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1.
Proc Natl Acad Sci U S A ; 119(6)2022 02 08.
Article in English | MEDLINE | ID: covidwho-1650946

ABSTRACT

The development of small-molecules targeting different components of SARS-CoV-2 is a key strategy to complement antibody-based treatments and vaccination campaigns in managing the COVID-19 pandemic. Here, we show that two thiol-based chemical probes that act as reducing agents, P2119 and P2165, inhibit infection by human coronaviruses, including SARS-CoV-2, and decrease the binding of spike glycoprotein to its receptor, the angiotensin-converting enzyme 2 (ACE2). Proteomics and reactive cysteine profiling link the antiviral activity to the reduction of key disulfides, specifically by disruption of the Cys379-Cys432 and Cys391-Cys525 pairs distal to the receptor binding motif in the receptor binding domain (RBD) of the spike glycoprotein. Computational analyses provide insight into conformation changes that occur when these disulfides break or form, consistent with an allosteric role, and indicate that P2119/P2165 target a conserved hydrophobic binding pocket in the RBD with the benzyl thiol-reducing moiety pointed directly toward Cys432. These collective findings establish the vulnerability of human coronaviruses to thiol-based chemical probes and lay the groundwork for developing compounds of this class, as a strategy to inhibit the SARS-CoV-2 infection by shifting the spike glycoprotein redox scaffold.


Subject(s)
Amino Alcohols/pharmacology , Angiotensin-Converting Enzyme 2/chemistry , Antiviral Agents/pharmacology , Phenyl Ethers/pharmacology , Receptors, Virus/chemistry , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/chemistry , Sulfhydryl Compounds/pharmacology , Allosteric Regulation , Amino Alcohols/chemistry , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/chemistry , Binding Sites , COVID-19/drug therapy , COVID-19/virology , Cell Line , Disulfides/antagonists & inhibitors , Disulfides/chemistry , Disulfides/metabolism , Dose-Response Relationship, Drug , Humans , Molecular Docking Simulation , Nasal Mucosa/drug effects , Nasal Mucosa/metabolism , Nasal Mucosa/virology , Oxidation-Reduction , Phenyl Ethers/chemistry , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/genetics , Receptors, Virus/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Sulfhydryl Compounds/chemistry
3.
PLoS Pathog ; 17(5): e1009229, 2021 05.
Article in English | MEDLINE | ID: covidwho-1239922

ABSTRACT

While MERS-CoV (Middle East respiratory syndrome Coronavirus) provokes a lethal disease in humans, camelids, the main virus reservoir, are asymptomatic carriers, suggesting a crucial role for innate immune responses in controlling the infection. Experimentally infected camelids clear infectious virus within one week and mount an effective adaptive immune response. Here, transcription of immune response genes was monitored in the respiratory tract of MERS-CoV infected alpacas. Concomitant to the peak of infection, occurring at 2 days post inoculation (dpi), type I and III interferons (IFNs) were maximally transcribed only in the nasal mucosa of alpacas, while interferon stimulated genes (ISGs) were induced along the whole respiratory tract. Simultaneous to mild focal infiltration of leukocytes in nasal mucosa and submucosa, upregulation of the anti-inflammatory cytokine IL10 and dampened transcription of pro-inflammatory genes under NF-κB control were observed. In the lung, early (1 dpi) transcription of chemokines (CCL2 and CCL3) correlated with a transient accumulation of mainly mononuclear leukocytes. A tight regulation of IFNs in lungs with expression of ISGs and controlled inflammatory responses, might contribute to virus clearance without causing tissue damage. Thus, the nasal mucosa, the main target of MERS-CoV in camelids, seems central in driving an efficient innate immune response based on triggering ISGs as well as the dual anti-inflammatory effects of type III IFNs and IL10.


Subject(s)
Camelids, New World , Coronavirus Infections/immunology , Interferon Type I/metabolism , Interferons/metabolism , Middle East Respiratory Syndrome Coronavirus/immunology , Animals , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Camelids, New World/immunology , Camelids, New World/metabolism , Camelids, New World/virology , Chlorocebus aethiops , Coronavirus Infections/metabolism , Coronavirus Infections/prevention & control , Coronavirus Infections/veterinary , Disease Reservoirs/veterinary , Disease Resistance/drug effects , Disease Resistance/genetics , Disease Resistance/immunology , Gene Expression Regulation , Immunity, Innate/physiology , Inflammation/immunology , Inflammation/metabolism , Inflammation/veterinary , Inflammation/virology , Interferon Type I/genetics , Interferon Type I/pharmacology , Interferons/genetics , Interferons/pharmacology , Middle East Respiratory Syndrome Coronavirus/drug effects , Middle East Respiratory Syndrome Coronavirus/physiology , Nasal Mucosa/drug effects , Nasal Mucosa/immunology , Nasal Mucosa/metabolism , Nasal Mucosa/virology , Respiratory System/drug effects , Respiratory System/immunology , Respiratory System/metabolism , Respiratory System/virology , Vero Cells , Viral Load/drug effects , Virus Replication/drug effects
4.
mBio ; 12(2)2021 04 27.
Article in English | MEDLINE | ID: covidwho-1206005

ABSTRACT

SARS-CoV-2 infection causing the COVID-19 pandemic calls for immediate interventions to avoid viral transmission, disease progression, and subsequent excessive inflammation and tissue destruction. Primary normal human bronchial epithelial cells are among the first targets of SARS-CoV-2 infection. Here, we show that ColdZyme medical device mouth spray efficiently protected against virus entry, excessive inflammation, and tissue damage. Applying ColdZyme to fully differentiated, polarized human epithelium cultured at an air-liquid interphase (ALI) completely blocked binding of SARS-CoV-2 and increased local complement activation mediated by the virus as well as productive infection of the tissue model. While SARS-CoV-2 infection resulted in exaggerated intracellular complement activation immediately following infection and a drop in transepithelial resistance, these parameters were bypassed by single pretreatment of the tissues with ColdZyme mouth spray. Crucially, our study highlights the importance of testing already evaluated and safe drugs such as ColdZyme mouth spray for maintaining epithelial integrity and hindering SARS-CoV-2 entry within standardized three-dimensional (3D) in vitro models mimicking the in vivo human airway epithelium.IMPORTANCE Although our understanding of COVID-19 continuously progresses, essential questions regarding prophylaxis and treatment remain open. A hallmark of severe SARS-CoV-2 infection is a hitherto-undescribed mechanism leading to excessive inflammation and tissue destruction associated with enhanced pathogenicity and mortality. To tackle the problem at the source, transfer of SARS-CoV-2, subsequent binding, infection, and inflammatory responses have to be avoided. In this study, we used fully differentiated, mucus-producing, and ciliated human airway epithelial cultures to test the efficacy of ColdZyme medical device mouth spray in terms of protection from SARS-CoV-2 infection. Importantly, we found that pretreatment of the in vitro airway cultures using ColdZyme mouth spray resulted in significantly shielding the epithelial integrity, hindering virus binding and infection, and blocking excessive intrinsic complement activation within the airway cultures. Our in vitro data suggest that ColdZyme mouth spray may have an impact in prevention of COVID-19.


Subject(s)
Antiviral Agents/pharmacology , Respiratory Mucosa/drug effects , SARS-CoV-2/drug effects , Bronchi/cytology , COVID-19/prevention & control , COVID-19/virology , Complement C3/immunology , Epithelial Cells , Humans , Immunity, Innate/drug effects , Nasal Mucosa/drug effects , Nasal Mucosa/immunology , Nasal Mucosa/virology , Oral Sprays , Respiratory Mucosa/immunology , Respiratory Mucosa/virology , SARS-CoV-2/physiology , Virus Attachment/drug effects
6.
Cell Rep Med ; 1(8): 100142, 2020 11 17.
Article in English | MEDLINE | ID: covidwho-894264

ABSTRACT

The acid sphingomyelinase/ceramide system plays an important role in bacterial and viral infections. Here, we report that either pharmacological inhibition of acid sphingomyelinase with amitriptyline, imipramine, fluoxetine, sertraline, escitalopram, or maprotiline or genetic downregulation of the enzyme prevents infection of cultured cells or freshy isolated human nasal epithelial cells with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or vesicular stomatitis virus (VSV) pseudoviral particles (pp-VSV) presenting SARS-CoV-2 spike protein (pp-VSV-SARS-CoV-2 spike), a bona fide system mimicking SARS-CoV-2 infection. Infection activates acid sphingomyelinase and triggers a release of ceramide on the cell surface. Neutralization or consumption of surface ceramide reduces infection with pp-VSV-SARS-CoV-2 spike. Treating volunteers with a low dose of amitriptyline prevents infection of freshly isolated nasal epithelial cells with pp-VSV-SARS-CoV-2 spike. The data justify clinical studies investigating whether amitriptyline, a safe drug used clinically for almost 60 years, or other antidepressants that functionally block acid sphingomyelinase prevent SARS-CoV-2 infection.


Subject(s)
Epithelial Cells/drug effects , SARS-CoV-2/drug effects , Sphingomyelin Phosphodiesterase/antagonists & inhibitors , Amitriptyline/pharmacology , Animals , Antidepressive Agents/pharmacology , Ceramides/antagonists & inhibitors , Ceramides/metabolism , Chlorocebus aethiops , Epithelial Cells/metabolism , Epithelial Cells/virology , Humans , Nasal Mucosa/drug effects , Nasal Mucosa/metabolism , Nasal Mucosa/virology , Neutral Ceramidase/pharmacology , SARS-CoV-2/physiology , Sphingomyelin Phosphodiesterase/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Vesicular stomatitis Indiana virus/genetics
7.
Hum Vaccin Immunother ; 16(12): 2921-2931, 2020 12 01.
Article in English | MEDLINE | ID: covidwho-759862

ABSTRACT

The sudden emergence of a highly transmissible and pathogenic coronavirus SARS-CoV-2 in December 2019 from China and its rapid global spread has posed an international health emergency. The rapid development of an effective vaccine is imperative to control the spread of SARS-CoV-2. A number of concurrent efforts to find an effective therapeutic agent or vaccine for COVID-19 (coronavirus disease 2019) are being undertaken globally. Oral and nasal mucosal surfaces serve as the primary portal of entry for pathogens like coronaviruses in the human body. As evidenced by studies on similar coronaviruses (SARS-CoV and MERS-CoV), mucosal vaccination can provide a safe and effective means for the induction of long-lasting systemic and mucosal immunity to confer protection against SARS-CoV-2. This article summarizes the approaches to an effective mucosal vaccine formulation which can be a rewarding approach to combat the unprecedented threat posed by this emerging global pandemic.


Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/prevention & control , Nasal Mucosa/immunology , SARS-CoV-2/immunology , Administration, Intranasal/methods , Animals , COVID-19/epidemiology , Humans , Nasal Mucosa/drug effects , Protein Structure, Secondary , Protein Structure, Tertiary , SARS-CoV-2/chemistry , SARS-CoV-2/drug effects
8.
Med Hypotheses ; 144: 110207, 2020 Nov.
Article in English | MEDLINE | ID: covidwho-726783

ABSTRACT

COVID-19 has been the talk of the year 2020, taking many lives and leaving others in critical conditions. It has clearly and severally been reported that the SARSCoV-2 uses the Angiotensin Converting Enzyme-2 receptors to penetrate and infect cells. Reports have also stated that the nasal and olfactory mucosa are overloaded with these receptors. We emphasize that anosmia in COVID-19 is secondary to the binding of the SARSCoV-2 to Angiotensin Converting Enzyme-2 receptors on the olfactory mucosa. A hypotheses pertaining to the presentation, diagnosis, management and possible prevention of SARS-CoV-2 is proposed. Given the high false negative rates of the polymerase chain reaction (PCR) tests, we suggest that COVID-19 negative patients with anosmia without any other nasal symptom should raise a high index of suspicion and should be further evaluated. We propose the formulation and use of Angiotensin Converting Enzyme-2 receptors agonist or angiotensin receptor blockers (ARBs) as nasal lavage, to reduce the viral load of confirmed positive patients, and as a mode of prevention, especially in high risk patients, until a vaccine is developed. These medications are readily available and testing this theory involves determination of the correct dosage of angiotensin receptor blockers or ACE inhibitors (via dilution in water) that can be used as nasal lavage and performing efficacy trials. Potential side effects to be monitored for include low blood pressure or changes in heart rate. Administration of a medicated nasal lavage may be easier and rapidly disseminated on the nasal mucosa.


Subject(s)
Angiotensin-Converting Enzyme 2/drug effects , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/drug therapy , Angiotensin Receptor Antagonists/administration & dosage , Anosmia/diagnosis , Anosmia/etiology , Antiviral Agents/administration & dosage , COVID-19/metabolism , COVID-19/virology , Humans , Models, Biological , Nasal Lavage , Nasal Mucosa/drug effects , Nasal Mucosa/metabolism , Nasal Mucosa/virology , Pandemics , Receptors, Virus/drug effects , Receptors, Virus/metabolism , SARS-CoV-2/drug effects , Viral Load
9.
Sci Rep ; 10(1): 10568, 2020 06 29.
Article in English | MEDLINE | ID: covidwho-618682

ABSTRACT

Topical intra-nasal sprays are amongst the most commonly prescribed therapeutic options for sinonasal diseases in humans. However, inconsistency and ambiguity in instructions show a lack of definitive knowledge on best spray use techniques. In this study, we have identified a new usage strategy for nasal sprays available over-the-counter, that registers an average 8-fold improvement in topical delivery of drugs at diseased sites, when compared to prevalent spray techniques. The protocol involves re-orienting the spray axis to harness inertial motion of particulates and has been developed using computational fluid dynamics simulations of respiratory airflow and droplet transport in medical imaging-based digital models. Simulated dose in representative models is validated through in vitro spray measurements in 3D-printed anatomic replicas using the gamma scintigraphy technique. This work breaks new ground in proposing an alternative user-friendly strategy that can significantly enhance topical delivery inside human nose. While these findings can eventually translate into personalized spray usage instructions and hence merit a change in nasal standard-of-care, this study also demonstrates how relatively simple engineering analysis tools can revolutionize everyday healthcare. Finally, with respiratory mucosa as the initial coronavirus infection site, our findings are relevant to intra-nasal vaccines that are in-development, to mitigate the COVID-19 pandemic.


Subject(s)
Administration, Inhalation , Administration, Intranasal/methods , Betacoronavirus , Coronavirus Infections/prevention & control , Drug Delivery Systems/methods , Nasal Sprays , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , COVID-19 , Computer Simulation , Coronavirus Infections/virology , Humans , Hydrodynamics , Nasal Cavity/anatomy & histology , Nasal Mucosa/drug effects , Nasal Mucosa/virology , Nebulizers and Vaporizers , Paranasal Sinuses/drug effects , Paranasal Sinuses/virology , Pneumonia, Viral/virology , SARS-CoV-2 , Viral Vaccines/administration & dosage
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