Screening of Natural Products Inhibitors of SARS-CoV-2 Entry.

The COVID-19 pandemic has led to the search for new molecules with antiviral activity against SARS-CoV-2. The entry of the virus into the cell is one of the main targets for inhibiting SARS-CoV-2 infection. Natural products are an important source of new therapeutic alternatives against diseases. Pseudotyped viruses allow the study of SARS-CoV-2 viral entry inhibitors, and due to their simplicity, they allow the screening of a large number of antiviral candidates in Biosafety Level 2 facilities. We used pseudotyped HIV-1 with the D614G SARS-CoV-2 spike glycoprotein to test its ability to infect ACE2-expressing HEK 293T cells in the presence of diverse natural products, including 21 plant extracts, 7 essential oils, and 13 compounds from plants and fungi. The 50% cytotoxic concentration (CC50) was evaluated using the resazurin method. From these analyses, we determined the inhibitory activity of the extract of Stachytarpheta cayennensis, which had a half-maximal inhibitory concentration (IC50) of 91.65 µg/mL, a CC50 of 693.5 µg/mL, and a selectivity index (SI) of 7.57, indicating its potential use as an inhibitor of SARS-CoV-2 entry. Moreover, our work indicates the usefulness of the pseudotyped-virus system in the screening of SARS-CoV-2 entry inhibitors.

Characterization of the orthodox Pinus occidentalis seed and pollen proteomes by using complementary gel-based and gel-free approaches.

UNLABELLED: This work presents an analysis of Pinus occidentalis pollen and seed proteomes, in which both gel-based and gel-free approaches have been used. Proteins were extracted from P. occidentalis seeds and pollen by using the TCA/acetone/phenol precipitation protocol, and protein extracts were subjected to 1- and 2-DE coupled to MALDI-TOF-TOF as well as to shotgun (nLC-LTQ-Orbitrap) analysis. All bands (1-DE) and the most abundant spots (2-DE) were excised, trypsin digested and the resulting peptides analyzed by MALDI TOF/TOF. In order to increase the proteome coverage, a gel free approach was used. Proteins were identified from mass spectra by using three different databases, including UniProtKB, NCBI and a Pinus spp. custom database [2]. The gel-based approach resulted in 42 (seeds) and 94 (pollen) protein identifications, while the shotgun approach permitted the identification of 187 (seed) and 960 (pollen) proteins. Proteins were classified based on their corresponding functional categories. In seeds, storage proteins were the most abundant ones, and some allergens and proteases were also identified. In pollen proteins related to general metabolism were the most predominant. Data are compared and discussed from a methodological and biological point of view, taking into account the particularities of the seed and pollen organs. BIOLOGICAL SIGNIFICANCE: In this work we characterized P. occidentalis proteome with seeds and pollen samples implementing two complementary approaches for the analysis. We found a high content of storage protein, stress response and metabolism related proteins in the seed proteome. Similarly, in the pollen proteome we found predominant groups of proteins related to metabolism and stress response.