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1.
Angewandte Chemie ; 134(9), 2022.
Article in English | ProQuest Central | ID: covidwho-1680266

ABSTRACT

The main protease (Mpro) and papain‐like protease (PLpro) play critical roles in SARS‐CoV‐2 replication and are promising targets for antiviral inhibitors. The simultaneous visualization of Mpro and PLpro is extremely valuable for SARS‐CoV‐2 detection and rapid inhibitor screening. However, such a crucial investigation has remained challenging because of the lack of suitable probes. We have now developed a dual‐color probe (3MBP5) for the simultaneous detection of Mpro and PLpro by fluorescence (or Förster) resonance energy transfer (FRET). This probe produces fluorescence from both the Cy3 and Cy5 fluorophores that are cleaved by Mpro and PLpro. 3MBP5‐activatable specificity was demonstrated with recombinant proteins, inhibitors, plasmid‐transfected HEK 293T cells, and SARS‐CoV‐2‐infected TMPRSS2‐Vero cells. Results from the dual‐color probe first verified the simultaneous detection and intracellular distribution of SARS‐CoV‐2 Mpro and PLpro. This is a powerful tool for the simultaneous detection of different proteases with value for the rapid screening of inhibitors.

2.
Angew Chem Int Ed Engl ; 61(9): e202113617, 2022 02 21.
Article in English | MEDLINE | ID: covidwho-1565164

ABSTRACT

The main protease (Mpro ) and papain-like protease (PLpro ) play critical roles in SARS-CoV-2 replication and are promising targets for antiviral inhibitors. The simultaneous visualization of Mpro and PLpro is extremely valuable for SARS-CoV-2 detection and rapid inhibitor screening. However, such a crucial investigation has remained challenging because of the lack of suitable probes. We have now developed a dual-color probe (3MBP5) for the simultaneous detection of Mpro and PLpro by fluorescence (or Förster) resonance energy transfer (FRET). This probe produces fluorescence from both the Cy3 and Cy5 fluorophores that are cleaved by Mpro and PLpro . 3MBP5-activatable specificity was demonstrated with recombinant proteins, inhibitors, plasmid-transfected HEK 293T cells, and SARS-CoV-2-infected TMPRSS2-Vero cells. Results from the dual-color probe first verified the simultaneous detection and intracellular distribution of SARS-CoV-2 Mpro and PLpro . This is a powerful tool for the simultaneous detection of different proteases with value for the rapid screening of inhibitors.


Subject(s)
Color , Coronavirus 3C Proteases/metabolism , Coronavirus Papain-Like Proteases/metabolism , Fluorescent Dyes/chemistry , Protease Inhibitors/pharmacology , SARS-CoV-2/enzymology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Fluorescence Resonance Energy Transfer , HEK293 Cells , Humans
3.
Nat Med ; 27(9): 1600-1606, 2021 09.
Article in English | MEDLINE | ID: covidwho-1526089

ABSTRACT

Clinical evidence suggests the central nervous system is frequently impacted by SARS-CoV-2 infection, either directly or indirectly, although the mechanisms are unclear. Pericytes are perivascular cells within the brain that are proposed as SARS-CoV-2 infection points. Here we show that pericyte-like cells (PLCs), when integrated into a cortical organoid, are capable of infection with authentic SARS-CoV-2. Before infection, PLCs elicited astrocytic maturation and production of basement membrane components, features attributed to pericyte functions in vivo. While traditional cortical organoids showed little evidence of infection, PLCs within cortical organoids served as viral 'replication hubs', with virus spreading to astrocytes and mediating inflammatory type I interferon transcriptional responses. Therefore, PLC-containing cortical organoids (PCCOs) represent a new 'assembloid' model that supports astrocytic maturation as well as SARS-CoV-2 entry and replication in neural tissue; thus, PCCOs serve as an experimental model for neural infection.


Subject(s)
Astrocytes/virology , Brain/virology , COVID-19/pathology , Pericytes/virology , Viral Tropism/physiology , Astrocytes/cytology , Brain/pathology , Cell Differentiation/physiology , Cells, Cultured , Humans , Interferon Type I/immunology , SARS-CoV-2 , Virus Replication/physiology
4.
mSystems ; 6(6): e0113621, 2021 Dec 21.
Article in English | MEDLINE | ID: covidwho-1494994

ABSTRACT

Environmental monitoring in public spaces can be used to identify surfaces contaminated by persons with coronavirus disease 2019 (COVID-19) and inform appropriate infection mitigation responses. Research groups have reported detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on surfaces days or weeks after the virus has been deposited, making it difficult to estimate when an infected individual may have shed virus onto a SARS-CoV-2-positive surface, which in turn complicates the process of establishing effective quarantine measures. In this study, we determined that reverse transcription-quantitative PCR (RT-qPCR) detection of viral RNA from heat-inactivated particles experiences minimal decay over 7 days of monitoring on eight out of nine surfaces tested. The properties of the studied surfaces result in RT-qPCR signatures that can be segregated into two material categories, rough and smooth, where smooth surfaces have a lower limit of detection. RT-qPCR signal intensity (average quantification cycle [Cq]) can be correlated with surface viral load using only one linear regression model per material category. The same experiment was performed with untreated viral particles on one surface from each category, with essentially identical results. The stability of RT-qPCR viral signal demonstrates the need to clean monitored surfaces after sampling to establish temporal resolution. Additionally, these findings can be used to minimize the number of materials and time points tested and allow for the use of heat-inactivated viral particles when optimizing environmental monitoring methods. IMPORTANCE Environmental monitoring is an important tool for public health surveillance, particularly in settings with low rates of diagnostic testing. Time between sampling public environments, such as hospitals or schools, and notifying stakeholders of the results should be minimal, allowing decisions to be made toward containing outbreaks of coronavirus disease 2019 (COVID-19). The Safer At School Early Alert program (SASEA) (https://saseasystem.org/), a large-scale environmental monitoring effort in elementary school and child care settings, has processed >13,000 surface samples for SARS-CoV-2, detecting viral signals from 574 samples. However, consecutive detection events necessitated the present study to establish appropriate response practices around persistent viral signals on classroom surfaces. Other research groups and clinical labs developing environmental monitoring methods may need to establish their own correlation between RT-qPCR results and viral load, but this work provides evidence justifying simplified experimental designs, like reduced testing materials and the use of heat-inactivated viral particles.

5.
ACS Infect Dis ; 7(11): 3096-3110, 2021 11 12.
Article in English | MEDLINE | ID: covidwho-1483084

ABSTRACT

The development of vaccines against coronaviruses has focused on the spike (S) protein, which is required for the recognition of host-cell receptors and thus elicits neutralizing antibodies. Targeting conserved epitopes on the S protein offers the potential for pan-beta-coronavirus vaccines that could prevent future pandemics. We displayed five B-cell epitopes, originally identified in the convalescent sera from recovered severe acute respiratory syndrome (SARS) patients, on the surface of the cowpea mosaic virus (CPMV) and evaluated these formulations as vaccines. Prime-boost immunization of mice with three of these candidate vaccines, CPMV-988, CPMV-1173, and CPMV-1209, elicited high antibody titers that neutralized the severe acute respiratory syndrome coronavirus (SARS-CoV) in vitro and showed an early Th1-biased profile (2-4 weeks) transitioning to a slightly Th2-biased profile just after the second boost (6 weeks). A pentavalent slow-release implant comprising all five peptides displayed on the CPMV elicited anti-S protein and epitope-specific antibody titers, albeit at a lower magnitude compared to the soluble formulations. While the CPMV remained intact when released from the PLGA implants, processing results in loss of RNA, which acts as an adjuvant. Loss of RNA may be a reason for the lower efficacy of the implants. Finally, although the three epitopes (988, 1173, and 1209) that were found to be neutralizing the SARS-CoV were 100% identical to the SARS-CoV-2, none of the vaccine candidates neutralized the SARS-CoV-2 in vitro suggesting differences in the natural epitope perhaps caused by conformational changes or the presence of N-linked glycans. While a cross-protective vaccine candidate was not developed, a multivalent SARS vaccine was developed. The technology discussed here is a versatile vaccination platform that can be pivoted toward other diseases and applications that are not limited to infectious diseases.


Subject(s)
COVID-19 , Comovirus , Nanoparticles , Vaccines , Animals , COVID-19/therapy , Comovirus/genetics , Epitopes, B-Lymphocyte , Humans , Immunization, Passive , Mice , Peptides , SARS-CoV-2 , Spike Glycoprotein, Coronavirus
6.
J Med Chem ; 65(4): 2866-2879, 2022 02 24.
Article in English | MEDLINE | ID: covidwho-1440451

ABSTRACT

The emergence of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presents an urgent public health crisis. Without available targeted therapies, treatment options remain limited for COVID-19 patients. Using medicinal chemistry and rational drug design strategies, we identify a 2-phenyl-1,2-benzoselenazol-3-one class of compounds targeting the SARS-CoV-2 main protease (Mpro). FRET-based screening against recombinant SARS-CoV-2 Mpro identified six compounds that inhibit proteolysis with nanomolar IC50 values. Preincubation dilution experiments and molecular docking determined that the inhibition of SARS-CoV-2 Mpro can occur by either covalent or noncovalent mechanisms, and lead E04 was determined to inhibit Mpro competitively. Lead E24 inhibited viral replication with a nanomolar EC50 value (844 nM) in SARS-CoV-2-infected Vero E6 cells and was further confirmed to impair SARS-CoV-2 replication in human lung epithelial cells and human-induced pluripotent stem cell-derived 3D lung organoids. Altogether, these studies provide a structural framework and mechanism of Mpro inhibition that should facilitate the design of future COVID-19 treatments.


Subject(s)
Antiviral Agents/pharmacology , Benzothiazoles/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Drug Discovery , SARS-CoV-2/drug effects , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Benzothiazoles/chemistry , COVID-19/drug therapy , COVID-19/metabolism , Chlorocebus aethiops , Coronavirus 3C Proteases/isolation & purification , Coronavirus 3C Proteases/metabolism , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Dose-Response Relationship, Drug , Fluorescence Resonance Energy Transfer , Humans , Microbial Sensitivity Tests , Molecular Docking Simulation , Molecular Structure , SARS-CoV-2/enzymology , Vero Cells , Virus Replication/drug effects
7.
Antimicrob Agents Chemother ; 65(10): e0115521, 2021 09 17.
Article in English | MEDLINE | ID: covidwho-1416580

ABSTRACT

Remdesivir (RDV; GS-5734) is currently the only FDA-approved antiviral drug for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The drug is approved for use in adults or children 12 years or older who are hospitalized for the treatment of COVID-19 on the basis of an acceleration of clinical recovery for inpatients with this disease. Unfortunately, the drug must be administered intravenously, restricting its use to those requiring hospitalization for relatively advanced disease. RDV is also unstable in plasma and has a complex activation pathway which may contribute to its highly variable antiviral efficacy in SARS-CoV-2-infected cells. Potent orally bioavailable antiviral drugs for early treatment of SARS-CoV-2 infection are urgently needed, and several, including molnupiravir and PF-07321332, are currently in clinical development. We focused on making simple, orally bioavailable lipid analogs of remdesivir nucleoside (RVn; GS-441524) that are processed to RVn monophosphate, the precursor of the active RVn triphosphate, by a single-step intracellular cleavage. In addition to high oral bioavailability, stability in plasma, and simpler metabolic activation, new oral lipid prodrugs of RVn had submicromolar anti-SARS-CoV-2 activity in a variety of cell types, including Vero E6, Calu-3, Caco-2, human pluripotent stem cell (PSC)-derived lung cells, and Huh7.5 cells. In Syrian hamsters, oral treatment with 1-O-octadecyl-2-O-benzyl-glycero-3-phosphate RVn (ODBG-P-RVn) was well tolerated and achieved therapeutic levels in plasma above the 90% effective concentration (EC90) for SARS-CoV-2. The results suggest further evaluation as an early oral treatment for SARS-CoV-2 infection to minimize severe disease and reduce hospitalizations.


Subject(s)
COVID-19 , Prodrugs , Adenosine/analogs & derivatives , Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , Caco-2 Cells , Cricetinae , Humans , Lipids , SARS-CoV-2
8.
PMC; 2020.
Preprint in English | PMC | ID: ppcovidwho-290515

ABSTRACT

We show that SARS-CoV-2 spike protein interacts with cell surface heparan sulfate and angiotensin converting enzyme 2 (ACE2) through its Receptor Binding Domain. Docking studies suggest a putative heparin/heparan sulfate-binding site adjacent to the domain that binds to ACE2. In vitro, binding of ACE2 and heparin to spike protein ectodomains occurs independently and a ternary complex can be generated using heparin as a template. Contrary to studies with purified components, spike protein binding to heparan sulfate and ACE2 on cells occurs codependently. Unfractionated heparin, non-anticoagulant heparin, treatment with heparin lyases, and purified lung heparan sulfate potently block spike protein binding and infection by spike protein-pseudotyped virus and SARS-CoV-2 virus. These findings support a model for SARS-CoV-2 infection in which viral attachment and infection involves formation of a complex between heparan sulfate and ACE2. Manipulation of heparan sulfate or inhibition of viral adhesion by exogenous heparin may represent new therapeutic opportunities. ### Competing Interest Statement J.D.E. is a co-founder of TEGA Therapeutics. J.D.E. and The Regents of the University of California have licensed a University invention to and have an equity interest in TEGA Therapeutics. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. C.A.G and B.E.T are employees of TEGA Therapeutics.

9.
ACS Chem Biol ; 16(4): 642-650, 2021 04 16.
Article in English | MEDLINE | ID: covidwho-1387141

ABSTRACT

Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50< 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC90 = 4.3 µM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC50 was >10 µM. There was no toxicity to any of the host cell lines at 10-100 µM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.


Subject(s)
Antiviral Agents/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Phenylalanine/pharmacology , Piperazines/pharmacology , SARS-CoV-2/drug effects , Tosyl Compounds/pharmacology , Animals , Cathepsin L/antagonists & inhibitors , Cathepsin L/metabolism , Cell Line, Tumor , Chlorocebus aethiops , Humans , Microbial Sensitivity Tests , Protein Domains , Proteolysis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virus Internalization/drug effects
10.
Sci Adv ; 7(34)2021 08.
Article in English | MEDLINE | ID: covidwho-1365115

ABSTRACT

Novel coronavirus disease 2019 (COVID-19) severity is highly variable, with pediatric patients typically experiencing less severe infection than adults and especially the elderly. The basis for this difference is unclear. We find that mRNA and protein expression of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19, increases with advancing age in distal lung epithelial cells. However, in humans, ACE2 expression exhibits high levels of intra- and interindividual heterogeneity. Further, cells infected with SARS-CoV-2 experience endoplasmic reticulum stress, triggering an unfolded protein response and caspase-mediated apoptosis, a natural host defense system that halts virion production. Apoptosis of infected cells can be selectively induced by treatment with apoptosis-modulating BH3 mimetic drugs. Notably, epithelial cells within young lungs and airways are more primed to undergo apoptosis than those in adults, which may naturally hinder virion production and support milder COVID-19 severity.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Apoptosis/genetics , COVID-19/genetics , Gene Expression Profiling/methods , Age Factors , Aged , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/metabolism , COVID-19/virology , Cells, Cultured , Chlorocebus aethiops , Female , Humans , Infant , Lung/cytology , Lung/metabolism , Lung/virology , Male , Mice, Inbred C57BL , Middle Aged , SARS-CoV-2/physiology , Severity of Illness Index , Vero Cells , Virus Internalization
11.
Data Brief ; 38: 107278, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1351628

ABSTRACT

We present supplementary data for the published article, "Hitting the diagnostic sweet spot: Point-of-care SARS-CoV-2 salivary antigen testing with an off-the-shelf glucometer" [1]. The assay described is designed to be performed at home or in a clinic without expensive instrumentation or professional training. SARS-CoV-2 is detected by an aptamer-based assay that targets the Nucleocapsid (N) or Spike (S) antigens. Binding of the N or S protein to their respective aptamer results in the competitive release of a complementary antisense-invertase enzyme complex. The released enzyme then catalyzes the conversion of sucrose to glucose that is measured by an off-the-shelf glucometer. The data presented here describe the optimization of the assay parameters and their contribution to developing this aptamer-based assay to detect SARS-CoV-2. The assay performance was checked in a standard buffer, contrived samples, and patient samples validated with well-established scientific methods. The resulting dataset can be used to further develop glucometer-based assays for diagnosing other communicable and non-communicable diseases.

12.
J Vitreoretin Dis ; 4(5): 420-429, 2020 Oct 01.
Article in English | MEDLINE | ID: covidwho-1295335

ABSTRACT

PURPOSE: To detail the rationale behind recommendations recently published by the American Society of Retina Specialists (ASRS) outlining best practices for safety of vitreoretinal surgeons and staff while performing vitreoretinal surgery during the coronavirus disease (COVID)-19 pandemic. METHODS: The committee for ASRS Best Practices for Retinal Surgery during the COVID-19 Pandemic reviewed existing evidence and information on SARS-CoV-2 transmission, and risk factors during vitreoretinal surgery. Recommendations were based on best available published data, cumulative clinical experiences, and recommendations and policies from other organizations. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach was used to assess the strength of recommendations and confidence in the evidence. These serve as interim recommendations which are routinely updated given gaps of knowledge and lack of high-quality data on this evolving subject. RESULTS: Relevant existing literature related to methods of transmission, and ocular manifestations of SARS-CoV-2 are summarized. The data and clinical experiences driving recommendations for pre-operative, intraoperative and post-operative surgical considerations, anesthesia choice, as well as considerations for intravitreal injections are provided. CONCLUSION: Recommendations are provided with the goal of protecting vitreoretinal surgeons and associated personnel from exposure to SARS-CoV-2 during interventional vitreoretinal procedures. This is a rapidly evolving topic with numerous remaining gaps in our current knowledge. As such, recommendations will evolve and the current manuscript is intended to serve as a foundation for continued dialogue on best practices.

13.
PLoS Pathog ; 17(5): e1009519, 2021 05.
Article in English | MEDLINE | ID: covidwho-1232468

ABSTRACT

SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.


Subject(s)
Amiloride/pharmacology , COVID-19/drug therapy , Coronavirus Envelope Proteins/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Amiloride/pharmacokinetics , Animals , Antiviral Agents/pharmacology , Binding Sites/drug effects , COVID-19/virology , Chlorocebus aethiops , Coronavirus Envelope Proteins/chemistry , Humans , Ion Channels/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Binding/drug effects , Protein Conformation/drug effects , Protein Domains , Vero Cells , Virus Assembly/drug effects
14.
Cell Rep ; 35(6): 109091, 2021 05 11.
Article in English | MEDLINE | ID: covidwho-1213072

ABSTRACT

It is urgent and important to understand the relationship of the widespread severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) with host immune response and study the underlining molecular mechanism. N6-methylation of adenosine (m6A) in RNA regulates many physiological and disease processes. Here, we investigate m6A modification of the SARS-CoV-2 gene in regulating the host cell innate immune response. Our data show that the SARS-CoV-2 virus has m6A modifications that are enriched in the 3' end of the viral genome. We find that depletion of the host cell m6A methyltransferase METTL3 decreases m6A levels in SARS-CoV-2 and host genes, and m6A reduction in viral RNA increases RIG-I binding and subsequently enhances the downstream innate immune signaling pathway and inflammatory gene expression. METTL3 expression is reduced and inflammatory genes are induced in patients with severe coronavirus disease 2019 (COVID-19). These findings will aid in the understanding of COVID-19 pathogenesis and the design of future studies regulating innate immunity for COVID-19 treatment.


Subject(s)
COVID-19/genetics , Methyltransferases/metabolism , SARS-CoV-2/genetics , Adenosine/metabolism , COVID-19/metabolism , Cell Line , DEAD Box Protein 58/genetics , DEAD Box Protein 58/metabolism , Humans , Immunity, Innate/genetics , Methylation , Methyltransferases/genetics , RNA, Viral/genetics , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , SARS-CoV-2/pathogenicity , Signal Transduction
15.
ACS Chem Biol ; 16(4): 642-650, 2021 04 16.
Article in English | MEDLINE | ID: covidwho-1160225

ABSTRACT

Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50< 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC90 = 4.3 µM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC50 was >10 µM. There was no toxicity to any of the host cell lines at 10-100 µM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.


Subject(s)
Antiviral Agents/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Phenylalanine/pharmacology , Piperazines/pharmacology , SARS-CoV-2/drug effects , Tosyl Compounds/pharmacology , Animals , Cathepsin L/antagonists & inhibitors , Cathepsin L/metabolism , Cell Line, Tumor , Chlorocebus aethiops , Humans , Microbial Sensitivity Tests , Protein Domains , Proteolysis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virus Internalization/drug effects
16.
Clin Case Rep ; 9(4): 2228-2235, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-1122024

ABSTRACT

An 83-year-old female had asymptomatic SARS-CoV-2 infection while taking ruxolitinib. She remained RT-PCR positive for viral RNA for >120 days, and Pegylated interferon for 4 weeks led to viral RNA clearance. The observations support combination therapy of ruxolitinib + interferon for COVID-19.

17.
Biosens Bioelectron ; 180: 113111, 2021 May 15.
Article in English | MEDLINE | ID: covidwho-1108095

ABSTRACT

Significant barriers to the diagnosis of latent and acute SARS-CoV-2 infection continue to hamper population-based screening efforts required to contain the COVID-19 pandemic in the absence of widely available antiviral therapeutics or vaccines. We report an aptamer-based SARS-CoV-2 salivary antigen assay employing only low-cost reagents ($3.20/test) and an off-the-shelf glucometer. The test was engineered around a glucometer as it is quantitative, easy to use, and the most prevalent piece of diagnostic equipment globally, making the test highly scalable with an infrastructure that is already in place. Furthermore, many glucometers connect to smartphones, providing an opportunity to integrate with contact tracing apps, medical providers, and electronic health records. In clinical testing, the developed assay detected SARS-CoV-2 infection in patient saliva across a range of viral loads - as benchmarked by RT-qPCR - within 1 h, with 100% sensitivity (positive percent agreement) and distinguished infected specimens from off-target antigens in uninfected controls with 100% specificity (negative percent agreement). We propose that this approach provides an inexpensive, rapid, and accurate diagnostic for distributed screening of SARS-CoV-2 infection at scale.


Subject(s)
Antigens, Viral/analysis , Biosensing Techniques/methods , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Point-of-Care Testing , SARS-CoV-2/immunology , Saliva/virology , Adult , COVID-19 Testing , Coronavirus Nucleocapsid Proteins/analysis , Female , Humans , Male , Phosphoproteins/analysis , SARS-CoV-2/isolation & purification , SELEX Aptamer Technique , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/analysis
18.
Stem Cell Reports ; 16(3): 437-445, 2021 03 09.
Article in English | MEDLINE | ID: covidwho-1084274

ABSTRACT

COVID-19 is a transmissible respiratory disease caused by a novel coronavirus, SARS-CoV-2, and has become a global health emergency. There is an urgent need for robust and practical in vitro model systems to investigate viral pathogenesis. Here, we generated human induced pluripotent stem cell (iPSC)-derived lung organoids (LORGs), cerebral organoids (CORGs), neural progenitor cells (NPCs), neurons, and astrocytes. LORGs containing epithelial cells, alveolar types 1 and 2, highly express ACE2 and TMPRSS2 and are permissive to SARS-CoV-2 infection. SARS-CoV-2 infection induces interferons, cytokines, and chemokines and activates critical inflammasome pathway genes. Spike protein inhibitor, EK1 peptide, and TMPRSS2 inhibitors (camostat/nafamostat) block viral entry in LORGs. Conversely, CORGs, NPCs, astrocytes, and neurons express low levels of ACE2 and TMPRSS2 and correspondingly are not highly permissive to SARS-CoV-2 infection. Infection in neuronal cells activates TLR3/7, OAS2, complement system, and apoptotic genes. These findings will aid in understanding COVID-19 pathogenesis and facilitate drug discovery.


Subject(s)
Brain/virology , COVID-19/virology , Induced Pluripotent Stem Cells/virology , Lung/virology , Neural Stem Cells/virology , Organoids/virology , SARS-CoV-2/pathogenicity , Apoptosis/physiology , Brain/metabolism , COVID-19/metabolism , Cells, Cultured , Complement System Proteins/metabolism , Epithelial Cells/metabolism , Epithelial Cells/virology , Humans , Induced Pluripotent Stem Cells/metabolism , Inflammation/metabolism , Inflammation/virology , Lung/metabolism , Neural Stem Cells/metabolism , Neurons/metabolism , Neurons/virology , Organoids/metabolism , Serine Endopeptidases/metabolism , Signal Transduction/physiology , Stem Cells/metabolism , Stem Cells/virology
19.
PLoS Pathog ; 17(2): e1009165, 2021 02.
Article in English | MEDLINE | ID: covidwho-1079380

ABSTRACT

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe, and not mild, infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of ACE2:RBD inhibition. B cell receptor (BCR) sequencing revealed that VH3-53 was enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against authentic SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and mutagenesis of RBD, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.


Subject(s)
Antibodies, Monoclonal , Antibodies, Neutralizing , Antibodies, Viral , COVID-19 , Convalescence , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Adult , Aged , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Antibodies, Viral/genetics , Antibodies, Viral/immunology , COVID-19/genetics , COVID-19/immunology , Chlorocebus aethiops , Cloning, Molecular , Epitope Mapping , Epitopes/genetics , Epitopes/immunology , Female , Humans , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Male , Middle Aged , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
20.
bioRxiv ; 2020 Oct 30.
Article in English | MEDLINE | ID: covidwho-915969

ABSTRACT

K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 µM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of ≤ 100 µM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2 , differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing.

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