Apresentação do PowerPoint

 Este estudo descreve a primeira triagem antibiofilme e antibacteriana de C. baccatum contra S. epidermidis e P. aeruginosa e os resultados aqui apresentados sugerem o potencial farmacológico da espécie como inibidor de biofilme;  O Esaq foi o extrato mais promissor contra o biofilme de S. epidermidis e P. aeruginosa sem inibir o crescimento bacteriano,indicando que a inibição do biofilme é independente da morte bacteriana.  Combater a adesão bacteriana e a formação de biofilme por uma via que não envolva a morte celular é uma característica marcante do novo conceito de terapias antivirulência.  A terapia antivirulência é uma das alternativas mais promissoras no combate á micro-organismos patogênicos. É importante explorar novos mecanismos de ação que dificultem o rápido desenvolvimento de resistência bacteriana, por exemplo: tornar esses microrganismos mais suscetíveis a outros antimicrobianos e para o sistema imunológico. Neste contexto, os produtos naturais são uma importante fonte de moléculas bioativas e os polifenóis têm recebido certa atenção recentemente em relação à sua ação antimicrobiana sobre microrganismos em biofilmes.


Results
The SARS-CoV-2 pandemic continues to cause major global morbidity, mortality and economic burden. The public health urgency led to the study of clinically approved drugs as repurposed medicines to treat individuals with COVID-19. The current approaches have not proven successful to date. The understanding of preclinical activity, mechanism of action, pharmacokinetics and safety are critical to achieving clinical benefits from repurposing drugs.
Direct-acting antivirals (DDA) against hepatitis C virus (HCV) are among the safest antiviral agents, since they become routinely used in the last five years. Due to their recent incorporation among therapeutic agents, drugs like daclatasvir (DCV) and sofosbuvir (SFV) have not been systematically tested against SARS-CoV or MERS-CoV. DCV inhibits HCV replication by binding to the Nterminus of non-structural protein (NS5A), affecting both viral RNA replication and virion assembly. NS5A is a multifunctional protein in the HCV replicative cycle, involved with recruitment of host cellular lipid droplets, RNA binding and replication, protein-phosphorylation, cell signaling and antagonism of interferon pathways. In large positive sense RNA viruses, such as SARS-CoV-2, these activities are executed by various viral proteins, especially the non-structural proteins (nsp) 1 to 14. SFV inhibits the HCV protein NS5B, its RNA polymerase. The similarities between the SARS-CoV-2 and HCV RNA polymerase provide a rational for studying sofosbuvir as an antiviral for COVID-19.
Taken collectively, current data provided a bases to investigate whether DCV and SFV could inhibit the production of infectious SARS-CoV-2 particles in physiologically relevant cells.   Resumed Methodology: Time-of-addition assay was performed to gain insights on the temporality of DCV`s activity against SARS-CoV-2. Vero cells were infected at MOI of 0.01 and treated at different time points, with DCV at 2-fold its EC50. Viruses present in the supernatants were titrated by PFU/mL. To confirm the rational that both SFV and DCV inhibit viral RNA synthesis, intracellular levels of SARS-CoV-2 genomic and sub genomic RNA were measured in Calu-3 cells through real time RT-PCR. Molecular docking methods were applied to predict the complexes with lowest energy interactions between the SARS-CoV-2 RNA polymerase and the active metabolite of SFV as well as DCV.

Conclusions
 Altogether, our data reveal that SFV and DCV inhibited SARS-CoV-2 replication in physiologically relevant cells, including type II pneumocytes. Besides, the drugs prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators by monocytes, respectively. Both drugs inhibited independent events during RNA synthesis and this was particularly the case for DCV, which also targeted secondary RNA structures in the SARS-CoV-2 genome.  In summary, effective early antiviral interventions are urgently required for the SARS-CoV-2 pandemic to improve patient clinical outcomes and disrupt transmission at population level. The presented data for two widely available anti-HCV drugs, particularly for DCV, provide a rational basis for further validation of these molecules for anti-SARS-CoV-2 interventions.
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Protective effect of SFV and DCV in non-permissive cells
Resumed Methodology: Neurons and monocytes do not present productive replication of SARS-CoV-2, however, infection of these cells is known to be associated with neuro-COVID-19 and cytokine storm, respectively. Therefore, these cell were used in inhibition assays. Neural Stem Cells (NSCs), NSCs-based neurospheres, and human primary monocytes were infected at MOI 0.1 for 2h at 37 °C, inoculum was removed and fresh medium containing the compounds was added. After 24h (monocytes) and 5 days (neurospheres), cell death was measured by TUNEL approach, virus levels in the supernatant were quantified by RT-PCR and cytokines measured by ELISA.

Physiologically based pharmacokinetic (PBPK) modeling for DCV
Resumed Methodology: Molecular modeling predictions and melting curves of extracted viral RNA was generated to assess whether DCV could affect the virus RNA folding. The thermal melting profiles of the RNA and RNA/DCV complexes were obtained by varying the temperature in a regular real time thermocycler. Continuous passages of SARS-CoV-2 in the presence of DCV were performed in order to evaluated the generation of mutations in viral RNA that may result in a change of its secondary structure. Viruses were passaged in Vero cells at the MOI of 0.1 during two months in the presence of increasing concentrations of DCV (up to 7 µM). The virus RNA was submitted to unbiased sequence using a MGI-2000 and a metatranscriptomics approach.
Resumed Methodology: PBPK model was constructed in Python 3.5 and simulated using a population of one hundred virtual healthy individuals (50% female) between 20-60 years and having weight and height as provided by the US national health statistics reports. A seven compartmental absorption and transit model representing the various parts of the duodenum, jejunum and ileum to capture effective absorption kinetics was used in the model. DCV PBPK model was validated in healthy individuals using available data in humans for various single doses -1, 10, 25, 50, 100 and 200 mg and for various multiple doses -1, 10, 30 and 60 mg at fasted state. For the inhibition of SARS-CoV-2, a mean target concentration (EC90) of 4.12 µM or 3079 ng/ml obtained from multiple in vitro studies was used