Xiangge Tian

Human transporters, PEPT1/2, facilitate melatonin transportation into mitochondria of cancer cells: an implication of the therapeutic potential

Melatonin is present in virtually all organisms from bacteria to mammals, and it exhibits a broad spectrum of biological functions, including synchronization of circadian rhythms and oncostatic activity. Several functions of melatonin are mediated by its membrane receptors, but others are receptor‐independent. For the latter, melatonin is required to penetrate membrane and enters intracellular compartments. However, the mechanism by which melatonin enters cells remains debatable. In this study, it was identified that melatonin and its sulfation metabolites were the substrates of oligopeptide transporter (PEPT) 1/2 and organic anion transporter (OAT) 3, respectively. The docking analysis showed that the binding of melatonin to PEPT1/2 was attributed to their low binding energy and suitable binding conformation in which melatonin was embedded in the active site of PEPT1/2 and fitted well with the cavity in three‐dimensional space. PEPT1/2 transporters play a pivotal role in melatonin uptake in cells. Melatonins membrane transportation via PEPT1/2 renders its oncostatic effect in malignant cells. For the first time, PEPT1/2 were identified to localize in the mitochondrial membrane of human cancer cell lines of PC3 and U118. PEPT1/2 facilitated the transportation of melatonin into mitochondria. Melatonin accumulation in mitochondria induced apoptosis of PC3 and U118 cells. Thus, PEPT1/2 can potentially be used as a cancer cell‐targeted melatonin delivery system to improve the therapeutic effects of melatonin in cancer treatment.

Sulfation of melatonin: enzymatic characterization, differences of organs, species and genders, and bioactivity variation.

Exogenous melatonin (Mel) is widely used in clinic for multiple therapeutic purposes. In metabolism pathways of Mel, 6-hydroxymelatonin-sulfate (S-O-Mel) and N-acetylserotonin sulfate (S-NAS) are the most abundant metabolites account for over 90% of total Mel metabolites in humans, indicating that sulfation plays an important role in reflecting the functions and clearance of Mel in vivo. In the present study, we characterized Mel sulfation using various human organ cytosols (liver, lung, kidney, small intestine and brain), liver cytosols from five different animal species, and cDNA-expressed human sulfotransferase (SULT) for the first time. Our results demonstrated that liver, lung, kidney and small intestine of humans had high catalytic efficiency for Mel sulfation, however, brain contained a very low reaction rate. Interestingly, organ cytosols prepared from females exhibited higher sulfation activity than those of males. SULT isoforms 1A1, 1A2, 1A3, 1B1 and 1E1 exhibited metabolic activities toward Mel. According to kinetic parameters (Km and Vmax), chemical inhibition, correlation analysis, molecular docking and sulfation assays with recombinant human SULTs isoforms, SULT1A1 was determined as the major enzyme responsible for Mel sulfation. Furthermore, considerable species differences in Mel sulfation were observed, and the total intrinsic clearance rate of Mel sulfation was as follows: monkey>rat>dog>human>pig>mouse. Additionally, the anti-inflammatory effects of Mel and its sulfated metabolites were evaluated by inhibiting nitric oxide (NO) production in RAW264.7 cells, and S-O-Mel as a bioactive form, exhibited potent bioactivity. Our investigation provided a global view of the enzyme-dependent sulfation of Mel that can guide biomedical research on Mel.

Regioselective Glucuronidation of Andrographolide and Its Major Derivatives: Metabolite Identification, Isozyme Contribution, and Species Differences

Andrographolide (AND) and two of its derivatives, deoxyandrographolide (DEO) and dehydroandrographolide (DEH), are widely used in clinical practice as anti-inflammatory agents. However, UDP-glucuronosyltransferase (UGT)-mediated phase II metabolism of these compounds is not fully understood. In this study, glucuronidation of AND, DEO, and DEH was characterized using liver microsomes and recombinant UGT enzymes. We isolated six glucuronides and identified them using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. We also systematically analyzed various kinetic parameters (Km, Vmax, and CLint) for glucuronidation of AND, DEO, and DEH. Among 12 commercially available UGT enzymes, UGT1A3, 1A4, 2B4, and 2B7 exhibited metabolic activities toward AND, DEO, and DEH. Further, UGT2B7 made the greatest contribution to glucuronidation of all three anti-inflammatory agents. Regioselective glucuronidation showed considerable species differences. 19-O-Glucuronides were present in liver microsomes from all species except rats. 3-O-Glucuronides were produced by pig and cynomolgus monkey liver microsomes for all compounds, and 3-O-glucuronide of DEH was detected in mouse and rat liver microsomes (RLM). Variations in Km values were 48.6-fold (1.93–93.6 μM) and 49.5-fold (2.01–99.1 μM) for 19-O-glucuronide and 3-O-glucuronide formation, respectively. Total intrinsic clearances (CLint) for 3-O- and 19-O-glucuronidation varied 4.8-fold (22.7–110 μL min−1 mg−1), 10.6-fold (94.2–991 μL min−1 mg−1), and 8.3-fold (122–1,010 μL min−1 mg−1), for AND, DEH, and DEO, respectively. Our results indicate that UGT2B7 is the major UGT enzyme involved in the metabolism of AND, DEO, and DEH. Metabolic pathways in the glucuronidation of AND, DEO, and DEH showed considerable species differences.

Novel protostane-type triterpenoids with inhibitory human carboxylesterase 2 activities

The rhizomes of Alisma orientalis have been used for centuries in China and other Asian countries as an effective herbal remedy. The phytochemical investigation of A. orientalis and biotransformation of two major triterpenoids alisols A (11) and B 23-acetate (13) by Cunninghamella elagans AS 3.2028 and Penicillium janthinellum AS 3.510 have led to the isolation of ten new protostane-type triterpenoids (1–5 and 18–22), including one novel 26-nor-protostane (1) and one unusual 17-nor-protostane (2), together with twelve known analogues. Their structures were determined by 1D and 2D NMR, and HRESIMS spectroscopic analyses. All the isolated compounds were assayed for their inhibitory activities against human carboxylesterase 2 (HCE-2). Compounds 1, 3–9, 12, 14–16, 19, and 20 showed significant inhibitory activities on HCE-2 with IC50 values from 0.51 ± 0.09 μM to 9.45 ± 0.73 μM. The inhibition kinetics of compound 5 toward HCE-2 were established, and its Ki value was determined as 0.57 μM. The interaction of compound 5 with HCE-2 was investigated using molecular docking.

Alismanoid A, an unprecedented 1,2-seco bisabolene from Alisma orientale, and its protective activity against H2O2-induced damage in SH-SY5Y cells

A pair of novel sesquiterpenoids, (8R)-alismanoid A (1a) and (8S)-alismanoid A (1b), possessing an unprecedented 1,2-seco bisabolene carbon skeleton, were isolated from rhizomes of Alisma orientale together with two new sesquiterpenoids: a 15-nor guaiane alismanoid B (2) and alismanoid C (3). Their structures were elucidated by 1D and 2D NMR, HRESIMS, electronic circular dichroism (ECD), and theoretical calculations, and a plausible biosynthetic pathway for compound 1 is discussed. Meanwhile, compounds 1–3 were investigated for their protective effects on H2O2-induced damage in human dopaminergic neuroblastoma cells (SH-SY5Y), and compounds 1b, 2, and 3 showed significantly protective activities at a certain concentration. Further, the action mechanism of compound 3 was proved to be through inhibition of H2O2-induced apoptosis in SH-SY5Y cells. Herein, these results suggest that sesquiterpenoids are potential candidate drugs for treating Parkinsons disease induced by reactive oxygen species.

Two new protostane-type triterpenoids from Alisma orientalis

Abstract Two new protostane-type triterpenoids, 17-epi alisolide (1) and 24-epi alismanol D (2), were isolated from Alisma orientalis together with one known compound. Their structural elucidations were conducted by NMR, UV and HRESIMS spectroscopic analyses, and comparison with the literature data. All the isolated compounds were evaluated for inhibitory effects on HCE-2. Compound 2 displayed moderate inhibitory activity against HCE-2 with IC50 value of 23.1 μM.

Activatable Near-Infrared Fluorescent Probe for Dipeptidyl Peptidase IV and Its Bioimaging Applications in Living Cells and Animals

Visualization of endogenous disease-associated enzymes is of great clinical significance, as it could allow earlier clinical diagnosis and timely intervention. Herein, we first synthesized and characterized an enzyme-activatable near-infrared fluorescent probe, GP-DM, for determining the activity of dipeptidyl peptidase IV (DPP IV), which is associated with various pathological processes, especially in diabetes and malignant tumors. GP-DM emitted significant turn-on NIR fluorescent signals simultaneously in response to DPP IV, making it favorable for accurately and dynamically monitoring DPP IV activity in vitro and in vivo. GP-DM exhibited excellent specificity and sensitivity in DPP IV imaging, as indicated by its higher catalytic activity than other human serine hydrolases and by its strong anti-interference ability to a complex biological matrix, which was fully characterized in a series of phenotyping reactions and inhibition assays. Encouraged by the advantages mentioned above, we successfully used GP-DM to evaluate endogenous DPP IV activity in various biological samples (plasma and tissue preparations) and living tumor cells and performed real-time in vivo bioimaging of DPP IV in zebrafish and tumor-bearing nude mice. All of the results reflected and highlighted the potential application value of GP-DM in the early detection of pathologies, individual tailoring of drug therapy, and image-guided tumor resection. Furthermore, our results revealed that DPP IV, a key target enzyme, is closely associated with the migration and proliferation of cancer cells and regulating the biological activity of DPP IV may be a useful approach for cancer therapy.

Alismanin A, a Triterpenoid with a C34 Skeleton from Alisma orientale as a Natural Agonist of Human Pregnane X Receptor

Alismanin A (1), a novel aromatic triterpenoid with a C34 skeleton, was isolated from Alisma orientale together with a rearranged nor-triterpenoid (2) and a 13,17-seco triterpenoid (3). Their structures were determined by a combination of HRESIMS, 2D NMR spectra, electronic circular dichroism (ECD), theoretical calculations, and X-ray diffraction analysis. Compounds 1 and 2 displayed significant activation effects on pregnane X receptor (PXR) at 10 nM. A plausible biosynthetic pathway for 1-3 is also discussed.

ent-Abietane and Tigliane Diterpenoids from the Roots of Euphorbia fischeriana and Their Inhibitory Effects against Mycobacterium smegmatis

An investigation on the bioactive chemical constituents of the roots of Euphorbia fischeriana has been conducted, with 21 diterpenoids obtained using various chromatographic techniques. On the basis of spectroscopic data analysis, the new compounds were elucidated as four ent-abietane-type diterpenoids (1-4) and four tigliane-type diterpenoids (13-16). Also obtained were eight known ent-abietane (5-12) and five known tigliane (17-21) diterpenoids. The potential antituberculosis effects of these diterpenoids were evaluated using a Mycobacterium smegmatis model. The most potent compound according to the in vitro bioassay used was 17-hydroxyjolkinolide B (12) (MIC 1.5 μg/mL).

Drug interaction study of natural steroids from herbs specifically toward human UDP-glucuronosyltransferase (UGT) 1A4 and their quantitative structure activity relationship (QSAR) analysis for prediction.

The wide application of herbal medicines and foods containing steroids has resulted in the high risk of herb-drug interactions (HDIs). The present study aims to evaluate the inhibition potential of 43 natural steroids from herb medicines toward human UDP- glucuronosyltransferases (UGTs). A remarkable structure-dependent inhibition toward UGT1A4 was observed in vitro. Some natural steroids such as gitogenin, tigogenin, and solasodine were found to be the novel selective inhibitors of UGT1A4, and did not inhibit the activities of major human CYP isoforms. To clarify the possibility of the in vivo interaction of common steroids and clinical drugs, the kinetic inhibition type and related kinetic parameters (Ki) were measured. The target compounds 2-6 and 15, competitively inhibited the UGT1A4-catalyzed trifluoperazine glucuronidation reaction, with Ki values of 0.6, 0.18, 1.1, 0.7, 0.8, and 12.3μM, respectively. And this inhibition of steroids towards UGT1A4 was also verified in human primary hepatocytes. Furthermore, a quantitative structure-activity relationship (QSAR) of steroids with inhibitory effects toward human UGT1A4 isoform was established using the computational methods. Our findings elucidate the potential for in vivo HDI effects of steroids in herbal medicine and foods, with the clinical dr ugs eliminated by UGT1A4, and reveal the vital pharamcophoric requirement of natural steroids for UGT1A4 inhibition activity.