A new coronavirus (CoV) identified as COVID-19 virus is the etiological agent responsible for the 2019-2020 viral pneumonia outbreak that commenced in Wuhan [1][2][3][4] . Currently there are no targeted therapeutics and effective treatment options remain very limited. In order to rapidly discover lead compounds for clinical use, we initiated a program of combined structure-assisted drug design, virtual drug screening and high-throughput screening to identify new drug leads that target the COVID-19 virus main protease (M pro ). M pro is a key CoV enzyme, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target for this virus 5,6 . Here, we identified a mechanism-based inhibitor, N3, by computer-aided drug design and subsequently determined the crystal structure of COVID-19 virus M pro in complex with this compound. Next, through a combination of structure-based virtual and high-throughput screening, we assayed over 10,000 compounds including approved drugs, drug candidates in clinical trials, and other pharmacologically active compounds as inhibitors of M pro . Six of these compounds inhibited M pro with IC 50 values ranging from 0.67 to 21.4 μM. Ebselen also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of this screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases for which no specific drugs or vaccines are available.CoVs infect humans and other animal species, causing a variety of highly prevalent and severe diseases, including Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) 7 . The COVID-19 virus genome is comprised of ~30,000 nucleotides; its replicase gene encodes two overlapping polyproteins, pp1a and pp1ab, required for viral replication and transcription 3,4 . The functional polypeptides are released from the polyproteins by extensive proteolytic processing, predominantly by a 33.8-kDa main protease (M pro ), also referred to as the 3C-like protease. M pro digests the polyprotein at no less than 11 conserved sites, starting with the autolytic cleavage of this enzyme itself from pp1a and pp1ab 8 . The functional importance of M pro in the viral life cycle, together with the absence of closely related homologues in humans, identify the M pro as an attractive target for antiviral drug design 9 .To facilitate the rapid discovery of antiviral compounds with clinical potential, we developed a strategy combining structure-assisted drug design, virtual drug screening and high-throughput screening to repurpose existing drugs to target COVID-19 virus M pro . Establishing a high-throughput activity assayRecombinant COVID-19 virus M pro with native N and C termini was expressed in Escherichia coli and subsequently purified (Extended Data Fig. 1a, b). The molecular weight of COVID-19 virus M pro as determined by mass spectroscopy is 33797.0 Da, consistent with its theoretical molecular weight 337...
The recent outbreak of coronavirus disease caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the molecular mechanism that underlies viral infection must first be defined. Therefore, we herein established a SARS-CoV-2 spike (S) protein-mediated cell-cell fusion assay and found that SARS-CoV-2 showed a superior plasma membrane fusion capacity compared to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in the SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be associated with enhanced interactions with the HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted the HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. Here we generated a series of lipopeptides derived from EK1 and found that EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241-and 149-fold more potent than the original EK1 peptide, respectively. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, and potently inhibited the replication of 5 live human coronaviruses examined, including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by the currently circulating SARS-CoV-2 and other emerging SARSr-CoVs.
Since the SARS outbreak 18 years ago, a large number of severe acute respiratory syndrome related coronaviruses (SARSr-CoV) have been discovered in their natural reservoir host, bats1-4. Previous studies indicated that some of those bat SARSr-CoVs have the potential to infect humans5-7. Here we report the identification and characterization of a novel coronavirus (nCoV-2019) which caused an epidemic of acute respiratory syndrome in humans, in Wuhan, China. The epidemic, started from December 12th, 2019, has caused 198 laboratory confirmed infections with three fatal cases by January 20th, 2020. Full-length genome sequences were obtained from five patients at the early stage of the outbreak. They are almost identical to each other and share 79.5% sequence identify to SARS-CoV. Furthermore, it was found that nCoV-2019 is 96% identical at the whole genome level to a bat coronavirus. The pairwise protein sequence analysis of seven conserved non-structural proteins show that this virus belongs to the species of SARSr-CoV. The nCoV-2019 virus was then isolated from the bronchoalveolar lavage fluid of a critically ill patient, which can be neutralized by sera from several patients. Importantly, we have confirmed that this novel CoV uses the same cell entry receptor, ACE2, as SARS-CoV.
Coronavirus disease 2019 , caused by the novel human coronavirus SARS-CoV-2, is currently a major threat to public health worldwide. The viral spike protein binds the host receptor angiotensin-converting enzyme 2 (ACE2) via the receptor-binding domain (RBD), and thus is believed to be a major target to block viral entry. Both SARS-CoV-2 and SARS-CoV share this mechanism.Here we functionally analyzed the key amino acid residues located within receptor binding motif of RBD that may interact with human ACE2 and available neutralizing antibodies. The in vivo experiments showed that immunization with either the SARS-CoV RBD or SARS-CoV-2 RBD was able to induce strong clade-specific neutralizing antibodies in mice; however, the cross-neutralizing activity was much weaker, indicating that there are distinct antigenic features in the RBDs of the two viruses. This finding was confirmed with the available neutralizing monoclonal antibodies against SARS-CoV or SARS-CoV-2. It is worth noting that a newly developed SARS-CoV-2 human antibody, HA001, was able to neutralize SARS-CoV-2, but failed to recognize SARS-CoV. Moreover, the potential epitope residues of HA001 were identified as A475 and F486 in the SARS-CoV-2 RBD, representing new binding sites for neutralizing antibodies. Overall, our study has revealed the presence of different key epitopes between SARS-CoV and SARS-CoV-2, which indicates the necessity to develop new prophylactic vaccine and antibody drugs for specific control of the COVID-19 pandemic although the available agents obtained from the SARS-CoV study are unneglectable.
Using confocal microscopy, X‐ray microanalysis and the scanning ion‐selective electrode technique, we investigated the signalling of H2O2, cytosolic Ca2+ ([Ca2+]cyt) and the PM H+‐coupled transport system in K+/Na+ homeostasis control in NaCl‐stressed calluses of Populus euphratica. An obvious Na+/H+ antiport was seen in salinized cells; however, NaCl stress caused a net K+ efflux, because of the salt‐induced membrane depolarization. H2O2 levels, regulated upwards by salinity, contributed to ionic homeostasis, because H2O2 restrictions by DPI or DMTU caused enhanced K+ efflux and decreased Na+/H+ antiport activity. NaCl induced a net Ca2+ influx and a subsequent rise of [Ca2+]cyt, which is involved in H2O2‐mediated K+/Na+ homeostasis in salinized P. euphratica cells. When callus cells were pretreated with inhibitors of the Na+/H+ antiport system, the NaCl‐induced elevation of H2O2 and [Ca2+]cyt was correspondingly restricted, leading to a greater K+ efflux and a more pronounced reduction in Na+/H+ antiport activity. Results suggest that the PM H+‐coupled transport system mediates H+ translocation and triggers the stress signalling of H2O2 and Ca2+, which results in a K+/Na+ homeostasis via mediations of K+ channels and the Na+/H+ antiport system in the PM of NaCl‐stressed cells. Accordingly, a salt stress signalling pathway of P. euphratica cells is proposed.
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