The haplotype-resolved genome of diploid Chrysanthemum indicum unveils new acacetin synthases genes and their evolutionary history.

Acacetin, a flavonoid compound, possesses a wide range of pharmacological effects, including antimicrobial, immune regulation, and anticancer effects. Some key steps in its biosynthetic pathway were largely unknown in flowering plants. Here, we present the first haplotype-resolved genome of Chrysanthemum indicum, whose dried flowers contain abundant flavonoids and have been utilized as traditional Chinese medicine. Various phylogenetic analyses revealed almost equal proportion of three tree topologies among three Chrysanthemum species (C. indicum, C. nankingense, and C. lavandulifolium), indicating that frequent gene flow among Chrysanthemum species or incomplete lineage sorting due to rapid speciation might contribute to conflict topologies. The expanded gene families in C. indicum were associated with oxidative functions. Through comprehensive candidate gene screening, we identified five flavonoid O-methyltransferase (FOMT) candidates, which were highly expressed in flowers and whose expressional levels were significantly correlated with the content of acacetin. Further experiments validated two FOMTs (CI02A009970 and CI03A006662) were capable of catalyzing the conversion of apigenin into acacetin, and these two genes are possibly responsible acacetin accumulation in disc florets and young leaves, respectively. Furthermore, combined analyses of ancestral chromosome reconstruction and phylogenetic trees revealed the distinct evolutionary fates of the two validated FOMT genes. Our study provides new insights into the biosynthetic pathway of flavonoid compounds in the Asteraceae family and offers a model for tracing the origin and evolutionary routes of single genes. These findings will facilitate in vitro biosynthetic production of flavonoid compounds through cellular and metabolic engineering and expedite molecular breeding of C. indicum cultivars.

Novel methyltransferase G9a inhibitor induces ferroptosis in multiple myeloma through Nrf2/HO-1 pathway.

Multiple myeloma (MM) is a common malignant hematologic neoplasm, and the involvement of epigenetic modifications in its development and drug resistance has received widespread attention. Ferroptosis, a new ferroptosis-dependent programmed death mode, is closely associated with the development of MM. The novel methyltransferase inhibitor DCG066 has higher cell activity, but its mechanism of action in MM has not been clarified. Here, we found that DCG066 (5µM) inhibited the proliferation and induced ferroptosis in MM cells; the intracellular levels of ROS, iron, and MDA were significantly elevated, and the level of GSH was reduced after the treatment of DCG066; The protein expression levels of SLC7A11, GPX4, Nrf2 and HO-1 were significantly reduced, and these phenomena could be reversed by ferroptosis inhibitor Ferrostatin-1 (Fer-1) and Nrf2 activator Tert-butyl hydroquinone (TBHQ). Meanwhile, the protein expression levels of Keap1 was increased, and heat shock proteins (HSP70, HSP90 and HSPB1) were reduced after DCG066 treatment. In conclusion, this study confirmed that DCG066 inhibits MM proliferation and induces ferroptosis via the Nrf2/HO-1 pathway.

Haplotype-phased genome unveils the butylphthalide biosynthesis and homoploid hybrid origin of Ligusticum chuanxiong.

Butylphthalide is one of the first-line drugs for ischemic stroke therapy, while no biosynthetic enzyme for butylphthalide has been reported. Here, we present a haplotype-resolved genome of Ligusticum chuanxiong, a long-cultivated and phthalide-rich medicinal plant in Apiaceae. On the basis of comprehensive screening, four Fe(II)- and 2-oxoglutarate-dependent dioxygenases and two CYPs were mined and further biochemically verified as phthalide C-4/C-5 desaturases (P4,5Ds) that effectively promoted the forming of (S)-3-n-butylphthalide and butylidenephthalide. The substrate promiscuity and functional redundancy featured for P4,5Ds may contribute to the high phthalide diversity in L. chuanxiong. Notably, comparative genomic evidence supported L. chuanxiong as a homoploid hybrid with Ligusticum sinense as a potential parent. The two haplotypes demonstrated exceptional structure variance and diverged around 3.42 million years ago. Our study is an icebreaker for the dissection of phthalide biosynthetic pathway and reveals the hybrid origin of L. chuanxiong, which will facilitate the metabolic engineering for (S)-3-n-butylphthalide production and breeding for L. chuanxiong.

Stellera chamaejasme L. extracts in the treatment of glioblastoma cell lines: Biological verification based on a network pharmacology approach.

Background: Stellera chamaejasme L (RXLD) has been demonstrated with good clinical effects and medicinal value in the treatment of cancer in vivo and in vitro. Specifically, RXLD can eliminate aggregation accumulation, which is depicted as a vital characteristic feature of intracranial tumors. The potential pharmacological mechanisms of anti-glioblastoma (GBM) have not been adequately identified. Methods: The 3D structures of the chemical ingredients in RXLD were imported into the PharmMapper database to construct the pharmacophore models. The gene targets of GBM were obtained from databases. The pharmacophore-targets network and the protein-protein interactions (PPI) were constructed using the String database and were visualized by using Cytoscape. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses were conducted using Bioconductor software. Cytoscape visualized the relationship of pathways and candidate genes to screen for key target genes. Software packages PyMOL, AutoDock, and Vina acquired the molecular docking results. In vitro experiments were undertaken to characterize RXLD extracts' effects on A172 cell line proliferation, viability, apoptosis, cell cycle, cell wound healing, cell migration, reactive oxygen species generation, and mitochondrial membrane potential. The expression of core genes in the related pathways was detected by Western blotting. Results: We identified 216 potential targets associated with GBM. The core components in RXLD were neochamaejasmin A, wikstrol A, isochamaejasmin, chamaejasmine, and subtoxin A. The undertaken GO enrichment analysis revealed that oxidative stress, cell proliferation, cell cycle, cell invasion, and cell migration were involved in the biological processes. The KEGG enrichment analysis revealed that the crucial pathway was MAPK pathway, while HRAS, PRKCB, MAPK9, CCND1, and TP53 were distributed in core locations. A total of seven RXLD pharmacophores demonstrated strong spontaneous docking activities with MAPK9. In vitro assays indicated that RXLD can induce apoptosis, block the cell cycle in the G2/M and S phases, inhibit cell migration via the Wnt/ß-catenin pathway, and inhibited p62/Nrf2 pathway. Conclusions: We speculate that the RAS/MAPK pathway might be an upstream pathway through which the RXLD exerts its anti-GBM effects and might be able to regulate further the Wnt/ß-catenin, the oxidative stress, and the ferroptosis pathways.

SNHG1 knockdown upregulates miR-376a and downregulates FOXK1/Snail axis to prevent tumor growth and metastasis in HCC.

Long non-coding RNAs (lncRNAs), microRNAs (miRNAs or miRs), and genes are emerging players in cancer progression. In the present study, we explored the roles and interactions of oncogenic lncRNA small nucleolar RNA host gene 1 (SNHG1), miR-376, forkhead box protein K1 (FOXK1), and Snail in hepatocellular carcinoma (HCC). Expression of SNHG1, miR-376, and FOXK1 in HCC was characterized in clinical HCC tissues of 75 patients with HCC. The interactions between SNHG1 and miR-376 and between miR-376 and FOXK1 were predicted and confirmed by dual-luciferase reporter gene and RNA immunoprecipitation assays. Overexpression and knockdown experiments were performed in HCC cells to examine the effects of the SNHG1/miR-376/FOXK1/Snail axis on viability, apoptosis, invasiveness, and migrating abilities. Their effects on tumor growth and metastasis were validated in nude mouse models. SNHG1 and FOXK1 were upregulated, and miR-376a was downregulated in HCC. SNHG1 knockdown contributed to suppression of HCC cell viability, invasion, and migration properties and promotion of apoptosis. SNHG1 could competitively bind to miR-376a to upregulate its target gene FOXK1, which upregulated Snail. SNHG1 knockdown delayed cancer progression both in vitro and in vivo by upregulating miR-376a and downregulating FOXK1 and Snail. SNHG1 knockdown exerts anti-tumor activity in HCC, suggesting a therapeutic target.

Potential new targets and drugs related to histone modifications in glioma treatment.

Glioma accounts for 40-50% of craniocerebral tumors, whose outcome rarely improves after standard treatment. The development of new therapeutic targets for glioma treatment has important clinical significance. With the deepening of research on gliomas, recent researchers have found that the occurrence and development of gliomas is closely associated with histone modifications, including methylation, acetylation, phosphorylation, and ubiquitination. Additionally, evidence has confirmed the close relationship between histone modifications and temozolomide (TMZ) resistance. Therefore, histone modification-related proteins have been widely recognized as new therapeutic targets for glioma treatment. In this review, we summarize the potential histone modification-associated targets and related drugs for glioma treatment. We have further clarified how histone modifications regulate the pathogenesis of gliomas and the mechanism of drug action, providing novel insights for the current clinical glioma treatment. Herein, we have also highlighted the limitations of current clinical therapies and have suggested future research directions and expected advances in potential areas of disease prognosis. Due to the complicated glioma pathogenesis, in the present review, we have acknowledged the limitations of histone modification applications in the related clinical treatment.

Potential Epigenetic-Based Therapeutic Targets for Glioma.

Glioma is characterized by a high recurrence rate, short survival times, high rates of mortality and treatment difficulties. Surgery, chemotherapy and radiation (RT) are the standard treatments, but outcomes rarely improve even after treatment. With the advancement of molecular pathology, recent studies have found that the development of glioma is closely related to various epigenetic phenomena, including DNA methylation, abnormal microRNA (miRNA), chromatin remodeling and histone modifications. Owing to the reversibility of epigenetic modifications, the proteins and genes that regulate these changes have become new targets in the treatment of glioma. In this review, we present a summary of the potential therapeutic targets of glioma and related effective treating drugs from the four aspects mentioned above. We further illustrate how epigenetic mechanisms dynamically regulate the pathogenesis and discuss the challenges of glioma treatment. Currently, among the epigenetic treatments, DNA methyltransferase (DNMT) inhibitors and histone deacetylase inhibitors (HDACIs) can be used for the treatment of tumors, either individually or in combination. In the treatment of glioma, only HDACIs remain a good option and they provide new directions for the treatment. Due to the complicated pathogenesis of glioma, epigenetic applications to glioma clinical treatment are still limited.

Synthesis and biological evaluation of novel N-aryl-ω-(benzoazol-2-yl)-sulfanylalkanamides as dual inhibitors of α-glucosidase and protein tyrosine phosphatase 1B.

α-Glucosidase is known to catalyze the digestion of carbohydrates and release free glucose into the digestive tract. Protein tyrosine phosphatase 1B (PTP1B) is engaged in the dephosphorylation of the insulin receptor and regulation of insulin sensitivity. Therefore, dual antagonists by targeting both α-glucosidase and PTP1B may be potential candidates for type 2 diabetes therapy. In this work, three series of novel N-aryl-ω-(benzoazol-2-yl)-sulfanylalkanamides were synthesized and assayed for their α-glucosidase and PTP1B inhibitory activities, respectively. Compound 3l, exhibiting the most effective α-glucosidase inhibitory activity (IC50  = 10.96 µm (3l), IC50  = 51.32 µm (Acarbose), IC50  = 18.22 µm (Ursolic acid)) and potent PTP1B inhibitory activity (IC50  = 13.46 µm (3l), IC50  = 14.50 µm (Ursolic acid)), was identified as a novel dual inhibitor of α-glucosidase and PTP1B. Furthermore, 3l is a highly selective PTP1B inhibitor because no inhibition was showed by 3l at 100 µm against PTP-MEG2, TCPTP, SHP2, or SHP1. Subsequent kinetic analysis revealed 3l inhibited α-glucosidase in a reversible and mixed manner. Molecular docking study indicated that hydrogen bonds, van der Waals, charge interactions and Pi-cation interactions all contributed to affinity between 3l and α-glucosidase/PTP1B.

B7-H6 expression is induced by lipopolysaccharide and facilitates cancer invasion and metastasis in human gliomas.

Although great progress has been made in treatment regimens, gliomas are still incurable and the 5-year survival remains poor. Studies focusing on molecules that regulate tumorigenesis or tumor immunity may provide potential therapeutic strategies for patients with glioma. B7-H6 is selectively expressed in tumor cells and plays vital roles in host immune responses. In this study, we demonstrated that B7-H6 was expressed in glioma cell lines, including CRT, U251, SHG-44, SF-295, TG-905 and U373, and tumor tissues isolated from glioma patients. Moreover, the expression levels of B7-H6 were significantly correlated with glioma grade. Previous studies reported that inflammatory mediators and cytokines induced the expression of B7 family members including programmed death-ligand 1, B7-H2 and B7-H4. Therefore, we explored the regulation of B7-H6 expression in gliomas and showed that lipopolysaccharide induced the expression of B7-H6 in glioma cells. To further analyze the roles of B7-H6 in gliomas, the expression of B7-H6 in glioma cells was knocked down. The results of cell counting kit-8, colony formation, wound healing, and transwell migration and invasion assays demonstrated that the proliferation, migration and invasion of glioma cells were inhibited after knocking down B7-H6. To elucidate the specific mechanisms of B7-H6 function in cancer progression, we examined the expression levels of proteins involved in cell apoptosis, migration and invasion. We demonstrated that the expression levels of E-cadherin and Bcl-2 associated X protein increased, and the expression levels of vimentin, N-cadherin, matrix metalloproteinase-2, matrix metalloproteinase-9 and survivin decreased after knocking down B7-H6. In conclusion, B7-H6 plays important roles in glioma, and targeting B7-H6 may provide a novel therapeutic strategy for glioma patients.

Production of Glycopeptide Derivatives for Exploring Substrate Specificity of Human OGA Toward Sugar Moiety.

O-GlcNAcase (OGA) is the only enzyme responsible for removing N-acetyl glucosamine (GlcNAc) attached to serine and threonine residues on proteins. This enzyme plays a key role in O-GlcNAc metabolism. However, the structural features of the sugar moiety recognized by human OGA (hOGA) remain unclear. In this study, a set of glycopeptides with modifications on the GlcNAc residue, were prepared in a recombinant full-length human OGT-catalyzed reaction, using chemoenzymatically synthesized UDP-GlcNAc derivatives. The resulting glycopeptides were used to evaluate the substrate specificity of hOGA toward the sugar moiety. This study will provide insights into the exploration of probes for O-GlcNAc modification, as well as a better understanding of the roles of O-GlcNAc in cellular physiology.

Structure based design, synthesis and activity studies of small hybrid molecules as HDAC and G9a dual inhibitors.

Aberrant enzymatic activities or expression profiles of epigenetic regulations are therapeutic targets for cancers. Among these, histone 3 lysine 9 methylation (H3K9Me2) and global de-acetylation on histone proteins are associated with multiple cancer phenotypes including leukemia, prostatic carcinoma, hepatocellular carcinoma and pulmonary carcinoma. Here, we report the discovery of the first small molecule capable of acting as a dual inhibitor targeting both G9a and HDAC. Our structure based design, synthesis, and screening for the dual activity of the small molecules led to the discovery of compound 14 which displays promising inhibition of both G9a and HDAC in low micro-molar range in cell based assays.

Discovery of novel small molecule inhibitors of lysine methyltransferase G9a and their mechanism in leukemia cell lines.

Lysine methyltransferase G9a regulates the transcription of multiple genes by primarily catalyzing mono- and di-methylation of histone H3 lysine 9, as well as several non-histone lysine sites. An attractive therapeutic target in treating leukemia, knockout studies of G9a in mice have found dramatically slowed proliferation and self-renewal of acute myeloid leukemia (AML) cells due to the attenuation of HoxA9-dependent transcription. In this study, a series of compounds were identified as potential inhibitors through structure-based virtual screening. Among these compounds, a new G9a inhibitor, DCG066, was confirmed by in vitro biochemical, and cell based enzyme assays. DCG066 has a novel molecular scaffold unlike other G9a inhibitors presently available. Similar to G9a's histone substrate, DCG066 can bind directly to G9a and inhibit methyltransferase activity in vitro. In addition to suppressing G9a methyltransferase activity and reducing histone H3 methylation levels, DCG066 displays low cytotoxicity in leukemia cell lines with high levels of G9a expression, including K562. This work presents DCG066 as an inhibitor of G9a with a novel structure, providing both a lead in G9a inhibitor design and a means for probing the functionality of G9a.

Efficient enzymatic synthesis of L-rhamnulose and L-fuculose.

L-Rhamnulose (6-deoxy-L-arabino-2-hexulose) and L-fuculose (6-deoxy-L-lyxo-2-hexulose) were prepared from L-rhamnose and L-fucose by a two-step strategy. In the first reaction step, isomerization of L-rhamnose to L-rhamnulose, or L-fucose to L-fuculose was combined with a targeted phosphorylation reaction catalyzed by L-rhamnulose kinase (RhaB). The by-products (ATP and ADP) were selectively removed by silver nitrate precipitation method. In the second step, the phosphate group was hydrolyzed to produce L-rhamnulose or L-fuculose with purity exceeding 99% in more than 80% yield (gram scale).

Facile Enzymatic Synthesis of Ketoses.

Studies of rare ketoses have been hampered by a lack of efficient preparation methods. A convenient, efficient, and cost-effective platform for the facile synthesis of ketoses is described. This method enables the preparation of difficult-to-access ketopentoses and ketohexoses from common and inexpensive starting materials with high yield and purity and without the need for a tedious isomer separation step.

SAHA-based novel HDAC inhibitor design by core hopping method.

The catalytic activity of the histone deacetylase (HDAC) is directly relevant to the pathogenesis of cancer, and HDAC inhibitors represented a promising strategy for cancer therapy. SAHA (suberoanilide hydroxamic acid), an effective HDAC inhibitor, is an anti-cancer agent against T-cell lymphoma. However, SAHA has adverse effects such as poor pharmacokinetic properties and severe toxicities in clinical use. In order to identify better HDAC inhibitors, a compound database was established by core hopping of SAHA, which was then docked into HDAC-8 (PDB ID: 1T69) active site to select a number of candidates with higher docking score and better interaction with catalytic zinc ion. Further ADMET prediction was done to give ten compounds. Molecular dynamics simulation of the representative compound 101 was performed to study the stability of HDAC8-inhibitor system. This work provided an approach to design novel high-efficiency HDAC inhibitors with better ADMET properties.