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1.
Proc Natl Acad Sci U S A ; 119(40): e2210990119, 2022 10 04.
Article in English | MEDLINE | ID: covidwho-2037061

ABSTRACT

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available coronavirus disease 2019 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors, which block formation of the so-called heptad repeat 1 and 2 (HR1HR2) six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. We performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based and virus-based assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ∼100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a prehairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the prehairpin intermediate of the S protein.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/drug therapy , Humans , Peptides/chemistry , Peptides/pharmacology , SARS-CoV-2/drug effects
2.
Proc Natl Acad Sci U S A ; 119(38): e2209514119, 2022 09 20.
Article in English | MEDLINE | ID: covidwho-2017036

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry starts with membrane attachment and ends with spike (S) protein-catalyzed membrane fusion depending on two cleavage steps, namely, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time three-dimensional single-virion tracking, we show that fusion and genome penetration require virion exposure to an acidic milieu of pH 6.2 to 6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2-overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2-expressing cells in the acidic milieu of the nasal cavity.


Subject(s)
COVID-19 , Nasal Cavity , SARS-CoV-2 , Serine Endopeptidases , Virus Internalization , COVID-19/virology , Furin/genetics , Furin/metabolism , Humans , Hydrogen-Ion Concentration , Nasal Cavity/chemistry , Nasal Cavity/virology , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism
3.
Sci Immunol ; 7(76): eadd5446, 2022 10 28.
Article in English | MEDLINE | ID: covidwho-1992933

ABSTRACT

SARS-CoV-2 Omicron subvariants have generated a worldwide health crisis due to resistance to most approved SARS-CoV-2 neutralizing antibodies and evasion of vaccination-induced antibodies. To manage Omicron subvariants and prepare for new ones, additional means of isolating broad and potent humanized SARS-CoV-2 neutralizing antibodies are desirable. Here, we describe a mouse model in which the primary B cell receptor (BCR) repertoire is generated solely through V(D)J recombination of a human VH1-2 heavy chain (HC) and, substantially, a human Vκ1-33 light chain (LC). Thus, primary humanized BCR repertoire diversity in these mice derives from immensely diverse HC and LC antigen-contact CDR3 sequences generated by nontemplated junctional modifications during V(D)J recombination. Immunizing this mouse model with SARS-CoV-2 (Wuhan-Hu-1) spike protein immunogens elicited several VH1-2/Vκ1-33-based neutralizing antibodies that bound RBD in a different mode from each other and from those of many prior patient-derived VH1-2-based neutralizing antibodies. Of these, SP1-77 potently and broadly neutralized all SARS-CoV-2 variants through BA.5. Cryo-EM studies revealed that SP1-77 bound RBD away from the receptor-binding motif via a CDR3-dominated recognition mode. Lattice light-sheet microscopy-based studies showed that SP1-77 did not block ACE2-mediated viral attachment or endocytosis but rather blocked viral-host membrane fusion. The broad and potent SP1-77 neutralization activity and nontraditional mechanism of action suggest that it might have therapeutic potential. Likewise, the SP1-77 binding epitope may inform vaccine strategies. Last, the type of humanized mouse models that we have described may contribute to identifying therapeutic antibodies against future SARS-CoV-2 variants and other pathogens.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Mice , Animals , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2 , Membrane Fusion , Antibodies, Viral , Antibodies, Neutralizing , Epitopes , Receptors, Antigen, B-Cell
4.
J Virol ; 95(21): e0097521, 2021 10 13.
Article in English | MEDLINE | ID: covidwho-1361966

ABSTRACT

Repurposing FDA-approved inhibitors able to prevent infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could provide a rapid path to establish new therapeutic options to mitigate the effects of coronavirus disease 2019 (COVID-19). Proteolytic cleavages of the spike (S) protein of SARS-CoV-2, mediated by the host cell proteases cathepsin and TMPRSS2, alone or in combination, are key early activation steps required for efficient infection. The PIKfyve kinase inhibitor apilimod interferes with late endosomal viral traffic and through an ill-defined mechanism prevents in vitro infection through late endosomes mediated by cathepsin. Similarly, inhibition of TMPRSS2 protease activity by camostat mesylate or nafamostat mesylate prevents infection mediated by the TMPRSS2-dependent and cathepsin-independent pathway. Here, we combined the use of apilimod with camostat mesylate or nafamostat mesylate and found an unexpected ∼5- to 10-fold increase in their effectiveness to prevent SARS-CoV-2 infection in different cell types. Comparable synergism was observed using both a chimeric vesicular stomatitis virus (VSV) containing S of SARS-CoV-2 (VSV-SARS-CoV-2) and SARS-CoV-2. The substantial ∼5-fold or higher decrease of the half-maximal effective concentrations (EC50s) suggests a plausible treatment strategy based on the combined use of these inhibitors. IMPORTANCE Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the coronavirus disease 2019 (COVID-2019) global pandemic. There are ongoing efforts to uncover effective antiviral agents that could mitigate the severity of the disease by controlling the ensuing viral replication. Promising candidates include small molecules that inhibit the enzymatic activities of host proteins, thus preventing SARS-CoV-2 entry and infection. They include apilimod, an inhibitor of PIKfyve kinase, and camostat mesylate and nafamostat mesylate, inhibitors of TMPRSS2 protease. Our research is significant for having uncovered an unexpected synergism in the effective inhibitory activity of apilimod used together with camostat mesylate or nafamostat mesylate.


Subject(s)
Antiviral Agents/pharmacology , Benzamidines/pharmacology , Esters/pharmacology , Guanidines/pharmacology , Hydrazones/pharmacology , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Pyrimidines/pharmacology , SARS-CoV-2/drug effects , Serine Endopeptidases/metabolism , Animals , COVID-19/drug therapy , Cell Line, Tumor , Chlorocebus aethiops , Drug Synergism , Humans , Phosphoinositide-3 Kinase Inhibitors/pharmacology , Protease Inhibitors/pharmacology , SARS-CoV-2/physiology , Vero Cells , Virus Internalization
5.
Proc Natl Acad Sci U S A ; 117(50): 32105-32113, 2020 12 15.
Article in English | MEDLINE | ID: covidwho-947594

ABSTRACT

Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export. Our results highlight one of the possible antiviral mechanisms of 25HC and provide the molecular basis for its therapeutic development.


Subject(s)
Endosomes/genetics , Hydroxycholesterols/pharmacology , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Antiviral Agents/pharmacology , COVID-19/metabolism , COVID-19/pathology , COVID-19/virology , Endosomes/metabolism , Humans , Interferons/metabolism , Membrane Fusion/drug effects , /metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization/drug effects , Virus Replication/drug effects
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