Discovery of 4'-trifluoromethylchalcones as novel, potent and selective hCYP1B1 inhibitors without concomitant AhR activation.

Human cytochrome P450 1B1 (hCYP1B1), an extrahepatic cytochrome P450 enzyme over-expressed in various tumors, has been validated as a promising target for preventing and treating cancers. Herein, two series of chalcone derivatives were synthesized to discover potent hCYP1B1 inhibitors without AhR agonist effect. Structure-activity relationship (SAR) studies demonstrated that 4'-trifluoromethyl on the B-ring strongly enhanced the anti-hCYP1B1 effects, identifying A9 as a promising lead compound. Further SAR analysis on A9 derivatives (modified A-ring of 4'-trifluoromethylchalcone) showed that introducing 2-methoxyl improved the anti-hCYP1B1 effect and selectivity, while introducing a methoxyl at the C-4 site was beneficial for avoiding AhR activation. Ultimately, five 4'-trifluoromethyl chalcones were identified as potent hCYP1B1 inhibitors (IC50 < 10 nM), while B18 exhibits the most potent anti-hCYP1B1 effect (IC50 = 3.6 nM), suitable metabolic stability and good cell-permeability. B18 also acted as an AhR antagonist and could down-regulate hCYP1B1 in living systems. Mechanistic studies showed that B18 potently inhibited hCYP1B1 in a competitive inhibition manner (Ki = 3.92 nM), while docking simulations revealed that B18 could tightly bind to the catalytic cavity of hCYP1B1 mainly via hydrophobic and hydrogen-bonding interactions. Furthermore, B18 could potently inhibit hCYP1B1 in living cells and showed remarkable anti-migration ability on MFC-7 cells. Taken together, this study deciphered the SARs of chalcones as hCYP1B1 inhibitors and provided several potent hCYP1B1 inhibitors as promising candidates for the development of more efficacious anti-migration agents.

Glycosaminoglycan from Ostrea rivularis attenuates hyperlipidemia and regulates gut microbiota in high-cholesterol diet-fed zebrafish.

Hyperlipidemia an immense group of acquired or genetic metabolic disorders that is characterized by an excess of lipids in the bloodstream. Altogether, they have a high prevalence worldwide and constitute a major threat to human health. Glycosaminoglycans (GAG) are natural biomolecules that have hypolipidemic activity. The purpose of this study was to investigate the potential hypolipidemic effect of glycosaminoglycans extracted from Ostrea rivularis (OGAG) on hyperlipidemic zebrafish, as well as the possible underlying mechanism of such effect. Dietary supplementation with OGAG during 4 weeks significantly reduced the serum and hepatic lipid levels and the hepatosomatic index in hyperlipidemic zebrafish. In addition, histopathological showed that OGAG supplementation decreases the volume and number of lipid droplets in hepatocytes. Transcriptome and real-time quantitative polymerase chain reaction analysis revealed that the gene expression levels of PPARγ, SCD, HMGRA, ACAT2, HMGCS, and HMGCR were significantly downregulated by OGAG treatment in hepatocytes, whereas those of CD36, FABP2, FABP6, ABCG5, and CYP7A1 were significantly upregulated. This suggests that the hypolipidemic effect of OGAG relies on increasing the ketogenic metabolism of fatty acids, inhibiting cholesterol synthesis, and enhancing the transformation of cholesterol to bile acid. Furthermore, OGAG treatment improved gut microbiota imbalance by reducing the Firmicutes-to-Bacteroidetes ratio, increasing the relative abundance of beneficial bacteria (Bacteroidetes, Verrucomicrobia, Acidobacteria, and Sphingomonas), and reducing the relative abundance of harmful bacteria (Proteobacteria, Cohaesibacter, Vibrio, and Terrisporobacter). These findings highlight the potential benefit of implementing OGAG as a dietary supplement to prevent and treat hyperlipidemia.

Cytochrome P450 3A Enzymes Are Key Contributors for Hepatic Metabolism of Bufotalin, a Natural Constitute in Chinese Medicine Chansu.

Bufotalin (BFT), one of the naturally occurring bufodienolides, has multiple pharmacological and toxicological effects including antitumor activity and cardiotoxicity. This study aimed to character the metabolic pathway(s) of BFT and to identify the key drug metabolizing enzyme(s) responsible for hepatic metabolism of BFT in human, as well as to explore the related molecular mechanism of enzymatic selectivity. The major metabolite of BFT in human liver microsomes (HLMs) was fully identified as 5ß-hydroxylbufotalin by LC-MS/MS and NMR techniques. Reaction phenotyping and chemical inhibition assays showed that CYP3A4 and CYP3A5 were key enzymes responsible for BFT 5ß-hydroxylation. Kinetic analyses demonstrated that BFT 5ß-hydroxylation in both HLMs and human CYP3A4 followed the biphasic kinetics, while BFT 5ß-hydroxylation in CYP3A5 followed substrate inhibition kinetics. Furthermore, molecular docking simulations showed that BFT could bind on two different ligand-binding sites on both CYP3A4 and CYP3A5, which partially explained the different kinetic behaviors of BFT in CYP3A4 and CYP3A5. These findings are very helpful for elucidating the phase I metabolism of BFT in human and for deeper understanding the key interactions between CYP3A enzymes and bufadienolides, as well as for the development of bufadienolide-type drugs with improved pharmacokinetic and safety profiles.

Biflavones from Ginkgo biloba as novel pancreatic lipase inhibitors: Inhibition potentials and mechanism.

Reduction of lipid absorption has been recognized as an attractive approach for the discovery of new drugs to treat obesity and overweight. The leave extract of Ginkgo biloba has been widely used for the treatment of metabolic diseases (such as hyperlipidemia) in both eastern and western countries, but the bioactive compounds in Ginkgo biloba and the underlying mechanism have not been fully characterized. This study aimed to investigate the inhibition potentials and mechanism of major biflavones from G. biloba on pancreatic lipase (PL), a key target regulating lipid absorption. The results clearly demonstrated that all tested biflavones in G. biloba including isoginkgetin, bilobetin, ginkgetin and sciadopitysin, displayed strong to moderate inhibitory effects on PL with the IC50 values ranging from 2.90 µM to 12.78 µM. Further investigations on both inhibition kinetic analyses and docking simulations demonstrated that isoginkgetin, bilobetin and ginkgetin were potent PL inhibitors (Ki < 2.5 µM), which could create strong interactions with the catalytic triad of PL via hydrogen bonding. These findings provided a new powerful evidence for explaining the hypolipidemic effects of G. biloba, while these newly identified PL inhibitors from G. biloba could serve as lead compounds for the development of biflavonoid-type PL inhibitors.

Natural constituents from Cortex Mori Radicis as new pancreatic lipase inhibitors.

Pancreatic lipase (PL), a key enzyme responsible for the hydrolysis of triacylglycerides in the gastrointestinal tract, has been identified as the therapeutic target for the regulation of lipid absorption. In the present study, six major constituents from a famous Chinese herbal medicine Cortex Mori Radicis (also named sangbaipi in Chinese), have been collected and their inhibitory effects on PL have been carefully investigated and well characterized by a fluorescence-based assay. The results clearly demonstrated that all tested bioactive constituents from Cortex Mori Radicis including sanggenone C (SC), sanggenone D (SD), kuwanon C (KC), kuwanon G (KG), morin and morusin displayed strong to moderate inhibitory effects towards PL with the IC50 values ranging from 0.77 µM to 20.56 µM. Further investigations on inhibition kinetics demonstrated that SC, SD, KC and KG functioned as potent and mixed inhibitors against PL-mediated 4-MU oleate hydrolysis, with the Ki values less than 5.0 µM. Furthermore, molecular docking simulations demonstrated that SD (the most potent PL inhibitor from Cortex Mori Radicis) could create strong interaction with Ser152 (the key amino acid in the catalytic triad) of PL via hydrogen bonding. All these findings provided a new powerful evidence for explaining the hypolipidemic effect of Cortex Mori Radicis, also suggested that some abundant natural compounds in this herbal medicine could be served as lead compounds for the development of new PL inhibitors.

Induction of CYP1A1 increases gefitinib-induced oxidative stress and apoptosis in A549 cells.

As the first selective EGFR tyrosine kinase inhibitor, gefitinib has been clinically demonstrated to be effective for certain cancer cell types with EGFR-active gene mutations. However, a number of gefitinib-associated adverse pulmonary events have been reported, which could lead to the discontinuation of gefitinib therapy. Although previous reports have implicated that CYP1A1-mediated bioactivation of gefitinib maybe a major reason for the pulmonary toxicity, the roles of CYP1A1 in gefitinib-associated toxicity and the related molecular mechanism have not been well-characterized. This study aimed to reveal whether the induction of CYP1A1 would contribute to the toxic effect of gefitinib in living cells and to investigate the underlying molecular mechanism. The results demonstrated that gefitinib led to the enhancement of the dose-dependent cytotoxicity and the percentage of gefitinib-induced apoptosis was significantly increased on CYP1A1-overexpressed A549 cells, which was accompanied with a substantial increase in the intracellular reactive oxygen species and a remarkable decrease in the mitochondrial membrane potential. These findings strongly suggest that CYP1A1 can enhance the cytotoxicity of gefitinib and gefitinib-induced oxidative stress, which may partially explain the occurrence of pulmonary toxicity in some patients administered with gefitinib.

A practical strategy to design and develop an isoform-specific fluorescent probe for a target enzyme: CYP1A1 as a case study.

The development of isoform-specific probe(s) for a target enzyme with multiple homologs is always challenging. Herein, a practical strategy was used to design and develop an isoform-specific probe for CYP1A1, a key cytochrome P450 isoenzyme involved in xenobiotic metabolism and bioactivation. On the basis of the subtle differences in 3D structure and substrate preference between CYP1A1 and its homolog CYP1A2, we proposed that it was possible to design a CYP1A1-specific probe via local modification of the reaction site on known CYP1A substrates. To validate this hypothesis, 4-hydroxy-1,8-naphthalimide (HN) was selected as the basic fluorophore due to its excellent optical properties, while a series of O-alkylated HN derivatives were prepared to evaluate their specificity towards CYP1A1. Our results revealed that the introduction of a chloroethyl to HN could get the best isoform selectivity towards CYP1A1 over other CYPs including CYP1A2. The newly developed probe NBCeN exhibited excellent specificity, high sensitivity, and a ratiometric fluorescence response following CYP1A1-catalyzed O-dechloroethylation. NBCeN was successfully used to real-time monitor the activity of CYP1A1 in complex biological samples and to rapidly screen CYP1A1 modulators in living systems. NBCeN could also be used for two-photon imaging of intracellular CYP1A1 in living cells and tissues with high ratiometric imaging resolution and deep tissue penetration. All these findings demonstrated that local modification of non-specific substrates was a practical strategy to develop an isoform-specific probe for a target isoenzyme, while NBCeN could serve as a specific imaging tool to explore the biological functions of CYP1A1 in complex biological systems.

Structure-activity relationships of flavonoids as natural inhibitors against E. coli ß-glucuronidase.

Bacterial ß-glucuronidases play key roles in the deconjugation of a variety of endogenous and drug glucuronides, thus have been recognized as important targets to modulate the enterohepatic circulation of various glucuronides. In this study, more than 30 natural flavonoids were collected and their inhibitory effects against E. coli ß-glucuronidase (EcGUS) were assayed. The results demonstrated that some flavonoids including scutellarein, luteolin, baicalein, quercetin and scutellarin displayed strong to moderate inhibitory effects against EcGUS, with the IC50 values ranging from 5.76 µM to 29.64 µM, while isoflavones and dihydroflavones displayed weak inhibitory effects against EcGUS. Further investigation on inhibition kinetics revealed that scutellarein and luteolin functioned as potent competitive inhibitors against EcGUS-mediated PNPG hydrolysis, with the Ki values less than 3.0 µM. Molecular docking simulations demonstrated that scutellarein and luteolin could be well-docked into the catalytic site of EcGUS, while the binding areas of these two natural inhibitors on EcGUS were highly overlapped with that of PNPG on EcGUS. Additionally, the structure-inhibition relationships of natural flavonoids against EcGUS are also summarized, which will be very helpful for the medicinal chemists to design and develop more potent flavonoid-type inhibitors against EcGUS.

A highly selective near-infrared fluorescent probe for carboxylesterase 2 and its bioimaging applications in living cells and animals.

A near-infrared fluorescent probe (DDAB) for highly selective and sensitive detection of carboxylesterase 2 (CE2) has been designed, synthesized, and systematically studied both in vitro and in vivo. Upon addition of CE2, the ester bond of DDAB could be rapidly cleaved and then release a near-infrared (NIR) fluorophore DDAO, which brings a remarkable yellow-to-blue color change and strong NIR fluorescence emission in physiological solutions. The newly developed probe exhibits excellent properties including good specificity, ultrahigh sensitivity and high imaging resolution. Moreover, DDAB has been applied to measure the real activities of CE2 in complex biological samples, as well as to screen CE2 inhibitors by using tissue preparations as the enzymes sources. The probe has also been successfully used to detect endogenous CE2 in living cells and in vivo for the first time, and the results demonstrate that such detection is highly reliable. All these prominent features of DDAB make it holds great promise for further investigation on CE2-associated biological process and for exploring the physiological functions of CE2 in living systems.

Gomisin A is a Novel Isoform-Specific Probe for the Selective Sensing of Human Cytochrome P450 3A4 in Liver Microsomes and Living Cells.

Nearly half of prescription medicines are metabolized by human cytochrome P450 (CYP) 3A. CYP3A4 and 3A5 are two major isoforms of human CYP3A and share most of the substrate spectrum. A very limited previous study distinguished the activity of CYP3A4 and CYP3A5, identifying the challenge in predicting CYP3A-mediated drug clearance and drug-drug interaction. In the present study, we introduced gomisin A (GA) with a dibenzocyclooctadiene skeleton as a novel selective probe of CYP3A4. The major metabolite of GA was fully characterized as 8-hydroxylated GA by LC-MS and NMR. CYP3A4 was assigned as the predominant isozyme involved in GA 8-hydroxylation by reaction phenotyping assays, chemical inhibition assays, and correlation studies. GA 8-hydroxylation in both recombinant human CYP3A4 and human liver microsomes followed classic Michaelis-Menten kinetics. The intrinsic clearance values indicated that CYP3A4 contributed 12.8-fold more than CYP3A5 to GA 8-hydroxylation. Molecular docking studies indicated different hydrogen bonds and π-π interactions between CYP3A4 and CYP3A5, which might result in the different catalytic activity for GA 8-hydroxylation. Furthermore, GA exhibited a stronger inhibitory activity towards CYP3A4 than CYP3A5, which further suggested a preferred selectivity of CYP3A4 for the transformation of GA. More importantly, GA has been successfully applied to selectively monitor the modulation of CYP3A4 activities by the inducer rifampin in hepG2 cells, which is consistent with the level change of CYP3A4 mRNA expression. In summary, our results suggested that GA could be used as a novel probe for the selective sensing of CYP3A4 in tissue and cell preparations.

A Two-Photon Ratiometric Fluorescent Probe for Imaging Carboxylesterase 2 in Living Cells and Tissues.

In this study, a two-photon ratiometric fluorescent probe NCEN has been designed and developed for highly selective and sensitive sensing of human carboxylesterase 2 (hCE2) based on the catalytic properties and substrate preference of hCE2. Upon addition of hCE2, the probe could be readily hydrolyzed to release 4-amino-1,8-naphthalimide (NAH), which brings remarkable red-shift in fluorescence (90 nm) spectrum. The newly developed probe exhibits good specificity, ultrahigh sensitivity, and has been successfully applied to determine the real activities of hCE2 in complex biological samples such as cell and tissue preparations. NCEN has also been used for two-photon imaging of intracellular hCE2 in living cells as well as in deep-tissues for the first time, and the results showed that the probe exhibited high ratiometric imaging resolution and deep-tissue imaging depth. All these findings suggested that this probe holds great promise for applications in bioimaging of endogenous hCE2 in living cells and in exploring the biological functions of hCE2 in complex biological systems.

A Highly Selective Ratiometric Two-Photon Fluorescent Probe for Human Cytochrome P450 1A.

Cytochrome P450 1A (CYP1A), one of the most important phase I drug-metabolizing enzymes in humans, plays a crucial role in the metabolic activation of procarcinogenic compounds to their ultimate carcinogens. Herein, we reported the development of a ratiometric two-photon fluorescent probe NCMN that allowed for selective and sensitive detection of CYP1A for the first time. The probe was designed on the basis of substrate preference of CYP1A and its high capacity for O-dealkylation, while 1,8-naphthalimide was selected as fluorophore because of its two-photon absorption properties. To achieve a highly selective probe for CYP1A, a series of 1,8-naphthalimide derivatives were synthesized and used to explore the potential structure-selectivity relationship, by using a panel of human CYP isoforms for selectivity screening. After screening and optimization, NCMN displayed the best combination of selectivity, sensitivity and ratiometric fluorescence response following CYP1A-catalyzed O-demetylation. Furthermore, the probe can be used to real-time monitor the enzyme activity of CYP1A in complex biological systems, and it has the potential for rapid screening of CYP1A modulators using tissue preparation as enzyme sources. NCMN has also been successfully used for two-photon imaging of intracellular CYP1A in living cells and tissues, and showed high ratiometric imaging resolution and deep-tissue imaging depth. In summary, a two-photon excited ratiometric fluorescent probe NCMN has been developed and well-characterized for sensitive and selective detection of CYP1A, which holds great promise for bioimaging of endogenous CYP1A in living cells and for further investigation on CYP1A associated biological functions in complex biological systems.

A Mechanism-Based Model for the Prediction of the Metabolic Sites of Steroids Mediated by Cytochrome P450 3A4.

Early prediction of xenobiotic metabolism is essential for drug discovery and development. As the most important human drug-metabolizing enzyme, cytochrome P450 3A4 has a large active cavity and metabolizes a broad spectrum of substrates. The poor substrate specificity of CYP3A4 makes it a huge challenge to predict the metabolic site(s) on its substrates. This study aimed to develop a mechanism-based prediction model based on two key parameters, including the binding conformation and the reaction activity of ligands, which could reveal the process of real metabolic reaction(s) and the site(s) of modification. The newly established model was applied to predict the metabolic site(s) of steroids; a class of CYP3A4-preferred substrates. 38 steroids and 12 non-steroids were randomly divided into training and test sets. Two major metabolic reactions, including aliphatic hydroxylation and N-dealkylation, were involved in this study. At least one of the top three predicted metabolic sites was validated by the experimental data. The overall accuracy for the training and test were 82.14% and 86.36%, respectively. In summary, a mechanism-based prediction model was established for the first time, which could be used to predict the metabolic site(s) of CYP3A4 on steroids with high predictive accuracy.

Interspecies variation in phase I metabolism of bufalin in hepatic microsomes from mouse, rat, dog, minipig, monkey, and human.

1. Bufalin (BF), one of the major bioactive compounds in traditional Chinese medicine (TCM) Chansu, has been found with various pharmacological and toxicological effects. This study aims to investigate the species differences in phase I metabolism of BF in hepatic microsomes from human and five common experimental animals. 2. Metabolite profiling demonstrated that two major metabolites were formed in liver microsomes from human and animal species in NADPH-generating system. Two major metabolites were identified as 5ß-hydroxyl-bufalin and 3-keto-bufalin, with the help of authentic standards. CYP3A was assigned as the main isoform involved in both 5ß-hydroxylation and 3-oxidation in all studied liver microsomes. The apparent kinetic parameters including substrate affinity and catalytic efficiency for 5ß-hydroxylation and 3-oxidation of BF were also determined. 3. In summary, CYP3A mediated 5ß-hydroxylation and 3-oxidation were two major metabolic pathways of BF in hepatic microsomes from human and five studied animals, but kinetic analysis demonstrated that the intrinsic clearances of these two metabolic pathways were much different among various species. The qualitative and quantitative interspecies study indicated that minipig exhibited the similar metabolic profile, kinetic behaviors and intrinsic metabolic clearances of BF phase I biotransformation in comparison with that of human.

A highly selective probe for human cytochrome P450 3A4: isoform selectivity, kinetic characterization and its applications.

Bufalin 5ß-hydroxylation was found to be an isoform-specific biotransformation probe substrate for cytochrome P450 3A4 (CYP3A4). The probe reaction was well-characterized and it can be used for measuring the real catalytic activities of CYP3A4 from different enzyme sources.

Immunomodulatory activities on macrophage of a polysaccharide from Sipunculus nudus L.

A water-soluble polysaccharide, named as SNP, was extracted and fractioned from the body wall of Sipunculus nudus L. by DEAE-Sepharose anion exchange and Sepharose CL-6B column chromatography. The structural characteristics of SNP investigated by high performance size exclusion chromatography (HP-SEC), Fourier transform infrared spectroscopy (FT-IR) and gas chromatography-mass spectrometry (GC-MS) indicated that SNP was a homogeneous polysaccharide with a molecular mass of 350kD and the monosaccharide composition was determined to be rhamnose (28%), fucose (16%) and galactose (56%). SNP was able to upregulate the expression of cytokines (IL-6 and TNF-α), but did not affect IL-10 secretion by murine macrophages and human peripheral blood mononuclear cells. RT-PCR analysis demonstrated that the SNP also induced the expression of iNOS and COX-2, responsible for the induction of NO and PGE2 respectively, and SNP suppressed the arginase activity. These results suggest that the polysaccharide isolated from S. nudus activates macrophages and has potent immunostimulating activity.

Anti-inflammatory and anti-nociceptive activities of Sipunculus nudus L. extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Sipunculus nudus has long been employed as traditional Chinese medicine in folk remedies for the treatment of carbuncles, tuberculosis and nocturia, regulating the functions of stomach and spleen, as well as for the restoration to health in debilities caused by various pathogens and aging. Decoction of Sipunculus nudus has traditionally been used to remedy sternalgia in folk medicine. AIM OF THE STUDY: This study aimed to assess the anti-inflammatory and anti-nociceptive activity of the water extract from Sipunculus nudus. MATERIALS AND METHODS: The water extract from the body wall of Sipunculus nudus was obtained with the yield of 14.1%. The anti-inflammatory effect in six animal models and anti-nociceptive effect in two animal models of the water extract were evaluated by oral for the study. RESULTS: Pretreatment with the extract (at the dose of 50, 100 and 200 mg/kg) produced significant dose-dependent anti-inflammatory and anti-nociceptive effects. At 200 mg/kg dose, the inhibition ratio of the extract on carrageenan-induced rat hind paw oedema, dextran-induced rat paw oedema, cotton pellet granuloma in rats, carrageenan-induced peritonitis and acetic acid-induced vascular permeability were 59.2%, 51.0%, 53.1%, 42.5%, 50.8%, better than that of indomethacin (5 mg/kg) 49.5%, 50%, 44.4%, 37.6%, 46.8%, respectively. The inhibition ratio of the extract (200 mg/kg dose) on xylene-induced mouse ear oedema was 61.5%, lower than 63.7% for indomethacin (5 mg/kg). At 200mg/kg, the extract decreased number of writhing 52.3% in acetic acid-induced writhing model and increased the response latency 25.24% in hot plate test. CONCLUSIONS: The water extract from the body wall of Sipunculus nudus possesses excellent anti-inflammatory activity as well as peripheral and central analgesic properties.

Anti-hypoxia activity of a polysaccharide extracted from the Sipunculus nudus L.

A water-soluble polysaccharide, named as SNP, was extracted and fractioned from the body wall of Sipunculus nudus L. by DEAE-Sepharose anion exchange and Sepharose CL-6B column chromatography. The evaluation for anti-hypoxia activity demonstrated that SNP had significant anti-hypoxic activity on normobarie hypoxia, chemical intoxicant hypoxia and acute cerebral ischemia hypoxia models in mice. SNP also enhanced the number of red blood cell count (RBC) and the concentration of hemoglobin (HGB). The structural characteristics of SNP investigated by high performance size exclusion chromatography, Fourier transform infrared spectroscopy and gas chromatography-mass spectrometry indicated that SNP was a homogeneous polysaccharide with a molecular mass of 350 kD and was composed of rhamnose (28%), fucose (16%) and galactose (56%). The results suggested that SNP could be explored as a novel potential anti-hypoxia agent.