The global health emergency generated by coronavirus disease 2019 (COVID-19) has prompted the search for preventive and therapeutic treatments for its pathogen, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). There are many potential targets for drug discovery and development to tackle this disease. One of these targets is the main protease, Mpro or 3CLpro, which is highly conserved among coronaviruses. 3CLpro is an essential player in the viral replication cycle, processing the large viral polyproteins and rendering the individual proteins functional. We report a biophysical characterization of the structural stability and the catalytic activity of 3CLpro from SARS-CoV-2, from which a suitable experimental in vitro molecular screening procedure has been designed. By screening of a small chemical library consisting of about 150 compounds, the natural product quercetin was identified as reasonably potent inhibitor of SARS-CoV-2 3CLpro (K i ~ 7 μM). Quercetin could be shown to interact with 3CLpro using biophysical techniques and bind to the active site in molecular simulations. Quercetin, with well-known pharmacokinetic and ADMET properties, can be considered as a good candidate for further optimization and development, or repositioned for COVID-19 therapeutic treatment.

The pandemic, due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has stimulated the search for antivirals to tackle COVID-19 infection. Molecules with known pharmacokinetics and already approved for human use have been demonstrated or predicted to be suitable to be used either directly or as a base for a scaffold-based drug design. Among these substances, quercetin is known to be a potent in vitro inhibitor of 3CLpro, the SARS-CoV-2 main protease. However, its low in vivo bioavailability calls for modifications to its molecular structure. In this work, this issue is addressed by using rutin, a natural flavonoid that is the most common glycosylated conjugate of quercetin, as a model. Combining experimental (spectroscopy and calorimetry) and simulation techniques (docking and molecular dynamics simulations), we demonstrate that the sugar adduct does not hamper rutin binding to 3CLpro, and the conjugated compound preserves a high potency (inhibition constant in the low micromolar range, Ki = 11 μM). Although showing a disruption of the pseudo-symmetry in the chemical structure, a larger steric volume and molecular weight, and a higher solubility compared to quercetin, rutin is able to associate in the active site of 3CLpro, interacting with the catalytic dyad (His41/Cys145). The overall results have implications in the drug-design of quercetin analogs, and possibly other antivirals, to target the catalytic site of the SARS-CoV-2 3CLpro.

The development of new antiviral drugs against SARS-CoV-2 is a valuable long-term strategy to protect the global population from the COVID-19 pandemic complementary to the vaccination. Considering this, the viral main protease (Mpro) is among the most promising molecular targets in light of its importance during the viral replication cycle. The natural flavonoid quercetin 1 has been recently reported to be a potent Mpro inhibitor in vitro, and we explored the effect produced by the introduction of organoselenium functionalities in this scaffold. In particular, we report here a new synthetic method to prepare previously inaccessible C-8 seleno-quercetin derivatives. By screening a small library of flavonols and flavone derivatives, we observed that some compounds inhibit the protease activity in vitro. For the first time, we demonstrate that quercetin (1) and 8-(p-tolylselenyl)quercetin (2d) block SARS-CoV-2 replication in infected cells at non-toxic concentrations, with an IC50 of 192 μM and 8 μM, respectively. Based on docking experiments driven by experimental evidence, we propose a non-covalent mechanism for Mpro inhibition in which a hydrogen bond between the selenium atom and Gln189 residue in the catalytic pocket could explain the higher Mpro activity of 2d and, as a result, its better antiviral profile.

Exosomes are small extracellular vesicles that act as intercellular messengers. Previous studies revealed that, during acute pancreatitis, circulating exosomes could reach the alveolar compartment and activate macrophages. However, proteomic analysis suggested that the most likely origin of these exosomes could be the liver instead of the pancreas. The present study aimed to characterize the exosomes released by pancreas to pancreatitis-associated ascitic fluid (PAAF) as well as those circulating in plasma in an experimental model of taurocholate-induced acute pancreatitis in rats. We provide evidence that during acute pancreatitis two different populations of exosomes are generated with relevant differences in cell distribution, protein and microRNA content as well as different implications in their physiological effects. During pancreatitis plasma exosomes, but not PAAF exosomes, are enriched in the inflammatory miR-155 and show low levels of miR-21 and miR-122. Mass spectrometry-based proteomic analysis showed that PAAF exosomes contains 10–30 fold higher loading of histones and ribosomal proteins compared to plasma exosomes. Finally, plasma exosomes have higher pro-inflammatory activity on macrophages than PAAF exosomes. These results confirm the generation of two different populations of exosomes during acute pancreatitis. Deep understanding of their specific functions will be necessary to use them as therapeutic targets at different stages of the disease.