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1.
Phys Chem Chem Phys ; 23(27): 14873-14888, 2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34223589

RESUMO

The COVID-19 disease caused by the virus SARS-CoV-2, first detected in December 2019, is still emerging through virus mutations. Although almost under control in some countries due to effective vaccines that are mitigating the worldwide pandemic, the urgency to develop additional vaccines and therapeutic treatments is imperative. In this work, the natural polyphenols corilagin and 1,3,6-tri-O-galloy-ß-d-glucose (TGG) are investigated to determine the structural basis of inhibitor interactions as potential candidates to inhibit SARS-CoV-2 viral entry into target cells. First, the therapeutic potential of the ligands are assessed on the ACE2/wild-type RBD. We first use molecular docking followed by molecular dynamics, to take into account the conformational flexibility that plays a significant role in ligand binding and that cannot be captured using only docking, and then analyze more precisely the affinity of these ligands using MMPBSA binding free energy. We show that both ligands bind to the ACE2/wild-type RBD interface with good affinities which might prevent the ACE2/RBD association. Second, we confirm the potency of these ligands to block the ACE2/RBD association using a combination of surface plasmon resonance and biochemical inhibition assays. These experiments confirm that TGG and, to a lesser extent, corilagin, inhibit the binding of RBD to ACE2. Both experiments and simulations show that the ligands interact preferentially with RBD, while weak binding is observed with ACE2, hence, avoiding potential physiological side-effects induced by the inhibition of ACE2. In addition to the wild-type RBD, we also study numerically three RBD mutations (E484K, N501Y and E484K/N501Y) found in the main SARS-CoV-2 variants of concerns. We find that corilagin could be as effective for RBD/E484K but less effective for the RBD/N501Y and RBD/E484K-N501Y mutants, while TGG strongly binds at relevant locations to all three mutants, demonstrating the significant interest of these molecules as potential inhibitors for variants of SARS-CoV-2.


Assuntos
Antivirais/química , Ácido Gálico/análogos & derivados , Glucose/análogos & derivados , Glucosídeos/química , Taninos Hidrolisáveis/química , SARS-CoV-2/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Sítios de Ligação , Ácido Gálico/química , Glucose/química , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas/genética , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus/efeitos dos fármacos
2.
PLoS Med ; 18(7): e1003660, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34228712

RESUMO

BACKGROUND: Development of an effective antiviral drug for Coronavirus Disease 2019 (COVID-19) is a global health priority. Although several candidate drugs have been identified through in vitro and in vivo models, consistent and compelling evidence from clinical studies is limited. The lack of evidence from clinical trials may stem in part from the imperfect design of the trials. We investigated how clinical trials for antivirals need to be designed, especially focusing on the sample size in randomized controlled trials. METHODS AND FINDINGS: A modeling study was conducted to help understand the reasons behind inconsistent clinical trial findings and to design better clinical trials. We first analyzed longitudinal viral load data for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) without antiviral treatment by use of a within-host virus dynamics model. The fitted viral load was categorized into 3 different groups by a clustering approach. Comparison of the estimated parameters showed that the 3 distinct groups were characterized by different virus decay rates (p-value < 0.001). The mean decay rates were 1.17 d-1 (95% CI: 1.06 to 1.27 d-1), 0.777 d-1 (0.716 to 0.838 d-1), and 0.450 d-1 (0.378 to 0.522 d-1) for the 3 groups, respectively. Such heterogeneity in virus dynamics could be a confounding variable if it is associated with treatment allocation in compassionate use programs (i.e., observational studies). Subsequently, we mimicked randomized controlled trials of antivirals by simulation. An antiviral effect causing a 95% to 99% reduction in viral replication was added to the model. To be realistic, we assumed that randomization and treatment are initiated with some time lag after symptom onset. Using the duration of virus shedding as an outcome, the sample size to detect a statistically significant mean difference between the treatment and placebo groups (1:1 allocation) was 13,603 and 11,670 (when the antiviral effect was 95% and 99%, respectively) per group if all patients are enrolled regardless of timing of randomization. The sample size was reduced to 584 and 458 (when the antiviral effect was 95% and 99%, respectively) if only patients who are treated within 1 day of symptom onset are enrolled. We confirmed the sample size was similarly reduced when using cumulative viral load in log scale as an outcome. We used a conventional virus dynamics model, which may not fully reflect the detailed mechanisms of viral dynamics of SARS-CoV-2. The model needs to be calibrated in terms of both parameter settings and model structure, which would yield more reliable sample size calculation. CONCLUSIONS: In this study, we found that estimated association in observational studies can be biased due to large heterogeneity in viral dynamics among infected individuals, and statistically significant effect in randomized controlled trials may be difficult to be detected due to small sample size. The sample size can be dramatically reduced by recruiting patients immediately after developing symptoms. We believe this is the first study investigated the study design of clinical trials for antiviral treatment using the viral dynamics model.


Assuntos
Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Ensaios Clínicos Controlados Aleatórios como Assunto , Tamanho da Amostra , Humanos , Modelos Biológicos , SARS-CoV-2 , Resultado do Tratamento , Carga Viral , Replicação Viral , Eliminação de Partículas Virais
3.
C R Biol ; 344(2): 145-156, 2021 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-34213852

RESUMO

Pregnancy complications occur frequently and are particularly prevalent during the first trimester. They are caused by a multitude of factors, including karyotypic, genetic or environmental conditions, congenital infections and inflammation. The molecular mechanisms leading to placental complications under inflammatory conditions remain unclear. In this review, we discuss how uncontrolled inflammation, triggered by viral infections or other diseases can lead to placental complications. We first highlight the importance of syncytins, ancestral retroviral genes co-opted by mammals including humans, millions of years ago for the process of placenta formation. We then focus on recent advances elucidating how interferon-induced transmembrane (IFITM) proteins, antiviral proteins rendering cells refractory to viral infections, interfere with placental development.


Assuntos
Interferons , Complicações na Gravidez , Animais , Antivirais , Feminino , Humanos , Placenta , Placentação , Gravidez
4.
Acta Gastroenterol Belg ; 84(2): 311-316, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34217181

RESUMO

Background: Hepatitis C is a viral infection caused by the hepatitis C virus (HCV) with people who inject drugs as the main group at risk worldwide. Aim: This study investigated the differences in uptake for HCV screening and treatment between persons in opioid substitution therapy (OST) and the other members of the Christian Health Insurance Fund in Belgium. Methods: Invoice data were retrospectively collected from the Christian Health Insurance Fund, representing 42% of the healthcare users. Information on demographics, screening, diagnostic tests, treatment and disease progression was obtained from 2008 till 2013. All people in this study were aged 20-65 year. Persons in the OST group were identified as having at least one prescription reimbursed for methadone. This group was compared to the other members of the Insurance Fund not on OST (NOST). Results: The Insurance Fund registered 8,409 unique OST and 3,525,190 members in the general group. HCV RNA screening rate was higher in the OST group after correction for age and gender (4.3% vs. 0.2%). Ribavirin reimbursement, did not differ between the OST and NOST group screened for HCV RNA (16.9% vs. 14.4%), though the probability of having ribavirin reimbursed was smaller for females than for males. Procedures concerning disease progression were reimbursed less frequently in the HCV RNA screened OST group compared to the NOST group (0.3% vs. 1.2%). Conclusion: People on OST were screened more often for HCV RNA. However, the general uptake for HCV screening and treatment in both populations remained suboptimal.


Assuntos
Hepatite C , Abuso de Substâncias por Via Intravenosa , Antivirais/uso terapêutico , Bélgica , Feminino , Hepacivirus , Hepatite C/tratamento farmacológico , Humanos , Masculino , Tratamento de Substituição de Opiáceos , Estudos Retrospectivos
5.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204256

RESUMO

The marine carotenoids fucoxanthin and siphonaxanthin are powerful antioxidants that are attracting focused attention to identify a variety of health benefits and industry applications. In this study, the binding energy of these carotenoids with the SARS-CoV-2 Spike-glycoprotein was predicted by molecular docking simulation, and their inhibitory activity was confirmed with SARS-CoV-2 pseudovirus on HEK293 cells overexpressing angiotensin-converting enzyme 2 (ACE2). Siphonaxanthin from Codium fragile showed significant antiviral activity with an IC50 of 87.4 µM against SARS-CoV-2 pseudovirus entry, while fucoxanthin from Undaria pinnatifida sporophyll did not. The acute toxicities were predicted to be relatively low, and pharmacokinetic predictions indicate GI absorption. Although further studies are needed to elucidate the inhibition of viral infection by siphonaxanthin, these results provide useful information in the application of these marine carotenoids for the treatment and prevention of COVID-19.


Assuntos
Antivirais/farmacologia , SARS-CoV-2/fisiologia , Internalização do Vírus/efeitos dos fármacos , Xantofilas/química , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Antivirais/química , Antivirais/uso terapêutico , Sítios de Ligação , COVID-19/tratamento farmacológico , COVID-19/patologia , COVID-19/virologia , Sobrevivência Celular/efeitos dos fármacos , Clorófitas/química , Clorófitas/metabolismo , Células HEK293 , Meia-Vida , Humanos , Simulação de Acoplamento Molecular , Feófitas/química , Feófitas/metabolismo , Ratos , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Xantofilas/metabolismo , Xantofilas/farmacologia , Xantofilas/uso terapêutico
6.
Molecules ; 26(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208050

RESUMO

Potential effects of tea and its constituents on SARS-CoV-2 infection were assessed in vitro. Infectivity of SARS-CoV-2 was decreased to 1/100 to undetectable levels after a treatment with black tea, green tea, roasted green tea, or oolong tea for 1 min. An addition of (-) epigallocatechin gallate (EGCG) significantly inactivated SARS-CoV-2, while the same concentration of theasinensin A (TSA) and galloylated theaflavins including theaflavin 3,3'-di-O-gallate (TFDG) had more remarkable anti-viral activities. EGCG, TSA, and TFDG at 1 mM, 40 µM, and 60 µM, respectively, which are comparable to the concentrations of these compounds in tea beverages, significantly reduced infectivity of the virus, viral RNA replication in cells, and secondary virus production from the cells. EGCG, TSA, and TFDG significantly inhibited interaction between recombinant ACE2 and RBD of S protein. These results suggest potential usefulness of tea in prevention of person-to-person transmission of the novel coronavirus.


Assuntos
Antivirais/farmacologia , Biflavonoides/química , Catequina/química , Ácido Gálico/análogos & derivados , SARS-CoV-2/fisiologia , Chá/química , Replicação Viral/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Antivirais/química , Biflavonoides/farmacologia , COVID-19/patologia , COVID-19/virologia , Catequina/análogos & derivados , Catequina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Ácido Gálico/química , Ácido Gálico/farmacologia , Humanos , Mapas de Interação de Proteínas/efeitos dos fármacos , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Chá/metabolismo , Células Vero
7.
Molecules ; 26(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207756

RESUMO

Since December 2019, humanity has faced an important global threat. Many studies have been published on the origin, structure, and mechanism of action of the SARS-CoV-2 virus and the treatment of its disease. The priority of scientists all over the world has been to direct their time to research this subject. In this review, we highlight chemical studies and therapeutic approaches to overcome COVID-19 with seven different sections. These sections are the structure and mechanism of action of SARS-CoV-2, immunotherapy and vaccine, computer-aided drug design, repurposing therapeutics for COVID-19, synthesis of new molecular structures against COVID-19, food safety/security and functional food components, and potential natural products against COVID-19. In this work, we aimed to screen all the newly synthesized compounds, repurposing chemicals covering antiviral, anti-inflammatory, antibacterial, antiparasitic, anticancer, antipsychotic, and antihistamine compounds against COVID-19. We also highlight computer-aided approaches to develop an anti-COVID-19 molecule. We explain that some phytochemicals and dietary supplements have been identified as antiviral bioproducts, which have almost been successfully tested against COVID-19. In addition, we present immunotherapy types, targets, immunotherapy and inflammation/mutations of the virus, immune response, and vaccine issues.


Assuntos
COVID-19/prevenção & controle , Antivirais/química , Antivirais/uso terapêutico , Produtos Biológicos/uso terapêutico , COVID-19/patologia , COVID-19/terapia , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/imunologia , Síndrome da Liberação de Citocina/prevenção & controle , Desenho de Fármacos , Reposicionamento de Medicamentos , Humanos , Imunoterapia , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/fisiologia
8.
C R Biol ; 344(1): 27-42, 2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-34213847

RESUMO

This review provides an update on the different therapeutic approaches that have been used to treat SARS-CoV-2 infection, as well as, the resulting paradoxical inflammation disorders.


Assuntos
COVID-19 , Preparações Farmacêuticas , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Humanos , SARS-CoV-2
9.
BMC Infect Dis ; 21(1): 635, 2021 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215210

RESUMO

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has changed our lives. The scientific community has been investigating re-purposed treatments to prevent disease progression in coronavirus disease (COVID-19) patients. OBJECTIVE: To determine whether ivermectin treatment can prevent hospitalization in individuals with early COVID-19. DESIGN, SETTING AND PARTICIPANTS: A randomized, double-blind, placebo-controlled study was conducted in non-hospitalized individuals with COVID-19 in Corrientes, Argentina. Patients with SARS-CoV-2 positive nasal swabs were contacted within 48 h by telephone to invite them to participate. The trial randomized 501 patients between August 19th 2020 and February 22nd 2021. INTERVENTION: Patients were randomized to ivermectin (N = 250) or placebo (N = 251) arms in a staggered dose, according to the patient's weight, for 2 days. MAIN OUTCOMES AND MEASURES: The efficacy of ivermectin to prevent hospitalizations was evaluated as primary outcome. We evaluated secondary outcomes in relationship to safety and other efficacy end points. RESULTS: The mean age was 42 years (SD ± 15.5) and the median time since symptom onset to the inclusion was 4 days [interquartile range 3-6]. The primary outcome of hospitalization was met in 14/250 (5.6%) individuals in ivermectin group and 21/251 (8.4%) in placebo group (odds ratio 0.65; 95% confidence interval, 0.32-1.31; p = 0.227). Time to hospitalization was not statistically different between groups. The mean time from study enrollment to invasive mechanical ventilatory support (MVS) was 5.25 days (SD ± 1.71) in ivermectin group and 10 days (SD ± 2) in placebo group, (p = 0.019). There were no statistically significant differences in the other secondary outcomes including polymerase chain reaction test negativity and safety outcomes. LIMITATIONS: Low percentage of hospitalization events, dose of ivermectin and not including only high-risk population. CONCLUSION: Ivermectin had no significant effect on preventing hospitalization of patients with COVID-19. Patients who received ivermectin required invasive MVS earlier in their treatment. No significant differences were observed in any of the other secondary outcomes. TRIAL REGISTRATION: ClinicalTrials.gov NCT04529525 .


Assuntos
Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Ivermectina/uso terapêutico , Adulto , Antivirais/efeitos adversos , COVID-19/etiologia , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19 , Método Duplo-Cego , Feminino , Hospitalização , Humanos , Ivermectina/efeitos adversos , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Placebos , Resultado do Tratamento
11.
BMJ Case Rep ; 14(7)2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226250

RESUMO

A 44-year-old man with a history of renal transplantation presented with right lower abdominal wall swelling, redness and pain. A bacterial abscess was drained, and he was discharged home with oral antibiotics. After failing to improve, he returned to the hospital, where he was briefly treated with intravenous antibiotics and discharged home again. The patient returned 5 days later, reporting worsening right groin swelling that extended into the ipsilateral scrotum. Imaging revealed a persistent fluid collection in the region, and he was taken for surgical debridement. Tissue immunochemistry and histopathological evaluation identified cytomegalovirus infection. Plasma quantitative PCR for cytomegalovirus demonstrated high viraemia. The patient was successfully treated with intravenous ganciclovir, followed by oral valganciclovir, with resolution of the skin changes. Persistent hydrocele with epididymitis on imaging suggests that this process may have been the source of the cutaneous cytomegalovirus infection.


Assuntos
Infecções por Citomegalovirus , Transplante de Rim , Dermatopatias , Adulto , Antivirais/uso terapêutico , Citomegalovirus , Infecções por Citomegalovirus/tratamento farmacológico , Ganciclovir/uso terapêutico , Humanos , Transplante de Rim/efeitos adversos , Masculino , Dermatopatias/tratamento farmacológico
12.
Biochem J ; 478(13): 2533-2535, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198320

RESUMO

In response to the COVID-19 pandemic, we began a project in March 2020 to identify small molecule inhibitors of SARS-CoV-2 enzymes from a library of chemical compounds containing many established pharmaceuticals. Our hope was that inhibitors we found might slow the replication of the SARS-CoV-2 virus in cells and ultimately be useful in the treatment of COVID-19. The seven accompanying manuscripts describe the results of these chemical screens. This overview summarises the main highlights from these screens and discusses the implications of our results and how our results might be exploited in future.


Assuntos
Antivirais/química , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Ensaios Enzimáticos , Humanos , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Proteínas não Estruturais Virais/metabolismo
13.
Biochem J ; 478(13): 2399-2403, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198321

RESUMO

The coronavirus pandemic has had a huge impact on public health with over 165 million people infected, 3.4 million deaths and a hugely deleterious effect on most economies. While vaccination effectively protects against the disease it is likely that viruses will evolve that can replicate in hosts immunised with the present vaccines. Thus, there is a great unmet need for effective antivirals that can block the development of serious disease in infected patients. The seven papers published in this issue of the Biochemical Journal address this need by expressing and purifying components required for viral replication, developing biochemical assays for these components and using the assays to screen a library of pre-existing pharmaceuticals for drugs that inhibited the target in vitro and inhibited viral replication in cell culture. The candidate drugs obtained are potential antivirals that may protect against SARS-CoV-2 infection. While not all the antiviral candidates will make it through to the clinic, they will be useful tool compounds and can act as the starting point for further drug discovery programmes.


Assuntos
Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/virologia , Avaliação Pré-Clínica de Medicamentos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/crescimento & desenvolvimento , Replicação Viral/efeitos dos fármacos , Humanos , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
14.
Biochem J ; 478(13): 2405-2423, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198322

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here, we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterized pharmaceuticals for nsp13 inhibitors using a fluorescence resonance energy transfer-based high-throughput screening approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.


Assuntos
Antivirais/química , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , RNA Helicases/antagonistas & inibidores , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Chlorocebus aethiops , Ensaios Enzimáticos , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , RNA Helicases/metabolismo , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Suramina/farmacologia , Células Vero , Proteínas não Estruturais Virais/metabolismo
15.
Biochem J ; 478(13): 2425-2443, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198323

RESUMO

The coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication-transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologues in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified three novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.


Assuntos
Antivirais/química , Antivirais/farmacologia , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Benzoatos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Chlorocebus aethiops , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Ensaios Enzimáticos , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Holoenzimas/metabolismo , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Suramina/farmacologia , Células Vero , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
16.
Biochem J ; 478(13): 2465-2479, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198324

RESUMO

SARS-CoV-2 is responsible for COVID-19, a human disease that has caused over 2 million deaths, stretched health systems to near-breaking point and endangered economies of countries and families around the world. Antiviral treatments to combat COVID-19 are currently lacking. Remdesivir, the only antiviral drug approved for the treatment of COVID-19, can affect disease severity, but better treatments are needed. SARS-CoV-2 encodes 16 non-structural proteins (nsp) that possess different enzymatic activities with important roles in viral genome replication, transcription and host immune evasion. One key aspect of host immune evasion is performed by the uridine-directed endoribonuclease activity of nsp15. Here we describe the expression and purification of nsp15 recombinant protein. We have developed biochemical assays to follow its activity, and we have found evidence for allosteric behaviour. We screened a custom chemical library of over 5000 compounds to identify nsp15 endoribonuclease inhibitors, and we identified and validated NSC95397 as an inhibitor of nsp15 endoribonuclease in vitro. Although NSC95397 did not inhibit SARS-CoV-2 growth in VERO E6 cells, further studies will be required to determine the effect of nsp15 inhibition on host immune evasion.


Assuntos
Antivirais/química , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Endorribonucleases/antagonistas & inibidores , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Regulação Alostérica , Animais , Chlorocebus aethiops , Endorribonucleases/isolamento & purificação , Endorribonucleases/metabolismo , Ensaios Enzimáticos , Fluorescência , Ensaios de Triagem em Larga Escala , Técnicas In Vitro , Cinética , Naftoquinonas/farmacologia , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/crescimento & desenvolvimento , Bibliotecas de Moléculas Pequenas/química , Soluções , Células Vero , Proteínas não Estruturais Virais/isolamento & purificação , Proteínas não Estruturais Virais/metabolismo
17.
Biochem J ; 478(13): 2517-2531, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198325

RESUMO

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom compound library from which we identified dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.


Assuntos
Antivirais/química , Antivirais/farmacologia , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Compostos de Anilina/farmacologia , Animais , Benzamidas/farmacologia , Chlorocebus aethiops , Proteases Semelhantes à Papaína de Coronavírus/genética , Proteases Semelhantes à Papaína de Coronavírus/isolamento & purificação , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Sinergismo Farmacológico , Ensaios Enzimáticos , Flavinas/farmacologia , Transferência Ressonante de Energia de Fluorescência , Furanos/farmacologia , Ensaios de Triagem em Larga Escala , Concentração Inibidora 50 , Naftalenos/farmacologia , Fenantrenos/farmacologia , Quinonas/farmacologia , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/crescimento & desenvolvimento , Bibliotecas de Moléculas Pequenas/química , Células Vero , Replicação Viral/efeitos dos fármacos
18.
Biochem J ; 478(13): 2445-2464, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198326

RESUMO

SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is an urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication. SARS-CoV-2 proliferation relies on the enzymatic activities of the non-structural proteins (nsp), which makes them interesting targets for the development of new antiviral treatments. With the aim to identify novel SARS-CoV-2 antivirals, we have purified the exoribonuclease/methyltransferase (nsp14) and its cofactor (nsp10) and developed biochemical assays compatible with high-throughput approaches to screen for exoribonuclease inhibitors. We have screened a library of over 5000 commercial compounds and identified patulin and aurintricarboxylic acid (ATA) as inhibitors of nsp14 exoribonuclease in vitro. We found that patulin and ATA inhibit replication of SARS-CoV-2 in a VERO E6 cell-culture model. These two new antiviral compounds will be valuable tools for further coronavirus research as well as potentially contributing to new therapeutic opportunities for COVID-19.


Assuntos
Antivirais/química , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Exorribonucleases/antagonistas & inibidores , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Animais , Ácido Aurintricarboxílico/farmacologia , Chlorocebus aethiops , Ensaios Enzimáticos , Exorribonucleases/metabolismo , Fluorescência , Ensaios de Triagem em Larga Escala , Patulina/farmacologia , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Células Vero , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo
19.
Biochem J ; 478(13): 2499-2515, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198327

RESUMO

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.


Assuntos
Antivirais/química , Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Azóis/farmacologia , Chlorocebus aethiops , Proteases 3C de Coronavírus/genética , Proteases 3C de Coronavírus/isolamento & purificação , Proteases 3C de Coronavírus/metabolismo , Ensaios Enzimáticos , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Leupeptinas/farmacologia , Compostos Organosselênicos/farmacologia , Peptidomiméticos , Proteínas de Ligação a RNA/metabolismo , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Células Vero , Proteínas não Estruturais Virais/metabolismo
20.
Biochem J ; 478(13): 2481-2497, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198328

RESUMO

The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2'-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir.


Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Exorribonucleases/antagonistas & inibidores , Metiltransferases/antagonistas & inibidores , Capuzes de RNA/metabolismo , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Antivirais/química , Clorobenzenos/farmacologia , Chlorocebus aethiops , Ensaios Enzimáticos , Exorribonucleases/genética , Exorribonucleases/isolamento & purificação , Exorribonucleases/metabolismo , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Indazóis/farmacologia , Indenos/farmacologia , Indóis/farmacologia , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Metiltransferases/metabolismo , Nitrilas/farmacologia , Fenotiazinas/farmacologia , Purinas/farmacologia , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Especificidade por Substrato , Trifluperidol/farmacologia , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/isolamento & purificação , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/isolamento & purificação , Proteínas Virais Reguladoras e Acessórias/metabolismo
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