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J Med Chem ; 64(8): 4991-5000, 2021 04 22.
Article in English | MEDLINE | ID: covidwho-1574766


The main protease (3CL Mpro) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is an essential enzyme for viral replication with no human counterpart, making it an attractive drug target. To date, no small-molecule clinical drugs are available that specifically inhibit SARS-CoV-2 Mpro. To aid rational drug design, we determined a neutron structure of Mpro in complex with the α-ketoamide inhibitor telaprevir at near-physiological (22 °C) temperature. We directly observed protonation states in the inhibitor complex and compared them with those in the ligand-free Mpro, revealing modulation of the active-site protonation states upon telaprevir binding. We suggest that binding of other α-ketoamide covalent inhibitors can lead to the same protonation state changes in the Mpro active site. Thus, by studying the protonation state changes induced by inhibitors, we provide crucial insights to help guide rational drug design, allowing precise tailoring of inhibitors to manipulate the electrostatic environment of SARS-CoV-2 Mpro.

Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Oligopeptides/chemistry , Binding Sites , Coronavirus 3C Proteases/metabolism , Crystallography/methods , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/metabolism , Models, Molecular , Neutrons , Oligopeptides/metabolism , Protein Conformation , Protons
ACS Chem Biol ; 16(8): 1469-1481, 2021 08 20.
Article in English | MEDLINE | ID: covidwho-1387143


The programmed -1 ribosomal frameshifting element (PFSE) of SARS-CoV-2 is a well conserved structured RNA found in all coronaviruses' genomes. By adopting a pseudoknot structure in the presence of the ribosome, the PFSE promotes a ribosomal frameshifting event near the stop codon of the first open reading frame Orf1a during translation of the polyprotein pp1a. Frameshifting results in continuation of pp1a via a new open reading frame, Orf1b, that produces the longer pp1ab polyprotein. Polyproteins pp1a and pp1ab produce nonstructural proteins NSPs 1-10 and NSPs 1-16, respectively, which contribute vital functions during the viral life cycle and must be present in the proper stoichiometry. Both drugs and sequence alterations that affect the stability of the -1 programmed ribosomal frameshifting element disrupt the stoichiometry of the NSPs produced, which compromise viral replication. For this reason, the -1 programmed frameshifting element is considered a promising drug target. Using chaperone assisted RNA crystallography, we successfully crystallized and solved the three-dimensional structure of the PFSE. We observe a three-stem H-type pseudoknot structure with the three stems stacked in a vertical orientation stabilized by two triple base pairs at the stem 1/stem 2 and stem 1/stem 3 junctions. This structure provides a new conformation of PFSE distinct from the bent conformations inferred from midresolution cryo-EM models and provides a high-resolution framework for mechanistic investigations and structure-based drug design.

Crystallography/methods , Frameshifting, Ribosomal/physiology , Molecular Chaperones , RNA, Viral/metabolism , SARS-CoV-2/metabolism , Humans , Models, Molecular , Nucleic Acid Conformation , RNA, Viral/genetics , Ribosomes/metabolism , SARS-CoV-2/genetics , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Replication/physiology
J Biol Chem ; 296: 100745, 2021.
Article in English | MEDLINE | ID: covidwho-1213326


Fifty years ago, the first landmark structures of antibodies heralded the dawn of structural immunology. Momentum then started to build toward understanding how antibodies could recognize the vast universe of potential antigens and how antibody-combining sites could be tailored to engage antigens with high specificity and affinity through recombination of germline genes (V, D, J) and somatic mutation. Equivalent groundbreaking structures in the cellular immune system appeared some 15 to 20 years later and illustrated how processed protein antigens in the form of peptides are presented by MHC molecules to T cell receptors. Structures of antigen receptors in the innate immune system then explained their inherent specificity for particular microbial antigens including lipids, carbohydrates, nucleic acids, small molecules, and specific proteins. These two sides of the immune system act immediately (innate) to particular microbial antigens or evolve (adaptive) to attain high specificity and affinity to a much wider range of antigens. We also include examples of other key receptors in the immune system (cytokine receptors) that regulate immunity and inflammation. Furthermore, these antigen receptors use a limited set of protein folds to accomplish their various immunological roles. The other main players are the antigens themselves. We focus on surface glycoproteins in enveloped viruses including SARS-CoV-2 that enable entry and egress into host cells and are targets for the antibody response. This review covers what we have learned over the past half century about the structural basis of the immune response to microbial pathogens and how that information can be utilized to design vaccines and therapeutics.

Adaptive Immunity , Antibodies, Viral/chemistry , Antigens, Viral/chemistry , Immunity, Innate , Receptors, Antigen, T-Cell/chemistry , Receptors, Cytokine/chemistry , SARS-CoV-2/immunology , Allergy and Immunology/history , Animals , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Antibody Specificity , Antigen Presentation , Antigens, Viral/genetics , Antigens, Viral/immunology , COVID-19/immunology , COVID-19/virology , Crystallography/history , Crystallography/methods , History, 20th Century , History, 21st Century , Humans , Protein Folding , Protein Interaction Domains and Motifs , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Receptors, Cytokine/genetics , Receptors, Cytokine/immunology , SARS-CoV-2/pathogenicity , V(D)J Recombination