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1.
J Virol ; 96(2): e0106021, 2022 01 26.
Article in English | MEDLINE | ID: covidwho-1759286

ABSTRACT

Rhinoviruses (RVs) cause recurrent infections of the nasal and pulmonary tracts, life-threatening conditions in chronic respiratory illness patients, predisposition of children to asthmatic exacerbation, and large economic cost. RVs are difficult to treat. They rapidly evolve resistance and are genetically diverse. Here, we provide insight into RV drug resistance mechanisms against chemical compounds neutralizing low pH in endolysosomes. Serial passaging of RV-A16 in the presence of the vacuolar proton ATPase inhibitor bafilomycin A1 (BafA1) or the endolysosomotropic agent ammonium chloride (NH4Cl) promoted the emergence of resistant virus populations. We found two reproducible point mutations in viral proteins 1 and 3 (VP1 and VP3), A2526G (serine 66 to asparagine [S66N]), and G2274U (cysteine 220 to phenylalanine [C220F]), respectively. Both mutations conferred cross-resistance to BafA1, NH4Cl, and the protonophore niclosamide, as identified by massive parallel sequencing and reverse genetics, but not the double mutation, which we could not rescue. Both VP1-S66 and VP3-C220 locate at the interprotomeric face, and their mutations increase the sensitivity of virions to low pH, elevated temperature, and soluble intercellular adhesion molecule 1 receptor. These results indicate that the ability of RV to uncoat at low endosomal pH confers virion resistance to extracellular stress. The data endorse endosomal acidification inhibitors as a viable strategy against RVs, especially if inhibitors are directly applied to the airways. IMPORTANCE Rhinoviruses (RVs) are the predominant agents causing the common cold. Anti-RV drugs and vaccines are not available, largely due to rapid evolutionary adaptation of RVs giving rise to resistant mutants and an immense diversity of antigens in more than 160 different RV types. In this study, we obtained insight into the cell biology of RVs by harnessing the ability of RVs to evolve resistance against host-targeting small chemical compounds neutralizing endosomal pH, an important cue for uncoating of normal RVs. We show that RVs grown in cells treated with inhibitors of endolysosomal acidification evolved capsid mutations yielding reduced virion stability against elevated temperature, low pH, and incubation with recombinant soluble receptor fragments. This fitness cost makes it unlikely that RV mutants adapted to neutral pH become prevalent in nature. The data support the concept of host-directed drug development against respiratory viruses in general, notably at low risk of gain-of-function mutations.


Subject(s)
Capsid/chemistry , Mutation/drug effects , Rhinovirus/physiology , Virus Uncoating/physiology , Antiviral Agents/pharmacology , Capsid/drug effects , Capsid Proteins/genetics , Capsid Proteins/metabolism , Drug Resistance, Viral/drug effects , Drug Resistance, Viral/genetics , Endosomes/chemistry , Endosomes/drug effects , Endosomes/metabolism , HeLa Cells , Humans , Hydrogen-Ion Concentration , Intercellular Adhesion Molecule-1/metabolism , Protein Conformation , Rhinovirus/chemistry , Rhinovirus/drug effects , Rhinovirus/genetics , Virion/chemistry , Virion/genetics , Virion/metabolism , Virus Internalization/drug effects , Virus Uncoating/drug effects , Virus Uncoating/genetics
2.
J Cell Biochem ; 123(2): 155-160, 2022 02.
Article in English | MEDLINE | ID: covidwho-1473858

ABSTRACT

Drug repurposing is an attractive option for identifying new treatment strategies, in particular in extraordinary situations of urgent need such as the current coronavirus disease 2019 (Covid-19) pandemic. Recently, the World Health Organization announced testing of three drugs as potential Covid-19 therapeutics that are known for their dampening effect on the immune system. Thus, the underlying concept of selecting these drugs is to temper the potentially life-threatening overshooting of the immune system reacting to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. This viewpoint discusses the possibility that the impact of these and other drugs on autophagy contributes to their therapeutic effect by hampering the SARS-CoV-2 life cycle.


Subject(s)
Antiviral Agents/pharmacology , Artesunate/pharmacology , Autophagy/drug effects , COVID-19/drug therapy , Drug Repositioning , Imatinib Mesylate/pharmacology , Infliximab/pharmacology , Pandemics , SARS-CoV-2/drug effects , Antidepressive Agents/pharmacology , Antiviral Agents/therapeutic use , Artesunate/therapeutic use , Chloroquine/pharmacology , Drug Development , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/physiology , Endoplasmic Reticulum/virology , Endosomes/drug effects , Endosomes/virology , Humans , Hydroxychloroquine/pharmacology , Imatinib Mesylate/therapeutic use , Infliximab/therapeutic use , Intracellular Membranes/drug effects , Intracellular Membranes/physiology , Intracellular Membranes/virology , Ivermectin/pharmacology , Macrolides/pharmacology , Middle East Respiratory Syndrome Coronavirus/drug effects , Niclosamide/pharmacology , Niclosamide/therapeutic use , RNA, Viral/metabolism , SARS-CoV-2/physiology , Virus Replication
3.
mBio ; 12(5): e0254221, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1462902

ABSTRACT

Damage in COVID-19 results from both the SARS-CoV-2 virus and its triggered overactive host immune responses. Therapeutic agents that focus solely on reducing viral load or hyperinflammation fail to provide satisfying outcomes in all cases. Although viral and cellular factors have been extensively profiled to identify potential anti-COVID-19 targets, new drugs with significant efficacy remain to be developed. Here, we report the potent preclinical efficacy of ALD-R491, a vimentin-targeting small molecule compound, in treating COVID-19 through its host-directed antiviral and anti-inflammatory actions. We found that by altering the physical properties of vimentin filaments, ALD-491 affected general cellular processes as well as specific cellular functions relevant to SARS-CoV-2 infection. Specifically, ALD-R491 reduced endocytosis, endosomal trafficking, and exosomal release, thus impeding the entry and egress of the virus; increased the microcidal capacity of macrophages, thus facilitating the pathogen clearance; and enhanced the activity of regulatory T cells, therefore suppressing the overactive immune responses. In cultured cells, ALD-R491 potently inhibited the SARS-CoV-2 spike protein and human ACE2-mediated pseudoviral infection. In aged mice with ongoing, productive SARS-CoV-2 infection, ALD-R491 reduced disease symptoms as well as lung damage. In rats, ALD-R491 also reduced bleomycin-induced lung injury and fibrosis. Our results indicate a unique mechanism and significant therapeutic potential for ALD-R491 against COVID-19. We anticipate that ALD-R491, an oral, fast-acting, and non-cytotoxic agent targeting the cellular protein with multipart actions, will be convenient, safe, and broadly effective, regardless of viral mutations, for patients with early- or late-stage disease, post-COVID-19 complications, and other related diseases. IMPORTANCE With the Delta variant currently fueling a resurgence of new infections in the fully vaccinated population, developing an effective therapeutic drug is especially critical and urgent in fighting COVID-19. In contrast to the many efforts to repurpose existing drugs or address only one aspect of COVID-19, we are developing a novel agent with first-in-class mechanisms of action that address both the viral infection and the overactive immune system in the pathogenesis of the disease. Unlike virus-directed therapeutics that may lose efficacy due to viral mutations, and immunosuppressants that require ideal timing to be effective, this agent, with its unique host-directed antiviral and anti-inflammatory actions, can work against all variants of the virus, be effective during all stages of the disease, and even resolve post-disease damage and complications. Further development of the compound will provide an important tool in the fight against COVID-19 and its complications, as well as future outbreaks of new viruses.


Subject(s)
Anti-Inflammatory Agents/therapeutic use , Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19/metabolism , Organic Chemicals/therapeutic use , Spike Glycoprotein, Coronavirus/metabolism , Vimentin/metabolism , Animals , Endocytosis/drug effects , Endosomes/drug effects , Endosomes/metabolism , Exosomes/drug effects , Exosomes/metabolism , HEK293 Cells , Humans , Mice , RAW 264.7 Cells
4.
Biochimie ; 179: 237-246, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-1326916

ABSTRACT

The anti-malarial drug Chloroquine (CQ) and its derivative hydroxychloroquine have shown antiviral activities in vitro against many viruses, including coronaviruses, dengue virus and the biosafety level 4 Nipah and Hendra paramyxoviruses. The in vivo efficacy of CQ in the treatment of COVID-19 is currently a matter of debate. CQ is a lysosomotrophic compound that accumulates in lysosomes, as well as in food vacuoles of Plasmodium falciparum. In the treatment of malaria, CQ impairs the digestion and growth of the parasite by increasing the pH of the food vacuole. Similarly, it is assumed that the antiviral effects of CQ results from the increase of lysosome pH and the inhibition of acidic proteases involved in the maturation of virus fusion protein. CQ has however other effects, among which phospholipidosis, characterized by the accumulation of multivesicular bodies within the cell. The increase in phospholipid species particularly concerns bis(monoacylglycero)phosphate (BMP), a specific lipid of late endosomes involved in vesicular trafficking and pH-dependent vesicle budding. It was shown previously that drugs like progesterone, the cationic amphiphile U18666A and the phospholipase inhibitor methyl arachidonyl fluoro phosphonate (MAFP) induce the accumulation of BMP in THP-1 cells and decrease cell infection by human immunodeficiency virus. HIV viral particles were found to be retained into large endosomal-type vesicles, preventing virus spreading. Since BMP was also reported to favour virus entry through hijacking of the endocytic pathway, we propose here that BMP could play a dual role in viral infection, with its antiviral effects triggered by lysosomotropic drugs like CQ.


Subject(s)
Antiviral Agents/pharmacology , Chloroquine/pharmacology , Endocytosis/drug effects , Endosomes/drug effects , Endosomes/metabolism , Lysophospholipids/metabolism , Monoglycerides/metabolism , SARS-CoV-2/drug effects , Humans , SARS-CoV-2/physiology
5.
PLoS Pathog ; 17(7): e1009706, 2021 07.
Article in English | MEDLINE | ID: covidwho-1305581

ABSTRACT

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.


Subject(s)
COVID-19/drug therapy , COVID-19/virology , SARS-CoV-2/drug effects , SARS-CoV-2/pathogenicity , Virus Internalization/drug effects , Ammonium Chloride/pharmacology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/physiology , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/pharmacology , Cell Line , Chlorocebus aethiops , Chloroquine/pharmacology , Clathrin/metabolism , Drug Synergism , Endocytosis/drug effects , Endocytosis/physiology , Endosomes/drug effects , Endosomes/metabolism , Humans , Hydrogen-Ion Concentration/drug effects , Hydroxychloroquine/administration & dosage , Macrolides/pharmacology , Niclosamide/administration & dosage , Niclosamide/pharmacology , Protein Binding/drug effects , Protein Domains , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/physiology , Vero Cells
6.
Cells ; 10(5)2021 05 07.
Article in English | MEDLINE | ID: covidwho-1223961

ABSTRACT

The flavonoid naringenin (Nar), present in citrus fruits and tomatoes, has been identified as a blocker of an emerging class of human intracellular channels, namely the two-pore channel (TPC) family, whose role has been established in several diseases. Indeed, Nar was shown to be effective against neoangiogenesis, a process essential for solid tumor progression, by specifically impairing TPC activity. The goal of the present review is to illustrate the rationale that links TPC channels to the mechanism of coronavirus infection, and how their inhibition by Nar could be an efficient pharmacological strategy to fight the current pandemic plague COVID-19.


Subject(s)
COVID-19/drug therapy , Calcium Channel Blockers/pharmacology , Calcium Channels/metabolism , Flavanones/pharmacology , Neoplasms/drug therapy , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Arabidopsis/metabolism , COVID-19/epidemiology , COVID-19/pathology , COVID-19/virology , Calcium Channel Blockers/therapeutic use , Drug Evaluation, Preclinical , Endosomes/drug effects , Endosomes/metabolism , Endosomes/virology , Flavanones/therapeutic use , Humans , Lysosomes/drug effects , Lysosomes/metabolism , Lysosomes/virology , Neoplasms/blood supply , Neoplasms/pathology , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/pathology , Pandemics/prevention & control , SARS-CoV-2/pathogenicity , Vacuoles/metabolism , Virus Internalization/drug effects
7.
J Biol Chem ; 296: 100306, 2021.
Article in English | MEDLINE | ID: covidwho-1152462

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, so understanding its biology and infection mechanisms is critical to facing this major medical challenge. SARS-CoV-2 is known to use its spike glycoprotein to interact with the cell surface as a first step in the infection process. As for other coronaviruses, it is likely that SARS-CoV-2 next undergoes endocytosis, but whether or not this is required for infectivity and the precise endocytic mechanism used are unknown. Using purified spike glycoprotein and lentivirus pseudotyped with spike glycoprotein, a common model of SARS-CoV-2 infectivity, we now demonstrate that after engagement with the plasma membrane, SARS-CoV-2 undergoes rapid, clathrin-mediated endocytosis. This suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system. Importantly, we further demonstrate that knockdown of clathrin heavy chain, which blocks clathrin-mediated endocytosis, reduces viral infectivity. These discoveries reveal that SARS-CoV-2 uses clathrin-mediated endocytosis to gain access into cells and suggests that this process is a key aspect of virus infectivity.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Clathrin Heavy Chains/genetics , Endocytosis/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization/drug effects , A549 Cells , Angiotensin-Converting Enzyme 2/metabolism , Animals , Chlorocebus aethiops , Clathrin Heavy Chains/antagonists & inhibitors , Clathrin Heavy Chains/metabolism , Endocytosis/drug effects , Endosomes/drug effects , Endosomes/metabolism , Endosomes/virology , Gene Expression Regulation , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HEK293 Cells , Host-Pathogen Interactions/genetics , Humans , Hydrazones/pharmacology , Lentivirus/genetics , Lentivirus/metabolism , Protein Binding/drug effects , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Signal Transduction , Spike Glycoprotein, Coronavirus/metabolism , Sulfonamides/pharmacology , Thiazolidines/pharmacology , Vero Cells
8.
Virol J ; 18(1): 46, 2021 02 27.
Article in English | MEDLINE | ID: covidwho-1105717

ABSTRACT

BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 and broke out as a global pandemic in late 2019. The acidic pH environment of endosomes is believed to be essential for SARS-CoV-2 to be able to enter cells and begin replication. However, the clinical use of endosomal acidification inhibitors, typically chloroquine, has been controversial with this respect. METHODS: In this study, RT-qPCR method was used to detect the SARS-CoV-2N gene to evaluate viral replication. The CCK-8 assay was also used to evaluate the cytotoxic effect of SARS-CoV-2. In situ hybridization was used to examine the distribution of the SARS-CoV-2 gene in lung tissues. Hematoxylin and eosin staining was also used to evaluate virus-associated pathological changes in lung tissues. RESULTS: In this study, analysis showed that endosomal acidification inhibitors, including chloroquine, bafilomycin A1 and NH4CL, significantly reduced the viral yields of SARS-CoV-2 in Vero E6, Huh-7 and 293T-ACE2 cells. Chloroquine and bafilomycin A1 also improved the viability and proliferation of Vero E6 cells after SARS-CoV-2 infection. Moreover, in the hACE2 transgenic mice model of SARS-CoV-2 infection, chloroquine and bafilomycin A1 reduced viral replication in lung tissues and alleviated viral pneumonia with reduced inflammatory exudation and infiltration in peribronchiolar and perivascular tissues, as well as improved structures of alveolar septum and pulmonary alveoli. CONCLUSIONS: Our research investigated the antiviral effects of endosomal acidification inhibitors against SARS-CoV-2 in several infection models and provides an experimental basis for further mechanistic studies and drug development.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/virology , Endosomes/drug effects , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Virus Replication/drug effects , Ammonium Chloride/pharmacology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/metabolism , COVID-19/pathology , Cell Survival/drug effects , Chlorocebus aethiops , Chloroquine/pharmacology , Endosomes/metabolism , Female , HEK293 Cells , Humans , Hydrogen-Ion Concentration , Lung/pathology , Macrolides/pharmacology , Mice , Mice, Transgenic , Random Allocation , SARS-CoV-2/genetics , Vero Cells
9.
Antiviral Res ; 186: 104990, 2021 02.
Article in English | MEDLINE | ID: covidwho-1064808

ABSTRACT

The endocytic pathway is a common strategy that several highly pathogenic viruses use to enter into the cell. To demonstrate the usefulness of this pathway as a common target for the development of broad-spectrum antivirals, the inhibitory effect of drug compounds targeting endosomal membrane proteins were investigated. This study entailed direct comparison of drug effectiveness against animal and human pathogenic viruses, namely Ebola (EBOV), African swine fever virus (ASFV), and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A panel of experimental and FDA-approved compounds targeting calcium channels and PIKfyve at the endosomal membrane caused potent reductions of entry up to 90% in SARS-CoV-2 S-protein pseudotyped retrovirus. Similar inhibition was observed against transduced EBOV glycoprotein pseudovirus and ASFV. SARS-CoV-2 infection was potently inhibited by selective estrogen receptor modulators in cells transduced with pseudovirus, among them Raloxifen inhibited ASFV with very low 50% inhibitory concentration. Finally, the mechanism of the inhibition caused by the latter in ASFV infection was analyzed. Overall, this work shows that cellular proteins related to the endocytic pathway can constitute suitable cellular targets for broad range antiviral compounds.


Subject(s)
African Swine Fever Virus/drug effects , Antiviral Agents/pharmacology , Ebolavirus/drug effects , Endosomes/drug effects , SARS-CoV-2/drug effects , Virus Internalization/drug effects , African Swine Fever Virus/physiology , Animals , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Cell Line , Cell Line, Tumor , Chlorocebus aethiops , Cholesterol/metabolism , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Ebolavirus/physiology , Endocytosis/drug effects , Endosomes/metabolism , Humans , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors/pharmacology , Raloxifene Hydrochloride/pharmacology , Receptors, Estrogen/metabolism , SARS-CoV-2/physiology , Selective Estrogen Receptor Modulators/pharmacology , Vero Cells
10.
Int J Mol Sci ; 21(16)2020 Aug 07.
Article in English | MEDLINE | ID: covidwho-1024585

ABSTRACT

Gitelman's syndrome (GS) and Bartter's syndrome (BS) are rare inherited salt-losing tubulopathies whose variations in genotype do not correlate well with either clinical course or electrolyte requirements. Using GS/BS patients as nature's experiments, we found them to be a human model of endogenous Ang II antagonism with activated Renin-Angiotensin System (RAS), resulting in high Ang II levels with blunted cardiovascular effects. These patients are also characterized by increased and directly correlated levels of both Angiotensin Converting Enzyme 2 (ACE2) and Ang 1-7. Understanding the myriad of distinctive and frequently overlapping clinical presentations of GS/BS arises remains challenging. Efforts to find a treatment for COVID-19 has fueled a recent surge in interest in chloroquine/hydroxychloroquine and its effects. Of specific interest are chloroquine/hydroxychloroquine's ability to inhibit SARS-CoV infection by impairing ACE2, the SARS-CoV2 entry point, through terminal glycosylation via effects on TGN/post-Golgi pH homeostasis. Several different studies with a GS or a BS phenotype, along with a nonsyndromic form of X-linked intellectual disability linked to a mutated SLC9A7, provide additional evidence that specific gene defects can act via misregulation of TGN/post-Golgi pH homeostasis, which leads to a common mechanistic basis resulting in overlapping phenotypes. We suggest that linkage between the specific gene defects identified in GS and BS and the myriad of distinctive and frequently overlapping clinical findings may be the result of aberrant glycosylation of ACE2 driven by altered TGN/endosome system acidification caused by the metabolic alkalosis brought about by these salt-losing tubulopathies in addition to their altered intracellular calcium signaling due to a blunted second messenger induced intracellular calcium release that is, in turn, amplified by the RAS system.


Subject(s)
Bartter Syndrome/genetics , Coronavirus Infections/drug therapy , Gitelman Syndrome/genetics , Peptidyl-Dipeptidase A/metabolism , Phenotype , Pneumonia, Viral/drug therapy , Angiotensin-Converting Enzyme 2 , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Animals , Bartter Syndrome/metabolism , Bartter Syndrome/pathology , COVID-19 , Endosomes/drug effects , Endosomes/metabolism , Gitelman Syndrome/metabolism , Gitelman Syndrome/pathology , Humans , Hydroxychloroquine/pharmacology , Hydroxychloroquine/therapeutic use , Pandemics
11.
FEBS J ; 287(17): 3664-3671, 2020 09.
Article in English | MEDLINE | ID: covidwho-960850

ABSTRACT

The quest for the effective treatment against coronavirus disease 2019 pneumonia caused by the severe acute respiratory syndrome (SARS)-coronavirus 2(CoV-2) coronavirus is hampered by the lack of knowledge concerning the basic cell biology of the infection. Given that most viruses use endocytosis to enter the host cell, mechanistic investigation of SARS-CoV-2 infection needs to consider the diversity of endocytic pathways available for SARS-CoV-2 entry in the human lung epithelium. Taking advantage of the well-established methodology of membrane trafficking studies, this research direction allows for the rapid characterisation of the key cell biological mechanism(s) responsible for SARS-CoV-2 infection. Furthermore, 11 clinically approved generic drugs are identified as potential candidates for repurposing as blockers of several potential routes for SARS-CoV-2 endocytosis. More broadly, the paradigm of targeting a fundamental aspect of human cell biology to protect against infection may be advantageous in the context of future pandemic outbreaks.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Repositioning , Endocytosis/drug effects , SARS-CoV-2/drug effects , Virus Internalization/drug effects , Alveolar Epithelial Cells/drug effects , Alveolar Epithelial Cells/virology , Amiloride/pharmacology , COVID-19/metabolism , COVID-19/pathology , COVID-19/virology , Caveolae/drug effects , Caveolae/virology , Chlorpromazine/pharmacology , Clathrin-Coated Vesicles/drug effects , Clathrin-Coated Vesicles/virology , Endosomes/drug effects , Endosomes/virology , Humans , Itraconazole/pharmacology , Lung/drug effects , Lung/metabolism , Lung/pathology , Lung/virology , Lysosomes/drug effects , Lysosomes/virology , Nystatin/pharmacology , Pinocytosis/drug effects , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Vinblastine/pharmacology
12.
Eur J Pharmacol ; 882: 173288, 2020 Sep 05.
Article in English | MEDLINE | ID: covidwho-959742

ABSTRACT

In December 2019, many pneumonia cases with unidentified sources appeared in Wuhan, Hubei, China, with clinical symptoms like viral pneumonia. Deep sequencing analysis of samples from lower respiratory tract revealed a novel coronavirus, called 2019 novel coronavirus (2019-nCoV). Currently there is a rapid global spread. World Health Organization declare the disease a pandemic condition. The pathologic source of this disease was a new RNA virus from Coronaviridae family, which was named COVID-19. SARS-CoV-2 entry starts with the binding of the spike glycoprotein expressed on the viral envelope to ACE2 on the alveolar surface followed by clathrin-dependent endocytosis of the SARS-CoV-2 and ACE2 complex. SARS-CoV-2 enters the cells through endocytosis process, which is possibly facilitated, via a pH dependent endosomal cysteine protease cathepsins. Once inside the cells, SARS-CoV-2 exploits the endogenous transcriptional machinery of alveolar cells to replicate and spread through the entire lung. Endosomal acidic pH for SARS-CoV-2 processing and internalization is critical. After entering the cells, it possibly activates or hijack many intracellular pathways in favor of its replication. In the current opinion article, we will explain the possible involvement of unfolded protein response as a cellular stress response to the SARS-CoV-2 infection.


Subject(s)
Alveolar Epithelial Cells/drug effects , Coronavirus Infections/drug therapy , Endoplasmic Reticulum/drug effects , Ionophores/pharmacology , Pneumonia, Viral/drug therapy , Alveolar Epithelial Cells/cytology , Alveolar Epithelial Cells/metabolism , Alveolar Epithelial Cells/virology , Angiotensin-Converting Enzyme 2 , Betacoronavirus/metabolism , COVID-19 , Clathrin-Coated Vesicles/drug effects , Clathrin-Coated Vesicles/metabolism , Coronavirus Infections/virology , Endocytosis/drug effects , Endoplasmic Reticulum/metabolism , Endosomes/drug effects , Endosomes/metabolism , Humans , Ionophores/therapeutic use , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/virology , SARS-CoV-2 , Unfolded Protein Response/drug effects
14.
Life Sci ; 262: 118541, 2020 Dec 01.
Article in English | MEDLINE | ID: covidwho-816772

ABSTRACT

The possibility is examined that immunomodulatory pharmacotherapy may be clinically useful in managing the pandemic coronavirus disease 2019 (COVID-19), known to result from infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense single-stranded RNA virus. The dominant route of cell entry of the coronavirus is via phagocytosis, with ensconcement in endosomes thereafter proceeding via the endosomal pathway, involving transfer from early (EEs) to late endosomes (LEs) and ultimately into lysosomes via endolysosomal fusion. EE to LE transportation is a rate-limiting step for coronaviruses. Hence inhibition or dysregulation of endosomal trafficking could potentially inhibit SARS-CoV-2 replication. Furthermore, the acidic luminal pH of the endolysosomal system is critical for the activity of numerous pH-sensitive hydrolytic enzymes. Golgi sub-compartments and Golgi-derived secretory vesicles also depend on being mildly acidic for optimal function and structure. Activation of endosomal toll-like receptors by viral RNA can upregulate inflammatory mediators and contribute to a systemic inflammatory cytokine storm, associated with a worsened clinical outcome in COVID-19. Such endosomal toll-like receptors could be inhibited by the use of pharmacological agents which increase endosomal pH, thereby reducing the activity of acid-dependent endosomal proteases required for their activity and/or assembly, leading to suppression of antigen-presenting cell activity, decreased autoantibody secretion, decreased nuclear factor-kappa B activity and decreased pro-inflammatory cytokine production. It is also noteworthy that SARS-CoV-2 inhibits autophagy, predisposing infected cells to apoptosis. It is therefore also suggested that further pharmacological inhibition of autophagy might encourage the apoptotic clearance of SARS-CoV-2-infected cells.


Subject(s)
Antiviral Agents/pharmacology , Autophagy/drug effects , COVID-19/drug therapy , COVID-19/virology , Endosomes/drug effects , Lysosomes/drug effects , SARS-CoV-2/drug effects , Azithromycin/adverse effects , Azithromycin/pharmacology , Azithromycin/therapeutic use , Humans , Hydroxychloroquine/adverse effects , Hydroxychloroquine/pharmacology , Hydroxychloroquine/therapeutic use , Pandemics
15.
Nat Commun ; 11(1): 4252, 2020 08 25.
Article in English | MEDLINE | ID: covidwho-741685

ABSTRACT

The 2019 novel respiratory virus (SARS-CoV-2) causes COVID-19 with rapid global socioeconomic disruptions and disease burden to healthcare. The COVID-19 and previous emerging virus outbreaks highlight the urgent need for broad-spectrum antivirals. Here, we show that a defensin-like peptide P9R exhibited potent antiviral activity against pH-dependent viruses that require endosomal acidification for virus infection, including the enveloped pandemic A(H1N1)pdm09 virus, avian influenza A(H7N9) virus, coronaviruses (SARS-CoV-2, MERS-CoV and SARS-CoV), and the non-enveloped rhinovirus. P9R can significantly protect mice from lethal challenge by A(H1N1)pdm09 virus and shows low possibility to cause drug-resistant virus. Mechanistic studies indicate that the antiviral activity of P9R depends on the direct binding to viruses and the inhibition of virus-host endosomal acidification, which provides a proof of concept that virus-binding alkaline peptides can broadly inhibit pH-dependent viruses. These results suggest that the dual-functional virus- and host-targeting P9R can be a promising candidate for combating pH-dependent respiratory viruses.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus/drug effects , Influenza A virus/drug effects , Peptides/pharmacology , Amino Acid Sequence , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/therapeutic use , Cell Line , Endosomes/chemistry , Endosomes/drug effects , Female , Humans , Hydrogen-Ion Concentration , Influenza A virus/metabolism , Mice , Mice, Inbred BALB C , Orthomyxoviridae Infections/drug therapy , Orthomyxoviridae Infections/metabolism , Peptides/chemistry , Peptides/metabolism , Peptides/therapeutic use , Protein Binding , Protein Conformation , Rhinovirus/drug effects , Rhinovirus/metabolism , Viral Load/drug effects , Virus Replication/drug effects
16.
Cells ; 9(9)2020 08 25.
Article in English | MEDLINE | ID: covidwho-730305

ABSTRACT

An outbreak of the novel coronavirus (CoV) SARS-CoV-2, the causative agent of COVID-19 respiratory disease, infected millions of people since the end of 2019, led to high-level morbidity and mortality and caused worldwide social and economic disruption. There are currently no antiviral drugs available with proven efficacy or vaccines for its prevention. An understanding of the underlying cellular mechanisms involved in virus replication is essential for repurposing the existing drugs and/or the discovery of new ones. Endocytosis is the important mechanism of entry of CoVs into host cells. Endosomal maturation followed by the fusion with lysosomes are crucial events in endocytosis. Late endosomes and lysosomes are characterized by their acidic pH, which is generated by a proton transporter V-ATPase and required for virus entry via endocytic pathway. The cytoplasmic cAMP pool produced by soluble adenylyl cyclase (sAC) promotes V-ATPase recruitment to endosomes/lysosomes and thus their acidification. In this review, we discuss targeting the sAC-specific cAMP pool as a potential strategy to impair the endocytic entry of the SARS-CoV-2 into the host cell. Furthermore, we consider the potential impact of sAC inhibition on CoV-induced disease via modulation of autophagy and apoptosis.


Subject(s)
Adenylyl Cyclase Inhibitors/therapeutic use , Adenylyl Cyclases/metabolism , Betacoronavirus/physiology , Coronavirus Infections/drug therapy , Coronavirus Infections/prevention & control , Cyclic AMP/antagonists & inhibitors , Pandemics/prevention & control , Pneumonia, Viral/drug therapy , Pneumonia, Viral/prevention & control , Antiviral Agents/pharmacology , Apoptosis/drug effects , Autophagy/drug effects , COVID-19 , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Endocytosis/drug effects , Endosomes/drug effects , Endosomes/metabolism , Humans , Lysosomes/drug effects , Lysosomes/metabolism , Pneumonia, Viral/metabolism , Pneumonia, Viral/virology , SARS-CoV-2 , Virus Internalization/drug effects , Virus Replication/drug effects
17.
Biomed Pharmacother ; 130: 110582, 2020 Oct.
Article in English | MEDLINE | ID: covidwho-688980

ABSTRACT

Given the speed of viral infection spread, repurposing of existing drugs has been given the highest priority in combating the ongoing COVID-19 pandemic. Only drugs that are already registered or close to registration, and therefore have passed lengthy safety assessments, have a chance to be tested in clinical trials and reach patients quickly enough to help in the current disease outbreak. Here, we have reviewed available evidence and possible ways forward to identify already existing pharmaceuticals displaying modest broad-spectrum antiviral activity which is likely linked to their high accumulation in cells. Several well studied examples indicate that these drugs accumulate in lysosomes, endosomes and biological membranes in general, and thereby interfere with endosomal pathway and intracellular membrane trafficking crucial for viral infection. With the aim to identify other lysosomotropic drugs with possible inherent antiviral activity, we have applied a set of clear physicochemical, pharmacokinetic and molecular criteria on 530 existing drugs. In addition to publicly available data, we have also used our in silico model for the prediction of accumulation in lysosomes and endosomes. By this approach we have identified 36 compounds with possible antiviral effects, also against coronaviruses. For 14 of them evidence of broad-spectrum antiviral activity has already been reported, adding support to the value of this approach. Presented pros and cons, knowledge gaps and methods to identify lysosomotropic antivirals, can help in the evaluation of many drugs currently in clinical trials considered for repurposing to target COVID-19, as well as open doors to finding more potent and safer alternatives.


Subject(s)
Antiviral Agents/therapeutic use , Betacoronavirus , Coronavirus Infections/drug therapy , Drug Repositioning , Lysosomes/drug effects , Pandemics , Pneumonia, Viral/drug therapy , Anti-Inflammatory Agents/pharmacokinetics , Antiviral Agents/adverse effects , Antiviral Agents/pharmacokinetics , Arrhythmias, Cardiac/chemically induced , Azithromycin/pharmacokinetics , Azithromycin/therapeutic use , COVID-19 , Chemical and Drug Induced Liver Injury/etiology , Chloroquine/pharmacokinetics , Chloroquine/therapeutic use , Computer Simulation , Drug Evaluation, Preclinical , Endosomes/drug effects , Humans , Hydrogen-Ion Concentration , Hydroxychloroquine/pharmacokinetics , Hydroxychloroquine/therapeutic use , Intracellular Membranes/physiology , Lysosomes/chemistry , Membrane Lipids/metabolism , Models, Biological , Phospholipids/metabolism , SARS-CoV-2 , Surface-Active Agents/pharmacokinetics , Virus Internalization
18.
Cell Signal ; 73: 109706, 2020 09.
Article in English | MEDLINE | ID: covidwho-625663

ABSTRACT

Chloroquine (CQ) and its analogue hydroxychloroquine (HCQ) have been thrust into our everyday vernacular because some believe, based on very limited basic and clinical data, that they might be helpful in preventing and/or lessening the severity of the pandemic coronavirus disease 2019 (COVID-19). However, lacking is a temperance in enthusiasm for their possible use as well as sufficient perspective on their effects and side-effects. CQ and HCQ have well-known properties of being diprotic weak bases that preferentially accumulate in acidic organelles (endolysosomes and Golgi apparatus) and neutralize luminal pH of acidic organelles. These primary actions of CQ and HCQ are responsible for their anti-malarial effects; malaria parasites rely on acidic digestive vacuoles for survival. Similarly, de-acidification of endolysosomes and Golgi by CQ and HCQ may block severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) integration into host cells because SARS-CoV-2 may require an acidic environment for its entry and for its ability to bud and infect bystander cells. Further, de-acidification of endolysosomes and Golgi may underly the immunosuppressive effects of these two drugs. However, modern cell biology studies have shown clearly that de-acidification results in profound changes in the structure, function and cellular positioning of endolysosomes and Golgi, in signaling between these organelles and other subcellular organelles, and in fundamental cellular functions. Thus, studying the possible therapeutic effects of CQ and HCQ against COVID-19 must occur concurrent with studies of the extent to which these drugs affect organellar and cell biology. When comprehensively examined, a better understanding of the Janus sword actions of these and other drugs might yield better decisions and better outcomes.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Chloroquine/pharmacology , Endosomes/drug effects , Golgi Apparatus/drug effects , Hydroxychloroquine/pharmacology , Antimalarials/pharmacology , Antimalarials/therapeutic use , Antiviral Agents/therapeutic use , Betacoronavirus/enzymology , Betacoronavirus/metabolism , Betacoronavirus/pathogenicity , COVID-19 , Chloroquine/therapeutic use , Coronavirus Infections/drug therapy , Cytokines/metabolism , Endocytosis/drug effects , Endosomes/metabolism , Golgi Apparatus/metabolism , Humans , Hydrogen-Ion Concentration , Hydroxychloroquine/therapeutic use , Lysosomes/drug effects , Lysosomes/metabolism , Malaria/drug therapy , Pandemics/prevention & control , Pneumonia, Viral/drug therapy , SARS-CoV-2
19.
J Transl Med ; 18(1): 261, 2020 06 29.
Article in English | MEDLINE | ID: covidwho-617269

ABSTRACT

Amino-bisphosphonates such as zoledronic acid (ZA) can possibly ameliorate or prevent severe COVID-19 disease by at least three distinct mechanisms: (1) as immunostimulants which could boost γδ T cell expansion, important in the acute response in the lung; (2) as DC modulators, limiting their ability to only partially activate T cells; and (3) as prenylation inhibitors of small GTPases in the endosomal pathway of the DC to prevent expulsion of lysosomes containing SARS-CoV-2 virions. Use of ZA or other amino-bisphosphonates as modulators of COVID-19 disease should be considered.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/immunology , Coronavirus Infections/virology , Dendritic Cells/virology , Diphosphonates/therapeutic use , Endosomes/metabolism , Immunologic Factors/pharmacology , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Animals , COVID-19 , Dendritic Cells/drug effects , Endosomes/drug effects , Humans , Pandemics , SARS-CoV-2
20.
Open Heart ; 7(1)2020 06.
Article in English | MEDLINE | ID: covidwho-595177

ABSTRACT

The high rate of thrombotic complications associated with COVID-19 seems likely to reflect viral infection of vascular endothelial cells, which express the ACE2 protein that enables SARS-CoV-2 to invade cells. Various proinflammatory stimuli can promote thrombosis by inducing luminal endothelial expression of tissue factor (TF), which interacts with circulating coagulation factor VII to trigger extrinsic coagulation. The signalling mechanism whereby these stimuli evoke TF expression entails activation of NADPH oxidase, upstream from activation of the NF-kappaB transcription factor that drives the induced transcription of the TF gene. When single-stranded RNA viruses are taken up into cellular endosomes, they stimulate endosomal formation and activation of NADPH oxidase complexes via RNA-responsive toll-like receptor 7. It is therefore proposed that SARS-CoV-2 infection of endothelial cells evokes the expression of TF which is contingent on endosomal NADPH oxidase activation. If this hypothesis is correct, hydroxychloroquine, spirulina (more specifically, its chromophore phycocyanobilin) and high-dose glycine may have practical potential for mitigating the elevated thrombotic risk associated with COVID-19.


Subject(s)
Betacoronavirus/pathogenicity , Blood Coagulation , Coronavirus Infections/virology , Endosomes/virology , Endothelial Cells/virology , NADPH Oxidases/metabolism , Pneumonia, Viral/virology , Thromboplastin/metabolism , Thrombosis/virology , Animals , Antiviral Agents/therapeutic use , Betacoronavirus/drug effects , Blood Coagulation/drug effects , COVID-19 , Coronavirus Infections/blood , Coronavirus Infections/drug therapy , Coronavirus Infections/enzymology , Endosomes/drug effects , Endosomes/enzymology , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Enzyme Activation , Fibrinolytic Agents/therapeutic use , Host-Pathogen Interactions , Humans , Pandemics , Pneumonia, Viral/blood , Pneumonia, Viral/drug therapy , Pneumonia, Viral/enzymology , SARS-CoV-2 , Signal Transduction , Thrombosis/blood , Thrombosis/enzymology , Thrombosis/prevention & control
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