Inhibition mechanism of SARS-CoV-2 main protease by ebselen and its derivatives.

The SARS-CoV-2 pandemic has triggered global efforts to develop therapeutics. The main protease of SARS-CoV-2 (Mpro), critical for viral replication, is a key target for therapeutic development. An organoselenium drug called ebselen has been demonstrated to have potent Mpro inhibition and antiviral activity. We have examined the binding modes of ebselen and its derivative in Mpro via high resolution co-crystallography and investigated their chemical reactivity via mass spectrometry. Stronger Mpro inhibition than ebselen and potent ability to rescue infected cells were observed for a number of derivatives. A free selenium atom bound with cysteine of catalytic dyad has been revealed in crystallographic structures of Mpro with ebselen and MR6-31-2 suggesting hydrolysis of the enzyme bound organoselenium covalent adduct and formation of a phenolic by-product, confirmed by mass spectrometry. The target engagement with selenation mechanism of inhibition suggests wider therapeutic applications of these compounds against SARS-CoV-2 and other zoonotic beta-corona viruses.

Correction to "A Novel G Protein-Biased and Subtype-Selective Agonist for a G Protein-Coupled Receptor Discovered from Screening Herbal Extracts".

[This corrects the article DOI: 10.1021/acscentsci.9b01125.].

Pathway engineering and medium optimization for α-farnesene biosynthesis in oleaginous yeast Yarrowia lipolytica.

Farnesene is a typical sesquiterpene with applications as fragrance, flavor and precursor for the synthesis of vitamin E/K1. In this study, a series of strategies were employed to facilitate α-farnesene accumulation in Yarrowia lipolytica. Among them, the promoter optimization of OptFSLERG20, Sc-tHMG1 and IDI resulted in more than 62 % increase in α-farnesene production. Together with the overexpression of Yl-HMGR and ERG19, α-farnesene content was significantly improved by more than 3.5 times. The best metabolic engineered strain obtained was therefore used for a uniform design in shake flasks to determine the optimal medium compositions. Furthermore, a maximum α-farnesene production of approximately 2.57 g/L (34 mg/g DCW) was obtained in fed-batch fermentation where glycerol was supplemented as the feeding carbon source when initial glucose was depleted. This study has laid a good foundation for the development of Y. lipolytica as a promising chassis microbial cell for heterologous biosynthesis of α-farnesene and other sesquiterpenes.

Antioxidants suppress radiation-induced apoptosis via inhibiting MAPK pathway in nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is relatively sensitive to ionizing radiation, and radiotherapy is the main treatment modality for non-metastatic NPC. Radiation therapy generates overproduction of reactive oxygen species (ROS), which can cause DNA damage and induce apoptosis in tumors, thereby killing the malignant cells. Although dietary antioxidant supplementation reduces oxidative stress and promotes tumor progression, the effects of antioxidants on the NPC cells upon radiation have not been reported. In the present study, we showed that antioxidants (ß-Carotene, NAC, GSH) played an anti-apoptotic role in response to radiation via decreasing ROS production and inhibiting MAPK pathway in NPC cells. Based on that, we conclude that the use of supplemental antioxidants during radiotherapy should be avoided because of the possibility of tumor protection and reduced treatment efficacy.

A Novel G Protein-Biased and Subtype-Selective Agonist for a G Protein-Coupled Receptor Discovered from Screening Herbal Extracts.

Subtype selectivity and functional bias are vital in current drug discovery for G protein-coupled receptors (GPCRs) as selective and biased ligands are expected to yield drug leads with optimal on-target benefits and minimal side-effects. However, structure-based design and medicinal chemistry exploration remain challenging in part because of highly conserved binding pockets within subfamilies. Herein, we present an affinity mass spectrometry approach for screening herbal extracts to identify active ligands of a GPCR, the 5-HT2C receptor. Using this method, we discovered a naturally occurring aporphine 1857 that displayed strong selectivity for activating 5-HT2C without activating the 5-HT2A or 5-HT2B receptors. Remarkably, this novel ligand exhibited exclusive bias toward G protein signaling for which key residues were identified, and it showed comparable in vivo efficacy for food intake suppression and weight loss as the antiobesity drug, lorcaserin. Our study establishes an efficient approach to discovering novel GPCR ligands by exploring the largely untapped chemical space of natural products.

Discovery of Potent Inhibitors of 11ß-Hydroxysteroid Dehydrogenase Type 1 Using a Novel Growth-Based Protocol of in Silico Screening and Optimization in CONTOUR.

With the continuous progress in ultralarge virtual libraries which are readily accessible, it is of great interest to explore this large chemical space for hit identification and lead optimization using reliable structure-based approaches. In this work, a novel growth-based screening protocol has been designed and implemented in the structure-based design platform CONTOUR. The protocol was used to screen the ZINC database in silico and optimize hits to discover 11ß-HSD1 inhibitors. In contrast to molecular docking, the virtual screening process makes significant improvements in computational efficiency without losing chemical equities through partitioning 1.8 million ZINC compounds into fragments, docking fragments to form key hydrogen bonds with anchor residues, reorganizing molecules into molecular fragment trees using matched fragments and common substructures, and then regrowing molecules with the help of developed intelligent growth features inside the protein binding site to find hits. The growth-base screening approach is validated by the high hit rate. A total of 50 compounds have been selected for testing; of these, 15 hits having diverse scaffolds are found to inhibit 11ß-HSD1 with IC50 values of less than 1 µM in a biochemical enzyme assay. The best hit which exhibits an enzyme IC50 of 33 nM is further developed to a novel series of bicyclic 11ß-HSD1 inhibitors with the best inhibition of enzyme IC50 of 3.1 nM. The final lead candidate exhibits IC50 values of 7.2 and 21 nM in enzyme and adipocyte assays, respectively, displayed greater than 1000-fold of selectivity over 11ß-HSD2 and two other related hydroxysteroid dehydrogenases, and can serve as good starting points for further optimization to develop clinical candidates.

Functionality-Independent DNA Encoding of Complex Natural Products.

DNA encoded chemical libraries (DELs) link the powers of genetics and chemical synthesis via combinatorial optimization. Through combinatorial chemistry, DELs can grow to the unprecedented size of billions to trillions. To take full advantage of the DEL approach, linking the power of genetics directly to chemical structures would offer even greater diversity in a finite chemical world. Natural products have evolved an incredible structural diversity along with their biological evolution. Herein, we used traditional Chinese medicines (TCMs) as examples in a late-stage modification toolbox approach to annotate these complex organic compounds with amplifiable DNA barcodes, which could be easily incorporated into a DEL. The method of end-products labeling also generates a cluster of isomers with a single DNA tag at different sites. These isomers provide an additional spatial diversity for multiple accessible pockets of targeted proteins. Notably, a novel PARP1 inhibitor from TCM has been identified from the natural products enriched DEL (nDEL).

Sanyang Xuedai enhances the radiosensitivity of human non-small cell lung cancer cells via increasing iNOS/NO production.

OBJECTIVE: In this research, we aimed at finding out how San Yang Xue Dai (SYKT) promotes the radiosensitivity of non-small cell lung cancer (NSCLC) cell line NCI-H460. METHODS: Survival rate of NSCLC cells (A549, NCI-H460, NCI-H1650 and NCI-H1975) after the SYKT treatment or irradiation (IR) was calculated by the MTT assay. The radiosensitization of SYKT (0.5 g/mL and 1.0 g/mL) on cell line NCI-H460 and the radioresistant cell line NCI-H460R was studied by MTT assay and clone formation assay. The protein expression levels of iNOS, Cyclin B1 and CDC2 were determined by western blot, and the expression of NO was measured by Griess method. Finally, cell cycle and apoptotic rate of NSCLC cell line NCI-H460 were accessed by flow cytometry assay. BrdU staining was also applied to detect the cell proliferation after IR with or without SYKT treatment. RESULTS: The IC10 value of SYKT for NCI-H460 cells was 1.03 g/mL. After 1.0 g/mL SYKT treatment, the radiosensitivity of NCI-H460R cells was enhanced. The level of iNOS in the cells was found decreased after IR. We also found that SYKT could enhance iNOS and NO expressions while inhibit cyclin B1 and CDC2 expressions in radiation resistant cells. Combining ß-irradiation with SYKT caused cell cycle arrest in G2/M phase and increased cell apoptosis. CONCLUSION: SYKT resensitized radioresistant NCI-H460R cells via increasing cell apoptosis and cell cycle arrest. This was due to an elevated NO level caused by accumulating iNOS and effects of SYKT on radiosensitization of NSCLC should be further investigated in clinical application.

A herbal formula, SYKT, reverses doxorubicin­induced myelosuppression and cardiotoxicity by inhibiting ROS­mediated apoptosis.

Doxorubicin (DOX) is an antineoplastic drug widely used for the treatment of various types of cancer; however, it can induce severe side effects, such as myelosuppression and cardiotoxicity. Sanyang Xuedai (SYKT) is a natural medicine originating from an ancient prescription of the Dai nationality in Southwest China. With eight Chinese herbal medicines, including sanguis draconis, radix et rhizoma notoginseng, radix et rhizoma glycyrrhizae and radix angelicae sinensis as the primary ingredients, SYKT has been reported to possess numerous biological functions. The present study investigated whether SYKT can confer protection against DOX­induced myelosuppression and cardiotoxicity, and explored the potential mechanism involved. Mice were treated with DOX, SYKT or a combination of the two; hematopoietic functions were assessed by measuring the number of peripheral blood cells, cluster of differentiation CD34+/CD44+ bone marrow cells and apoptotic cells. Myocardial enzymes, including aspartate aminotransferase, lactate dehydrogenase, creatine kinase (CK) and its isoform CK­MB, were assessed using a biochemical analyzer. The apoptotic rate of cardiomyocytes was assessed using flow cytometry. Histopathological analysis was conducted using hematoxylin­eosin staining. Intracellular reactive oxygen species (ROS) production was evaluated using a dichlorofluorescein intensity assay. The mice treated with DOX exhibited a reduced survival rate, reduced peripheral blood and CD34+/CD44+ cell counts, elevated myocardial enzymes and apoptotic indices in bone marrow cells and cardiomyocytes, all of which were effectively prevented by SYKT co­administration. Furthermore, bone marrow cells and myocytes from mice treated with DOX demonstrated increased dichlorofluorescein intensity, which was attenuated by SYKT. Notably, SYKT did not interfere with the effects of DOX on tumor volume or the induction of tumor cell apoptosis in tumor­bearing mice. The present study indicated that SYKT may counteract DOX­induced myelosuppression and cardiotoxicity through inhibiting ROS­mediated apoptosis. These findings suggested that SYKT may have potential as a means to counteract the potentially fatal hematopoietic and cardiac complications associated with DOX treatment.

Combination Therapy of LysGH15 and Apigenin as a New Strategy for Treating Pneumonia Caused by Staphylococcus aureus.

Pneumonia is one of the most prevalent Staphylococcus aureus-mediated diseases, and the treatment of this infection is becoming challenging due to the emergence of multidrug-resistant S. aureus, especially methicillin-resistant S. aureus (MRSA) strains. It has been reported that LysGH15, the lysin derived from phage GH15, displays high efficiency and a broad lytic spectrum against MRSA and that apigenin can markedly diminish the alpha-hemolysin of S. aureus. In this study, the combination therapy of LysGH15 and apigenin was evaluated in vitro and in a mouse S. aureus pneumonia model. No mutual adverse influence was detected between LysGH15 and apigenin in vitro. In animal experiments, the combination therapy showed a more effective treatment effect than LysGH15 or apigenin monotherapy (P < 0.05). The bacterial load in the lungs of mice administered the combination therapy was 1.5 log units within 24 h after challenge, whereas the loads in unprotected mice or mice treated with apigenin or LysGH15 alone were 10.2, 4.7, and 2.6 log units, respectively. The combination therapy group showed the best health status, the lowest ratio of wet tissue to dry tissue of the lungs, the smallest amount of total protein and cells in the lung, the fewest pathological manifestations, and the lowest cytokine level compared with the other groups (P < 0.05). With regard to its better protective efficacy, the combination therapy of LysGH15 and apigenin exhibits therapeutic potential for treating pneumonia caused by MRSA. This paper reports the combination therapy of lysin and natural products derived from traditional Chinese medicine.

Identification and characterization of Argonaute gene family and meiosis-enriched Argonaute during sporogenesis in maize.

Argonaute (AGO) proteins play a key role in regulation of gene expression through small RNA-directed RNA cleavage and translational repression, and are essential for multiple developmental processes. In the present study, 17 AGO genes of maize (Zea mays L., ZmAGOs) were identified using a Hidden Markov Model and validated by rapid amplification of cDNA ends assay. Subsequently, quantitative PCR revealed that expressions of these genes were higher in reproductive than in vegetative tissues. AGOs presented five temporal and spatial expression patterns, which were likely modulated by DNA methylation, 5'-untranslated exons and microRNA-mediated feedback loops. Intriguingly, ZmAGO18b was highly expressed in tassels during meiosis. Furthermore, in situ hybridization and immunofluorescence showed that ZmAGO18b was enriched in the tapetum and germ cells in meiotic anthers. We hypothesized that ZmAGOs are highly expressed in reproductive tissues, and that ZmAGO18b is a tapetum and germ cell-specific member of the AGO family in maize.

Protein-protein complexation in bioluminescence.

In this review we summarize the progress made towards understanding the role of protein-protein interactions in the function of various bioluminescence systems of marine organisms, including bacteria, jellyfish and soft corals, with particular focus on methodology used to detect and characterize these interactions. In some bioluminescence systems, protein-protein interactions involve an "accessory protein" whereby a stored substrate is efficiently delivered to the bioluminescent enzyme luciferase. Other types of complexation mediate energy transfer to an "antenna protein" altering the color and quantum yield of a bioluminescence reaction. Spatial structures of the complexes reveal an important role of electrostatic forces in governing the corresponding weak interactions and define the nature of the interaction surfaces. The most reliable structural model is available for the protein-protein complex of the Ca(2+)-regulated photoprotein clytin and green-fluorescent protein (GFP) from the jellyfish Clytia gregaria, solved by means of Xray crystallography, NMR mapping and molecular docking. This provides an example of the potential strategies in studying the transient complexes involved in bioluminescence. It is emphasized that structural studies such as these can provide valuable insight into the detailed mechanism of bioluminescence.

Cloning and characterization of a cDNA clone encoding calreticulin from Haemaphysalis qinghaiensis (Acari: Ixodidae).

The application of anti-tick vaccine has been shown to be the most promising alternative strategy compared to the current use of acaricides that suffer from a number of serious limitations. The success of this method is dependent upon identification and cloning of potential tick vaccine antigens. Previously, we have cloned 21 positive clones (named from Hq02 to Hq22) by immunoscreening complimentary DNA (cDNA) libraries of Haemaphysalis qinghaiensis; however, some of those clones did not contain open reading frames (ORF). In this study, we amplified the entire sequence of Hq07 by using rapid amplification of the cDNA ends. Hq07 contains an ORF of 1,233 bp that encodes for 410 amino acid residues with a coding capacity of 47 kDa. Search of the cloned sequences against GenBank revealed that Hq07 is a calreticulin (CRT)-similar clone and designated HqCRT. Expression analysis by reverse transcription-polymerase chain reaction showed that this gene is ubiquitously expressed at different developmental stages and in different tissues of H. qinghaiensis. The gene was expressed as glutathione S-transferase-fused proteins in a prokaryotic system. Western blot analysis revealed that native HqCRT was secreted into their hosts by ticks during blood sucking. Vaccination of sheep with rHqCRT conferred protective immunity against ticks, resulting in 54.3% mortality in adult ticks, compared to the 38.7% death rate in the control group. These results demonstrated that rHqCRT might be a useful vaccine candidate antigen for biological control of H. qinghaiensis.