism of 2019-nCoV and fusion inhibitors targeting HR1 domain in spike protein
  2. Perspectives on therapeutic neutralizing antibodies against the Novel Coronavirus SARS-CoV-2
  3. Insight derived from molecular docking and molecular dynamics simulations into the binding interactions between HIV-1 protease inhibitors and SARS-CoV-2 CLpro
  4. A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike
  5. SARS-CoV-2 Cell Entry Depends on ACE2 andTMPRSS2 and Is Blocked by a Clinically ProvenProtease InhibitorGraphical AbstractHighlightsdSARS-CoV-2 uses the SARS-CoV receptor ACE2 for hostcell entrydThe spike protein of SARS-CoV-2 is primed by TMPRSS2dAntibodies against SARS-CoV spike may offer someprotection against SARS-CoV-2AuthorsMarkus Hoffmann, Hannah Kleine-Weber,Simon Schroeder, …, Marcel A. Mu ̈ller,Christian Drosten, Stefan Po ̈hlmannCorrespondencemhoffmann@dpz.eu (M.H.),spoehlmann@dpz.eu (S.P.)In BriefThe emerging SARS-coronavirus 2(SARS-CoV-2) threatens public health.Hoffmann and coworkers show thatSARS-CoV-2 infection depends on thehost cell factors ACE2 and TMPRSS2 andcan be blocked by a clinically provenprotease inhibitor. These findings mighthelp to establish options for preventionand treatment.Hoffmann et al., 2020, Cell181, 1–10April 16, 2020ª2020 Elsevier Inc. https://doi.org/10.1016/j.cell.2020.02.052
  6. A human monoclonal antibody blocking SARS-CoV-2 infection

 

Yazarlar:

  • Simge Ceyhan
  • Çağla Arslan

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