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Introduction: Treatment of influenza A virus infections is currently limited to few direct acting antiviral substances. Repurposing other established pharmaceuticals as antivirals could aid in improving treatment options. Methods: This study investigates the antiviral properties of ProcCluster® and procaine hydrochloride, two derivatives of the local anesthetic procaine, in influenza A virus infection of A549, Calu-3 and MDCK cells. Results: Both substances inhibit replication in all three of these cell lines in multi-cycle experiments. However, cell line-dependent differences in the effects of the substances on viral RNA replication and subsequent protein synthesis, as well as release of progeny viruses in single-cycle experiments can be observed. Both ProcCluster® and procaine hydrochloride delay endosome fusion of the virus early in the replication cycle, possibly due to the alkaline nature of the active component procaine. In A549 and Calu-3 cells an additional effect of the substances can be observed at late stages in the first replication cycle. Interestingly, this effect is absent in MDCK cells. We demonstrate that ProcCluster® and procaine hydrochloride inhibit phospholipase A2 (PLA2) enzymes from A549 but not MDCK cells and confirm that specific inhibition of calcium independent PLA2 but not cytosolic PLA2 has antiviral effects. Discussion: We show that ProcCluster® and procaine hydrochloride inhibit influenza A virus infection at several stages of the replication cycle and have potential as antiviral substances.
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As vaccination efforts against SARS-CoV-2 progress in many countries, there is still an urgent need for efficient antiviral treatment strategies for those with severer disease courses, and lately, considerable efforts have been undertaken to repurpose existing drugs as antivirals. The local anaesthetic procaine has been investigated for antiviral properties against several viruses over the past decades. Here, we present data on the inhibitory effect of the procaine prodrugs ProcCluster® and procaine hydrochloride on SARS-CoV-2 infection in vitro. Both procaine prodrugs limit SARS-CoV-2 progeny virus titres as well as reduce interferon and cytokine responses in a proportional manner to the virus load. The addition of procaine during the early stages of the SARS-CoV-2 replication cycle in a cell culture first limits the production of subgenomic RNA transcripts, and later affects the replication of the viral genomic RNA. Interestingly, procaine additionally exerts a prominent effect on SARS-CoV-2 progeny virus release when added late during the replication cycle, when viral RNA production and protein production are already largely completed.
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COVID-19 , Pró-Fármacos , Animais , Chlorocebus aethiops , SARS-CoV-2 , Antivirais/farmacologia , Anestésicos Locais/farmacologia , Pró-Fármacos/farmacologia , Células Vero , Procaína/farmacologia , Replicação ViralRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the coronavirus disease-19 (COVID-19) is still challenging healthcare systems and societies worldwide. While vaccines are available, therapeutic strategies are developing and need to be adapted to each patient. Many clinical approaches focus on the repurposing of approved therapeutics against other diseases. However, the efficacy of these compounds on viral infection or even harmful secondary effects in the context of SARS-CoV-2 infection are sparsely investigated. Similarly, adverse effects of commonly used therapeutics against lifestyle diseases have not been studied in detail. Using mono cell culture systems and a more complex chip model, we investigated the effects of the acetylsalicylic acid (ASA) salt D,L-lysine-acetylsalicylate + glycine (LASAG) on SARS-CoV-2 infection in vitro. ASA is commonly known as Aspirin® and is one of the most frequently used medications worldwide. Our data indicate an inhibitory effect of LASAG on SARS-CoV-2 replication and SARS-CoV-2-induced expression of pro-inflammatory cytokines and coagulation factors. Remarkably, our data point to an additive effect of the combination of LASAG and the antiviral acting drug remdesivir on SARS-CoV-2 replication in vitro.
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Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , Antivirais/uso terapêutico , Aspirina/farmacologia , Aspirina/uso terapêutico , Glicina/farmacologia , Glicina/uso terapêutico , Humanos , LisinaRESUMO
Influenza virus (IV) infections are considered to cause severe diseases of the respiratory tract. Beyond mild symptoms, the infection can lead to respiratory distress syndrome and multiple organ failure. Occurrence of resistant seasonal and pandemic strains against the currently licensed antiviral medications points to the urgent need for new and amply available anti-influenza drugs. Interestingly, the virus-supportive function of the cellular phosphatidylinositol 3-kinase (PI3K) suggests that this signaling module may be a potential target for antiviral intervention. In the sense of repurposing existing drugs for new indications, we used Pictilisib, a known PI3K inhibitor to investigate its effect on IV infection, in mono-cell-culture studies as well as in a human chip model. Our results indicate that Pictilisib is a potent inhibitor of IV propagation already at early stages of infection. In a murine model of IV pneumonia, the in vitro key findings were verified, showing reduced viral titers as well as inflammatory response in the lung after delivery of Pictilisib. Our data identified Pictilisib as a promising drug candidate for anti-IV therapies that warrant further studying. These results further led to the conclusion that the repurposing of previously approved substances represents a cost-effective and efficient way for development of novel antiviral strategies.
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Indazóis/farmacologia , Vírus da Influenza A/metabolismo , Pulmão , Infecções por Orthomyxoviridae , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Pneumonia Viral , Sulfonamidas/farmacologia , Células A549 , Animais , Cães , Humanos , Pulmão/enzimologia , Pulmão/virologia , Células Madin Darby de Rim Canino , Camundongos , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/enzimologia , Infecções por Orthomyxoviridae/virologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/enzimologia , Pneumonia Viral/virologiaRESUMO
Clinical observations indicate that COVID-19 is a systemic disease. An investigation of the viral distribution within the human body and its correlation with tissue damage can aid in understanding the pathophysiology of SARS-CoV-2 infection. We present a detailed mapping of the viral RNA in 61 tissues and organs of 11 deceased patients with COVID-19. The autopsies were performed within the early postmortem interval (between 1.5 and 15 hr, mean: 5.6 hr) to minimize the bias due to viral RNA and tissue degradation. Very high viral loads (>104copies/ml) were detected in most patients' lungs, and the presence of intact viral particles in the lung tissue could be verified by transmission electron microscopy. Interestingly, viral RNA was detected throughout various extrapulmonary tissues and organs without visible tissue damage. The dissemination of SARS-CoV-2-RNA throughout the body supports the hypothesis that there is a maladaptive host response with viremia and multiorgan dysfunction.
Since the discovery of the new coronavirus that causes COVID-19, scientists have been scrambling to understand the different features of the virus. While a lot more is now known about SARS-CoV-2, several key questions have proved more difficult to answer. For example, it remained unclear where the virus travels to in the body and causes the most harm. To help answer this question, Deinhardt-Emmer, Wittschieber et al. performed postmortem examinations on 11 patients who had recently died of COVID-19. After sampling 61 different organs and tissues from each patient, several tests were used to detect traces of SARS-CoV-2. The experiments showed that the largest pool of SARS-CoV-2 was present in the lungs, where it had caused severe damage to the alveolae, the delicate air sacs at the end of the lungs' main air tubes. Small amounts of the virus were also detected in other organs and tissues, but no severe tissue damage was seen. In addition, Deinhardt-Emmer, Wittschieber et al. found that each patient had increased levels of some of the proteins involved in inflammation and blood clotting circulating their bloodstream. This suggests that the inflammation caused by SARS-CoV-2 leads to an excessive immune reaction throughout the entire body. This research provides important new insights into which areas of the body are most impacted by SARS-CoV-2. These findings may help to design more effective drug treatments that target the places SARS-CoV-2 is most likely to accumulate and help patients fight off the infection at these regions.
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Autopsia , COVID-19/patologia , SARS-CoV-2/isolamento & purificação , Idoso , Idoso de 80 Anos ou mais , COVID-19/sangue , COVID-19/imunologia , COVID-19/terapia , Causas de Morte , Comorbidade , Feminino , Humanos , Inflamação/sangue , Inflamação/imunologia , Pulmão/patologia , Pulmão/virologia , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , RNA Viral/sangue , Carga ViralRESUMO
Infections with SARS-CoV-2 can be asymptomatic, but they can also be accompanied by a variety of symptoms that result in mild to severe coronavirus disease-19 (COVID-19) and are sometimes associated with systemic symptoms. Although the viral infection originates in the respiratory system, it is unclear how the virus can overcome the alveolar barrier, which is observed in severe COVID-19 disease courses. To elucidate the viral effects on the barrier integrity and immune reactions, we used mono-cell culture systems and a complex human chip model composed of epithelial, endothelial, and mononuclear cells. Our data show that SARS-CoV-2 efficiently infected epithelial cells with high viral loads and inflammatory response, including interferon expression. By contrast, the adjacent endothelial layer was neither infected nor did it show productive virus replication or interferon release. With prolonged infection, both cell types were damaged, and the barrier function was deteriorated, allowing the viral particles to overbear. In our study, we demonstrate that although SARS-CoV-2 is dependent on the epithelium for efficient replication, the neighboring endothelial cells are affected, e.g., by the epithelial cytokines or components induced during infection, which further results in the damage of the epithelial/endothelial barrier function and viral dissemination.IMPORTANCESARS-CoV-2 challenges healthcare systems and societies worldwide in unprecedented ways. Although numerous new studies have been conducted, research to better understand the molecular pathogen-host interactions are urgently needed. For this, experimental models have to be developed and adapted. In the present study we used mono cell-culture systems and we established a complex chip model, where epithelial and endothelial cells are cultured in close proximity. We demonstrate that epithelial cells can be infected with SARS-CoV-2, while the endothelium did not show any infection signs. Since SARS-CoV-2 is able to establish viremia, the link to thromboembolic events in severe COVID-19 courses is evident. However, whether the endothelial layer is damaged by the viral pathogens or whether other endothelial-independent homeostatic factors are induced by the virus is essential for understanding the disease development. Therefore, our study is important as it demonstrates that the endothelial layer could not be infected by SARS-CoV-2 in our in vitro experiments, but we were able to show the destruction of the epithelial-endothelial barrier in our chip model. From our experiments we can assume that virus-induced host factors disturbed the epithelial-endothelial barrier function and thereby promote viral spread.