Discovery of TCMs and derivatives against the main protease of SARS-CoV-2 via high throughput screening, ADMET analysis, and inhibition assay in vitro.

The rapidly evolving Coronavirus Disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide with thousands of deaths and infected cases. For the identification of effective treatments against this disease, the main protease (Mpro) of SARS­CoV­2 was found to be an attractive drug target, as it played a central role in viral replication and transcription. Here, we report the results of high-throughput molecular docking with 1,045,468 ligands' structures from 116 kinds of traditional Chinese medicine (TCM). Subsequently, 465 promising candidates were obtained, showing high binding affinities. The dynamic simulation, ADMET (absorption, distribution, metabolism, excretion and toxicity) and drug-likeness properties were further analyzed the screened docking results. Basing on these simulation results, 23 kinds of Chinese herbal extracts were employed to study their inhibitory activity for Mpro of SARS­CoV­2. Plants extracts from Forsythiae Fructus, Radix Puerariae, Radix astragali, Anemarrhenae Rhizoma showed acceptable inhibitory efficiencies, which were over 70%. The best candidate was Anemarrhenae Rhizoma, reaching 78.9%.

Production of an Anise- and Woodruff-like Aroma by Monokaryotic Strains of Pleurotus sapidus Grown on Citrus Side Streams.

The production of natural flavors by means of microorganisms is of great interest for the food and flavor industry, and by-products of the agro-industry are particularly suitable as substrates. In the present study, Citrus side streams were fermented using monokaryotic strains of the fungus Pleurotus sapidus. Some of the cultures exhibited a pleasant smell, reminiscent of woodruff and anise, as well as herbaceous notes. To evaluate the composition of the overall aroma, liquid/liquid extracts of submerged cultures of a selected monokaryon were prepared, and the volatiles were isolated via solvent-assisted flavor evaporation. Aroma extract dilution analyses revealed p-anisaldehyde (sweetish, anisic- and woodruff-like) with a flavor dilution factor of 218 as a character impact compound. The coconut-like, herbaceous, and sweetish smelling acyloin identified as (2S)-hydroxy-1-(4-methoxyphenyl)-1-propanone also contributed to the overall aroma and was described as an aroma-active substance with an odor threshold in air of 0.2 ng L-1 to 2.4 ng L-1 for the first time. Supplementation of the culture medium with isotopically substituted l-tyrosine elucidated this phenolic amino acid as precursor of p-anisaldehyde as well as of (2S)-hydroxy-1-(4-methoxyphenyl)-1-propanone. Chiral analysis via HPLC revealed an enantiomeric excess of 97% for the isolated product produced by P. sapidus.

Monokaryotic Pleurotus sapidus Strains with Intraspecific Variability of an Alkene Cleaving DyP-Type Peroxidase Activity as a Result of Gene Mutation and Differential Gene Expression.

The basidiomycete Pleurotus sapidus produced a dye-decolorizing peroxidase (PsaPOX) with alkene cleavage activity, implying potential as a biocatalyst for the fragrance and flavor industry. To increase the activity, a daughter-generation of 101 basidiospore-derived monokaryons (MK) was used. After a pre-selection according to the growth rate, the activity analysis revealed a stable intraspecific variability of the strains regarding peroxidase and alkene cleavage activity of PsaPOX. Ten monokaryons reached activities up to 2.6-fold higher than the dikaryon, with MK16 showing the highest activity. Analysis of the PsaPOX gene identified three different enzyme variants. These were co-responsible for the observed differences in activities between strains as verified by heterologous expression in Komagataella phaffii. The mutation S371H in enzyme variant PsaPOX_high caused an activity increase alongside a higher protein stability, while the eleven mutations in variant PsaPOX_low resulted in an activity decrease, which was partially based on a shift of the pH optimum from 3.5 to 3.0. Transcriptional analysis revealed the increased expression of PsaPOX in MK16 as reason for the higher PsaPOX activity in comparison to other strains producing the same PsaPOX variant. Thus, different expression profiles, as well as enzyme variants, were identified as crucial factors for the intraspecific variability of the PsaPOX activity in the monokaryons.

Virtual screening for functional foods against the main protease of SARS-CoV-2.

The special attention was paid on the interaction between functional foods and the main protease of severe acute respiratory syndrome coronavirus (SARS-CoV-2). Here, 10,870 ligands were employed and screened by the molecular docking, which involved 12 kinds of functional foods (carbohydrates, fatty acids, phospholipids, vitamin, ß-sitosterol, flavonoids, nordihydroguaiaretic acid, curcumin, nootkatone, ß-pinene, tincturoid, betulinic acid, and their isomers/analogs/derivatives). Then, 60 ligands were obtained with the good docking affinity. Most of them belong to quercetrin and its isomers/analogs/derivatives, which also showed the highest affinity for the main protease of SARS-CoV-2. The dynamic simulation indicated that quercetrin-protease and quercetrin-analog-protease showed the excellent stability. Compared with reported docking results, quercetrin should be the best inhibitor for the main protease of SARS-CoV-2. Considering the green and white tea are rich in quercetrin and its isomers/analogs/derivatives, tea and relative beverages may become a good option to regulate our metabolism and help us to overcome this special time. PRACTICAL APPLICATIONS: The docking and molecular dynamics technology were combined to screen the functional foods, which would be the potential candidate of the inhibitor for SARS-CoV-2. Many functional foods screened in this work belong to necessary nutrients for body. Thus, SARS-CoV-2 would consume some necessary nutrients, and thus, damage our body. It should be further consideration whether exogenous nutrients should be provided to slow, halt, or reverse biochemical alterations and structural deterioration in our body. On the contrary, this work also provided a new possibility to design a functional food or drug to help us overcome this special time.

Biotransformation of rice and sunflower side-streams by dikaryotic and monokaryotic strains of Pleurotus sapidus: Impact on phenolic profiles and bioactive properties.

Fungi are known to modify the properties of lignocellulosic materials during solid-state fermentation (SSF). In this study, agricultural side-streams (sunflower seed hulls, rice husks and rice straw) were used as substrates for SSF with dikaryotic and monokaryotic strains of Pleurotus sapidus. The phenolic profiles of the mentioned substrates were characterized by LC-DAD/ESI-MSn pre- and post- fermentation. Moreover, antioxidant, cytotoxic and antimicrobial activities were screened against oxidizable cellular substrates, tumour and primary cell lines, and different bacteria and fungi, respectively. The concentration of phenolic compounds in the crop side-streams was reduced after fermentation with both strains of the fungus. The fermented extracts also displayed lower antioxidant and cytotoxic activities and had no hepatotoxicity. The antimicrobial activity depended upon the crop side-stream and/or SSF conditions. These results indicate that P. sapidus represent a good candidate to modify the phenolic fraction presents in crop side-streams with a consequent decrease in its bioactivities. However, the SSF with P. sapidus strains play an interesting role in the detoxification of plant materials which can be used for different applications according to the "reduce - reuse - recycle" concept contributing with the sustainable land use and circular economy.

Long-Term Monokaryotic Cultures of Pleurotus ostreatus var. florida Produce High and Stable Laccase Activity Capable to Degrade ß-Carotene.

An extracellular laccase (Lacc10) was discovered in submerged cultures of Pleurotus ostreatus var. florida bleaching ß-carotene effectively without the addition of a mediator (650 mU/L, pH 4). Heterologous expression in P. pastoris confirmed the activity and structural analyses revealed a carotenoid-binding domain, which formed the substrate-binding pocket and is reported here for the first time. In order to increase activity, 106 basidiospore-derived monokaryons and crosses of compatible progenies were generated. These showed high intraspecific variability in growth rate and enzyme formation. Seventy-two homokaryons exhibited a higher activity-to-growth-rate-relation than the parental dikaryon, and one isolate produced a very high activity (1800 mU/L), while most of the dikaryotic hybrids showed lower activity. The analysis of the laccase gene of the monokaryons revealed two sequences differing in three amino acids, but the primary sequences gave no clue for the diversity of activity. The enzyme production in submerged cultures of monokaryons was stable over seven sub-cultivation cycles.

Biorefining via solid-state fermentation of rice and sunflower by-products employing novel monosporic strains from Pleurotus sapidus.

Pleurotus sapidus monokaryotic strains (Mk) were screened as a novel source of mycelia to valorize rice straw (RS), rice husks (RH) and sunflower seed hulls (SSH) into value-added products through solid-state fermentation (SSF). P. sapidus Dk3174 basidiospores were cultured in the presence of Remazol Brillant Blue R for strain selection, revealing the ligninolytic ability of emerging colonies. Further screening demonstrated the intraspecific variability in dye degradation and enzyme production of 63 strains. Growth rate, biomass and enzyme production in plates containing RS, RH or SSH pointed at MkP6 as a suitable strain for pilot-scale SSF. MkP6 presented a similar laccase profile as the parental Dk3174, being greater in pasteurized substrates (300-1200 U/Kg) than in sterilized substrates (30-250 U/Kg). Peroxidase represented 25% of the total ligninolytic activity measured. The SSH fermented biomass with MkP6 obtained good yields of nanocellulose (67%) and the saccharide release for ethanol production increased by 3-4 times.

Crosses between monokaryons of Pleurotus sapidus or Pleurotus florida show an improved biotransformation of (+)-valencene to (+)-nootkatone.

Several hundred monokaryotic and new dikaryotic strains derived thereof were established from (+)-valencene tolerant Pleurotus species. When grouped according to their growth rate on agar plates and compared to the parental of Pleurotus sapidus 69, the slowly growing monokaryons converted (+)-valencene more efficiently to the grapefruit flavour compound (+)-nootkatone. The fast growing monokaryons and the slow×slow and the fast×fast dikaryotic crosses showed similar or inferior yields. Some slow×fast dikaryons, however, exceeded the biotransformation capability of the parental dikaryon significantly. The activity of the responsible enzyme, lipoxygenase, showed a weak correlation with the yields of (+)-nootkatone indicating that the determination of enzyme activity using the primary substrate linoleic acid may be misleading in predicting the biotransformation efficiency. This exploratory study indicated that a classical genetics approach resulted in altered and partly improved terpene transformation capability (plus 60%) and lipoxygenase activity of the strains.