ABSTRACT
We present in this work a first X-ray Absorption Spectroscopy study of the interactions of Zn with human BST2/tetherin and SARS-CoV-2 orf7a proteins as well as with some of their complexes. The analysis of the XANES region of the measured spectra shows that Zn binds to BST2, as well as to orf7a, thus resulting in the formation of BST2-orf7a complexes. This structural information confirms the the conjecture, recently put forward by some of the present Authors, according to which the accessory orf7a (and possibly also orf8) viral protein are capable of interfering with the BST2 antiviral activity. Our explanation for this behavior is that, when BST2 gets in contact with Zn bound to the orf7a Cys15 ligand, it has the ability of displacing the metal owing to the creation of a new disulfide bridge across the two proteins. The formation of this BST2-orf7a complex destabilizes BST2 dimerization, thus impairing the antiviral activity of the latter.
Subject(s)
Antigens, CD/metabolism , SARS-CoV-2/chemistry , Viral Proteins/metabolism , Zinc/metabolism , Cysteine/chemistry , GPI-Linked Proteins/metabolism , Histidine/chemistry , Humans , Molecular Dynamics Simulation , Protein Binding , X-Ray Absorption SpectroscopyABSTRACT
Site-specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a non-enzymatic, post-translational modification of N-terminal HisTags. We report high-yield, site-selective inâ vitro α-aminoacylation of peptides, glycoproteins, antibodies, and virus-like particles (VLPs) with azidogluconolactone at pHâ 7.5 in 1â h. Conjugates slowly hydrolyse, but diol-masking with borate esters inhibits reversibility. In an example, we multimerise azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) onto VLPs via click-chemistry, to give a COVID-19 vaccine. Compared to yeast antigen, HEK-derived RBD was immunologically superior, likely due to observed differences in glycosylation. We show the benefits of ordered over randomly oriented multimeric antigen display, by demonstrating single-shot seroconversion and best virus-neutralizing antibodies. Azidogluconoylation is simple, fast and robust chemistry, and should accelerate research and development.
Subject(s)
Azides/chemistry , COVID-19 Vaccines/chemistry , Gluconates/chemistry , Glycine/chemistry , Histidine/chemistry , Lactones/chemistry , Vaccines, Virus-Like Particle/chemistry , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Azides/immunology , COVID-19 Vaccines/immunology , Gluconates/immunology , Glycine/immunology , Histidine/immunology , Humans , Lactones/immunology , Models, Molecular , Molecular Structure , Vaccines, Virus-Like Particle/immunologyABSTRACT
The question of whether COVID protease (SARS-CoV-2 Mpro) can be blocked by inhibitors has been examined, with a particularly successful performance exhibited by α-ketoamide derivative substrates like 13b of Hilgenfeld and co-workers (Zhang, L., et al. Science 2020, 368, 409-412). After the biological characterization, here density functional theory calculations explain not only how inhibitor 13b produces a thermodynamically favorable interaction but also how to reach it kinetically. The controversial and unprovable concept of aromaticity here enjoys being the agent that rationalizes the seemingly innocent role of histidine (His41 of Mpro). It has a hydrogen bond with the hydroxyl group and is the proton carrier of the thiol of Cys145 at almost zero energy cost that favors the interaction with the inhibitor that acts as a Michael acceptor.
Subject(s)
Antiviral Agents/metabolism , Betacoronavirus , Coronavirus Infections/drug therapy , Cysteine Proteinase Inhibitors/metabolism , Histidine/chemistry , Pneumonia, Viral/drug therapy , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/chemistry , Betacoronavirus/enzymology , Binding Sites , COVID-19 , Coronavirus 3C Proteases , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/chemistry , Density Functional Theory , Hydrogen Bonding , Ketones/chemistry , Ketones/metabolism , Models, Chemical , Pandemics , Protein Binding , SARS-CoV-2 , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolismABSTRACT
BACKGROUND/AIM: Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One drug that has attracted interest is the antiparasitic compound ivermectin, a macrocyclic lactone derived from the bacterium Streptomyces avermitilis. We carried out a docking study to determine if ivermectin might be able to attach to the SARS-CoV-2 spike receptor-binding domain bound with ACE2. MATERIALS AND METHODS: We used the program AutoDock Vina Extended to perform the docking study. RESULTS: Ivermectin docked in the region of leucine 91 of the spike and histidine 378 of the ACE2 receptor. The binding energy of ivermectin to the spike-ACE2 complex was -18 kcal/mol and binding constant was 5.8 e-08. CONCLUSION: The ivermectin docking we identified may interfere with the attachment of the spike to the human cell membrane. Clinical trials now underway should determine whether ivermectin is an effective treatment for SARS-Cov2 infection.
Subject(s)
Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Ivermectin/chemistry , Peptidyl-Dipeptidase A/chemistry , Pneumonia, Viral/drug therapy , Angiotensin-Converting Enzyme 2 , Betacoronavirus/chemistry , Betacoronavirus/pathogenicity , Binding Sites/drug effects , COVID-19 , Cell Membrane/drug effects , Coronavirus Infections/virology , Drug Repositioning , Histidine/chemistry , Humans , Ivermectin/therapeutic use , Leucine/chemistry , Molecular Docking Simulation , Pandemics , Peptidyl-Dipeptidase A/drug effects , Pneumonia, Viral/virology , SARS-CoV-2 , Streptomyces/chemistryABSTRACT
We have investigated the structural stability of the SARS (Severe acute respiratory syndrome)-CoV-2 main protease monomer (Mpro). We quantified the spatial and angular changes in the structure using two independent analyses, one based on a spatial metrics (δ, ratio), the second on angular metrics. The order of unfolding of the 10 helices in Mpro is characterized by beta vs alpha plots similar to those of cytochromes and globins. The longest turning region is anomalous in the earliest stage of unfolding. In an investigation of excluded-volume effects, we found that the maximum spread in average molecular-volume values for Mpro, cytochrome c-b562, cytochrome c', myoglobin, and cytoglobin is ~10 Å3. This apparent universality is a consequence of the dominant contributions from six residues: ALA, ASP, GLU, LEU, LYS and VAL. Of the seven Mpro histidines, residues 41, 163, 164, and 246 are in stable H-bonded regions; metal ion binding to one or more of these residues could break up the H-bond network, thereby affecting protease function. Our analysis also indicated that metal binding to cysteine residues 44 and 145 could disable the enzyme.