RESUMEN
Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target-inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.
Asunto(s)
Antivirales/farmacología , Proteasas 3C de Coronavirus/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , SARS-CoV-2/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antivirales/metabolismo , Antivirales/farmacocinética , Dominio Catalítico , Chlorocebus aethiops , Proteasas 3C de Coronavirus/química , Inhibidores de Cisteína Proteinasa/metabolismo , Inhibidores de Cisteína Proteinasa/farmacocinética , Evaluación Preclínica de Medicamentos , Humanos , Pruebas de Sensibilidad Microbiana , Microsomas Hepáticos/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , SARS-CoV-2/enzimología , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacocinética , Células VeroRESUMEN
We assessed the in vitro antiviral activity of remdesivir and its parent nucleoside GS-441524, molnupiravir and its parent nucleoside EIDD-1931 and the viral protease inhibitor nirmatrelvir against the ancestral SARS-CoV2 strain and the five variants of concern including Omicron. VeroE6-GFP cells were pre-treated overnight with serial dilutions of the compounds before infection. The GFP signal was determined by high-content imaging on day 4 post-infection. All molecules have equipotent antiviral activity against the ancestral virus and the VOCs Alpha, Beta, Gamma, Delta and Omicron. These findings are in line with the observation that the target proteins of these antivirals (respectively the viral RNA dependent RNA polymerase and the viral main protease Mpro) are highly conserved.
Asunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/uso terapéutico , Tratamiento Farmacológico de COVID-19 , Citidina/análogos & derivados , Hidroxilaminas/uso terapéutico , Lactamas/uso terapéutico , Leucina/uso terapéutico , Nitrilos/uso terapéutico , Prolina/uso terapéutico , SARS-CoV-2/efectos de los fármacos , Adenosina/análogos & derivados , Adenosina/uso terapéutico , Adenosina Monofosfato/uso terapéutico , Alanina/uso terapéutico , Animales , Línea Celular , Chlorocebus aethiops , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Citidina/uso terapéutico , Humanos , Pruebas de Sensibilidad Microbiana , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
There are, besides remdesivir, no approved antivirals for the treatment of SARS-CoV-2 infections. To aid in the search for antivirals against this virus, we explored the use of human tracheal airway epithelial cells (HtAEC) and human small airway epithelial cells (HsAEC) grown at the air-liquid interface (ALI). These cultures were infected at the apical side with one of two different SARS-CoV-2 isolates. Each virus was shown to replicate to high titers for extended periods of time (at least 8 days) and, in particular an isolate with the D614G in the spike (S) protein did so more efficiently at 35 °C than 37 °C. The effect of a selected panel of reference drugs that were added to the culture medium at the basolateral side of the system was explored. Remdesivir, GS-441524 (the parent nucleoside of remdesivir), EIDD-1931 (the parent nucleoside of molnupiravir) and IFN (ß1 and λ1) all resulted in dose-dependent inhibition of viral RNA and infectious virus titers collected at the apical side. However, AT-511 (the free base form of AT-527 currently in clinical testing) failed to inhibit viral replication in these in vitro primary cell models. Together, these results provide a reference for further studies aimed at selecting SARS-CoV-2 inhibitors for further preclinical and clinical development.
Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , SARS-CoV-2/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Línea Celular , Chlorocebus aethiops , Evaluación Preclínica de Medicamentos/métodos , Células Epiteliales/virología , Humanos , ARN Viral , SARS-CoV-2/aislamiento & purificación , Células VeroRESUMEN
BACKGROUND: The antifungal drug itraconazole exerts in vitro activity against SARS-CoV-2 in Vero and human Caco-2 cells. Preclinical and clinical studies are required to investigate if itraconazole is effective for the treatment and/or prevention of COVID-19. METHODS: Due to the initial absence of preclinical models, the effect of itraconazole was explored in a clinical, proof-of-concept, open-label, single-center study, in which hospitalized COVID-19 patients were randomly assigned to standard of care with or without itraconazole. Primary outcome was the cumulative score of the clinical status until day 15 based on the 7-point ordinal scale of the World Health Organization. In parallel, itraconazole was evaluated in a newly established hamster model of acute SARS-CoV-2 infection and transmission, as soon as the model was validated. FINDINGS: In the hamster acute infection model, itraconazole did not reduce viral load in lungs, stools or ileum, despite adequate plasma and lung drug concentrations. In the transmission model, itraconazole failed to prevent viral transmission. The clinical trial was prematurely discontinued after evaluation of the preclinical studies and because an interim analysis showed no signal for a more favorable outcome with itraconazole: mean cumulative score of the clinical status 49 vs 47, ratio of geometric means 1.01 (95% CI 0.85 to 1.19) for itraconazole vs standard of care. INTERPRETATION: Despite in vitro activity, itraconazole was not effective in a preclinical COVID-19 hamster model. This prompted the premature termination of the proof-of-concept clinical study. FUNDING: KU Leuven, Research Foundation - Flanders (FWO), Horizon 2020, Bill and Melinda Gates Foundation.
Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Itraconazol/farmacología , Animales , Antivirales/administración & dosificación , Antivirales/farmacocinética , Antivirales/uso terapéutico , COVID-19/etiología , COVID-19/transmisión , Chlorocebus aethiops , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Itraconazol/administración & dosificación , Itraconazol/farmacocinética , Itraconazol/uso terapéutico , Masculino , Mesocricetus , Persona de Mediana Edad , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/patología , Neumonía Viral/virología , Prueba de Estudio Conceptual , SARS-CoV-2/efectos de los fármacos , Resultado del Tratamiento , Células VeroRESUMEN
Objectives: We report the use of reconstituted 3D human airway epithelium cells (HuAECs) of bronchial origin in an air-liquid interface to study respiratory syncytial virus (RSV) infection and to assess the efficacy of RSV inhibitors in (pre-)clinical development. Methods: HuAECs were infected with RSV-A Long strain (0.01 CCID50/cell, where CCID50 represents 50% cell culture infectious dose in HEp2 cells) on the apical compartment of the culture. At the time of infection or at 1 or 3 days post-infection, selected inhibitors were added and refreshed daily on the basal compartment of the culture. Viral shedding was followed up by apical washes collected daily and quantifying viral RNA by RT-qPCR. Results: RSV-A replicates efficiently in HuAECs and viral RNA is shed for weeks after infection. RSV infection reduces the ciliary beat frequency of the ciliated cells as of 4 days post-infection, with complete ciliary dyskinesia observed by day 10. Treatment with RSV fusion inhibitors resulted in an antiviral effect only when added at the time of infection. In contrast, the use of replication inhibitors (both nucleoside and non-nucleoside) elicited a marked antiviral effect even when the start of treatment was delayed until 1 day or even 3 days after infection. Levels of the inflammation marker RANTES (mRNA) increased â¼200-fold in infected, untreated cultures (at 3 weeks post-infection), but levels were comparable to those of uninfected cultures in the presence of PC786, an RSV replication inhibitor, suggesting that an efficient antiviral treatment might inhibit virus-induced inflammation in this model. Conclusions: Overall, HuAECs offer a robust and physiologically relevant model to study RSV replication and to assess the efficacy of antiviral compounds.
Asunto(s)
Antivirales/farmacología , Mucosa Respiratoria/virología , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Benzamidas , Benzazepinas , Técnicas de Cultivo de Célula , Evaluación Preclínica de Medicamentos , Células Epiteliales/virología , Humanos , Técnicas de Cultivo de Órganos , ARN Viral/genética , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Virus Sincitial Respiratorio Humano/genética , Compuestos de Espiro/farmacologíaRESUMEN
The discovery of novel antivirals for HIV and HCV has been a focus of intensive research for many years. Where the inhibition of critical viral enzymes by small molecules has proven effective for many viruses, there is considerable merit in pursuing protein-protein interactions (PPIs) as targets for therapeutic intervention. The mammalian protein-protein interaction trap (MAPPIT) is a two-hybrid system used for the study of PPIs. The bait and prey proteins are linked to deficient cytokine receptor chimeras, where the bait and prey interaction and subsequent ligand stimulation restores JAK-STAT signaling, resulting in reporter gene expression controlled by a STAT3-responsive promoter. We report the use of MAPPIT as a high-throughput screening assay for the discovery of inhibitors or stimulators of the Vif-APOBEC3G interaction and the reverse transcriptase heterodimerization (RTp66-RTp51) for HIV and the NS4A-NS3 interaction for HCV.