Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 7 de 7
Filter
1.
iScience ; : 104925, 2022.
Article in English | ScienceDirect | ID: covidwho-1983262

ABSTRACT

Summary Pharmacologically active compounds with known biological targets were evaluated for inhibition of SARS-CoV-2 infection in cell and tissue models to help identify potent classes of active small molecules and to better understand host-virus interactions. We evaluated 6710 clinical and preclinical compounds targeting 2183 host proteins by immunocytofluorescence-based screening to identify SARS-CoV-2 infection inhibitors. Computationally integrating relationships between small molecule structure, dose-response antiviral activity, host target, and cell interactome produced cellular networks important for infection. This analysis revealed 389 small molecules with micromolar to low nanomolar activities, representing >12 scaffold classes and 813 host targets. Representatives were evaluated for mechanism of action in stable and primary human cell models with SARS-CoV-2 variants and MERS-CoV. One promising candidate, obatoclax, significantly reduced SARS-CoV-2 viral lung load in mice. Ultimately, this work establishes a rigorous approach for future pharmacological and computational identification of host factor dependencies and treatments for viral diseases.

2.
SSRN; 2022.
Preprint in English | SSRN | ID: ppcovidwho-330033

ABSTRACT

Identification of host factors contributing to replication of viruses and resulting disease progression remains a promising approach for development of new therapeutics. Here, we evaluated 6710 clinical and preclinical compounds targeting 2183 host proteins by immunocytofluorescence-based screening to identify SARS-CoV-2 infection inhibitors. Computationally integrating relationships between small molecule structure, dose-response antiviral activity, host target and cell interactome networking produced cellular networks important for infection. This analysis revealed 389 small molecules, >12 scaffold classes and 813 host targets with micromolar to low nanomolar activities. From these classes, representatives were extensively evaluated for mechanism of action in stable and primary human cell models, and additionally against Beta and Delta SARS-CoV-2 variants and MERS-CoV. One promising candidate, obatoclax, significantly reduced SARS-CoV-2 viral lung load in mice. Ultimately, this work establishes a rigorous approach for future pharmacological and computational identification of novel host factor dependencies and treatments for viral diseases. Funding: JJP, CJD, HM, PK, KGB, RB, SHS and RAD were supported by NIH grants P01AI120943, 5R01AI128364-02, 5R01AI125453-05, UC7AI095321 and by a grant from the Massachusetts Consortium on Pathogen Readiness. JLB, SJ and SG were supported by NIH grants 3RO1AI064099-15. ALB is supported by NIH grant 1P01HL132825, American Heart Association grant 151708, and ERC grant 810115-DYNASET. MZ is supported, in part, by NSF grants IIS2030459 and IIS-2033384, and by Harvard Data Science Initiative. JL was supported, in part, by NIH grants HG007690, HL108630, and HL119145, and by American Heart Association grants D700382 and CV-19;MN was supported by a grant from the Massachusetts Consortium on Pathogen Readiness. SJE is an investigator with the Howard Hughes Medical Institute. We acknowledge the hard work and dedication of the NEIDL veterinary services team who performed disease treatment testing in the high biological containment laboratory under institutionally approved IACUC protocols to RAD. \We thank the Center for the Development of Therapeutics and Repurposing Hub at the Broad Institute for providing the compound library. We thank Tim Mitichson for critical reading of the manuscript. We thank John H. Connor, Jacquelyn Turcinovic, Scott Seitz, Florian Douam, and Devin Kenney for isolation and production of SARS-CoV-2 Delta (B.1.617), and Anthony Griffiths and Anna Honko for production of MERS-CoV.

3.
Cell ; 185(4): 614-629.e21, 2022 02 17.
Article in English | MEDLINE | ID: covidwho-1676664

ABSTRACT

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.


Subject(s)
Adjuvants, Immunologic/pharmacology , Antigens, Viral/immunology , Candida albicans/chemistry , Mannans/immunology , Aluminum Hydroxide/chemistry , Animals , Antibodies, Neutralizing/immunology , Antibody Specificity/immunology , B-Lymphocytes/immunology , COVID-19/immunology , COVID-19/prevention & control , COVID-19/virology , Chlorocebus aethiops , Epitopes/immunology , Immunity, Innate , Immunization , Inflammation/pathology , Interferons/metabolism , Lectins, C-Type/metabolism , Ligands , Lung/immunology , Lung/pathology , Lung/virology , Lymph Nodes/immunology , Lymph Nodes/metabolism , Macrophages/metabolism , Mice, Inbred C57BL , Paranasal Sinuses/metabolism , Protein Subunits/metabolism , Sialic Acid Binding Ig-like Lectin 1/metabolism , Solubility , Spike Glycoprotein, Coronavirus/metabolism , T-Lymphocytes/immunology , Transcription Factor RelB/metabolism , Vero Cells , beta-Glucans/metabolism
4.
Mol Cell ; 81(17): 3650-3658.e5, 2021 09 02.
Article in English | MEDLINE | ID: covidwho-1356368

ABSTRACT

CRISPR-inspired systems have been extensively developed for applications in genome editing and nucleic acid detection. Here, we introduce a CRISPR-based peptide display technology to facilitate customized, high-throughput in vitro protein interaction studies. We show that bespoke peptide libraries fused to catalytically inactive Cas9 (dCas9) and barcoded with unique single guide RNA (sgRNA) molecules self-assemble from a single mixed pool to programmable positions on a DNA microarray surface for rapid, multiplexed binding assays. We develop dCas9-displayed saturation mutagenesis libraries to characterize antibody-epitope binding for a commercial anti-FLAG monoclonal antibody and human serum antibodies. We also show that our platform can be used for viral epitope mapping and exhibits promise as a multiplexed diagnostics tool. Our CRISPR-based peptide display platform and the principles of complex library self-assembly using dCas9 could be adapted for rapid interrogation of varied customized protein libraries or biological materials assembly using DNA scaffolding.


Subject(s)
Epitopes/genetics , Gene Editing/methods , Peptide Library , RNA, Guide/genetics , CRISPR-Cas Systems/genetics , CRISPR-Cas Systems/immunology , Epitopes/immunology , Humans , Mutagenesis/genetics , Protein Binding/genetics , Protein Binding/immunology , RNA, Guide/immunology
5.
Proc Natl Acad Sci U S A ; 118(17)2021 04 27.
Article in English | MEDLINE | ID: covidwho-1172591

ABSTRACT

In order to understand the transmission and virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is necessary to understand the functions of each of the gene products encoded in the viral genome. One feature of the SARS-CoV-2 genome that is not present in related, common coronaviruses is ORF10, a putative 38-amino acid protein-coding gene. Proteomic studies found that ORF10 binds to an E3 ubiquitin ligase containing Cullin-2, Rbx1, Elongin B, Elongin C, and ZYG11B (CRL2ZYG11B). Since CRL2ZYG11B mediates protein degradation, one possible role for ORF10 is to "hijack" CRL2ZYG11B in order to target cellular, antiviral proteins for ubiquitylation and subsequent proteasomal degradation. Here, we investigated whether ORF10 hijacks CRL2ZYG11B or functions in other ways, for example, as an inhibitor or substrate of CRL2ZYG11B While we confirm the ORF10-ZYG11B interaction and show that the N terminus of ORF10 is critical for it, we find no evidence that ORF10 is functioning to inhibit or hijack CRL2ZYG11B Furthermore, ZYG11B and its paralog ZER1 are dispensable for SARS-CoV-2 infection in cultured cells. We conclude that the interaction between ORF10 and CRL2ZYG11B is not relevant for SARS-CoV-2 infection in vitro.


Subject(s)
COVID-19/metabolism , Cell Cycle Proteins/metabolism , Cullin Proteins/metabolism , Multiprotein Complexes/metabolism , Open Reading Frames , SARS-CoV-2/metabolism , Viral Proteins/metabolism , COVID-19/genetics , Cell Cycle Proteins/genetics , Cullin Proteins/genetics , HEK293 Cells , Humans , Multiprotein Complexes/genetics , SARS-CoV-2/genetics , Viral Proteins/genetics
6.
J Clin Invest ; 131(7)2021 04 01.
Article in English | MEDLINE | ID: covidwho-1166661

ABSTRACT

SARS-CoV-2 (CoV2) antibody therapies, including COVID-19 convalescent plasma (CCP), monoclonal antibodies, and hyperimmune globulin, are among the leading treatments for individuals with early COVID-19 infection. The functionality of convalescent plasma varies greatly, but the association of antibody epitope specificities with plasma functionality remains uncharacterized. We assessed antibody functionality and reactivities to peptides across the CoV2 and the 4 endemic human coronavirus (HCoV) genomes in 126 CCP donations. We found strong correlation between plasma functionality and polyclonal antibody targeting of CoV2 spike protein peptides. Antibody reactivity to many HCoV spike peptides also displayed strong correlation with plasma functionality, including pan-coronavirus cross-reactive epitopes located in a conserved region of the fusion peptide. After accounting for antibody cross-reactivity, we identified an association between greater alphacoronavirus NL63 antibody responses and development of highly neutralizing antibodies against CoV2. We also found that plasma preferentially reactive to the CoV2 spike receptor binding domain (RBD), versus the betacoronavirus HKU1 RBD, had higher neutralizing titer. Finally, we developed a 2-peptide serosignature that identifies plasma donations with high anti-spike titer, but that suffer from low neutralizing activity. These results suggest that analysis of coronavirus antibody fine specificities may be useful for selecting desired therapeutics and understanding the complex immune responses elicited by CoV2 infection.


Subject(s)
Antibodies, Viral/blood , COVID-19/immunology , COVID-19/therapy , COVID-19/virology , Coronavirus/immunology , SARS-CoV-2/immunology , Antibodies, Neutralizing/blood , Antibody Specificity , Coronavirus/classification , Coronavirus/genetics , Cross Reactions , Endemic Diseases , Genome, Viral , Humans , Immunization, Passive , Immunodominant Epitopes/chemistry , Immunodominant Epitopes/genetics , Immunodominant Epitopes/immunology , Models, Molecular , Pandemics , SARS-CoV-2/genetics , Species Specificity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
7.
Science ; 370(6520)2020 11 27.
Article in English | MEDLINE | ID: covidwho-809284

ABSTRACT

Understanding humoral responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for improving diagnostics, therapeutics, and vaccines. Deep serological profiling of 232 coronavirus disease 2019 (COVID-19) patients and 190 pre-COVID-19 era controls using VirScan revealed more than 800 epitopes in the SARS-CoV-2 proteome, including 10 epitopes likely recognized by neutralizing antibodies. Preexisting antibodies in controls recognized SARS-CoV-2 ORF1, whereas only COVID-19 patient antibodies primarily recognized spike protein and nucleoprotein. A machine learning model trained on VirScan data predicted SARS-CoV-2 exposure history with 99% sensitivity and 98% specificity; a rapid Luminex-based diagnostic was developed from the most discriminatory SARS-CoV-2 peptides. Individuals with more severe COVID-19 exhibited stronger and broader SARS-CoV-2 responses, weaker antibody responses to prior infections, and higher incidence of cytomegalovirus and herpes simplex virus 1, possibly influenced by demographic covariates. Among hospitalized patients, males produce stronger SARS-CoV-2 antibody responses than females.


Subject(s)
COVID-19/immunology , Epitope Mapping , Epitopes/immunology , SARS-CoV-2/immunology , Severity of Illness Index , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibody Formation , COVID-19/blood , COVID-19 Serological Testing , Cross Reactions , Cryoelectron Microscopy , Epitopes/chemistry , Epitopes/genetics , Female , Humans , Male , Protein Conformation , Seroconversion
SELECTION OF CITATIONS
SEARCH DETAIL