Potential of plant extracts in targeting SARS-CoV-2 main protease: an in vitro and in silico study.

The deaths caused by the covid-19 pandemic have recently decreased due to a worldwide effort in vaccination campaigns. However, even vaccinated people can develop a severe form of the disease that requires ICU admission. As a result, the search for antiviral drugs to treat these severe cases has become a necessity. In this context, natural products are an interesting alternative to synthetic medicines used in drug repositioning, as they have been consumed for a long time through traditional medicine. Many natural compounds found in plant extracts have already been shown to be effective in treating viral and bacterial diseases, making them possible hits to exploit against covid-19. The objective of this work was to evaluate the antiviral activity of different plant extracts available in the library of natural products of the Universidade Estadual de Maringá, by inhibiting the SARS-CoV-2 main protease (Mpro), and by preventing viral infection in a cellular model. As a result, the extract of Cytinus hypocistis, obtained by ultrasound, showed a Mpro inhibition capacity greater than 90%. In the infection model assays using Vero cells, an inhibition of 99.6% was observed, with a selectivity index of 42.7. The in silico molecular docking simulations using the extract compounds against Mpro, suggested Tellimagrandin II as the component of C. hypocistis extract most likely to inhibit the viral enzyme. These results demonstrate the potential of C. hypocistis extract as a promising source of natural compounds with antiviral activity against covid-19.Communicated by Ramaswamy H. Sarma.

Antiviral activity of Cenostigma pluviosum var. peltophoroides extract and fractions against SARS-CoV-2.

Few extracts of plant species from the Brazilian flora have been validated from a pharmacological and clinical point of view, and it is important to determine whether their traditional use is proven by pharmacological effects. Cenostigma pluviosum var. peltophoroides is one of those plants, which belongs to the Fabaceae family that is widely used in traditional medicine and is very rich in tannins. Due to the lack of effective drugs to treat severe cases of Covid-19, the main protease of SARS-CoV-2 (Mpro) becomes an attractive target in the research for new antivirals since this enzyme is crucial for virus replication and does not have homologs in humans. This study aimed to prospect inhibitor candidates among the compounds from C. pluviosum extract, by virtual screening simulations using SARS-CoV-2 Mpro as target. Experimental validation was made by inhibitory proteolytic assays of recombinant Mpro and by antiviral activity with infected Vero cells. Docking simulations identify four compounds with potential inhibitory activity of Mpro present in the extract. The compound pentagalloylglucose showed the best result in proteolytic kinetics experiments, with suppression of recombinant Mpro activity by approximately 60%. However, in experiments with infected cells ethyl acetate fraction and sub-fractions, F2 and F4 of C. pluviosum extract performed better than pentagalloylglucose, reaching close to 100% of antiviral activity. The prominent activity of the extract fractions in infected cells may be a result of a synergistic effect from the different hydrolyzable tannins present, performing simultaneous action on Mpro and other targets from SARS-CoV-2 and host.Communicated by Ramaswamy H. Sarma.

Interactome Analysis of KIN (Kin17) Shows New Functions of This Protein.

KIN (Kin17) protein is overexpressed in a number of cancerous cell lines, and is therefore considered a possible cancer biomarker. It is a well-conserved protein across eukaryotes and is ubiquitously expressed in all cell types studied, suggesting an important role in the maintenance of basic cellular function which is yet to be well determined. Early studies on KIN suggested that this nuclear protein plays a role in cellular mechanisms such as DNA replication and/or repair; however, its association with chromatin depends on its methylation state. In order to provide a better understanding of the cellular role of this protein, we investigated its interactome by proximity-dependent biotin identification coupled to mass spectrometry (BioID-MS), used for identification of protein-protein interactions. Our analyses detected interaction with a novel set of proteins and reinforced previous observations linking KIN to factors involved in RNA processing, notably pre-mRNA splicing and ribosome biogenesis. However, little evidence supports that this protein is directly coupled to DNA replication and/or repair processes, as previously suggested. Furthermore, a novel interaction was observed with PRMT7 (protein arginine methyltransferase 7) and we demonstrated that KIN is modified by this enzyme. This interactome analysis indicates that KIN is associated with several cell metabolism functions, and shows for the first time an association with ribosome biogenesis, suggesting that KIN is likely a moonlight protein.

Biophysical characterization and molecular phylogeny of human KIN protein.

The DNA/RNA-binding KIN protein was discovered in 1989, and since then, it has been found to participate in several processes, e.g., as a transcription factor in bacteria, yeasts, and plants, in immunoglobulin isotype switching, and in the repair and resolution of double-strand breaks caused by ionizing radiation. However, the complete three-dimensional structure and biophysical properties of KIN remain important information for clarifying its function and to help elucidate mechanisms associated with it not yet completely understood. The present study provides data on phylogenetic analyses of the different domains, as well as a biophysical characterization of the human KIN protein (HSAKIN) using bioinformatics techniques, circular dichroism spectroscopy, and differential scanning calorimetry to estimate the composition of secondary structure elements; further studies were performed to determine the biophysical parameters ΔHm and Tm. The phylogenetic analysis indicated that the zinc-finger and winged helix domains are highly conserved in KIN, with mean identity of 90.37% and 65.36%, respectively. The KOW motif was conserved only among the higher eukaryotes, indicating that this motif emerged later on the evolutionary timescale. HSAKIN has more than 50% of its secondary structure composed by random coil and ß-turns. The highest values of ΔHm and Tm were found at pH 7.4 suggesting a stable structure at physiological conditions. The characteristics found for HSAKIN are primarily due to its relatively low composition of α-helices and ß-strands, making up less than half of the protein structure.

Identification and characterization of stem-bulge RNAs in Drosophila melanogaster.

Non-coding Y RNAs and stem-bulge RNAs are homologous small RNAs in vertebrates and nematodes, respectively. They share a conserved function in the replication of chromosomal DNA in these two groups of organisms. However, functional homologues have not been found in insects, despite their common early evolutionary history. Here, we describe the identification and functional characterization of two sbRNAs in Drosophila melanogaster, termed Dm1 and Dm2. The genes coding for these two RNAs were identified by a computational search in the genome of D. melanogaster for conserved sequence motifs present in nematode sbRNAs. The predicted secondary structures of Dm1 and Dm2 partially resemble nematode sbRNAs and show stability in molecular dynamics simulations. Both RNAs are phylogenetically closer related to nematode sbRNAs than to vertebrate Y RNAs. Dm1, but not Dm2 sbRNA is abundantly expressed in D. melanogaster S2 cells and adult flies. Only Dm1, but not Dm2 sbRNA can functionally replace Y RNAs in a human cell-free DNA replication initiation system. Therefore, Dm1 is the first functional sbRNA described in insects, allowing future investigations into the physiological roles of sbRNAs in the genetically tractable model organism D. melanogaster.

Identification and molecular structure analysis of a new noncoding RNA, a sbRNA homolog, in the silkworm Bombyx mori genome.

The small noncoding group of RNAs called stem-bulge RNAs (sbRNAs), first reported in Caenorhabditis elegans, is described as molecules homologous to the Y RNAs, a specific class of noncoding RNAs that is present in vertebrates. This homology indicates the possibility of the existence of sbRNAs in other invertebrate organisms. In this work, we used bioinformatic tools and conserved sequences of sbRNAs from C. Elegans and Y RNAs to search for homologous sbRNA sequences in the Bombyx mori genome. This analysis led to the discovery of one noncoding gene, which was translated into RNA segments and comparatively analysed with segments from human and hamster Y RNAs and C. elegans sbRNAs in molecular dynamic simulations. This gene represents the first evidence for a new sbRNA-like noncoding RNA, the BmsbRNA gene, in this Lepidoptera genome.