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...

A novel coronavirus (COVID-19 virus) outbreak has caused a global pandemic resulting in tens of thousands of infections and thousands of deaths worldwide. The RNA-dependent RNA polymerase (RdRp, also named nsp12) is the central component of coronaviral replication/transcription machinery and appears to be a primary target for the antiviral drug, remdesivir. We report the cryo-EM structure of COVID-19 virus fulllength nsp12 in complex with cofactors nsp7 and nsp8 at 2.9-Å resolution. In addition to the conserved architecture of the polymerase core of the viral polymerase family, nsp12 possesses a newly identified βhairpin domain at its N terminus. A comparative analysis model shows how remdesivir binds to this polymerase. The structure provides a basis for the design of new antiviral therapeutics targeting viral RdRp.

SUMMARYA new coronavirus (CoV) identified as COVID-19 virus is the etiological agent responsible for the 2019-2020 viral pneumonia outbreak that commenced in Wuhan1–4. Currently there is 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 (Mpro). Mpro is a key CoV enzyme, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target for this virus5,6. Here, we identified a mechanism-based inhibitor, N3, by computer-aided drug design and subsequently determined the crystal structure of COVID-19 virus Mpro 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 Mpro. Six of these inhibit Mpro with IC50 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 where no specific drugs or vaccines are available.

Highlights d Structures of SARS-CoV-2 RNA polymerase in complexes with RNA revealed d Conformational changes in nsp8 and its interaction with the exiting RNA are observed d Incorporation and delayed-chain-termination mechanism of remdesivir is elucidated d Transition model from primase complex to polymerase complex is proposed

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the r eplication and t ranscription c omplex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transition towards cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp8 2 -nsp12-nsp13 2 -RNA and a single RNA binding protein nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N-terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5’ extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC towards mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.