Structural investigations of proteins encoded by SARS-CoV-2.

It is hard to overestimate the influence of the COVID-19 pandemic on scientific research in the last two and a half years. Within a few weeks after the first cases of the disease were reported, the causative agent, now known as SARS-CoV-2, was identified, its genome was sequenced, individual proteins were expressed and purified, and structural work commenced. The originally described SARS-CoV-2 isolate (GenBank: MN908947.3) has a positive-sense single-stranded (ss) RNA genome consisting of 29,903 bases. The genome encodes 29 proteins falling into structural and nonstructural categories, expressed as polyproteins that have to be cleaved into the final products by two virally encoded cysteine proteases. This "In the Limelight" special issue of FEBS Open Bio includes three review articles, focused on different aspects of the structure and other properties of selected examples of SARS-CoV-2 proteins: (a) the properties of the Nsp14 and Nsp15 ribonucleases; (b) the current state of knowledge of the molecular mechanisms for the translation of both viral transcripts and cellular messenger RNAs, with a focus on the properties of the Nsp1 protein; and (c) the structural properties and evolution of the spike proteins in SARS-CoV-2 and other coronaviruses. These three reviews describe very different aspects of work that ultimately should lead to the development of more vaccines, antibodies, and small molecule drugs, necessary to combat this pandemic, as well as to counter future variants of this coronavirus.

An open chat with Alexander Wlodawer.

Alexander Wlodawer has been a member of the FEBS Open Bio Editorial Board since the journal's launch in 2011. Currently, he is Senior Investigator at the Center for Structural Biology, National Cancer Institute in Frederick, Maryland, USA. He received his Ph.D. from the University of California, Los Angeles in 1974, completed postdoctoral training at Stanford University and has also worked at the National Bureau of Standards, the ABL-Basic Research Program at the NCI-FCRDC and the University of Cambridge, UK. He is Doctor Honoris Causa of the Technical University of Lodz, Poland. Alexander Wlodawer is also a recipient of the 2006 NCI Mentor of Merit Award, was awarded the Heyrovsky Honorary Medal by the Czech Academy of Sciences in 2008, was elected Foreign Member of the Polish Academy of Sciences in 2005, and has been member of the Editorial Board of The FEBS Journal since 2007. He is currently Editor-in-Chief of the journal Current Research in Structural Biology. In this compelling interview, he shares with us his experiences on solving the structures of IL-4 and retroviral proteases, advice on how to deal with being scooped, and his thoughts on open data sharing and AlphaFold.

Control of SARS-CoV-2 infection after Spike DNA or Spike DNA+Protein co-immunization in rhesus macaques.

The speed of development, versatility and efficacy of mRNA-based vaccines have been amply demonstrated in the case of SARS-CoV-2. DNA vaccines represent an important alternative since they induce both humoral and cellular immune responses in animal models and in human trials. We tested the immunogenicity and protective efficacy of DNA-based vaccine regimens expressing different prefusion-stabilized Wuhan-Hu-1 SARS-CoV-2 Spike antigens upon intramuscular injection followed by electroporation in rhesus macaques. Different Spike DNA vaccine regimens induced antibodies that potently neutralized SARS-CoV-2 in vitro and elicited robust T cell responses. The antibodies recognized and potently neutralized a panel of different Spike variants including Alpha, Delta, Epsilon, Eta and A.23.1, but to a lesser extent Beta and Gamma. The DNA-only vaccine regimens were compared to a regimen that included co-immunization of Spike DNA and protein in the same anatomical site, the latter of which showed significant higher antibody responses. All vaccine regimens led to control of SARS-CoV-2 intranasal/intratracheal challenge and absence of virus dissemination to the lower respiratory tract. Vaccine-induced binding and neutralizing antibody titers and antibody-dependent cellular phagocytosis inversely correlated with transient virus levels in the nasal mucosa. Importantly, the Spike DNA+Protein co-immunization regimen induced the highest binding and neutralizing antibodies and showed the strongest control against SARS-CoV-2 challenge in rhesus macaques.

Crystallographic models of SARS-CoV-2 3CLpro: in-depth assessment of structure quality and validation.

The appearance at the end of 2019 of the new SARS-CoV-2 coronavirus led to an unprecedented response by the structural biology community, resulting in the rapid determination of many hundreds of structures of proteins encoded by the virus. As part of an effort to analyze and, if necessary, remediate these structures as deposited in the Protein Data Bank (PDB), this work presents a detailed analysis of 81 crystal structures of the main protease 3CLpro, an important target for the design of drugs against COVID-19. The structures of the unliganded enzyme and its complexes with a number of inhibitors were determined by multiple research groups using different experimental approaches and conditions; the resulting structures span 13 different polymorphs representing seven space groups. The structures of the enzyme itself, all determined by molecular replacement, are highly similar, with the exception of one polymorph with a different inter-domain orientation. However, a number of complexes with bound inhibitors were found to pose significant problems. Some of these could be traced to faulty definitions of geometrical restraints for ligands and to the general problem of a lack of such information in the PDB depositions. Several problems with ligand definition in the PDB itself were also noted. In several cases extensive corrections to the models were necessary to adhere to the evidence of the electron-density maps. Taken together, this analysis of a large number of structures of a single, medically important protein, all determined within less than a year using modern experimental tools, should be useful in future studies of other systems of high interest to the biomedical community.

A small molecule compound with an indole moiety inhibits the main protease of SARS-CoV-2 and blocks virus replication.

Except remdesivir, no specific antivirals for SARS-CoV-2 infection are currently available. Here, we characterize two small-molecule-compounds, named GRL-1720 and 5h, containing an indoline and indole moiety, respectively, which target the SARS-CoV-2 main protease (Mpro). We use VeroE6 cell-based assays with RNA-qPCR, cytopathic assays, and immunocytochemistry and show both compounds to block the infectivity of SARS-CoV-2 with EC50 values of 15 ± 4 and 4.2 ± 0.7 µM for GRL-1720 and 5h, respectively. Remdesivir permitted viral breakthrough at high concentrations; however, compound 5h completely blocks SARS-CoV-2 infection in vitro without viral breakthrough or detectable cytotoxicity. Combination of 5h and remdesivir exhibits synergism against SARS-CoV-2. Additional X-ray structural analysis show that 5h forms a covalent bond with Mpro and makes polar interactions with multiple active site amino acid residues. The present data suggest that 5h might serve as a lead Mpro inhibitor for the development of therapeutics for SARS-CoV-2 infection.

Rapid response to emerging biomedical challenges and threats.

As part of the global mobilization to combat the present pandemic, almost 100 000 COVID-19-related papers have been published and nearly a thousand models of macromolecules encoded by SARS-CoV-2 have been deposited in the Protein Data Bank within less than a year. The avalanche of new structural data has given rise to multiple resources dedicated to assessing the correctness and quality of structural data and models. Here, an approach to evaluate the massive amounts of such data using the resource https://covid19.bioreproducibility.org is described, which offers a template that could be used in large-scale initiatives undertaken in response to future biomedical crises. Broader use of the described methodology could considerably curtail information noise and significantly improve the reproducibility of biomedical research.

Covid-19.bioreproducibility.org: A web resource for SARS-CoV-2-related structural models.

The COVID-19 pandemic has triggered numerous scientific activities aimed at understanding the SARS-CoV-2 virus and ultimately developing treatments. Structural biologists have already determined hundreds of experimental X-ray, cryo-EM, and NMR structures of proteins and nucleic acids related to this coronavirus, and this number is still growing. To help biomedical researchers, who may not necessarily be experts in structural biology, navigate through the flood of structural models, we have created an online resource, covid19.bioreproducibility.org, that aggregates expert-verified information about SARS-CoV-2-related macromolecular models. In this article, we describe this web resource along with the suite of tools and methodologies used for assessing the structures presented therein.

Ligand-centered assessment of SARS-CoV-2 drug target models in the Protein Data Bank

Abstract A bright spot in the SARS-CoV-2 (CoV-2) coronavirus pandemic has been the immediate mobilization of the biomedical community, working to develop treatments and vaccines for COVID-19. Rational drug design against emerging threats depends on well-established methodology, mainly utilizing X-ray crystallography, to provide accurate structure models of the macromolecular drug targets and of their complexes with candidates for drug development. In the current crisis the structural biological community has responded by presenting structure models of CoV-2 proteins and depositing them in the Protein Data Bank (PDB), usually without time embargo and before publication. Since the structures from the first-line research are produced in an accelerated mode, there is an elevated chance of mistakes and errors, with the ultimate risk of hindering, rather than speeding-up, drug development. In the present work, we have used model-validation metrics and examined the electron density maps for the deposited models of CoV-2 proteins and a sample of related proteins available in the PDB as of 1 April 2020. We present these results with the aim of helping the biomedical community establish a better-validated pool of data. The proteins are divided into groups according to their structure and function. In most cases, no major corrections were necessary. However, in several cases significant revisions in the functionally sensitive area of protein-inhibitor complexes or for bound ions justified correction, re-refinement, and eventually re-versioning in the PDB. The re-refined coordinate files and a tool for facilitating model comparisons are available at https://covid-19.bioreproducibility.org.