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.

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Several carrier proteins are involved in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin α, of which there are several human isoforms; among them, importin α3 (Impα3) has a high flexibility. The protein NUPR1, a nuclear protein involved in the cell-stress response and cell cycle regulation, is an intrinsically disordered protein (IDP) that has a nuclear localization sequence (NLS) to allow for nuclear translocation. NUPR1 does localize through the whole cell. In this work, we studied the affinity of the isolated wild-type NLS region (residues 54–74) of NUPR1 towards Impα3 and several mutants of the NLS region by using several biophysical techniques and molecular docking approaches. The NLS region of NUPR1 interacted with Impα3, opening the way to model the nuclear translocation of disordered proteins. All the isolated NLS peptides were disordered. They bound to Impα3 with low micromolar affinity (1.7–27 μM). Binding was hampered by removal of either Lys65 or Lys69 residues, indicating that positive charges were important; furthermore, binding decreased when Thr68 was phosphorylated. The peptide phosphorylated at Thr68, as well as four phospho-mimetic peptides (all containing the Thr68Glu mutation), showed the presence of a sequential NN(i,i + 1) nuclear Overhauser effect (NOE) in the 2D-1H-NMR (two-dimensional–proton NMR) spectra, indicating the presence of turn-like conformations. Thus, the phosphorylation of Thr68 modulates the binding of NUPR1 to Impα3 by a conformational, entropy-driven switch from a random-coil conformation to a turn-like structure.

Artificial enzymes with modulated enzyme-mimicking activities of natural systems represent a challenge in catalytic applications. Here, we show the creation of artificial Cu metalloenzymes based on the generation of Cu nanoparticles in an enzyme matrix. Different enzymes were used, and the structural differences between the enzymes especially influenced the controlled the size of the nanoparticles and the environment that surrounds them. Herein, we demonstrated that the oxidase-like catalytic activity of these copper nanozymes was rationally modulated by enzyme used as a scaffold, with a special role in the nanoparticle size and their environment. In this sense, these nanocopper hybrids have confirmed the ability to mimic a unique enzymatic activity completely different from the natural activity of the enzyme used as a scaffold, such as tyrosinase-like activity or as Fenton catalyst, which has extremely higher stability than natural mushroom tyrosinase. More interestingly, the oxidoreductase-like activity of nanocopper hybrids was cooperatively modulated with the synergistic effect between the enzyme and the nanoparticles improving the catalase activity (no peroxidase activity). Additionally, a novel dual (metallic and enzymatic activity) of the nanozyme made the highly improved catechol-like activity interesting for the design of 3,4-dihydroxy- l -phenylalanine ( l -DOPA) biosensor for detection of tyrosinase. These hybrids also showed cytotoxic activity against different tumor cells, interesting in biocatalytic tumor therapy.

Inhibiting the main protease 3CLpro is the most common strategy in the search for antiviral drugs to fight the infection from SARS-CoV-2. We report that the natural compound eugenol is able to hamper in vitro the enzymatic activity of 3CLpro, the SARS-CoV-2 main protease, with an inhibition constant in the sub-micromolar range (Ki = 0.81 μM). Two phenylpropene analogs were also tested: the same effect was observed for estragole with a lower potency (Ki = 4.1 μM), whereas anethole was less active. The binding efficiency index of these compounds is remarkably favorable due also to their small molecular mass (MW < 165 Da). We envision that nanomolar inhibition of 3CLpro is widely accessible within the chemical space of simple natural compounds.