Xinhua Lu

Elaiophylin, a novel autophagy inhibitor, exerts antitumor activity as a single agent in ovarian cancer cells.

Currently, targeting the autophagic pathway is regarded as a promising new strategy for cancer drug discovery. Here, we screened the North China Pharmaceutical Group Corporations pure compound library of microbial origin using GFP-LC3B-SKOV3 cells and identified elaiophylin as a novel autophagy inhibitor. Elaiophylin promotes autophagosome accumulation but blocks autophagic flux by attenuating lysosomal cathepsin activity, resulting in the accumulation of SQSTM1/p62 in various cell lines. Moreover, elaiophylin destabilizes lysosomes as indicated by LysoTracker Red staining and CTSB/cathepsin B and CTSD/ cathepsin D release from lysosomes into the cytoplasm. Elaiophylin eventually decreases cell viability, especially in combination with cisplatin or under hypoxic conditions. Furthermore, administration of a lower dose (2 mg/kg) of elaiophylin as a single agent achieves a significant antitumor effect without toxicity in an orthotopic ovarian cancer model with metastasis; however, high doses (8 mg/kg) of elaiophylin lead to dysfunction of Paneth cells, which resembles the intestinal phenotype of ATG16L1-deficient mice. Together, these results provide a safe therapeutic window for potential clinical applications of this compound. Our results demonstrate, for the first time, that elaiophylin is a novel autophagy inhibitor, with significant antitumor efficacy as a single agent or in combination in human ovarian cancer cells, establishing the potential treatment of ovarian cancer by this compound.

Mycophenolic acid induces adipocyte-like differentiation and reversal of malignancy of breast cancer cells partly through PPARγ

Mycophenolic acid (MPA) has been known for decades to be an anticancer and immunosuppressive agent and has significant anticancer properties, but its underlying molecular mechanisms are poorly characterized. Peroxisome proliferator-activated receptor gamma (PPARγ) has a central role in adipocyte differentiation, and MPA has been shown to be a potent PPARγ agonist. Whether PPARγ activation has a putative role in the anticancer efficacy of MPA via induction of adipocyte-like differentiation has not been elucidated. In the present study, MPA was demonstrated to dose-dependently activate PPARγ transcription in the GAL4-hPPARγ (LBD) chimeric receptor assay and PPRE-luc reporter gene assay with an EC(50) of 5.2-9.3 μM. Treatment of the breast cancer cell lines MDA-MB-231 and MCF-7 with MPA resulted in differentiation of adipose tissue that was characterized by accumulation of intracellular lipids, enlargement of cell volume, and permanent withdrawal from the cell cycle at the G1/G0 stage. At a molecular level, the expression of three adipocyte differentiation markers (PPARγ, adipsin D, and aP2) was remarkably induced in differentiated breast cancer cells. However, RNA interference experiments showed that PPARγ-knockdown cannot completely reverse the differentiated state of MDA-MB-231 cells after MPA treatment. These data suggest that the effects of MPA on adipocyte-like terminal differentiation of breast cancer cells are (at least in part) due to PPARγ activation, which is a novel anticancer mechanism of MPA.

Impacts of different promoters on the mammalian one-hybrid assay for detecting nuclear receptor agonists

AbstractNuclear receptors are a superfamily of ligand-activated transcription factors that play key roles in many biological processes, and have become one class of the most important targets in drug discovery. Mammalian one-hybrid system has been used to develop a cell-based functional transactivation high-throughput screening (HTS) assay for detecting nuclear receptors ligands. In the present study, we proved that different promoters used in the reporter vector had significant different impacts on the performance of HTS assays. The assay using the SV40 promoter in the reporter vector showed the characteristics of much higher signal/noise ratios, acceptable Z′ factors (>0.6), low coefficient variation (<12.5%) and higher hits rate, which could be more robust, reproducible, and sensitive. In contrast, utilizing a TATA box promoter in the assay resulted in higher variance and low sensitivity. In addition, it was found that the assay using SV40 had longer signal decay time and was easier to be miniaturized in 384-well format. It has been confirmed that the choice of a promoter is a critical factor in developing a reporter gene HTS assay. However, the SV40 promoter used in the present study has been shown to be more adaptable than the minimal promoter TATA box in the Mammalian one-hybrid HTS assays for detecting nuclear receptor agonists. FigureDuring construction of a cell-based functional transactivation high-throughput screening (HTS) assay for detecting nuclear receptors ligands, different promoters used in the reporter vector had significant different impacts on the performance of HTS assays. The assay using the SV40 promoter in reporter vector showed the characteristics of much higher signal/noise ratios, acceptable Z′ factors (>0.6%), low coefficient variation (<12.5%) and higher hits rate, which could be more robust, reproducible and sensitive. In contrast, utilizing TATA box promoter in the assay resulted in higher variance and low sensitivity

Three novel members of angucycline group from Streptomyces sp. N05WA963.

Three novel members of angucycline family named N05WA963A (1), B (2) and D (4), together with a new anthracycline named N05WA963C (3) were isolated from the culture broth of Streptomyces sp. N05WA963. The structures were elucidated on the basis of comprehensive spectral data analysis. All four compounds have shown antiproliferative effects on a panel of cancer cell lines such as SW620, K-562, MDA-MB-231, YES-4, T-98 and U251SP.

Altenusin, a Nonsteroidal Microbial Metabolite, Attenuates Nonalcoholic Fatty Liver Disease by Activating the Farnesoid X Receptor

Nonalcoholic fatty liver disease (NAFLD) is a prevalent chronic liver disease. The incidence of NAFLD has increased steadily due to its close association with the global epidemic of obesity and type 2 diabetes. However, there is no effective pharmacological therapy approved for NAFLD. Farnesoid X receptor (FXR), a member of the nuclear receptor subfamily, plays important roles in maintaining the homeostasis of bile acids, glucose, and lipids. FXR agonists have shown promise for the treatment of NAFLD. In this study, we report altenusin (2076A), a natural nonsteroidal fungal metabolite, as a novel selective agonist of FXR with an EC50 value of 3.2 ± 0.2 μM. Administration of 2076A protected mice from high-fat diet (HFD)–induced obesity by reducing the body weight and fat mass by 22.9% and 50.0%, respectively. Administration of 2076A also decreased the blood glucose level from 178.3 ± 12.4 mg/dl to 116.2 ± 4.1 mg/dl and the serum insulin level from 1.4 ± 0.6 ng/dl to 0.4 ± 0.1 ng/dl. Moreover, 2076A treatment nearly reversed HFD-induced hepatic lipid droplet accumulation and macrovesicular steatosis. These metabolic effects were abolished in FXR knockout mice. Mechanistically, the metabolic benefits of 2076A might have been accounted for by the increased insulin sensitivity and suppression of genes that are involved in hepatic gluconeogenesis and lipogenesis. In summary, we have uncovered a new class of nonsteroidal FXR agonist that shows promise in treating NAFLD and the associated metabolic syndrome.

Trivaric acid, a new inhibitor of PTP1b with potent beneficial effect on diabetes

Aim: To screen a potential PTP1b inhibitor from the microbial origin‐based compound library and to investigate the potential anti‐diabetic effects of the inhibitor in vivo and determine its primary anti‐diabetic mechanism in vitro and in silico. Methods: PTP1b inhibitory activity was measured using recombination protein as the enzyme and p‐NPP as the substrate. The binding of the inhibitor to PTP1b was analysed by docking in silico and confirmed by ITC experiments. The intracellular signalling pathway was detected by Western blot analysis in HepG2 cells. The anti‐diabetic effects were evaluated using a diabetic mice model in vivo. Results: Among 545 microbial origin‐based pure compounds tested, trivaric acid, a tridepside, was selected as a PTP1B inhibitor exhibiting strong inhibitory activity with an IC50 of 173 nM. Docking and ITC studies showed that trivaric acid was able to spontaneously bind to PTP1b and may inhibit PTP1b by blocking the catalytic domain of the phosphatase. Trivaric acid also enhanced the ability of insulin to stimulate the IR/IRS/Akt/GLUT2 pathway and increase the glucose consumption in HepG2 cells. In diabetic mice, trivaric acid that had been encapsulated into Eudrgit L100‐5.5 showed significant anti‐diabetic effects, improving insulin resistance, leptin resistance and lipid profile and weight control at doses of 5 mg/kg and 50 mg/kg. Significance: Trivaric acid is a potential lead compound in the search for anti‐diabetic agents targeting PTP1b.

Isolation and characterization of N98-1272 A, B and C, selective acetylcholinesterase inhibitors from metabolites of an actinomycete strain

A high throughput screening was carried out in order to search for inhibitors of acetylcholinesterase (AChE) from microorganism metabolites. An actinomycete strain was found to produce active compounds named N98-1272 A, B and C with IC50 of 15.0, 11.5, 12.5 μM, respectively. Structural studies revealed that the three compounds are identical to the known antibiotics, Manumycin C, B and A. Kinetic analyses showed that N98-1272 C (Manumycin A) acted as a reversible noncompetitive inhibitor of acetylcholinesterase, with a Ki value of 7.2 μM. The cyclohexenone epoxide part of the structure plays a crucial role in the inhibitory activity against AChE. Compared with Tacrine, N98-1272 A, B, and C exhibit much better selectivity toward AChE over BuChE.

Two new members of mycophenolic acid family from Penicillium brevicompactum Dierckx.

Inosine-5¢-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205) is an essential rate-limiting enzyme in the purine metabolic pathway, catalyzing NAD-dependent oxidation of inosine-5¢-monophosphate (IMP) to xanthosine-5¢-monophosphate (XMP) of the de novo synthesis of guanine nucleotides.1 The activity of IMPDH is correlated with the growth of cells, and IMPDH has become one of the important targets of antiviral, antimicrobial, anti-cancer and immunosuppressive therapy drugs.2 Several classes of IMPDH inhibitors are now in clinical use or are under development.3–7 However, some of them suffer from a certain degree of toxicity and/or susceptibility to metabolic inactivation.8 In the program of searching for IMPDH inhibitors, the culture broth of a fungal strain Penicillium brevicompactum F01-1358 showed a strong inhibitory activity against IMPDH. Bioassay-guided fractionation of the crude extract of F01-1358 resulted in the isolation of two new mycophenolic compounds, F01-1358A (2) and B (3) (Figure 1), as well as mycophenolic acid (MPA) (1). In this study, we report the fermentation, isolation, structure elucidation and preliminary IMPDH inhibitory activity of compounds 2 and 3. Fungus F01-1358 was isolated from a soil sample collected in the Jiangjin district of Chongqing, China. The strain was identified as P. brevicompactum Dierckx by morphological and cultural characteristics, and was deposited in the China General Microbiological Culture Collection (accession number: CGMCC No. 2038). The strain F01-1358 was cultivated at 27 1C on a rotary shaker at 220 r.p.m. in the medium consisting of 1.0% starch, 2.0% glucose, 0.8% malt extracts, 0.4% yeast extracts, 0.3% bean powder, 0.13% NaCl and 0.15% CaCO3 (pH 7.0 before sterilization). The 5-day-old whole broth (5.0 l) was adjusted to pH of 3.0–3.5 by 2.0 N HCl and centrifuged at 3000 r.p.m. for 15 min. The mycelia were extracted with 75% acetone. After the insoluble mycelia were removed by centrifugation, the acetone was evaporated under normal pressure, the remaining aqueous solution was extracted with ethyl acetate and the organic layer was dried over Na2SO4 and concentrated under reduced pressure to yield a brown residue (8.0 g). The residue was subjected to a silica gel flash column chromatography and eluted with gradient of chloroform–methanol from 100:1 to 10:1. The fractions showing an inhibitory activity against IMPDH were collected and concentrated to yield a pale yellow solid. A white crystal (4.8 g, compound 1) was obtained by recrystallization in isopropanol. Further purification of the mother solution was carried out on a preparative reverse phase (RP)-HPLC (detection: UV at 250 nm; column: Phenomenex (Torrance, CA, USA) C18, 10mm, 21.2 250 mm; mobile phase: 70% CH3CN–water with 0.1% acetic acid; flow rate: 16.0 ml min 1) and yielded 2 (23.0 mg) and a fraction (16.0 mg) containing compound 3. Pure compound 3 (4.0 mg) was obtained by another run of preparative RP-HPLC with a mobile phase of 65% CH3CN–water with 0.1% acetic acid. Compound 1, a white crystal with UV lmax (MeOH) 249 and 304 nm, was identified as MPA (1) (Figure 1) by the analysis of its spectral data (electrospray ionization (ESI)-MS, 1Hand 13C-NMR) and by comparing them with that reported in the literature.9 F01-1358A (2) was obtained as a white crystalline powder with a molecular formula C20H26O6 (high resolution fast atom bombardment mass spectra (HRFABMS) m/z 363.1808, M+H+, calcd for 363.1808, D0.0 mmu error). The IR (KBr) spectrum suggested that 2 had a hydroxyl group (3423 cm 1), two carbonyl groups (1734 and 1705 cm 1) and an isolated alkenyl group (1622 cm 1). The 1H-NMR spectrum of 2 showed signals for a secondary aliphatic methyl group at d 0.98 (3H, d, J1⁄46.5 Hz), two aromatic methyl groups at d 2.05 (3H, s) and d 1.67 (3H, s), an aromatic methoxyl group at d 3.67 (3H, s), as well as a singlet at d 5.14 (2H) corresponding to an aliphatic methylene with an oxygen atom attached. 13C-NMR and DEPT spectra confirmed the presence of 20 carbons, containing 4 primary, 5 secondary, 2 tertiary including an olefinic carbon, 7 aromatic

Discovery of a new structural class of competitive hDHODH inhibitors with in vitro and in vivo anti-inflammatory, immunosuppressive effects

Human dihydroorotate dehydrogenase (hDHODH) is an inner mitochondrial membrane enzyme that involves in the fourth step of the biosynthesis of pyrimidine base. Inhibitors of hDHODH have been proven efficacy for the treatments of inflammation, rheumatoid arthritis, multiple sclerosis and cancer. In the present study, ascochlorin (ASC) and its derivatives, natural compounds from fungal metabolites, were discovered as hDHODH inhibitors by high-throughput screening. Enzyme kinetics studies showed that ASC competitively binds to hDHODH at the site of coenzyme Q substrate. In ex vivo study, ASC significantly inhibited the ConA-stimulated T lymphocytes proliferation and interleukin-2, interferon-γ production. Furthermore, ASC showed significant in vivo anti-inflammatory and immunosuppressive effects on the mice ears swelling, allogenic skin grafts and rat collagen-induced arthritis animal disease models. ASC significantly reduced ears edema level of mice, increased the survival time of allogenic skin implanted on the mice and attenuated arthritis severity of rat model. In conclusion, ASC was identified as a new structural class of hDHODH inhibitors with efficient anti-inflammatory, immunosuppressive activity, and may be a promising candidate for the development of new therapy in the treatment of autoimmune diseases.

Wortmannilactones I–L, new NADH-fumarate reductase inhibitors, induced by adding suberoylanilide hydroxamic acid to the culture medium of Talaromyces wortmannii

With the aim of finding more potential inhibitors against NADH-fumarate reductase (specific target for treating helminthiasis and cancer) from natural resources, Talaromyces wortmannii was treated with the epigenome regulatory agent suberoylanilide hydroxamic acid, which resulted in the isolation of four new wortmannilactones derivatives (wortmannilactones I-L, 1-4). The structures of these new compounds were elucidated based on IR, HRESIMS and NMR spectroscopic data analyses. These four new compounds showed potent inhibitory activity against NADH-fumarate reductase with the IC50 values ranging from 0.84 to 1.35μM.