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1.
Nat Commun ; 12(1): 6055, 2021 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-34663813

RESUMO

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.

2.
J Virol ; : JVI0106321, 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34669512

RESUMO

COVID-19 affects multiple organs. Clinical data from the Mount Sinai Health System shows that substantial numbers of COVID-19 patients without prior heart disease develop cardiac dysfunction. How COVID-19 patients develop cardiac disease is not known. We integrated cell biological and physiological analyses of human cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) infected with SARS-CoV-2 in the presence of interleukins, with clinical findings related to laboratory values in COVID-19 patients, to identify plausible mechanisms of cardiac disease in COVID-19 patients. We infected hiPSC-derived cardiomyocytes, from healthy human subjects, with SARS-CoV-2 in the absence and presence of IL-6 and IL-1ß. Infection resulted in increased numbers of multinucleated cells. Interleukin treatment and infection resulted in disorganization of myofibrils, extracellular release of troponin-I, and reduced and erratic beating. Infection resulted in decreased expression of mRNA encoding key proteins of the cardiomyocyte contractile apparatus. Although interleukins did not increase the extent of infection, they increased the contractile dysfunction associated with viral infection of cardiomyocytes resulting in cessation of beating. Clinical data from hospitalized patients from the Mount Sinai Health System show that a significant portion of COVID-19 patients without prior history of heart disease, have elevated troponin and interleukin levels. A substantial subset of these patients showed reduced left ventricular function by echocardiography. Our laboratory observations, combined with the clinical data, indicate that direct effects on cardiomyocytes by interleukins and SARS-CoV-2 infection might underlie heart disease in COVID-19 patients. Importance SARS-CoV-2 infects multiple organs including the heart. Analyses of hospitalized patients show that a substantial number without prior indication of heart disease or comorbidities show significant injury to heart tissue assessed by increased levels of troponin in blood. We studied the cell biological and physiological effects of virus infection of healthy human iPSC cardiomyocytes in culture. Virus infection with interleukins disorganizes myofibrils, increases cell size and the numbers of multinucleated cells, suppresses the expression of proteins of the contractile apparatus. Viral infection of cardiomyocytes in culture triggers release of troponin similar to elevation in levels of COVID-19 patients with heart disease. Viral infection in the presence of interleukins slows down and desynchronizes the beating of cardiomyocytes in culture. The cell level physiological changes are similar to decreases in left ventricular ejection seen in imaging of patients' hearts. These observations suggest that direct injury to heart tissue by virus can be one underlying cause of heart disease in COVID-19.

3.
Science ; 373(6554): 541-547, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34326236

RESUMO

Repurposing drugs as treatments for COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drawn much attention. Beginning with sigma receptor ligands and expanding to other drugs from screening in the field, we became concerned that phospholipidosis was a shared mechanism underlying the antiviral activity of many repurposed drugs. For all of the 23 cationic amphiphilic drugs we tested, including hydroxychloroquine, azithromycin, amiodarone, and four others already in clinical trials, phospholipidosis was monotonically correlated with antiviral efficacy. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the physicochemical properties of drugs and does not reflect specific target-based activities-rather, it may be considered a toxic confound in early drug discovery. Early detection of phospholipidosis could eliminate these artifacts, enabling a focus on molecules with therapeutic potential.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Reposicionamento de Medicamentos , Lipidoses/induzido quimicamente , Fosfolipídeos/metabolismo , SARS-CoV-2/efeitos dos fármacos , Células A549 , Animais , Antivirais/química , Antivirais/uso terapêutico , Antivirais/toxicidade , COVID-19/virologia , Cátions , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Feminino , Humanos , Camundongos , Testes de Sensibilidade Microbiana , SARS-CoV-2/fisiologia , Tensoativos/química , Tensoativos/farmacologia , Tensoativos/toxicidade , Células Vero , Replicação Viral/efeitos dos fármacos
4.
Sci Rep ; 11(1): 12310, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112877

RESUMO

The novel SARS-CoV-2 virus emerged in December 2019 and has few effective treatments. We applied a computational drug repositioning pipeline to SARS-CoV-2 differential gene expression signatures derived from publicly available data. We utilized three independent published studies to acquire or generate lists of differentially expressed genes between control and SARS-CoV-2-infected samples. Using a rank-based pattern matching strategy based on the Kolmogorov-Smirnov Statistic, the signatures were queried against drug profiles from Connectivity Map (CMap). We validated 16 of our top predicted hits in live SARS-CoV-2 antiviral assays in either Calu-3 or 293T-ACE2 cells. Validation experiments in human cell lines showed that 11 of the 16 compounds tested to date (including clofazimine, haloperidol and others) had measurable antiviral activity against SARS-CoV-2. These initial results are encouraging as we continue to work towards a further analysis of these predicted drugs as potential therapeutics for the treatment of COVID-19.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Reposicionamento de Medicamentos/métodos , SARS-CoV-2/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , COVID-19/genética , Biologia Computacional/métodos , Humanos , SARS-CoV-2/fisiologia
5.
medRxiv ; 2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34075384

RESUMO

Plitidepsin is a marine-derived cyclic-peptide that inhibits SARS-CoV-2 replication at low nanomolar concentrations by the targeting of host protein eEF1A (eukaryotic translation-elongation-factor-1A). We evaluated a model of intervention with plitidepsin in hospitalized COVID-19 adult patients where three doses were assessed (1.5, 2 and 2.5 mg/day for 3 days, as a 90-minute intravenous infusion) in 45 patients (15 per dose-cohort). Treatment was well tolerated, with only two Grade 3 treatment-related adverse events observed (hypersensitivity and diarrhea). The discharge rates by Days 8 and 15 were 56.8% and 81.8%, respectively, with data sustaining dose-effect. A mean 4.2 log10 viral load reduction was attained by Day 15. Improvement in inflammation markers was also noted in a seemingly dose-dependent manner. These results suggest that plitidepsin impacts the outcome of patients with COVID-19. One-Sentence Summary: Plitidepsin, an inhibitor of SARS-Cov-2 in vitro , is safe and positively influences the outcome of patients hospitalized with COVID-19.

6.
Res Sq ; 2021 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-33821262

RESUMO

The novel SARS-CoV-2 virus emerged in December 2019 and has few effective treatments. We applied a computational drug repositioning pipeline to SARS-CoV-2 differential gene expression signatures derived from publicly available data. We utilized three independent published studies to acquire or generate lists of differentially expressed genes between control and SARS-CoV-2-infected samples. Using a rank-based pattern matching strategy based on the Kolmogorov-Smirnov Statistic, the signatures were queried against drug profiles from Connectivity Map (CMap). We validated sixteen of our top predicted hits in live SARS-CoV-2 antiviral assays in either Calu-3 or 293T-ACE2 cells. Validation experiments in human cell lines showed that 11 of the 16 compounds tested to date (including clofazimine, haloperidol and others) had measurable antiviral activity against SARS-CoV-2. These initial results are encouraging as we continue to work towards a further analysis of these predicted drugs as potential therapeutics for the treatment of COVID-19.

7.
Front Pharmacol ; 12: 633680, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33833683

RESUMO

SARS-CoV-2 infection is required for COVID-19, but many signs and symptoms of COVID-19 differ from common acute viral diseases. SARS-CoV-2 infection is necessary but not sufficient for development of clinical COVID-19 disease. Currently, there are no approved pre- or post-exposure prophylactic COVID-19 medical countermeasures. Clinical data suggest that famotidine may mitigate COVID-19 disease, but both mechanism of action and rationale for dose selection remain obscure. We have investigated several plausible hypotheses for famotidine activity including antiviral and host-mediated mechanisms of action. We propose that the principal mechanism of action of famotidine for relieving COVID-19 symptoms involves on-target histamine receptor H2 activity, and that development of clinical COVID-19 involves dysfunctional mast cell activation and histamine release. Based on these findings and associated hypothesis, new COVID-19 multi-drug treatment strategies based on repurposing well-characterized drugs are being developed and clinically tested, and many of these drugs are available worldwide in inexpensive generic oral forms suitable for both outpatient and inpatient treatment of COVID-19 disease.

8.
bioRxiv ; 2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33791693

RESUMO

Repurposing drugs as treatments for COVID-19 has drawn much attention. A common strategy has been to screen for established drugs, typically developed for other indications, that are antiviral in cells or organisms. Intriguingly, most of the drugs that have emerged from these campaigns, though diverse in structure, share a common physical property: cationic amphiphilicity. Provoked by the similarity of these repurposed drugs to those inducing phospholipidosis, a well-known drug side effect, we investigated phospholipidosis as a mechanism for antiviral activity. We tested 23 cationic amphiphilic drugs-including those from phenotypic screens and others that we ourselves had found-for induction of phospholipidosis in cell culture. We found that most of the repurposed drugs, which included hydroxychloroquine, azithromycin, amiodarone, and four others that have already progressed to clinical trials, induced phospholipidosis in the same concentration range as their antiviral activity; indeed, there was a strong monotonic correlation between antiviral efficacy and the magnitude of the phospholipidosis. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the gross physical properties of drugs, and does not reflect specific target-based activities, rather it may be considered a confound in early drug discovery. Understanding its role in infection, and detecting its effects rapidly, will allow the community to better distinguish between drugs and lead compounds that more directly impact COVID-19 from the large proportion of molecules that manifest this confounding effect, saving much time, effort and cost. One Sentence Summary: Drug-induced phospholipidosis is a single mechanism that may explain the in vitro efficacy of a wide-variety of therapeutics repurposed for COVID-19.

9.
Science ; 371(6532): 926-931, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33495306

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins interact with the eukaryotic translation machinery, and inhibitors of translation have potent antiviral effects. We found that the drug plitidepsin (aplidin), which has limited clinical approval, possesses antiviral activity (90% inhibitory concentration = 0.88 nM) that is more potent than remdesivir against SARS-CoV-2 in vitro by a factor of 27.5, with limited toxicity in cell culture. Through the use of a drug-resistant mutant, we show that the antiviral activity of plitidepsin against SARS-CoV-2 is mediated through inhibition of the known target eEF1A (eukaryotic translation elongation factor 1A). We demonstrate the in vivo efficacy of plitidepsin treatment in two mouse models of SARS-CoV-2 infection with a reduction of viral replication in the lungs by two orders of magnitude using prophylactic treatment. Our results indicate that plitidepsin is a promising therapeutic candidate for COVID-19.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Depsipeptídeos/farmacologia , Fator 1 de Elongação de Peptídeos/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/farmacologia , Alanina/uso terapêutico , Animais , Antivirais/uso terapêutico , COVID-19/prevenção & controle , COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/biossíntese , Proteínas do Nucleocapsídeo de Coronavírus/genética , Depsipeptídeos/administração & dosagem , Depsipeptídeos/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HEK293 , Humanos , Pulmão/virologia , Camundongos Endogâmicos C57BL , Mutação , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , RNA Viral/biossíntese , RNA Viral/genética , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Replicação Viral/efeitos dos fármacos
10.
bioRxiv ; 2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33501437

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths worldwide and massive societal and economic burden. Recently, a new variant of SARS-CoV-2, known as B.1.1.7, was first detected in the United Kingdom and is spreading in several other countries, heightening public health concern and raising questions as to the resulting effectiveness of vaccines and therapeutic interventions. We and others previously identified host-directed therapies with antiviral efficacy against SARS-CoV-2 infection. Less prone to the development of therapy resistance, host-directed drugs represent promising therapeutic options to combat emerging viral variants as host genes possess a lower propensity to mutate compared to viral genes. Here, in the first study of the full-length B.1.1.7 variant virus , we find two host-directed drugs, plitidepsin (aplidin; inhibits translation elongation factor eEF1A) and ralimetinib (inhibits p38 MAP kinase cascade), as well as remdesivir, to possess similar antiviral activity against both the early-lineage SARS-CoV-2 and the B.1.1.7 variant, evaluated in both human gastrointestinal and lung epithelial cell lines. We find that plitidepsin is over an order of magnitude more potent than remdesivir against both viruses. These results highlight the importance of continued development of host-directed therapeutics to combat current and future coronavirus variant outbreaks.

11.
Science ; 370(6523): 1473-1479, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33154106

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryo-electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Afinidade de Anticorpos , Chlorocebus aethiops , Microscopia Crioeletrônica , Humanos , Testes de Neutralização , Ligação Proteica , Estabilidade Proteica , Anticorpos de Domínio Único/química , Glicoproteína da Espícula de Coronavírus/química , Células Vero
12.
medRxiv ; 2020 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-33200140

RESUMO

COVID-19 affects multiple organs. Clinical data from the Mount Sinai Health System shows that substantial numbers of COVID-19 patients without prior heart disease develop cardiac dysfunction. How COVID-19 patients develop cardiac disease is not known. We integrate cell biological and physiological analyses of human cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) infected with SARS-CoV-2 in the presence of interleukins, with clinical findings, to investigate plausible mechanisms of cardiac disease in COVID-19 patients. We infected hiPSC-derived cardiomyocytes, from healthy human subjects, with SARS-CoV-2 in the absence and presence of interleukins. We find that interleukin treatment and infection results in disorganization of myofibrils, extracellular release of troponin-I, and reduced and erratic beating. Although interleukins do not increase the extent, they increase the severity of viral infection of cardiomyocytes resulting in cessation of beating. Clinical data from hospitalized patients from the Mount Sinai Health system show that a significant portion of COVID-19 patients without prior history of heart disease, have elevated troponin and interleukin levels. A substantial subset of these patients showed reduced left ventricular function by echocardiography. Our laboratory observations, combined with the clinical data, indicate that direct effects on cardiomyocytes by interleukins and SARS-CoV-2 infection can underlie the heart disease in COVID-19 patients. One Sentence Summary: Cardiomyocytes derived from human induced pluripotent stem cells treated with interleukins and infected with SARS-CoV-2 in cultures, show increased release of troponin, disorganization of myofibrils, and changes in beating mirroring specific pathologies in some COVID-19 patients.

13.
Proc Natl Acad Sci U S A ; 117(45): 28344-28354, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33097660

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.


Assuntos
COVID-19/metabolismo , Interferons/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/metabolismo , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Sítios de Ligação , Chlorocebus aethiops , Células HEK293 , Humanos , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ligação Proteica , Transdução de Sinais , Células Vero
14.
bioRxiv ; 2020 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-32817938

RESUMO

Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.

15.
Nature ; 586(7827): 113-119, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32707573

RESUMO

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19)1. The development of a vaccine is likely to take at least 12-18 months, and the typical timeline for approval of a new antiviral therapeutic agent can exceed 10 years. Thus, repurposing of known drugs could substantially accelerate the deployment of new therapies for COVID-19. Here we profiled a library of drugs encompassing approximately 12,000 clinical-stage or Food and Drug Administration (FDA)-approved small molecules to identify candidate therapeutic drugs for COVID-19. We report the identification of 100 molecules that inhibit viral replication of SARS-CoV-2, including 21 drugs that exhibit dose-response relationships. Of these, thirteen were found to harbour effective concentrations commensurate with probable achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod2-4 and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825 and ONO 5334. Notably, MDL-28170, ONO 5334 and apilimod were found to antagonize viral replication in human pneumocyte-like cells derived from induced pluripotent stem cells, and apilimod also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, their known pharmacological and human safety profiles will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19.


Assuntos
Antivirais/análise , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/efeitos dos fármacos , Betacoronavirus/crescimento & desenvolvimento , COVID-19 , Linhagem Celular , Inibidores de Cisteína Proteinase/análise , Inibidores de Cisteína Proteinase/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrazonas , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Biológicos , Morfolinas/análise , Morfolinas/farmacologia , Pandemias , Pirimidinas , Reprodutibilidade dos Testes , SARS-CoV-2 , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Triazinas/análise , Triazinas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
16.
Res Sq ; 2020 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-32702719

RESUMO

SARS-CoV-2 infection is required for COVID-19, but many signs and symptoms of COVID-19 differ from common acute viral diseases. Currently, there are no pre- or post-exposure prophylactic COVID-19 medical countermeasures. Clinical data suggest that famotidine may mitigate COVID-19 disease, but both mechanism of action and rationale for dose selection remain obscure. We explore several plausible avenues of activity including antiviral and host-mediated actions. We propose that the principal famotidine mechanism of action for COVID-19 involves on-target histamine receptor H 2 activity, and that development of clinical COVID-19 involves dysfunctional mast cell activation and histamine release.

17.
Cell ; 182(3): 685-712.e19, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32645325

RESUMO

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Pneumonia Viral/metabolismo , Proteômica/métodos , Células A549 , Enzima de Conversão de Angiotensina 2 , Animais , Antivirais/farmacologia , COVID-19 , Células CACO-2 , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Fosforilação , Pneumonia Viral/virologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
18.
Curr Protoc Microbiol ; 58(1): e108, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32585083

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the city of Wuhan, Hubei Province, China, in late 2019. Since then, the virus has spread globally and caused a pandemic. Assays that can measure the antiviral activity of antibodies or antiviral compounds are needed for SARS-CoV-2 vaccine and drug development. Here, we describe in detail a microneutralization assay, which can be used to assess in a quantitative manner if antibodies or drugs can block entry and/or replication of SARS-CoV-2 in vitro. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Microneutralization assay to test inhibition of virus by antibodies (purified antibodies or serum/plasma) Basic Protocol 2: Screening of anti-SARS-CoV-2 compounds in vitro Support Protocol: SARS-CoV-2 propagation.


Assuntos
Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Avaliação Pré-Clínica de Medicamentos/métodos , Testes de Neutralização/métodos , Animais , COVID-19 , Chlorocebus aethiops , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Humanos , Camundongos , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , SARS-CoV-2 , Células Vero , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
19.
bioRxiv ; 2020 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-32511357

RESUMO

The emergence of novel SARS coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). To date, more than 2.1 million confirmed cases and 139,500 deaths have been reported worldwide, and there are currently no medical countermeasures available to prevent or treat the disease. As the development of a vaccine could require at least 12-18 months, and the typical timeline from hit finding to drug registration of an antiviral is >10 years, repositioning of known drugs can significantly accelerate the development and deployment of therapies for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we profiled a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules. Here, we report the identification of 30 known drugs that inhibit viral replication. Of these, six were characterized for cellular dose-activity relationships, and showed effective concentrations likely to be commensurate with therapeutic doses in patients. These include the PIKfyve kinase inhibitor Apilimod, cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334, and the CCR1 antagonist MLN-3897. Since many of these molecules have advanced into the clinic, the known pharmacological and human safety profiles of these compounds will accelerate their preclinical and clinical evaluation for COVID-19 treatment.

20.
Nature ; 583(7816): 459-468, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32353859

RESUMO

A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein-protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/metabolismo , Reposicionamento de Medicamentos , Terapia de Alvo Molecular , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/metabolismo , Mapas de Interação de Proteínas , Proteínas Virais/metabolismo , Animais , Antivirais/classificação , Antivirais/farmacologia , Betacoronavirus/genética , Betacoronavirus/metabolismo , Betacoronavirus/patogenicidade , COVID-19 , Chlorocebus aethiops , Clonagem Molecular , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Imunidade Inata , Espectrometria de Massas , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Domínios Proteicos , Mapeamento de Interação de Proteínas , Receptores sigma/metabolismo , SARS-CoV-2 , Proteínas Ligases SKP Culina F-Box/metabolismo , Células Vero , Proteínas Virais/genética
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