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.

Synthesis and evaluation of 3-(4-(phenoxymethyl)phenyl)propanoic acid and N-phenylbenzenesulfonamide derivatives as FFA4 agonists.

Free fatty acid receptor 4 (FFA4) has been recognized as an attractive target in metabolic diseases. To find potent and selective FFA4 agonist, 28 compounds of 3-(4-(phenoxymethyl)phenyl)propanoic acid and N-phenylbenzenesulfonamide derivatives were designed and synthesized, featuring OC and SO2-N linkage. For the OC linkage compounds, 1g showed the most potent FFA4 agonistic activity with a pEC50 of 5.81 ± 0.04 and exhibited at least 64-fold selectivity against FFA1. For SO2-N linkage agonists, 2m had a pEC50 of 5.66 ± 0.04 and displayed>46-fold selectivity against FFA1. Among these two series of compounds, 1g was the most potent agonist at FFA4 and the best selectivity against FFA1, demonstrated by docking simulation. Moreover, 1g showed receptor selectivity on other seven GPCRs. In anti-diabetic evaluation, 1g dose-dependently reduced blood glucose, which was better than a clinical phase III drug TAK875. This study provides guidance for FFA4 ligand design and drug optimization.

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.

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.

Label-free cell phenotypic study of FFA4 and FFA1 and discovery of novel agonists of FFA4 from natural products.

In this article, pharmacological studies of the free fatty acid receptor (FFA) 4 and FFA1 were conducted in transfected CHO cells (FFA4&FFA1) and HT29 cells with application of a label-free dynamic mass redistribution (DMR) assay. Commercially available compounds including α-linolenic acid (ALA), GW9508, TUG891, GSK137647A, TAK875, MEDICA16, AH7614 and GW1100, were used to validate the assay; real-time tracing of ligand-induced cell responses elucidated pharmacological properties of ligand-receptor interactions. A pool of 140 natural compounds was screened using the CHO-FFA4 cells. Three new FFA4 agonists with novel skeletons were discovered and they were dihydrotanshinone, emodin and acetylshikonin (EC50 values were 32.88, 38.18 and 10.17 µM, respectively). Ligand selectivity was compared between FFA4 and FFA1; dihydrotanshinone and emodin displayed FFA4 selectivity, while acetylshikonin shared FFA1 and FFA4 agonist activities with EC50 values comparable to the endogenous ligand ALA. The three novel FFA4 agonists provide a promising chemical starting point for identification and optimization of drugs used for treating metabolic and inflammatory diseases. Besides, this work will help to explain the mechanism of actions of natural products.

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.

SAR Studies of N-[2-(1H-Tetrazol-5-yl)phenyl]benzamide Derivatives as Potent G Protein-Coupled Receptor-35 Agonists.

G protein-coupled receptor-35 (GPR35) has emerged as a potential target in the treatment of pain and inflammatory and metabolic diseases. We have discovered a series of potent GPR35 agonists based on a coumarin scaffold and found that the introduction of a 1H-tetrazol-5-yl group significantly increased their potency. We designed and synthesized a new series of N-[2-(1H-tetrazol-5-yl)phenyl]benzamide derivatives through a two-step synthetic approach, and characterized their agonistic activities against GPR35 using a dynamic mass redistribution (DMR) assay. N-(5-bromo-2-(1H-tetrazol-5-yl)phenyl)-4-methoxybenzamide (56) and N-(5-bromo-2-(1H-tetrazol-5-yl)phenyl)-2-fluoro-4-methoxybenzamide (63) displayed the highest agonistic potency agonist GPR35 with an EC50 of 0.059 µM and 0.041 µM, respectively. The physicochemical properties of selected compounds were calculated to evaluate their druglikeness, suggesting that compounds 56 and 63 have good druglike properties. Together, N-[2-(1H-tetrazol-5-yl)phenyl]benzamide derivatives are potentially great candidates for developing potent GPR35 agonists.

Discovery of 2H-Chromen-2-one Derivatives as G Protein-Coupled Receptor-35 Agonists.

A family of 2H-chromen-2-one derivatives were identified as G protein-coupled receptor-35 (GPR35) agonists using dynamic mass redistribution assays in HT-29 cells. The compounds with 1H-tetrazol-5-yl in 3-substituted position displayed higher potency than the corresponding carboxyl analogs, and the hydroxyl group in the 7-position also played an important role in GPR35 agonistic activity. 6-Bromo-7-hydroxy-8-nitro-3-(1H-tetrazol-5-yl)-2H-chromen-2-one (50) was found to be the most potent GPR35 agonist with an EC50 of 5.8 nM. Calculating the physicochemical properties of compounds with moderate to high potency suggested that compounds 30, 50, and 51 showed good druggability. This study provides a novel series of GPR35 agonists, and compound 50 may be a powerful tool to study GPR35.

Anti-gastric cancer activity in three-dimensional tumor spheroids of bufadienolides.

Multicellular spheroids of cancer cells have been increasingly used to screen anti-tumor compounds, owing to their in vivo like microenvironment and structure as well as compatibility to high-throughput/high-content screening. Here we report the potency and efficacy of a family of bufadienolides to inhibit the growth of gastric cancer cell line HGC-27 in three-dimensional (3D) spheroidal models. Examining the morphological and growth patterns of several cell lines in round-bottomed ultra-low attachment microplate suggested that HGC-27 cells formed reproducibly multicellular spheroidal structures. Profiling of 15 natural bufadienolides isolated from toad skin indicated that 8 14-hydroxy bufadienolides displayed inhibitory activity of the growth of HGC-27 spheroids in a dose-dependent manner. Notably, compared to clinical drugs taxol and epirubicin, active bufadienolides were found to penetrate more effectively into the HGC-27 spheroids, but with a narrower effective concentration range and a shorter lasting inhibitory effect. Furthermore, compared to two-dimensional (2D) cell monolayer assays, active bufadienolides exhibited weaker efficacy and different potency in 3D spheroid model, demonstrating the great potential of 3D multicellular cell spheroid models in anti-cancer drug discovery and development.