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1.
SLAS Discovery ; 2022.
Article in English | ScienceDirect | ID: covidwho-1586501

ABSTRACT

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.

2.
The FASEB Journal ; 35(S1), 2021.
Article in English | Wiley | ID: covidwho-1233926

ABSTRACT

Understanding the SARS-CoV-2 virus? routes of infection, virus?host?protein interactions, and mechanisms of virus-induced cytopathic effects will greatly aid in the discovery and design of new therapeutics to treat COVID-19. Chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including alkalizing lysosomes and blocking autophagy as well as exhibiting dose-limiting toxicities in patients. To identify an alternative lysosome-based drug repurposing opportunity we evaluated additional lysosomotropic compounds . We found that six of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero E6 cells with half-maximal effective concentration (EC50) values ranging from 2.0 to 13 ?M and selectivity indices (SIs;SI = CC50/EC50) ranging from 1.5- to >10-fold. We demonstrate how the compounds (1) blocked lysosome functioning and autophagy, (2) prevented pseudotyped particle entry, (3) increased lysosomal pH, and (4) that ROC-325 reduced viral titers in the EpiAirway 3D tissue model. Consistent with these findings, the siRNA knockdown of ATP6V0D1 blocked the HCoV-NL63 cytopathic effect in LLC-MK2 cells. Moreover, an analysis of SARS-CoV-2 infected Vero E6 cell lysate revealed significant dysregulation of autophagy and lysosomal function, suggesting a contribution of the lysosome to the life cycle of SARS-CoV-2. Our findings support targeting the lysosome to combat SARS-CoV-2 infections and inhibitors of lysosomal function could become an important component of drug combination therapies aimed at improving treatment and outcomes for COVID-19.

3.
The FASEB Journal ; 35(S1), 2021.
Article in English | Wiley | ID: covidwho-1233885

ABSTRACT

The SARS-CoV-2 virus binds to host cell surface ACE2 on the plasma membrane via the spike protein's receptor binding domain. Our work has resulted in the generation of a versatile imaging probe using recombinant Spike receptor binding domain conjugated to fluorescent quantum dots (QDs). This probe is capable of engaging in energy transfer quenching with ACE2-conjugated gold nanoparticles enabling biochemical monitoring of binding. Neutralizing antibodies and recombinant human ACE2 blocked quenching, demonstrating a specific binding interaction. In cell-based assays, we observed immediate binding of the probe on the cell surface of ACE2-expressing cells followed by endocytosis. Neutralizing antibodies and ACE2-Fc fully prevented binding and endocytosis with low nanomolar potency. Importantly, we can use this QD nanoparticle probe to identify and validate inhibitors of the SARS-CoV-2 Spike and ACE2 receptor binding in human cells. This work enables facile, rapid, and high-throughput biochemical- and cell-based screening of inhibitors for coronavirus Spike-mediated cell recognition and entry.

4.
ACS Infect Dis ; 7(6): 1389-1408, 2021 06 11.
Article in English | MEDLINE | ID: covidwho-989665

ABSTRACT

Understanding the SARS-CoV-2 virus' pathways of infection, virus-host-protein interactions, and mechanisms of virus-induced cytopathic effects will greatly aid in the discovery and design of new therapeutics to treat COVID-19. Chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including alkalizing lysosomes and blocking autophagy as well as exhibiting dose-limiting toxicities in patients. Therefore, we evaluated additional lysosomotropic compounds to identify an alternative lysosome-based drug repurposing opportunity. We found that six of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero E6 cells with half-maximal effective concentration (EC50) values ranging from 2.0 to 13 µM and selectivity indices (SIs; SI = CC50/EC50) ranging from 1.5- to >10-fold. The compounds (1) blocked lysosome functioning and autophagy, (2) prevented pseudotyped particle entry, (3) increased lysosomal pH, and (4) reduced (ROC-325) viral titers in the EpiAirway 3D tissue model. Consistent with these findings, the siRNA knockdown of ATP6V0D1 blocked the HCoV-NL63 cytopathic effect in LLC-MK2 cells. Moreover, an analysis of SARS-CoV-2 infected Vero E6 cell lysate revealed significant dysregulation of autophagy and lysosomal function, suggesting a contribution of the lysosome to the life cycle of SARS-CoV-2. Our findings suggest the lysosome as a potential host cell target to combat SARS-CoV-2 infections and inhibitors of lysosomal function could become an important component of drug combination therapies aimed at improving treatment and outcomes for COVID-19.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Drug Repositioning , Humans , Lysosomes
5.
Cell Discov ; 6(1): 80, 2020 Nov 04.
Article in English | MEDLINE | ID: covidwho-912895

ABSTRACT

The cell entry of SARS-CoV-2 has emerged as an attractive drug repurposing target for COVID-19. Here we combine genetics and chemical perturbation to demonstrate that ACE2-mediated entry of SARS-Cov and CoV-2 requires the cell surface heparan sulfate (HS) as an assisting cofactor: ablation of genes involved in HS biosynthesis or incubating cells with a HS mimetic both inhibit Spike-mediated viral entry. We show that heparin/HS binds to Spike directly, and facilitates the attachment of Spike-bearing viral particles to the cell surface to promote viral entry. We screened approved drugs and identified two classes of inhibitors that act via distinct mechanisms to target this entry pathway. Among the drugs characterized, Mitoxantrone is a potent HS inhibitor, while Sunitinib and BNTX disrupt the actin network to indirectly abrogate HS-assisted viral entry. We further show that drugs of the two classes can be combined to generate a synergized activity against SARS-CoV-2-induced cytopathic effect. Altogether, our study establishes HS as an attachment factor that assists SARS coronavirus cell entry and reveals drugs capable of targeting this important step in the viral life cycle.

6.
ACS Pharmacol Transl Sci ; 3(6): 1165-1175, 2020 Dec 11.
Article in English | MEDLINE | ID: covidwho-872651

ABSTRACT

While vaccine development will hopefully quell the global pandemic of COVID-19 caused by SARS-CoV-2, small molecule drugs that can effectively control SARS-CoV-2 infection are urgently needed. Here, inhibitors of spike (S) mediated cell entry were identified in a high throughput screen of an approved drugs library with SARS-S and MERS-S pseudotyped particle entry assays. We discovered six compounds (cepharanthine, abemaciclib, osimertinib, trimipramine, colforsin, and ingenol) to be broad spectrum inhibitors for spike-mediated entry. This work could contribute to the development of effective treatments against the initial stage of viral infection and provide mechanistic information that might aid the design of new drug combinations for clinical trials for COVID-19 patients.

7.
Cell Stem Cell ; 27(6): 937-950.e9, 2020 12 03.
Article in English | MEDLINE | ID: covidwho-779663

ABSTRACT

Neurological complications are common in patients with COVID-19. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function is not well understood. Here, we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found that neurons and astrocytes were sparsely infected, but choroid plexus epithelial cells underwent robust infection. We optimized a protocol to generate choroid plexus organoids from hiPSCs and showed that productive SARS-CoV-2 infection of these organoids is associated with increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our findings provide evidence for selective SARS-CoV-2 neurotropism and support the use of hiPSC-derived brain organoids as a platform to investigate SARS-CoV-2 infection susceptibility of brain cells, mechanisms of virus-induced brain dysfunction, and treatment strategies.


Subject(s)
Choroid Plexus/virology , Neural Stem Cells/virology , Organoids/virology , Pluripotent Stem Cells/virology , SARS-CoV-2/physiology , Viral Tropism , Animals , Astrocytes/virology , Brain/cytology , Brain/virology , COVID-19/genetics , COVID-19/virology , Cells, Cultured , Gene Expression Regulation , Humans , Neurons/virology
8.
ACS Nano ; 14(9): 12234-12247, 2020 09 22.
Article in English | MEDLINE | ID: covidwho-741668

ABSTRACT

The first step of SARS-CoV-2 infection is binding of the spike protein's receptor binding domain to the host cell's ACE2 receptor on the plasma membrane. Here, we have generated a versatile imaging probe using recombinant Spike receptor binding domain conjugated to fluorescent quantum dots (QDs). This probe is capable of engaging in energy transfer quenching with ACE2-conjugated gold nanoparticles to enable monitoring of the binding event in solution. Neutralizing antibodies and recombinant human ACE2 blocked quenching, demonstrating a specific binding interaction. In cells transfected with ACE2-GFP, we observed immediate binding of the probe on the cell surface followed by endocytosis. Neutralizing antibodies and ACE2-Fc fully prevented binding and endocytosis with low nanomolar potency. Importantly, we will be able to use this QD nanoparticle probe to identify and validate inhibitors of the SARS-CoV-2 Spike and ACE2 receptor binding in human cells. This work enables facile, rapid, and high-throughput cell-based screening of inhibitors for coronavirus Spike-mediated cell recognition and entry.


Subject(s)
Endocytosis , Metal Nanoparticles/chemistry , Peptidyl-Dipeptidase A/metabolism , Quantum Dots/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2 , Betacoronavirus/metabolism , COVID-19 , Coronavirus Infections/metabolism , Gold , Humans , Pandemics , Peptidyl-Dipeptidase A/physiology , Pneumonia, Viral/metabolism , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Virion
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