Joseph Terracciano

Use of red-shifted dyes in a fluorescence polarization AKT kinase assay for detection of biological activity in natural product extracts

Kinases are an important therapeutic target for drug discovery, and many cancer chemotherapeutic agents have been derived from natural product sources. Natural product samples, however, have the likelihood of assay interference, particularly at elevated test concentrations. The authors developed a competitive fluorescence polarization (FP) assay using red-shifted fluorophores for the AKT kinase and demonstrated utility for testing concentrated natural product extracts. A set of 7 actinomycetes cultures containing indolocarbazoles, known nonselective kinase inhibitors, and a control set of 22 nonproducing indolocarbazole cultures were evaluated. Using red-shifted dyes (Cy3B™ or Cy5™), the authors identified active samples with minimal interference up to the extract concentrations that are 3 times nonextracted culture levels. In contrast, a significant number of interferences were observed using either a fluorescein competitive FP assay or a [33P]ATP Flashplate assay. This work demonstrates that one can screen natural product extracts at high concentrations successfully using FP technology with red-shifted dyes. (Journal of Biomolecular Screening 2004:52-61)

A new fungal metabolite, Sch 202596, with inhibitory activity in the galanin receptor GALR1 assay

Abstract A novel spirocoumaranone, Sch 202596 ( 1 ), was isolated from the fermentation broth of Aspergillus sp. The isolation, structure elucidation and stereochemistry of 1 are described.

Structure Elucidation of Sch 725674 from Aspergillus sp.

A new macrolide Sch725674 (1) was isolated and identified from the culture of an Aspergillus sp. The structure elucidation of 1 was accomplished based on extensive NMR spectroscopic analyses. Compound 1 showed inhibitory activity against Saccharomyces cerevisiae (PM503) and Candida albicans (C43) with MICs of 8 and 32 µg/ml, respectively.

Sch 213766, A Novel Chemokine Receptor CCR-5 Inhibitor from Chaetomium globosum

A novel fungal secondary metabolite, Sch 213766 was isolated from the fungal fermentation broth of Chaetomium globosum as the chemokine receptor CCR-5 inhibitor and shown to be the methyl ester of the previously described tetramic acid Sch 210972 on the basis of UV, MS and NMR spectral data analyses. Sch213766 exhibited an IC50 value of 8.6 μM in the CCR-5 receptor in vitro binding assay.

A New Hydrogenated Azaphilone Sch 725680 from Aspergillus sp.

A new hydrogenated azaphilone Sch725680 (1) was isolated and identified from the culture of an Aspergillus sp. The structure elucidation of 1 was achieved based on extensive NMR spectroscopic analyses. Compound 1 showed inhibitory activity against Saccharomyces cerevisiae (PM503) and Candida albicans (C43) with MICs of 8 and 64 μg/ml, respectively.

A new potent antifungal agent from Actinoplanes sp

Abstract A novel antifungal agent, Sch 56036 ( 1 ), was isolated from the fermentation culture broth of an Actinoplanes sp. Structure elucidation of 1 was accomplished by the analysis of spectroscopic data. Compound 1 was identified as a new polycyclic xanthone. Biological evaluation of 1 indicated that the compound possesses broad spectrum antifungal activity against various yeasts and dermatophytes with geometric mean MIC values ranging from 0.017∼0.031 μg/mL, respectively.

Isolation and structure elucidation of two novel deformylase inhibitors produced by Streptomyces sp.

Abstract Sch 382582 ( 1 ) and Sch 382583 ( 2 ), two novel pseudopeptides, were isolated from fermentation broth of Streptomyces sp. as bacteria peptide deformylase inhibitors. Structure elucidation of 1 and 2 was accomplished by extensive 2D NMR spectroscopic studies including NOESY, HMQC-TOCSY and HMBC experiments, and the relative stereochemistry was determined by X-ray crystallography. Both compounds displayed potent inhibitory activity against E. coli deformylase.

Sch 1385568, a new azaphilone from Aspergillus sp.

In the course of our continuing search for novel antimicrobial agents,1,2 we have identified a novel azaphilone Sch 1385568 (1) (Scheme 1) from an Aspergillus sp. culture (SPRI-0814). Various azaphilones and hydrogenated azaphilones have been isolated mainly from fungal species, such as Emericella sp.,3–5 Penicillium sp.,6–8 Phomopsis sp.,9 Chaetomium sp.,10 Pseudohalonectria sp.11 and Anuulohypoxylon sp.12 Some of them have been described to show biological activities against various targets related to the different therapeutic areas, including cardiovascular, lipid metabolism, inflammatory, antiinfectious and antitumor areas. More specifically, azaphilones have been reported to display inhibitory activity against the following targets: acyl-CoA: cholesterol acyltransferase,6 endothelin receptor,8 cholesteryl ester transfer protein,13 platelet-derived growth factor,14 gp120-CD4,15 monoamine oxidase,16 phospholipase A2 17 and nitric oxide production.18 Some azaphilones have also been reported to display antitumor10,19 and antimicrobial activities.10–12 In this communication, we describe the fermentation, isolation, structure elucidation and antimicrobial activity of 1. Fermentation of Aspergillus sp. culture SPRI-0814 was conducted in shake flasks. Stock cultures were maintained as frozen whole broths at 80 1C in a final concentration of 10% glycerol. The germination medium contained proteus peptone 5.0 g, NaCl 5.0 g, KH2PO4 5.0 g, yeast extract 3.0 g, cerelose 20 g and soybean grits 5.0 g in 1.0 l tap water with pH 7.0 before autoclaving. Each 250 ml flask containing 70 ml of this medium was inoculated with 2 ml of the stock culture. The flasks were incubated at 24 1C on a rotary shaker at 250 rpm for 4 days to obtain the first stage seed. The above procedure was repeated using the first stage seed to obtain the second stage seed. This second stage seed was then used to inoculate the fermentation medium at 5% v/v. The fermentation was carried out in 500 ml flasks, each containing 100 ml of the fermentation medium, which consisted of neopeptone 10 g and cerelose 40 g in 1.0 l tap water. The pH was adjusted to 7.4, and CaCO3 (4 g l 1) was added. The flasks were incubated at 24 1C in a rotary shaker at 250 rpm for 7 days. The harvested fermentation broth (10 l) was mixed with NaCl (2 kg) and acetonitrile (MeCN, 20 l) for 15 min. The organic layer was separated and concentrated to a slurry, and then the slurry material was absorbed onto the polymeric resin, CG161 (B200 ml, Tosoh Biosep LLC, Montgomeryville, PA, USA). The salts and hydrophilic substances were removed by washing with water (20 l). Then, the absorbed organic material was eluted with 85% aq. MeOH (4 l) to yield B2.4 g of dried material after concentration in vacuo. Part of this organic material was purified on a semi-preparative ODS-A HPLC column (YMC, 120 Å, S-7, 20 250 mm; Waters HPLC, Millennium System (Milford, MA, USA), equipped with a photodiode array detector). The column was eluted with a gradient of MeCN-H2O: 5–100% MeCN in 50 min, and then held isocratically with 100% MeCN for an additional 15 min with a flow rate of 15 ml min 1. All fractions were collected and analyzed on the basis of a UV chromatogram. Pure 1 (B5 mg) was obtained from three injections of the enriched material (40 mg each). The structure of 1 was mainly elucidated by extensive oneand two-dimensional NMR analyses. In the 1H-NMR spectrum, a total of 17 carbon-attached protons were detected. Three methyl and one methine signals were observed in the aliphatic region, and eight resonances were observed in the low-field region. In the 13C NMR spectrum, 21 carbon signals were detected, in which a conjugated ketone functionality (C-6, d 200.0) was identified. The molecular ion m/z 385, [M+H]+ was observed on an electrospray ionisation-MS instrument (Applied Biosystem, Foster City, CA, USA, API-150Ex spectrometer), and therefore the molecular formula of 1 was calculated as C21H20O7. From the analyses of NMR and MS data, three hydroxyl groups were proposed to be present in the molecule based on only 17 protons observed in the 1H-NMR spectrum. The azaphilone skeleton was mainly determined by 1H–13C long-range correlations measured in a heteronuclear multiple bond correlation (HMBC) experiment. The methyl group showing a doublet–doublet resonance (H3-3¢, d 1.88, dd, J1⁄47.0, 1.7 Hz) in 1H-NMR was determined to be

Caryophyllenes from a fungal culture of Chrysosporium pilosum.

Four new caryophyllene derivatives, Sch 725432 (1), Sch 601253 (2), Sch 601254 (3), and Sch 725434 (4), were isolated from the fungal fermentation broth of Chrysosporium pilosum by reversed-phase HPLC purification. The structure elucidation of trioxygenated caryophyllenes 1-4 was accomplished on the basis of spectroscopic data interpretation. Sch 725434 (4) possesses a dihydrofuran-3-one ring, forming a tricyclic ring skeleton, which represents an unprecedented ring skeleton for the caryophyllene-type of sesquiterpenes. Compounds 1-4 were evaluated for their antifungal activity.

New Antibiotic Sch 725424 and Its Dehydration Product Sch 725428 from Kitasatospora sp.

A new microbial metabolite Sch 725424 (1) was isolated from the culture of Kitasatospora sp. The structure elucidation of 1 was accomplished based on NMR spectroscopic analyses as well as extensive structure elucidation of its dehydration product Sch 725428 (2). Compound 1 showed inhibitory activity against Staphylococcus aureus with MIC values 1∼2 µg/ml, and also displayed weak antifungal activity against Saccharomyces cerevisiae (PM503) with an MIC 32 µg/ml.