Inhibitory Activity of Flavonoid Scaffolds on SARS-CoV-2 3CLpro: Insights from the Computational and Experimental Investigations.

The emergence of the COVID-19 situation has become a global issue due to the lack of effective antiviral drugs for treatment. Flavonoids are a class of plant secondary metabolites that have antiviral activity against SARS-CoV-2 through inhibition of the main protease (3CLpro). In this study, 22 flavonoids obtained from natural sources and semisynthetic approaches were investigated for their inhibitory activity against SARS-CoV-2 3CLpro, along with cytotoxicity on Vero cells. The protein-ligand interactions were examined using molecular dynamics simulation. Moreover, QSAR analysis was conducted to clarify the structural effects on bioactivity. Accordingly, the in vitro investigation demonstrated that four flavonoids, namely, tectochrysin (7), 6″,6″-dimethylchromeno-[2″,3″:7,8]-flavone (9), panduratin A (19), and genistein (20), showed higher protease inhibitory activity compared to the standard flavonoid baicalein. Finally, our finding suggests that genistein (20), an isoflavone discovered in Millettia brandisiana, has potential for further development as a SARS-CoV-2 3CLpro inhibitor.

Purification and characterization of crude fructooligosaccharides extracted from red onion (Allium cepa var. viviparum) by yeast treatment.

BACKGROUND: Yeast treatment has been used for purification of fructooligosaccharides (FOSs). However, the main drawback of this approach is that yeast can only partially remove sucrose from crude FOSs. The main objective of this research was to screen yeast strains for the capability of selectively consuming unwanted sugars, namely fructose, glucose, and sucrose, in crude FOSs extracted from red onion (Allium cepa var. viviparum) with minimal effect on FOS content. RESULTS: Among 43 yeast species isolated from Miang, ethnic fermented tea leaves, and Assam tea flowers, Candida orthopsilosis FLA44.2 and Priceomyces melissophilus FLA44.8 exhibited the greatest potential to specifically consume these unwanted sugars. In a shake flask, direct cultivation of C. orthopsilosis FLA44.2 was achieved in the original crude FOSs containing an initial FOSs concentration of 88.3 ± 1.2 g/L and 52.9 ± 1.2 g/L of the total contents of fructose, glucose, and sucrose. This was successful with 93.7% purity and 97.8% recovery after 24 h of cultivation. On the other hand, P. melissophilus FLA48 was limited by initial carbohydrate concentration of crude FOSs in terms of growth and sugar utilization. However, it could directly purify two-fold diluted crude FOSs to 95.2% purity with 92.2% recovery after 72 h of cultivation. Purification of crude FOSs in 1-L fermenter gave similar results to the samples purified in a shake flask. Extracellular ß-fructosidase was assumed to play a key role in the effective removal of sucrose. Both Candida orthopsilosis FLA44.2 and P. melissophilus FLA44.8 showed γ-hemolytic activity, while their culture broth had no cytotoxic effect on viability of small intestinal epithelial cells, preliminarily indicating their safety for food processing. The culture broth obtained from yeast treatment was passed through an activated charcoal column for decolorization and deodorization. After being freeze dried, the final purified FOSs appeared as a white granular powder similar to refined sugar and was odorless since the main sulfur-containing volatile compounds, including dimethyl disulfide and dipropyl trisulfide, were almost completely removed. CONCLUSION: The present purification process is considered simple and straight forward, and provides new and beneficial insight into utilization of alternative yeast species for purification of FOSs.

2',4'-Dihydroxy-6'­methoxy-3',5'-dimethylchalcone and its amino acid-conjugated derivatives induce G0/G1 cell cycle arrest and apoptosis via BAX/BCL2 ratio upregulation and in silico insight in SiHa cell lines.

We modified the chemical structure of 2',4'-dihydroxy-6'­methoxy-3',5'-dimethylchalcone (DMC, 1), a phytochemical found in the seed of Syzygium nervosum A.Cunn. ex DC., by conjugation with the amino acid L-alanine (compound 3a) or L-valine (compound 3b) to enhance anticancer activity and water solubility. Compounds 3a and 3b had antiproliferative activity in human cervical cancer cell lines (C-33A, SiHa and HeLa), with half-maximal inhibitory concentrations (IC50) of 7.56 ± 0.27 and 8.24 ± 0.14 µM, respectively in SiHa cells; these values were approximately two-fold greater than DMC. We investigated the biological activities of compounds 3a and 3b based on a wound healing assay, a cell cycle assay and messenger RNA (mRNA) expression analysis to determine the possible mechanism of anticancer activity. Compounds 3a and 3b inhibited SiHa cell migration in the wound healing assay. After treatment with compounds 3a and 3b, there was an increase in SiHa cells in the G1 phase, indicative of cell cycle arrest. Moreover, compound 3a showed potential anticancer activity by upregulating TP53 and CDKN1A that resulted in upregulation of BAX and downregulation of CDK2 and BCL2, leading to apoptosis and cell cycle arrest. The BAX/BCL2 expression ratio was increased after treatment with compound 3avia the intrinsic apoptotic pathway. In silico molecular dynamics simulation and binding free energy calculation shed light on how these DMC derivatives interact with the HPV16 E6 protein, a viral oncoprotein associated with cervical cancer. Our findings suggest that compound 3a is a potential candidate for anti-cervical cancer drug development.

Discovery of Natural Bisbenzylisoquinoline Analogs from the Library of Thai Traditional Plants as SARS-CoV-2 3CLPro Inhibitors: In Silico Molecular Docking, Molecular Dynamics, and In Vitro Enzymatic Activity.

The emergence of SARS-CoV-2 in December 2019 has become a global issue due to the continuous upsurge in patients and the lack of drug efficacy for treatment. SARS-CoV-2 3CLPro is one of the most intriguing biomolecular targets among scientists worldwide for developing antiviral drugs due to its relevance in viral replication and transcription. Herein, we utilized computer-assisted drug screening to investigate 326 natural products from Thai traditional plants using structure-based virtual screening against SARS-CoV-2 3CLPro. Following the virtual screening, the top 15 compounds based on binding energy and their interactions with key amino acid Cys145 were obtained. Subsequently, they were further evaluated for protein-ligand complex stability via molecular dynamics simulation and binding free energy calculation using molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approaches. Following drug-likeness and ADME/Tox assessments, seven bisbenzylisoquinolines were obtained, including neferine (3), liensinine (4), isoliensinine (5), dinklacorine (8), tiliacorinine (13), 2'-nortiliacorinine (14), and yanangcorinine (15). These compounds computationally showed a higher binding affinity than native N3 and GC-373 inhibitors and attained stable interactions on the active site of 3CLpro during 100 ns in molecular dynamics (MD) simulation. Moreover, the in vitro enzymatic assay showed that most bisbenzylisoquinolines could experimentally inhibit SARS-CoV-2 3CLPro. To our delight, isoliensinine (5) isolated from Nelumbo nucifera demonstrated the highest inhibition of protease activity with the IC50 value of 29.93 µM with low toxicity on Vero cells. Our findings suggested that bisbenzylisoquinoline scaffolds could be potentially used as an in vivo model for the development of effective anti-SARS-CoV-2 drugs.

Effects of 2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone from Syzygium nervosum Seeds on Antiproliferative, DNA Damage, Cell Cycle Arrest, and Apoptosis in Human Cervical Cancer Cell Lines.

2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), a natural product derived from Syzygium nervosum A. Cunn. ex DC., was investigated for its inhibitory activities against various cancer cell lines. In this work, we investigated the effects of DMC and available anticervical cancer drugs (5-fluorouracil, cisplatin, and doxorubicin) on three human cervical cancer cell lines (C-33A, HeLa, and SiHa). DMC displayed antiproliferative cervical cancer activity in C-33A, HeLa, and SiHa cells, with IC50 values of 15.76 ± 1.49, 10.05 ± 0.22, and 18.31 ± 3.10 µM, respectively. DMC presented higher antiproliferative cancer activity in HeLa cells; therefore, we further investigated DMC-induced apoptosis in this cell line, including DNA damage, cell cycle arrest, and apoptosis assays. As a potential anticancer agent, DMC treatment increased DNA damage in cancer cells, observed through fluorescence inverted microscopy and a comet assay. The cell cycle assay showed an increased number of cells in the G0/G1 phase following DMC treatment. Furthermore, DMC treatment-induced apoptosis cell death was approximately three- to four-fold higher compared to the untreated group. Here, DMC represented a compound-induced apoptosis for cell death in the HeLa cervical cancer cell line. Our findings suggest that DMC, a phytochemical agent, is a potential candidate for antiproliferative cervical cancer drug development.