Repurposing the Ebola and Marburg Virus Inhibitors Tilorone, Quinacrine, and Pyronaridine: In Vitro Activity against SARS-CoV-2 and Potential Mechanisms

Severe acute respiratory coronavirus 2 (SARS-CoV-2) is a newly identified virus that has resulted in over 2.5 million deaths globally and over 116 million cases globally in March, 2021. Small-molecule inhibitors that reverse disease severity have proven difficult to discover. One of the key approaches that has been widely applied in an effort to speed up the translation of drugs is drug repurposing. A few drugs have shown in vitro activity against Ebola viruses and demonstrated activity against SARS-CoV-2 in vivo. Most notably, the RNA polymerase targeting remdesivir demonstrated activity in vitro and efficacy in the early stage of the disease in humans. Testing other small-molecule drugs that are active against Ebola viruses (EBOVs) would appear a reasonable strategy to evaluate their potential for SARS-CoV-2. We have previously repurposed pyronaridine, tilorone, and quinacrine (from malaria, influenza, and antiprotozoal uses, respectively) as inhibitors of Ebola and Marburg viruses in vitro in HeLa cells and mouse-adapted EBOV in mice in vivo. We have now tested these three drugs in various cell lines (VeroE6, Vero76, Caco-2, Calu-3, A549-ACE2, HUH-7, and monocytes) infected with SARS-CoV-2 as well as other viruses (including MHV and HCoV 229E). The compilation of these results indicated considerable variability in antiviral activity observed across cell lines. We found that tilorone and pyronaridine inhibited the virus replication in A549-ACE2 cells with IC50 values of 180 nM and IC50 198 nM, respectively. We used microscale thermophoresis to test the binding of these molecules to the spike protein, and tilorone and pyronaridine bind to the spike receptor binding domain protein with Kd values of 339 and 647 nM, respectively. Human Cmax for pyronaridine and quinacrine is greater than the IC50 observed in A549-ACE2 cells. We also provide novel insights into the mechanism of these compounds which is likely lysosomotropic

<i>In vitro</i> antiviral activity of the anti-HCV drugs daclatasvir and sofosbuvir against SARS-CoV-2, the aetiological agent of COVID-19

BackgroundCurrent approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity.MethodsSARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir's dose and schedule to maximize the probability of success for COVID-19.ResultsDaclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 μM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans.ConclusionsDaclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy

Imunomodulação da replicação do HIV-1 pela hemaglutinina do vírus Influenza

Made available in DSpace on 2018-05-08T14:14:51Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) natalia_rodrigues_ioc_dout_2017.pdf: 2983688 bytes, checksum: b870156a4d64d7ada8eb12266e8d9ee1 (MD5)Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.Diversos agentes infecciosos de etiologia viral vêm afetando as populações durante séculos. A grande dispersão dos vírus HIV-1 por todo o mundo possibilita inúmeras coinfecções clínicas com outros patógenos, como por exemplo, o vírus Influenza, principalmente por sua distribuição geográfica ao longo do globo e por ser responsável por epidemias sazonais. A infecção pelo HIV-1 leva a uma ativação crônica do sistema imune, gerando uma resposta policlonal, reduzindo a chance de resposta específica contra infecções oportunistas, além de gerar um estado de imunocomprometimento nos pacientes. Surpreendemente, na pandemia de 2009, causada pelo vírus influenza A(H1N1)pdm09, diversos trabalhos na literatura descreveram que o desfecho clinico dos pacientes portadores do HIV em relação ao vírus influenza foi muito semelhante ao observado em indivíduos na população em geral. Nesse mesmo contexto, após a pandemia do Influenza, um estudo analisando o esquema de vacinação contra influenza de indivíduos HIV-1 demonstrou que após a vacinação os participantes mantiveram ou reduziram a carga viral Fato curioso, já que a vacinação de indivíduos portadores do HIV pode promover uma ativação do sistema imune e consequentemente aumento da replicação viral. É possível que durante a coinfecção HIV-1/influenza tanto sinais positivos quanto negativos para a replicação de ambos os vírus possam existir, portanto durante a coinfecção um novo equilíbrio deve ser imposto ao hospedeiro. Sendo assim, visamos investigar o potencial inibitório do vírus influenza, ou da hemaglutinina (HA), principal componente da vacina antiinfluenz, sobre a replicação do HIV-1. Nossos resultados mostraram que o tratamento com HA em diferentes tipos celulares, como macrófagos e PBMCs, de doadores saudáveis, leva a inibição da replicação do HIV-1. Em adição, a exposição a HA também promove o aumento dos níveis de IL-10, uma citocina regulatória, assim como de APOBEC3G, um importante fator de restrição celular. Além disso, corroborando o dado anterior, o bloqueio do receptor para IL-10 preveniu o efeito inibitório da replicação do HIV-1 pela HA. Sendo assim, nossos dados mostram a participação da IL-10 e do APOBEC3G nessa modulação da replicação do HIV-1 pela HA.Several viral agents have been affecting populations for centuries. The worldwide dispersion of HIV-1 viruses allows for numerous clinical co-infections with other pathogens, such as Influenza virus, mainly due to its geographical distribution across the globe and for being responsible for seasonal epidemics. HIV-1 infection leads to a chronic activation of the immune system, generating a polyclonal response, reducing the chance of specific response against opportunistic infections, and also promote immunocompromised status of the patients. Surprisingly, in the 2009 Influenza pandemic, the literature have described that the A (H1N1)pdm09 clinical outcome of HIV patients was very similar to that observed in individuals of general population. In the same context, after Influenza pandemic wave, a study analyzing the influenza vaccination schedule of HIV individuals demonstrated that after influenza vaccination the HIV viral load was maintained or reduced Curiously, HIV individuals vaccination can promote an immune system activation and consequently increase viral replication. It is possible that during HIV-1/influenza coinfection, positive and negative replication signals of both viruses may exist, therefore a new equilibrium should be imposed on the host. Thus, we decided to investigate the HIV-1 inhibition, in vitro, by Influenza virus, as well as HA, the main component of the anti-influenza vaccine. Our results showed that HA treatment of different cell types, such as macrophages and PBMCs, leads to inhibition of HIV-1 replication. . In addition, HA exposure also promotes increased levels of IL-10, a regulatory cytokine, as well as APOBEC3G, an important cell restriction factor. Furthermore, corroborating the above data, blocking IL-10 receptor prevented the inhibitory effect of HIV-1 replication by HA. Thus, our data show the participation of IL-10 and APOBEC3G in this HA modulation of HIV-1 replication

Fluorine Atoms on C6H5-Corrole Affect the Interaction with Mpro and PLpro Proteases of SARS-CoV-2: Molecular Docking and 2D-QSAR Approaches

The chymotrypsin-like cysteine protease (3CLpro, also known as main protease&mdash;Mpro) and papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been used as the main targets for screening potential synthetic inhibitors for posterior in vitro evaluation of the most promising compounds. In this sense, the present work reports for the first time the evaluation of the interaction between Mpro/PLpro with a series of 17 porphyrin analogues-corrole (C1), meso-aryl-corrole (C2), and 15 fluorinated-meso-aryl-corrole derivatives (C3&ndash;C17) via molecular docking calculations. The impact of fluorine atoms on meso-aryl-corrole structure was also evaluated in terms of binding affinity and physical-chemical properties by two-dimensional quantitative structure&ndash;activity relationship (2D-QSAR). The presence of phenyl moieties increased the binding capacity of corrole for both proteases and depending on the position of fluorine atoms might impact positively or negatively the binding capacity. For Mpro the para-fluorine atoms might decrease drastically the binding capacity, while for PLpro there was a certain increase in the binding affinity of fluorinated-corroles with the increase of fluorine atoms into meso-aryl-corrole structure mainly from tri-fluorinated insertions. The 2D-QSAR models indicated two separated regions of higher and lower affinity for Mpro:C1&ndash;C17 based on dual electronic parameters (&sigma;I and &sigma;R), as well as one model was obtained with a correlation between the docking score value of Mpro:C2&ndash;C17 and the corresponding 13C nuclear magnetic resonance (NMR) chemical shifts of the sp2 carbon atoms (&delta;C-1 and &delta;C-2) of C2&ndash;C17. Overall, the fluorinated-meso-aryl-corrole derivatives showed favorable in silico parameters as potential synthetic compounds for future in vitro assays on the inhibition of SARS-CoV-2 replication

Detection of the influenza A(H1N1)pdm09 virus carrying the K-15E, P83S and Q293H mutations in patients who have undergone bone marrow transplant.

The 2009 pandemic influenza A(H1N1)pdm09 virus emerged and caused considerable morbidity and mortality in the third world, especially in Brazil. Although circulating strains of A(H1N1)pdm09 are A/California/04/2009-like (CA-04-like) viruses, various studies have suggested that some mutations in the viral hemagglutinin (HA) may be associated with enhanced severity and fatality. This phenomenon is particularly challenging for immunocompromised individuals, such as those who have undergone bone marrow transplant (BMT), because they are more likely to display worse clinical outcomes to influenza infection than non-immunocompromised individuals. We studied the clinical and viral aspects of post-BMT patients with confirmed A(H1N1)pdm09 diagnosis in the largest cancer hospital in Brazil. We found a viral strain with K-15E, P83S and Q293H polymorphisms in the HA, which is presumably more virulent, in these individuals. Despite that, these patients showed only mild symptoms of infection. Our findings complement the discovery of mild cases of infection with the A(H1N1)pdm09 virus with the K-15E, P83S and Q293H mutations in Brazil and oppose other studies that have linked these changes with increased disease severity. These results could be important for a better comprehension of the impact of the pandemic influenza in the context of BMT

Detection of Oseltamivir-Resistant Pandemic Influenza A(H1N1)pdm2009 in Brazil: Can Community Transmission Be Ruled Out?

Made available in DSpace on 2015-09-21T17:25:44Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) thiago_souza_etal_IOC_2013.pdf: 213154 bytes, checksum: 9252ced13a5ff88a2364d36d24c35fb6 (MD5) Previous issue date: 2013Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Vírus Respiratórios e do Sarampo. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Vírus Respiratórios e do Sarampo. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Vírus Respiratórios e do Sarampo. Rio de Janeiro, RJ, Brasil.Fundação Estadual de Produção e Pesquisa em Saúde. Laboratório Central de Saúde Pública do Estado do Rio de Grande do Sul. Seção de Virologia. Porto Alegre, RS, Brasil.Universidade Luterana do Brasil. Porto Alegre, RS, Brasil.Laboratório Central de Saúde Pública do Estado de Santa Catarina. Florianópolis, SC, Brasil.Fundação Ezequiel Dias. Instituto Octávio Magalhães. Laboratório Central de Saúde Pública do Estado de Minas Gerais. Belo Horizonte, MG, Brasil.Laboratório Central de Saúde Pública do Estado do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Vírus Respiratórios e do Sarampo. Rio de Janeiro, RJ, Brasil.Although surveillance efforts that monitor the emergence of drug-resistant strains of influenza are critical, systematic analysis is overlooked in most developing countries. We report on the occurrence of strains of pandemic influenza A(H1N1)pdm09 with resistance and decreased susceptibility to oseltamivir (OST) in Brazil in 2009, 2011 and 2012. We found 7 mutant viruses, 2 with the mutation S247N and other 5 with the mutation H275Y. Most of these viruses were from samples concentrated in the southern region of Brazil. Some of these resistant viruses were detected prior to the initiation of OST treatment, suggesting that community transmission of mutant viruses may exist. Moreover, we show that one of these OST-resistant (H275Y) strains of A(H1N1)pdm09 was discovered in the tri-border region between Brazil, Argentina and Paraguay, highlighting that this strain could also be found in other Latin American countries. Our findings reinforce the importance of enhanced antiviral resistance surveillance in Brazil and in other Latin American countries to confirm or rule out the community transmission of OST-resistant strains of A(H1N1)pdm09

Oseltamivir-resistant influenza A(H1N1)pdm2009 strains found in Brazil are endowed with permissive mutations, which compensate the loss of fitness imposed by antiviral resistance

Submitted by sandra infurna ([email protected]) on 2015-11-16T14:59:50Z No. of bitstreams: 1 paola_resende_etal_IOC_2015.pdf: 1146844 bytes, checksum: 7920ec697e0b03b42cb30ad41a2714f7 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2015-11-16T15:57:03Z (GMT) No. of bitstreams: 1 paola_resende_etal_IOC_2015.pdf: 1146844 bytes, checksum: 7920ec697e0b03b42cb30ad41a2714f7 (MD5)Made available in DSpace on 2015-11-16T15:57:03Z (GMT). No. of bitstreams: 1 paola_resende_etal_IOC_2015.pdf: 1146844 bytes, checksum: 7920ec697e0b03b42cb30ad41a2714f7 (MD5) Previous issue date: 2015Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Virus Respiratórios e do Sarampo. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunofarmacologia. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto de Pesquisas Clínicas Evandro Chagas. Laboratório de Medicina Intensiva. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Virus Respiratórios e do Sarampo. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Virus Respiratórios e do Sarampo. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Virus Respiratórios e do Sarampo. Rio de Janeiro, RJ, BrasilLaboratório Central de Saúde Pública do Estado do Rio de Grande do Sul. Seção de Virologia. Porto Alegre, RS, Brasil. ,Fundação Estadual de Produção e Pesquisa em Saúde. Porto Alegre, RS, Brasil.The 2009 pandemic influenza A virus outbreak led to the systematic use of the neuraminidase (NA) inhibitor oseltamivir (OST). Consequently, OST-resistant strains, carrying the mutation H275Y, emerged in the years after the pandemics, with a prevalence of 1-2%. Currently, OST-resistant strains have been found in community settings, in untreated individuals. To spread in community settings, H275Y mutants must contain additional mutations, collectively called permissive mutations. We display the permissive mutations in NA of OST-resistant A(H1N1)pdm09 virus found in Brazilian community settings. The NAs from 2013 are phylogenetically distinct from those of 2012, indicating a tendency of positive selection of NAs with better fitness. Some previously predicted permissive mutations, such as V241I and N369K, found in different countries, were also detected in Brazil. Importantly, the change D344N, also predicted to compensate loss of fitness imposed by H275Y mutation, was found in Brazil, but not in other countries in 2013. Our results reinforce the notion that OST-resistant A(H1N1)pdm09 strains with compensatory mutations may arise in an independent fashion, with samples being identified in different states of Brazil and in different countries. Systematic circulation of these viral strains may jeopardise the use of the first line of anti-influenza drugs in the future