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1.
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.

2.
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
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