Antitumor potential of tricyclic pyridone analogues from an entomopathogenic fungus, Fusarium avenaceum SYKC02-P-1.

Two new tricyclic pyridone analogues, fusapyridons C (1) and D (2), were isolated along with structurally related known compounds 3 - 5 from the entomopathogenic fungus, F. avenaceum SYKC02-P-1. The structures of compounds 1 and 2 were elucidated by analyzing the spectral data of UV, 1 D and 2 D NMR as well as HRESIMS. The absolute configurations of fusapyridons C and D were established by means of single crystal X-ray diffraction and electronic circular dichroism calculation. And antitumor testing of all the isolates showed that compounds 4 and 5 exhibited significant inhibitory activity against the human prostate cancer cells (PC-3 cell lines) with IC50 values of 2.76 and 1.86 µM, respectively.

Dual roles of drug or its metabolite-protein conjugate: Cutting-edge strategy of drug discovery using shotgun proteomics.

Many drugs can bind directly to proteins or be bioactivated by metabolizing enzymes to form reactive metabolites (RMs) that rapidly bind to proteins to form drug-protein conjugates or metabolite-protein conjugates (DMPCs). The close relationship between DMPCs and idiosyncratic adverse drug reactions (IADRs) has been recognized; drug discovery teams tend to avoid covalent interactions in drug discovery projects. Covalent interactions in DMPCs can provide high potency and long action duration and conquer the intractable targets, inspiring drug design, and development. This forms the dual role feature of DMPCs. Understanding the functional implications of DMPCs in IADR control and therapeutic applications requires precise identification of these conjugates from complex biological samples. While classical biochemical methods have contributed significantly to DMPC detection in the past decades, the low abundance and low coverage of DMPCs have become a bottleneck in this field. An emerging transformation toward shotgun proteomics is on the rise. The evolving shotgun proteomics techniques offer improved reproducibility, throughput, specificity, operability, and standardization. Here, we review recent progress in the systematic discovery of DMPCs using shotgun proteomics. Furthermore, the applications of shotgun proteomics supporting drug development, toxicity mechanism investigation, and drug repurposing processes are also reviewed and prospected.

Quantification of Osimertinib and Metabolite-Protein Modification Reveals Its High Potency and Long Duration of Effects on Target Organs.

Covalent drugs are newly developed and proved to be successful therapies in past decades. However, the pharmacokinetics (PK) and pharmacodynamic (PD) studies of covalent drugs now ignore the drug and metabolite-protein modification. The low abundance of modified proteins also prevents its investigation. Herein, a simple, selective, and sensitive liquid chromatography-mass spectrometry (LC-MS)/MS quantitative method was established based on the mechanism of a drug and its metabolite-protein adducts using osimertinib as an example. Five metabolites with covalent modification potential were identified. The drug and its metabolite-cysteine adducts released from modified proteins by a mixed hydrolysis method were developed to characterize the level of the modified proteins. This turned the quantitative objects from proteins or peptides to small molecules, which increased the sensitivity and throughput of the quantitative approach. Accumulation of protein adducts formed by osimertinib and its metabolites in target organs was observed in vivo and long-lasting modifications were noted. These results interpreted the long duration of the covalent drugs' effect from the perspective of both parent and the metabolites. In addition, the established method could also be applied in blood testing as noninvasive monitoring. This newly developed approach showed great feasibility for PK and PD studies of covalent drugs.

MS-FINDER Assisted in Understanding the Profile of Flavonoids in Temporal Dimension during the Fermentation of Pu-erh Tea.

Flavonoid represents a significant class of secondary metabolites in Pu-erh tea with benefits to human health. For a rapid and complete discovery of such compounds, we established a data mining workflow that integrates software MS-DIAL, MS-FINDER, and molecular networking analysis. As a result, 181 flavonoids were tentatively annotated including 22 first found in Pu-erh tea, and two of them were potentially new molecules. The dynamic alteration of these flavonoids during Pu-erh fermentation was further investigated. They all showed a trend of first increasing and then decreasing. Moreover, statistical analysis showed that the first to third pile turnings of the fermentation process had a greater impact on the changes of flavonoids. Partial metabolic pathways were proposed. This study provides a quick and automatic strategy for flavonoid profiling. The temporal dimension of flavonoids during fermentation may serve as a theoretical basis for Pu-erh tea manufacturing technology and study on substance foundation.

Investigation and dynamic profiling of oligopeptides, free amino acids and derivatives during Pu-erh tea fermentation by ultra-high performance liquid chromatography tandem mass spectrometry.

Microbial fermentation is the critical step of Pu-erh tea manufacture, which will induce dramatic changes in the chemical composition and content of tea. In this research, we applied multi-methods based on UHPLC-Q-TOF/MS to profile the dynamic changes of oligopeptides, free amino acids, and derivatives (OPADs) during Pu-erh fermentation and predicted the potential bioactivities in silico. A total of 60 oligopeptides, 18 free amino acids, and 42 amino acid derivatives were identified, and the contents of most of them decreased after fermentation. But several N-acetyl amino acids increased 7-36 times after fermentation, and they might be the potential inhibitors of neurokinin-1 receptor. Moreover, the results of metamicrobiology showed Aspergillus niger and Aspergillus luchuensis were more prominent to metabolize protein, oligopeptides, and amino acids. Overall, these findings provide valuable insights about dynamic variations of OPADs during Pu-erh tea fermentation and are beneficial for guiding practical fermentation and quality control of Pu-erh tea.

Celastrol Niosome Hydrogel Has Anti-Inflammatory Effect on Skin Keratinocytes and Circulation without Systemic Drug Exposure in Psoriasis Mice.

PURPOSE: Psoriasis is an inflammatory skin disease, where keratinocytes play pivotal roles in its pathogenesis. We prepared Celastrol Noisome hydrogel (Cel Nio gel) for the treatment of psoriasis and aimed to study its target site as well as the mechanism. METHODS: Cel Nio was fabricated with thin-film hydration and sonication, then topically administered to imiquimod (IMQ)-induced psoriasis mice. The concentrations of Cel in the skin, blood and lymphatic system were determined using LC-MS. The anti-psoriasis effect of Cel Nio gel was studied, and the levels of inflammatory cytokines in blood were evaluated by flow cytometry. For the in vitro study, the uptake of Nio by HaCaT cells was quantified with flow cytometry, and the anti-inflammatory effect of Cel on HaCaT cells was detected with qPCR. The expressions of inflammatory factors and Ki-67 in skin were observed by immunofluorescence. RESULTS: Cel Nio possessed a particle size of 133 nm with encapsulation efficacy (EE%) of 83.2%. After topical administration of Cel Nio gel to mice, Cel was mainly accumulated in the skin instead of exposure in blood or lymphatic system, while the levels of inflammatory factors in blood had a significant decline. In addition, the preparation of Nio enhanced the uptake by HaCaT cells, and Cel obviously reduced the mRNA levels of inflammatory cytokines in HaCaT cells. Moreover, Cel Nio gel significantly decreased the expression of inflammatory cytokines and Ki-67 in the skin. CONCLUSION: Cel Nio gel achieved the anti-psoriatic effect by inhibiting the inflammation and hyperproliferation of keratinocytes in the skin and further suppressing the systemic inflammation, thus could be a novel topical drug delivery system to treat psoriasis with topical and systemic effects.

Discovery of the bioactive peptides secreted by Bifidobacterium using integrated MCX coupled with LC-MS and feature-based molecular networking.

Probiotics can release many bioactive peptides that confer a myriad of benefits to the host health. However, exploring new bioactive peptides secreted by probiotics is hampered by lots of matrix-related interference peptides from the medium, and the low abundance. To this end, a new approach integrating mixed-mode cationic exchange based solid-phase extraction (MCX-SPE) coupled with liquid chromatography-mass spectrometry (LC-MS) and feature-based molecular networking (FBMN) was developed. FBMN's intuitive visualization results enabled twenty-five novel peptides to be quickly discovered and characterized from the cultures of three strains of Bifidobacterium, B. animalis, B. longum, and B. pseudolongum. Interestingly, four were uniquely secreted by B. animalis treated with gypenosides, and one showed ACE inhibitory effect with an IC50 value of 193.22 µM. Consequently, this approach could serve as a powerful tool for quickly discovering bioactive peptides from the complex metabolites of probiotics, which contributes to the development of functional foods and nutraceuticals.

Bifidobacterium animalis: the missing link for the cancer-preventive effect of Gynostemma pentaphyllum.

Colorectal cancer (CRC) ranks the third most common cancer type in both men and women. Besides the known genetic and epigenetic changes in the gut epithelial cells, we now know that disturbed gut microbes could also contribute to the onset and progression of CRC. Hence, keeping a balanced gut microbiota (GM) has become a novel pursue in the medical field, particularly in the area of gastrointestinal disorders. Gynostemma pentaphyllum (Gp) is a dietary herbal medicine. In our previous study, Gp saponins (GpS) displayed prebiotic and cancer-preventive properties through the modulation of GM in ApcMin/+ mice. However, the specific group(s) of GM links to the health effects of GpS remains unknown. To track down the missing link, we first investigated and found that inoculation with fecal materials from GpS-treated ApcMin/+ mice effectively reduces polyps in ApcMin/+ mice. From the same source of the fecal sample, we successfully isolated 16 bacterial species. Out of the 16 bacteria, Bifidobacterium animalis stands out as the responder to the GpS-growth stimulus. Biochemical and RNAseq analysis demonstrated that GpS enhanced expressions of a wide range of genes encoding biogenesis and metabolic pathways in B. animalis culture. Moreover, we found that colonization of B. animalis markedly reduces the polyp burden in ApcMin/+ mice. These findings reveal a mutualistic interaction between the prebiotic and a probiotic to achieve anticancer and cancer-preventive activities. Our result, for the first time, unveils the anticancer function of B. animalis and extend the probiotic horizon of B. animalis.

New benzyl-aporphine alkaloids from Thalictrum cultratum.

Two new rare benzyl-aporphine alkaloids, thaliculine (1) and 6a,7-dehydrothaliculine (2), were isolated from the roots of Thalictrum cultratum Wall. Their structures were determined based on spectroscopic analysis including 1D, 2D NMR, and HR-ESI-MS. The stereochemistry of 1 was assigned by ECD spectroscopy. Compound 1 exhibited weak cytotoxicity against HL-60 cell line with IC50 value of 31.40 µM.