Structural Contributions to SARS-CoV2 and the COVID-19 Pandemic

Conference: 2020: 70th ACA Annual Meeting
08/04/2020: 11:30 AM  - 3:00 PM 
5.1.1 
Oral Session 
Virtual  

Presentations

Protein Data Bank in the Time of Coronavirus: How open-access 3D structures are enabling research and development in fundamental biology, biomedicine, biotechnology, and drug discovery as we confront the global SARS-CoV-2 pandemic.

11:30 AM - 11:55 AM 
The Protein Data Bank (PDB) archive currently holds >165,000 atomic level three-dimensional (3D) structures of biomolecules experimentally determined using macromolecular crystallography (MX), NMR spectroscopy, electron microscopy (3DEM), and electron diffraction. The archive was established in 1971 as the first open-access, digital-data resource in biology, and is now managed by the Worldwide Protein Data Bank partnership (wwPDB; wwPDB.org). US PDB operations are the responsibility of the RCSB Protein Data Bank (RCSB PDB; RCSB.org; based at Rutgers, The State University of New Jersey, UC San Diego, and UC San Francisco). The RCSB PDB serves many millions of users worldwide by delivering PDB data integrated with ~40 external biodata resources via RCSB.org, APIs, and FTP download, providing rich structural views of fundamental biology, biomedicine, biotechnology, and energy sciences. In addition, the RCSB PDB outreach/education portal serves more than half a million PDB101.RCSB.org users worldwide, who are primarily university educators and their undergraduate students. Not counted in these usage metrics are the many PDB users working in biopharmaceutical companies, wherein copies of the PDB archive are retained within firewalls for interoperation with proprietary structures. Discovery/development of nearly 90% of the 210 new drugs approved by the US Food and Drug Administration 2010-2016 was facilitated by open access to target protein structures in the PDB [1]. With public release of the SARS-CoV-2 genome sequence on January 10th 2020, structural biologists joined the fight against the global COVID-19 pandemic. The first atomic-level 3D structure of a COVID-19 protein (Nsp5 or Main Protease, a promising drug target; PDB ID 6lu7) [2] was released by the PDB on February 5th 2020. Since then, more than 200 additional COVID-19 protein structures have been released by PDB, including those of the Spike Protein (illuminating receptor binding to Angiotensin Converting Enzyme 2 and neutralization by monoclonal antibodies), Papain-like Protease (part of Nsp3, and another promising drug target), RNA-dependent RNA polymerase (Nsp7, Nsp8, and Nsp12 heterotrimer; including complexes with RNA and the US FDA approved small-molecule inhibitor remdesivir), ADP Ribose Phosphatase (part of Nsp3), and Endoribonuclease (Nsp15). Of particular significance for global structure-guided drug discovery efforts are more than 100 PDB structures of the Main Protease bound to small-chemical fragments (or scaffolds) generated using the XChem Fragment Screening Pipeline at the UK Diamond Light Source. Select examples drawn from PDB holdings will be presented, highlighting the impact of MX and 3DEM on our understanding of SARS-CoV-2 biology, evolution, infection, and pathogenesis, and discovery of vaccines and small- and large-molecule therapeutics. Released COVID-19 PDB structures and related resources are regularly updated at http://rcsb.org/covid19. Acknowledgements: RCSB Protein Data Bank is funded by the National Science Foundation (DBI-1832184), the US Department of Energy (DE-SC0019749), and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institutes of Health under grant R01GM133198. References: 1. Westbrook, J.W., and Burley, S.K. (2019) Structure. https://doi.org/10.1016/j.str.2018.11.007 2. Jin, Z. et al. (2020) Nature. https://doi.org/10.1038/s41586-020-2223-y Coronavirus 2020. Portrait painted by David S. Goodsell, RCSB Protein Data Bank. 

View Proposal 212

Author

Stephen Burley, RCSB Protein Data Bank, Rutgers University Piscataway, NJ 

Crystallography of SARS-CoV-2 Non-structural Proteins

12:00 PM - 12:25 PM 
The coronavirus SARS-CoV-2 is an agent causing COVID-19 pandemic affecting millions of people. At present there is no effective vaccine or proven drug to prevent infections and stop virus proliferation. Although the virus is similar to SARS- and MERS-CoVs, the detailed information about SARS-CoV-2 proteins structures and functions is urgently needed to rapidly develop effective therapeutics. We have applied high-throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS-CoV-2 proteins and determine high resolution crystals structures. We have focused on nonstructural proteins (Nsps) expressed as polyproteins 1a and 1ab that are processed and assemble into a large membrane-bound replicase-transcriptase complex exhibiting multiple enzymatic and binding activities. Thus far we have determined 28 structures for 10 CoV-2 proteins. These structures include Nsp3 ADP-ribose phosphatase domain (ADRP, also known as macrodomain) and PLpro papain-like protease, Nsp5 main protease Mpro, Nsp7/Nsp8 primase complex, Nsp9 RNA-binding protein, Nsp10/Nsp16 2'-O-ribose methyltransferase complex and Nsp15 uridylate-specific endoribonuclease. We compare these structures with previously reported homologs from SARS and MERS coronaviruses and point to similarities and differences. We have also determined structures of complexes with ligands and inhibitors, including FDA approved drugs. We deposit all structures to the Protein Data Bank and release the coordinates to scientific community prior to publication. We also share all reagents and protocols. These structures provide basis for structure-based drug development. 

View Proposal 442

Author

Andrzej Joachimiak, Argonne National Laboratory

Additional Author(s)

Changsoo Chang, Center for Structural Genomics of Infectious Diseases, University of Chicago
Michael Endres, Center for Structural Genomics of Infectious Diseases
Robert Jedrzejczak, Center for Structural Genomics of Infectious Diseases
Youngchang Kim, Argonne National Lab Lemont, IL 
Natalia Maltseva, Center for Structural Genomics of Infectious Diseases
Karolina Michalska, Argonne National Laboratory
Jerzy Osipiuk, Center for Structural Genomics of Infectious Diseases
Lucy Stols, Center for Structural Genomics of Infectious Diseases
Kemin Tan, Argonne National Laboratory Argonne, IL 
Christine Tesar, Center for Structural Genomics of Infectious Diseases
Mateusz Wilamowski, Center for Structural Genomics of Infectious Diseases

Crowdsourcing inhibitor discovery against the SARS-CoV-2 main protease

12:30 PM - 12:55 PM 
COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease. The results with more than 71 co-crystal structures that span the entire active site, were released to the public and we implement a crowed-sourcing platform to solicit next generation design ideas. Thousands of suggestions were submitted, of which we made and tested hundreds, through a global consortium including academic labs, chemical vendors, pharma advisors and more. This effort resulted in sub-uM inhibitors with crystallographic confirmation that are now being further developed and assessed by live virus assays. We hope this initiative can serve as a template for an alternative drug discovery pipeline for areas that are unappealing to traditional pharma such as pandemic preparedness and antibiotic development. 

View Proposal 460

Author

Nir London, Weizmann Institute of Science

Coffee Break

1:00 PM - 1:15 PM 

Structural insights into the feline coronavirus drug GC376, which inhibits the main protease of SARS-CoV-2 and blocks virus replication

1:15 PM - 1:40 PM 
The COVID-19 pandemic, attributed to the SARS-CoV-2 coronavirus infection, resulted in millions of cases of pneumonia and mortality worldwide. Hence, there is an immediate need for antiviral therapies to block the replication and spread of the virus. The main protease, Mpro (or 3CLpro) in SARS-CoV-2 is a viable drug target because of its essential role in the cleavage of the virus polypeptide and subsequent viral replication. Feline infectious peritonitis, a fatal infection in cats caused by a coronavirus, was successfully treated previously with a prodrug GC376, a dipeptide-based protease inhibitor. Here we show the prodrug and its parent GC373, are effective inhibitors of the Mpro from both SARS-CoV and SARS-CoV-2 with IC50 values in the nanomolar range. Crystal structures of SARS-CoV-2 Mpro with these inhibitors have a covalent modification of the nucleophilic Cys145. NMR analysis reveals that inhibition proceeds via reversible formation of a hemithioacetal. GC373 and GC376 are potent inhibitors of SARS-CoV-2 replication in cell culture, with EC50 values near one micromolar and little to no toxicity. These protease inhibitors are soluble, non-toxic, and bind reversibly. They are strong drug candidates for the treatment of human coronavirus infections because they have already been successful in animals (cats). The work here lays the framework for their use in human trials for the treatment of COVID-19. 

View Proposal 427

Author

Joanne Lemieux, University of Alberta Edmonton, AB 

Additional Author(s)

Wayne Vuong, Department fo Chemsitry Edmonton, Alberta 
Muhammad Bashir Khan, Department of Biochemistry Edmonton, Alberta 
Conrad Fischer, Department of Chemistry Edmonton, Alberta 
Elena Arutyunova, Department of Biochemistry Edmonton, Alberta 
Tess Lamer, Department of Chemistry Edmotnon, Alberta 
Justin Shields, Department of Medical Microbiology and Immunology Edmonton, Alberta 
Holly A. Saffran, Department of Medical Microbiology and Immunology Edmonton, Alberta 
Ryan T. McKay, Department of Chemistry Edmonton, Alberta 
Marco J. van Belkum, Department of Chemistry Edmonton, Alberta 
Michael Joyce, Department of Medical Microbiology and Immunology Edmonton, Alberta 
Howard S. Young, Department of Biochemistry Edmonton, Alberta 
D Lorne Tyrrell, Department of Medical Microbiology and Immunology Edmonton, Alberta 
John C. Vederas, Department of Chemistry Edmonton, Alberta 

Structural basis of recognition of SARS-CoV-2 by neutralizing antibodies isolated from convalescent patients

1:45 PM - 2:10 PM 
SARS-CoV-2 has emerged as a global pandemic in 2020 with devastating health and socioeconomic consequences. Effective vaccines and therapeutics are urgently needed to combat this novel coronavirus and protect not only against this pandemic, but also potentially against related coronaviruses that may arise in the future. Many human neutralizing monoclonal antibodies (nAbs) have now been isolated from COVID-19 convalescent patients by research groups worldwide. Many of these nAbs target the receptor binding domain (RBD) of the spike protein, which engages the ACE2 receptor on human cells for viral entry. We have been determining crystal structures of nAbs bound to the spike protein RBD to determine their binding sites (epitopes) and possible mechanisms of neutralization. The nAb-RBD structural information can reveal the key sites of vulnerability on the virus. Recurring motifs as well as novel binding modes used by antibodies for recognition of the RBD have been delineated, including which antibody germline genes in the human immune repertoire are preferentially used to target the RBD. Such structural and functional information can be utilized to assess vaccine responses against SARS CoV-2, aid in modifications and improvement of vaccine immunogens, and for design of novel therapeutics that inhibit entry of SARS CoV-2 into host cells. 

View Proposal 424

Author

Ian Wilson, Dept of Integrative Structural & Computational Biology, The Scripps ResearchInst La Jolla, CA 

Additional Author(s)

Meng Yuan, The Scripps Research Institute La Jolla, CA 
Nicholas C. Wu, The Scripps Research Institute La Jolla, CA 
Hejun Liu, The Scripps Research Institute La Jolla, CA 
Chang-Chun D. Lee, The Scripps Research Institute La Jolla, CA 
Xueyong Zhu, The Scripps Research Institute La Jolla, CA 

Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes

2:15 PM - 2:40 PM 
The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion protein that is metastable and difficult to produce recombinantly in large quantities. Here, we designed and expressed over 100 structure-guided spike variants based upon a previously determined cryo-EM structure of the prefusion SARS-CoV-2 spike. Biochemical, biophysical and structural characterization of these variants identified numerous individual substitutions that increased protein yields and stability. The best variant, HexaPro, has six beneficial proline substitutions leading to ~10-fold higher expression than its parental construct and is able to withstand heat stress, storage at room temperature, and multiple freeze-thaws. A 3.2 Å-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2.   

View Proposal 433

Author

Jason McLellan Austin, TX