benzofurans and phyllanthoside

The National Cancer Institute (NCI) Human Tumor Cell Line Anti-Cancer Drug Screen has evaluated the cytotoxicity profiles of a large number of synthetic compounds, natural products, and plant extracts on 60 different cell lines. The data for each compound/extract can be assessed for similarity of cytotoxicity pattern, relative to a given test compound, using an algorithm called COMPARE. In applying a chemical biology approach to better understand the mechanism of eukaryotic protein synthesis, we used these resources to search for novel inhibitors of translation. The cytotoxicity profiles of 31 known protein synthesis inhibitors were used to identify compounds from the NCI database with similar activity profiles. Using this approach, two natural products, phyllanthoside and nagilactone C, were identified and characterized as novel protein synthesis inhibitors. Both compounds are specific for the eukaryotic translation apparatus, function in vivo and in vitro, and interfere with translation elongation. Our results demonstrate the feasibility of utilizing cytotoxicity profiles to identify new inhibitors of translation.

Topics: Algorithms; Benzofurans; Diterpenes; Dose-Response Relationship, Drug; Eukaryotic Cells; Genes, Reporter; Glycosides; HeLa Cells; Humans; Polyribosomes; Protein Biosynthesis; Protein Synthesis Inhibitors; Sesquiterpenes; Spiro Compounds

The antitumor agent phyllanthoside is rapidly metabolized in vitro by mouse plasma. This metabolite has now been isolated from mouse plasma and its structural properties and cytotoxicity characterized. The isolated metabolite was estimated to be greater than 98% pure by HPLC analysis. Mass spectral analysis (fast atom bombardment and tandem mass spectrometry) indicated that the metabolite was the aglycone of phyllanthoside that resulted from the cleavage of the ester bond linking the aglycone and the disaccharide moieties of phyllanthoside. This identification was based on identical collision-induced dissociation spectra of both phyllanthoside and the metabolite. The aglycone was not formed by mouse plasma that had been boiled, filtered to remove proteins, or treated with 1.0 mM diisopropyl fluorophosphate. These results suggest that aglycone formation occurs as a result of plasma esterase activity. Michaelis-Menten constants, Vmax and Km, for conversion of phyllanthoside to the aglycone at 22 degrees C were estimated to be 1.1 mmol/ml plasma/min and 2.0 mM, respectively. Concentrations of phyllanthoside and metabolite required to inhibit cell-colony formation by human A204 rhabdomyosarcoma in vitro were 0.47 nM and 24 microM, respectively. The toxicity of phyllanthoside, and perhaps its efficacy as an antitumor agent in mice, may depend on its rate of conversion to the aglycone.

Topics: Animals; Benzofurans; Cell Line; Chromatography, High Pressure Liquid; Drug Screening Assays, Antitumor; Glycosides; Humans; Male; Mass Spectrometry; Mice; Mice, Inbred Strains; Rhabdomyosarcoma; Sesquiterpenes; Spiro Compounds; Tumor Cells, Cultured; Tumor Stem Cell Assay

Phyllanthoside is a naturally occurring glycoside with activity against IP transplantable murine tumors. Phyllanthoside administered IV, to mice at a nontoxic dose of 16 mg/kg could not be detected in blood or plasma even 30 s after administration. There was rapid formation of a less polar metabolite, which disappeared with a half-life of about 10 min. When phyllanthoside was administered as an IV bolus to beagle dogs at doses of 0.1, 0.5, and 3.0 mg/kg the mean half-life of phyllanthoside elimination from plasma was 1.3 min and total body clearance 85.8 ml min-1 kg-1. A second phase of elimination was seen but could not be accurately defined. Only trace amounts of the less polar metabolite were detected in dog plasma. Infusion of phyllanthoside to beagle dogs at doses of 0.5 and 3.0 mg/kg over 70 min gave values for an initial half-life of 0.3 and 0.6 min, a terminal half-life of 99.4 and 16.5 min, and a total body clearance of 11.2 and 49.2 ml min-1 kg-1, respectively. The highest nontoxic dose of phyllanthoside in dog was 0.1 mg/kg, while doses of 0.5 mg/kg and 3.0 mg/kg resulted in ataxia and death of the dog. There was no difference in toxicity to dog according to whether phyllanthoside was given by IV bolus or continuous infusion. Isolated hepatocytes from rat metabolized phyllanthoside at a rate of 4.4 micrograms/min per 10(6) cells to form the less polar metabolite. Coculture with isolated hepatocytes decreased the cytotoxicity of phyllanthoside to A204 human rhabdomyosarcoma cell line growing in soft agarose. It is suggested that rapid metabolism of phyllanthoside in mouse as against dog might account for the lower toxicity of phyllanthoside in mouse, and might also account for the reported poor antitumor activity of IV-administered phyllanthoside in the mouse.

Topics: Animals; Antineoplastic Agents, Phytogenic; Benzofurans; Dogs; Glycosides; Half-Life; In Vitro Techniques; Injections, Intravenous; Kinetics; Liver; Male; Mice; Mice, Inbred Strains; Rats; Rats, Inbred Strains; Sesquiterpenes; Species Specificity; Spiro Compounds

Phyllanthoside is a glycoside isolated from the roots of the Central American tree Phyllanthus acuminatus Vahl with antitumor activity against murine B-16 melanoma and P-388 leukemia. We report a reversed-phase high-performance liquid chromatographic assay for phyllanthoside in plasma using a 25-cm RP-18, 5-micron column with a linear 10-min gradient of 50% to 100% methanol in 0.3 M sodium acetate, pH 4.0, at a flow-rate of 1.5 ml/min. Eluting peaks were detected at 270 nm. The lower limit of sensitivity of the assay for phyllanthoside in 0.5 ml plasma following ethyl acetate extraction at pH 7.0 was 0.25 micrograms/ml and the coefficient of variation at 1 microgram/ml was +/- 7.4%. Phyllanthoside was very rapidly broken down by mouse and rat plasma in vitro to an unidentified less polar metabolite. Formation of this metabolite was completely inhibited by preheating mouse plasma to 100 degrees C for 10 min. When mouse plasma was diluted 1:50 with water the half-life of phyllanthoside disappearance at 37 degrees C was 2.0 min. Breakdown of phyllanthoside in plasma from other species was slower than in mouse and the initial half-life at 37 degrees C in dog plasma was 30 min, in monkey plasma 33 min and in human plasma 38 min. The same less polar metabolite as in mouse plasma was formed slowly by plasma of monkey and dog. Phyllanthoside did not accumulate in human red blood cells. Binding of phyllanthoside to human plasma protein determined by ultrafiltration at 4 degrees C was 70%.

Topics: Animals; Antineoplastic Agents, Phytogenic; Benzofurans; Chromatography, High Pressure Liquid; Dogs; Drug Stability; Glycosides; Humans; Macaca fascicularis; Mice; Mice, Inbred Strains; Rats; Rats, Inbred Strains; Sesquiterpenes; Species Specificity; Spiro Compounds

Topics: Antineoplastic Agents, Phytogenic; Benzofurans; Glycosides; Plants, Medicinal; Sesquiterpenes; Spiro Compounds