Design, semi-synthesis and bioevaluation of koumine-like derivatives as potential antitumor agents in vitro and in vivo.

Aims: Five series of novel koumine-like compounds were designed, semi-synthesized and systematically evaluated for antitumor activities.Methods: All compounds were evaluated for antiproliferative activity against four human cancer cell lines, including HT-29, HCT-116, HCT-15 and Caco-2.Results: Most compounds exhibited much higher antiproliferation activities (IC50 <10 µM) than koumine. Two selected compounds A4 and C5 showed comparable antitumor effects to 5-FU in vivo, as well as better safety profiles. Further studies suggested that A4 and C5 could arrest HT-29 cell cycle in G2 phase and raise reactive oxygen species level, thus inducing cell apoptosis related to Erk MAPK and NF-κB signaling pathways inhibition.Conclusion: These results will greatly promote the druggability study of these koumine-like compounds.

[Box: see text].

N-desmethyldauricine from Menispermum dauricum DC suppresses triple-negative breast cancer growth in 2D and 3D models by downregulating the NF-κB signaling pathway.

Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, for which targeted therapy regimens are lacking. The traditional Chinese medicine Menispermum dauricum DC (M. dauricum) and its compounds have been reported to have antitumor activity against various cancers; however, their anti-TNBC activity is unknown. In this work, dauricine and N-desmethyldauricine from M. dauricum were separated and identified to have anti-TNBC via a multi-component bioactivity and structure-guided method. The cell counting kit 8 assay showed that dauricine and N-desmethyldauricine inhibited the proliferation of four tested TNBC cell lines, with half maximal inhibitory concentration values ranging from 5.01 µM to 13.16 µM. Further research suggested that N-desmethyldauricine induced cell apoptosis, arrested cell cycle progression in the G0/G1 phase, and inhibited cell migration. Western blot analysis revealed that the proapoptotic protein cleaved-poly-ADP-ribose polymerase 1 was upregulated, and the G0/G1 phase-related proteins cyclin-dependent kinase 2 and cyclin D1 and the migration-related protein matrix metallopeptidase 9 were downregulated. Furthermore, N-desmethyldauricine decreased the protein expression of p65, an important subunit of nuclear factor kappa-beta (NF-κB). Moreover, an antiproliferation assay of three-dimensional (3D) tumor spheroids showed that N-desmethyldauricine diminished cell‒cell adhesion and suppressed the growth of TNBC 3D spheroids. Taken together, these findings indicate that N-desmethyldauricine inhibited the proliferation of TNBC cells and decreased the expression of p65 in the NF-κB pathway.

Precise Capture and Dynamic Release of Circulating Liver Cancer Cells with Dual-Histidine-Based Cell Imprinted Hydrogels.

Circulating tumor cells (CTCs) detection presents significant advantages in diagnosing liver cancer due to its noninvasiveness, real-time monitoring, and dynamic tracking. However, the clinical application of CTCs-based diagnosis is largely limited by the challenges of capturing low-abundance CTCs within a complex blood environment while ensuring them alive. Here, an ultrastrong ligand, l-histidine-l-histidine (HH), specifically targeting sialylated glycans on the surface of CTCs, is designed. Furthermore, HH is integrated into a cell-imprinted polymer, constructing a hydrogel with precise CTCs imprinting, high elasticity, satisfactory blood compatibility, and robust anti-interference capacities. These features endow the hydrogel with excellent capture efficiency (>95%) for CTCs in peripheral blood, as well as the ability to release CTCs controllably and alive. Clinical tests substantiate the accurate differentiation between liver cancer, cirrhosis, and healthy groups using this method. The remarkable diagnostic accuracy (94%), lossless release of CTCs, material reversibility, and cost-effectiveness ($6.68 per sample) make the HH-based hydrogel a potentially revolutionary technology for liver cancer diagnosis and single-cell analysis.

Discovery and characterization of novel ATP citrate lyase inhibitors from Acanthopanax senticosus (Rupr. & Maxim.) Harms.

ATP citrate lyase (ACLY) is a key enzyme in glucolipid metabolism, and abnormally high expression of ACLY occurs in many diseases, including cancers, dyslipidemia and cardiovascular diseases. ACLY inhibitors are prospective treatments for these diseases. However, the scaffolds of ACLY inhibitors are insufficient with weak activity. The discovery of inhibitors with structural novelty and high activity continues to be a research hotpot. Acanthopanax senticosus (Rupr. & Maxim.) Harms is used for cardiovascular disease treatment, from which no ACLY inhibitors have ever been found. In this work, we discovered three novel ACLY inhibitors, and the most potent one was isochlorogenic acid C (ICC) with an IC50 value of 0.14 ± 0.04 µM. We found dicaffeoylquinic acids with ortho-dihydroxyphenyl groups were important features for inhibition by studying ten phenolic acids. We further investigated interactions between the highly active compound ICC and ACLY. Thermal shift assay revealed that ICC could directly bind to ACLY and improve its stability in the heating process. Enzymatic kinetic studies indicated ICC was a noncompetitive inhibitor of ACLY. Our work discovered novel ACLY inhibitors, provided valuable structure-activity patterns and deepened knowledge on the interactions between this targe tand its inhibitors.

Elucidation of active components and target mechanism in Jinqiancao granules for the treatment of prostatitis and benign prostatic hyperplasia.

ETHNOPHARMACOLOGICAL RELEVANCE: Prostatitis and benign prostatic hyperplasia (BPH) are inflammations of the prostate gland, which surrounds the urethra in males. Jinqiancao granules are a traditional Chinese medicine used to treat kidney stones and this medicine consists of four herbs: Desmodium styracifolium (Osbeck) Merr., Pyrrosia calvata (Baker) Ching, Plantago asiatica L. and stigma of Zea mays L. AIM OF THE STUDY: We hypothesized that Jinqiancao granules could be a potential therapy for prostatitis and BPH, and this work aimed to elucidate active compounds in Jinqiancao granules and their target mechanisms for the potential treatment of the two diseases. MATERIALS AND METHODS: Jinqiancao granules were commercially available and purchased. Database-driven data mining and networking were utilized to establish a general correlation between Jinqiancao granules and the two diseases above. Ultra-performance liquid chromatography-mass spectrometry was used for compound separation and characterization. The characterized compounds were evaluated on four G-protein coupled receptors (GPCRs: GPR35, muscarinic acetylcholine receptor M3, alpha-1A adrenergic receptor α1A and cannabinoid receptor CB2). A dynamic mass redistribution technique was applied to evaluate compounds on four GPCRs. Nitric acid (NO) inhibition was tested on the macrophage cell line RAW264.7. Molecular docking was conducted on GPR35-active compounds and GPR35 crystal structure. Statistical analysis using GEO datasets was conducted. RESULTS: Seventy compounds were isolated and twelve showed GPCR activity. Three compounds showed potent GPR35 agonistic activity (EC50 < 10 µM) and the GPR35 agonism action of PAL-21 (Scutellarein) was reported for the first time. Docking results revealed that the GPR35-targeting compounds interacted at the key residues for the agonist-initiated activation of GPR35. Five compounds showed weak antagonistic activity on M3, which was confirmed to be a disease target by statistical analysis. Seventeen compounds showed NO inhibitory activity. Several compounds showed multi-target properties. An experiment-based network reflected a pharmacological relationship between Jinqiancao granules and the two diseases. CONCLUSIONS: This study identified active compounds in Jinqiancao granules that have synergistic mechanisms, contributing to anti-inflammatory effects. The findings provide scientific evidence for the potential use of Jinqiancao granules as a treatment for prostatitis and BPH.

The dual-targeting mechanism of an anti-inflammatory diarylheptanoid from Curcuma zedoaria (Christm.) Roscoe with the capacity for ß2-adrenoreceptor agonism and NLRP3 inhibition.

Chronic obstructive pulmonary disease (COPD) is a common respiratory disease characterized by symptoms of shortness of breath and chronic inflammation. Curcuma zedoaria (Christm.) Roscoe is a well-documented traditional medical herb that is frequently used in the treatment of COPD. Previously, we identified a diarylheptanoid compound (1-(4-hydroxy-5-methoxyphenyl)-7-(4,5-dihydroxyphenyl)-3,5-dihydroxyheptane; abbreviated as HMDD) from this herb that exhibited potent agonistic activity on ß2-adrenergic receptors (ß2 adrenoreceptor) that are present on airway smooth muscle cells. In this work, we used chemically synthesized HMDD compound, and confirmed its bioactivity on ß2 adrenoreceptors. Then by a proteomics study and anti-inflammatory evaluation detections, we found that HMDD downregulated the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) signaling pathway and suppressed the NLRP3 receptor expression in RAW264.7 macrophages and in a COPD model in A549 lung carcinoma cells. HMDD also decreased nitric oxide production levels, and impacted other interleukins and the phosphorylation of NF-κB and ERK pathways. We performed molecular docking of HMDD on ß2 adrenoreceptor and NLRP3 protein models. This work reports the anti-inflammatory effects of HMDD and suggests a dual-targeting mechanism of ß2-adrenoreceptor agonism and NLRP3 inhibition. Such a mechanism scientifically supports the clinical uses of Curcuma zedoaria (Christm.) Roscoe in treating COPD, as it can simultaneously relieve persistent breathlessness and inflammation. HMDD can be considered as a potential non-steroidal anti-inflammatory drug in novel therapy design for the treatment of COPD and other inflammatory diseases.

Aminosalicylates target GPR35, partly contributing to the prevention of DSS-induced colitis.

GPR35, a class A G-protein-coupled receptor, is considered an orphan receptor; the endogenous ligand and precise physiological function of GPR35 remain obscure. GPR35 is expressed relatively highly in the gastrointestinal tract and immune cells. It plays a role in colorectal diseases like inflammatory bowel diseases (IBDs) and colon cancer. More recently, the development of GPR35 targeting anti-IBD drugs is in solid request. Nevertheless, the development process is in stagnation due to the lack of a highly potent GPR35 agonist that is also active comparably in both human and mouse orthologs. Therefore, we proposed to find compounds for GPR35 agonist development, especially for the human ortholog of GPR35. As an efficient way to pick up a safe and effective GPR35 targeting anti-IBD drug, we screened Food and Drug Administration (FDA)-approved 1850 drugs using a two-step DMR assay. Interestingly, we found aminosalicylates, first-line medicine for IBDs whose precise target remains unknown, exhibited activity on both human and mouse GPR35. Among these, pro-drug olsalazine showed the most potency on GPR35 agonism, inducing ERK phosphorylation and ß-arrestin2 translocation. In dextran sodium sulfate (DSS)-induced colitis, the protective effect on disease progression and inhibitory effect on TNFα mRNA expression, NF-κB and JAK-STAT3 pathway of olsalazine are compromised in GPR35 knock-out mice. The present study identified a target for first-line medicine aminosalicylates, highlighted that uncleaved pro-drug olsalazine is effective, and provided a new concept for the design of aminosalicylic GPR35 targeting anti-IBD drug.

Integration of Two-Dimensional Liquid Chromatography-Mass Spectrometry and Molecular Docking to Characterize and Predict Polar Active Compounds in Curcuma kwangsiensis.

Curcuma kwangsiensis, one species of Curcumae zedoaria Ros. c, is a commonly used traditional Chinese medicine (TCM) for treating cardiovascular disease, cancer, asthma and inflammation. Polar compounds are abundant in water decoction, which would be responsible for critical pharmacological effects. However, current research on polar compounds in Curcumae zedoaria Ros. c remains scarce. In this study, the polar fraction from Curcuma kwangsiensis was firstly profiled on G protein-coupled receptor 109A (GPR109A), ß2-adrenergic receptor (ß2-AR), neurotensin receptor (NTSR), muscarinic-3 acetylcholine receptor (M3) and G protein-coupled receptor 35 (GPR35), which were involved in its clinical indications and exhibited excellent ß2-AR and GPR109A receptor activities. Then, an offline two-dimensional reversed-phase liquid chromatography (RPLC) coupled with the hydrophilic interaction chromatography (HILIC) method was developed to separate polar compounds. By the combination of a polar-copolymerized XAqua C18 column and an amide-bonded XAmide column, an orthogonality of 47.6% was achieved. As a result of coupling with the mass spectrometry (MS), a four-dimensional data plot was presented in which 373 mass peaks were detected and 22 polar compounds tentatively identified, including the GPR109A agonist niacin. Finally, molecular docking of these 22 identified compounds to ß2-AR, M3, GPR35 and GPR109A receptors was performed to predict potential active ingredients, and compound 9 was predicted to have a similar interaction to the ß2-AR partial agonist salmeterol. These results were supplementary to the material basis of Curcuma kwangsiensis and facilitated the bioactivity research of polar compounds. The integration of RPLC×HILIC-MS and molecular docking can be a powerful tool for characterizing and predicting polar active components in TCM.

Discovery of Flavonoids as Novel Inhibitors of ATP Citrate Lyase: Structure-Activity Relationship and Inhibition Profiles.

ATP citrate lyase (ACLY) is a key enzyme in glucolipid metabolism and its aberrantly high expression is closely associated with various cancers, hyperlipemia and atherosclerotic cardiovascular diseases. Prospects of ACLY inhibitors as treatments of these diseases are excellent. To date, flavonoids have not been extensively reported as ACLY inhibitors. In our study, 138 flavonoids were screened and 21 of them were subjected to concentration-response curves. A remarkable structure-activity relationship (SAR) trend was found: ortho-dihydroxyphenyl and a conjugated system maintained by a pyrone ring were critical for inhibitory activity. Among these flavonoids, herbacetin had a typical structure and showed a non-aggregated state in solution and a high inhibition potency (IC50 = 0.50 ± 0.08 µM), and therefore was selected as a representative for the ligand-protein interaction study. In thermal shift assays, herbacetin improved the thermal stability of ACLY, suggesting a direct interaction with ACLY. Kinetic studies determined that herbacetin was a noncompetitive inhibitor of ACLY, as illustrated by molecular docking and dynamics simulation. Together, this work demonstrated flavonoids as novel and potent ACLY inhibitors with a remarkable SAR trend, which may help design high-potency ACLY inhibitors. In-depth studies of herbacetin deepened our understanding of the interactions between flavonoids and ACLY.

Discovery and characterization of novel ATP citrate lyase inhibitors from natural products by a luminescence-based assay.

ATP citrate lyase (ACLY) is a key enzyme in glucolipid metabolism with therapeutic prospect for treating hyperlipidemia and various cancers. Much effort has been put into discovering ACLY inhibitors. However, current screening approaches have limitations in sensitivity, portability and high-throughput. To develop a general screening assay, we investigated series of conditions affecting the enzymatic reaction based on the ADP-Glo luminescence assay. Bovine serum albumin (0.001%) added triggered strong and stable fluorescence signal. The optimized assay was validated and applied to screen our natural product library. Two novel inhibitors were identified with IC50 values of 3.86 ± 0.62 µM (2) and 15.48 ± 2.51 µM (4). Their aggregations and target specificities were also examined. 2 was characterized as a noncompetitive inhibitor of ACLY, while 4 was a competitive inhibitor of CoA, which was also elucidated by docking studies. In anticancer activity evaluation, 2 with higher inhibition potency did not exhibit anticancer effect, probably owing to its insufficient cell-permeability. 4 showed moderate inhibition in the proliferation of A549 and PC3 cells. This study not only developed a general approach for ACLY inhibitor discovery, but also identified a new scaffold ACLY inhibitor, which could be served as a hit compound in drug design.

Mechanism deconvolution of Qing Fei Pai Du decoction for treatment of Coronavirus Disease 2019 (COVID-19) by label-free integrative pharmacology assays.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) has a long history in the prevention and treatment of pandemics. The TCM formula Lung Cleansing and Detoxifying Decoction (LCDD), also known as Qing Fei Pai Du Decoction, has been demonstrated effective against Coronavirus Disease 2019 (COVID-19). AIM OF THE STUDY: This work aimed to elucidate the active ingredients, targets and pathway mechanism of LCDD related to suppression of inflammatory, immunity regulation and relaxation of airway smooth muscle for the treatment of COVID-19. MATERIALS AND METHODS: Mining chemical ingredients reported in LCDD, 144 compounds covering all herbs were selected and screened against inflammatory-, immunity- and respiratory-related GPCRs including GPR35, H1, CB2, B2, M3 and ß2-adrenoceptor receptor using a label-free integrative pharmacology method. Further, all active compounds were detected using liquid chromatography-tandem mass spectrometry, and an herb-compound-target network based on potency and content of compounds was constructed to elucidate the multi-target and synergistic effect. RESULTS: Thirteen compounds were identified as GPR35 agonists, including licochalcone B, isoliquiritigenin, etc. Licochalcone B, isoliquiritigenin and alisol A exhibited bradykinin receptor B2 antagonism activities. Atractyline and shogaol showed as a cannabinoid receptor CB2 agonist and a histamine receptor H1 antagonist, respectively. Tectorigenin and aristofone acted as muscarinic receptor M3 antagonists, while synephrine, ephedrine and pseudoephedrine were ß2-adrenoceptor agonists. Pathway deconvolution assays suggested activation of GPR35 triggered PI3K, MEK, JNK pathways and EGFR transactivation, and the activation of ß2-adrenoceptor mediated MEK and Ca2+. The herb-compound-target network analysis found that some compounds such as licochalcone B acted on multiple targets, and multiple components interacted with the same target such as GPR35, reflecting the synergistic mechanism of Chinese medicine. At the same time, some low-abundance compounds displayed high target activity, meaning its important role in LCDD for anti-COVID-19. CONCLUSIONS: This study elucidates the active ingredients, targets and pathways of LCDD. This is useful for elucidating multitarget synergistic action for its clinical therapeutic efficacy.

Structure-Activity Relationship Studies of Coumarin-like Diacid Derivatives as Human G Protein-Coupled Receptor-35 (hGPR35) Agonists and a Consequent New Design Principle.

A series of coumarin-like diacid derivatives were designed and synthesized as novel agonists of human G-protein-coupled receptor 35 (hGPR35). Active compounds were characterized to possess one acidic group on both sides of a fused tricyclic aromatic scaffold. Most of them functioned as full agonists selective to hGPR35 and exhibited excellent potency at low nanomolar concentrations. Substitution on the middle ring of the scaffold could effectively regulate compound potency. Structure-activity relationship studies and docking simulation indicated that compounds that carried two acidic groups with a proper special distance and attached to a rigid aromatic scaffold would most likely show a potent agonistic activity on hGPR35. Following this principle, we screened a list of known compounds and some were found to be potent GPR35 agonists, and compound 24 even had an EC50 of 8 nM. Particularly, a dietary supplement pyrroloquinoline quinone (PQQ) was identified as a potent agonist (EC50 = 71.4 nM). To some extent, this principle provides a general strategy to design and recognize GPR35 agonists.

Identification and target-pathway deconvolution of FFA4 agonists with anti-diabetic activity from Arnebia euchroma (Royle) Johnst.

FFA4 is a novel therapeutic target for the treatment of metabolic diseases, such as type II diabetes. However, there are still few ligands with structural diversity, selectivity and high potency, and the signaling pathway downstream of FFA4 remains to be poorly characterized. In this study, a high performance liquid chromatography-corona charged aerosol detector (HPLC-CAD) combined with label-free dynamic mass redistribution (DMR) method was introduced to guide the discovery of FFA4 agonists from Arnebia euchroma (Royle) Johnst. Ten compounds were identified as FFA4 agonists and structure-activity relationship was obtained. Among them, shikonin displayed the most potent activity with pEC50 value of 6.02 ± 0.19. The activity of shikonin was confirmed by FLIPR (fluorometric imaging plate reader) assay. Signaling pathways of FFA4 were explored in HT-29 cells endogenously expressing FFA4 using shikonin and known FFA4 agonists α-linolenic acid (ALA) and TUG891. Multiple pathways included Gq/11-PLC-Ca2+-PKC, RohA, JNK, p38 MAPK, Gi/o and PI3K signaling but may not involve Gs signaling triggered by shikonin, ALA and TUG891. Besides, shikonin, TUG891 and ALA could induce ERK1/2 and AKT phosphorylation in HT-29 cells. Moreover, anti-diabetes effects of shikonin were evaluated on the glucose intolerance in diabetic db/db mice. Shikonin reduced plasma glucose level, suggesting that it had the potential in treatment of type II diabetes. The agonists identified in this study provided structure guidance for FFA4 drug design. This study was also useful for understanding FFA4 pharmacology and its biological function.

Systematic characterization of AT1 receptor antagonists with label-free dynamic mass redistribution assays.

INTRODUCTION: In the drug discovery field, the binding affinities and binding kinetics of drug candidates are very important. Angiotensin II type 1 (AT1) receptor antagonists, e.g., candesartan, telmisartan, irbesartan, losartan and valsartan, show high affinities and long-lasting bindings to the receptor, making them preferred medications for hypertension treatment. However, the molecular binding properties of AT1 receptor antagonists are controversial. METHODS: In this work, we established a profile to study the phenotypic properties of AT1 receptor antagonists with label-free dynamic mass redistribution (DMR) assays in native human cells. With noninvasive features, DMR assay were conducted in multiple formats. Eleven antagonists were systematically evaluated with angiotensin II as an agonist probe in the Hep G2 cell line, which endogenously expresses the AT1 receptor. RESULTS: The IC50 values to the AT1 receptor of individual antagonist varied with different incubation times. The antagonists showed competitive behavior with angiotensin II. Schild analysis was used to analyze the competitive behavior of the antagonist. All of the antagonist showed long-lasting possession of the AT1 receptor, except telmisartan. The systematic evaluation of the antagonists implied that 11 antagonists showed high binding affinity but distinct binding modes to AT1 receptor. DISCUSSION: This study demonstrated that the DMR assay has great potential for determining the pharmacological parameters of ligands. This work may serve as guidance for other receptor and ligand assays.

Phenotypic assessment and ligand screening of ETA/ETB receptors with label-free dynamic mass redistribution assay.

Endothelin receptors, consisting of two subtypes, ETA and ETB, are expressed in various tissues and widely regulate cardiovascular systems. The two receptors show distinct biological characteristics and are involved in different downstream pathways. Hence, to evaluate the ETA and ETB receptors on the same platform is helpful to display their pharmacological features. In this study, we developed a label-free dynamic mass redistribution (DMR) assay to investigate the phenotypic features of the ETA and ETB receptors in native cell lines. Meanwhile, specific agonists and antagonists were investigated for their pharmacological parameters. Results indicated that the DMR response of endothelin 1 (ET-1, an endogenous ETA/ETB agonist) was cell line dependent on ETA receptors and this ligand generated a biphasic dose-response curve in SH-SY5Y as well as PC3 cell lines. ET-1 and IRL 1620 (an ETB agonist) showed different DMR responses in U251 cells. IC50 values of antagonists were consistent with the Ki values previously reported. Furthermore, a list of compounds was screened on the ETA and ETB receptor models established by the high-throughput DMR assays. This study demonstrated that the DMR assay had great potential in the phenotypic-based investigation and ligand screening of GPCRs.

Isolation and bioactive evaluation of flavonoid glycosides from Lobelia chinensis Lour using two-dimensional liquid chromatography combined with label-free cell phenotypic assays.

Flavonoid glycosides are widespread in herbs and often used as medicines and nutraceuticals because of good bioactivities and low toxicities. However, due to their structural complexity and diversity, isolation of flavonoid glycosides and evaluation of their bioactivities are still highly challenging. To solve this problem, a new method for separation and preparation of novel flavonoid glycosides from Lobelia chinensis Lour (L. chinensis) was developed. To avoid the interference of non-flavonoids, a solid phase extraction method was used to selectively enrich the flavonoids from the total extract. Based on hydrophilic and hydrophobic properties of the flavonoid chemical structure consisting of sugar residue and diphenylpropane (C6C3C6) skeleton, a structure-guided method development strategy was employed to design a 2D-HILIC × RPLC system in the first time. After optimization of chromatographic conditions, high selectivity and symmetric peaks of flavonoids were obtained on a zwitterionic Click XIon column and a polar-modified Atlantis T3 column. Based on these two columns, a 2 D-HILIC × RPLC system was constructed and successfully enlarged from the analytical level to the preparative level. In the first dimension, 20 fractions were obtained with good peak shapes at high sample loading. In the second-dimensional preparation, nine compounds were isolated and identified. Seven of them were novel flavonoid glycosides, lobelitin A-G, and two other known compounds were linatin and diosmin, respectively. Their target activities were evaluated via label-free cell phenotypic assays. Four novel flavonoid glycosides lobelitin A-D were found to have agonistic activities at G protein-coupled receptor 35 (GPR35). These results demonstrated that this method was effective to orthogonally separate flavonoids at the preparative level, especially for novel active flavonoid glycosides. The discovery of flavonoid glycosides with novel agonistic activity on GPR35 also sheds light on the mechanisms of action of L. chinensis relevant to its clinical application.

Offline preparative 2-D polar-copolymerized reversed-phase chromatography × zwitterionic hydrophilic interaction chromatography for effective purification of polar compounds from Caulis Polygoni Multiflori.

Polar compounds are abundant in the water decoction of Traditional Chinese Medicines (TCMs) and possess important biological activities. However, purifying these compounds has faced great difficulties largely due to poor retention and insufficient selectivity. To solve this problem, an offline orthogonal 2-D RPLC coupled with hydrophilic interaction chromatography (HILIC) method was developed to achieve purification of polar compounds from Caulis Polygoni Multiflori. A polar-copolymerized XAqua C18 column and a zwitterionic Click XIon column exhibited satisfactory retention and separation for polar compounds. Therefore, they were adopted to construct the offline 2-D LC system. Furthermore, the method presented a high orthogonality, which was calculated to be 72%. The XAqua C18 column was used in the first dimension to fractionate the 5.1 g polar fraction with a recovery of 85% within 10 h. In the second dimension, three representative fractions were purified using the Click XIon column. Finally, three compounds with purity higher than 95% were identified for the first time from this plant. This offline 2-D RPLC/HILIC method was shown to be an effective approach to purifying polar compounds from Caulis Polygoni Multiflori.

Saikosaponin D from Radix Bupleuri suppresses triple-negative breast cancer cell growth by targeting ß-catenin signaling.

BACKGROUND: Triple-negative breast cancer (TNBC) is one of the most aggressive and poor prognosis breast cancers. Currently, chemotherapy with conventional cytotoxic agents is the only available option to treat TNBC. Hence, we identified new therapeutic agents against TNBC from traditional Chinese medicine Radix Bupleuri and unveiled the molecule mechanism of anti-TNBC effects. METHODS: Multi-component bioactivity and structure-guided methods were used to identify the most effective anti-TNBC compound Saikosaponin D (SSD) from Radix Bupleuri. Cell viability and apoptosis assays were employed to demonstrate the effect of SSD on the proliferation and apoptosis of TNBC cells. Dynamic mass redistribution assay, TopFlash assay, western blotting, and special agonist were applied to dissect the potential molecular mechanisms of SSD. RESULTS: We screened twenty fractions in Radix Bupleuri and identified SSD as the most effective component to inhibit the proliferation of TNBC cells. Investigating the interaction of SSD with the frequently overexpressed targets in TNBC led to the identification that it markedly suppressed Wnt/ß-catenin signaling, but did not act on epidermal growth factor receptor and neurotensin receptor-1. Moreover, we demonstrated that SSD significantly repressed ß-catenin and its downstream target genes, resulting in TNBC cell apoptosis. Specifically, docking of SSD to the crystal structure of ß-catenin suggested that SSD interacted with ß-catenin via hydrogen bonds and hydrophobic interaction. CONCLUSION: We identified the most effective component SSD from Radix Bupleuri in inhibiting the proliferation of TNBC cells by targeting ß-catenin signaling. Given the important role of Wnt/ß-catenin signaling in breast cancer, SSD may present an opportunity to discover new therapeutics for the treatment of TNBC.

Discovery of novel antagonists on ß2-adrenoceptor from natural products using a label-free cell phenotypic assay.

Label-free cell phenotypic assays were performed to establish a ß2-adrenoceptor (ß2-AR) target model in A431 cells and a ß1-AR target model in transfected HEK293-ß1 cells, using known ß2-AR and ß1-AR agonists and antagonists. A list of natural compounds was screened on the target models, among which seven new compounds were found to be antagonistically active against ß2-AR. After receptor specificity evaluations on hydroxyl carboxylic acid receptor-2 (ΗΧΑ-2), histamine receptor (H1R), and ß1-adrenoceptor (ß1-AR), six out of the seven compounds, including nuciferine, epiberberine, harmaline, harmine, palmatine, and columbamine, exhibited specific antagonistic activity against ß2-AR. Epiberberine and palmatine showed the strongest antagonistic activities against ß2-AR with IC50 values of 2.3 ± 0.2 µM and 2.6 ± 0.3 µM, respectively. Docking palmatine to the crystal structure of human ß2-AR (PDB 5X7D) suggested that the ligand forms a hydrogen bond with N312 and hydrophobic interaction with several amino acid residues in the binding pocket, such as D113 and V114. The kinetic binding profile of palmatine was further investigated using co-stimulation assays. Results suggested that palmatine was a competitive antagonist for ß2-AR. The six novel ß2-AR antagonists provide a promising chemical starting point for identification and optimization of drugs used for treating hypertension, glaucoma, and infantile hemangiomas. This study also lays the foundation for the in-depth investigation of biochemical mechanisms and pharmacological properties of natural compounds.

Label-free cell phenotypic profiling and pathway deconvolution of neurotensin receptor-1.

Neurotensin (NT), an endogenous peptide found in the central nervous system and in peripheral tissues, contributes to the pathophysiology of neurodegenerative and psychiatric diseases, cancer, inflammation, and immunomodulatory disease. NT exerts its physiological effects predominantly through its cognate high-affinity neurotensin receptor-1 (NTS1). NTS1 emerges as a druggable target; however, there are limited numbers of NTS1 active compounds reported to date. Here we reported a label-free cell phenotypic profiling model for screening NTS1 ligands and differentiating their biased agonism. Resonant waveguide grating enabled dynamic mass redistribution (DMR) assay was first optimized against cell confluency and then used to characterize the endogenous NTS1 in HT-29 cell using known agonists and antagonists. Pathway modulators were also used to deconvolute the signaling pathways of endogenous NTS1. Results showed that the NTS1 DMR assay is robust for screening and can differentiate biased agonism; and the activation of NTS1 in HT-29 triggers multiple pathways including Gq signaling and epidermal growth factor receptor transactivation. This study highlighted the power of label-free DMR assay to characterize receptor signaling and pharmacology of distinct classes of ligands for NTS1, G protein-coupled receptors in general.