Proteasome Inbibitors From Metabolites Of Microorganisms

Ubiquitin-proteasome pathway (UPP) play an important role in selective degradation the key proteins involved in a variety of basic physiological processes including cell transcription, protein stability, DNA repair, cell cycle regulation and stress response. It has been reported that proteasome is implicated in various diseases such as rheumatic diseases, diabetes and leukemia, and proteasome inhibitors have effects on anti-tumor, anti-fungal, immune suppression, et al. Therefore, proteasome has been developed as an important target for drug discovery.In the course of our screening program for high-efficiency and low toxicity proteasome inhibitors, metabolites from 4467 fungi and 1200 actinomycetes were tested by the high throughput screeening targeted to proteasome and three active strains numbered F04W2166, F05B151 and F05ZA896 were selected. They were identified as Verticillium, Aspergillus and Chaetomium by morphological characteristics, respectively.Nine compounds, F04W2166A, B, F05ZA896A and F05B151A, B, C, D, E, F, were obtained from the metabolites of the three active strains by solvent extraction, silica gel chromatography, Sephadex LH-20 gel filtration, Medium-pressure ODS column chromatography and HPLC preparation. The structures of them were determined by analysis of their physicochemical properties, UV, MS and NMR data, among them F04W2166A and B were identified as pullularin C and verticillin D. F05ZA896A was identified as chaetomin. F F05B151A, B, C, D and F were conformed to be the known compounds 9-hydroxymicroperfuranone, clavatol,3-(1-methylethyl)-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione, C10-deoxy derivative of gerfelin and soyasaponin1, respectively. F05B151E were complex of two compounds shared same planar structure but different in stereo configuration. They were determined as (1R,4R)-4-(((9R,9aR)-1,9-dihydroxy-2,2-dimethyl- 3-oxo-2,3,9,9a-tetrahydro-1H-imidazo[1,2-a]indol-9-yl)methyl)-1-isopropyl-[1,4]oxazino[3,4-b]quin azoline-3,6(1H,4H)-dione and (1R,4S)-4-(((9S,9aR)-1,9-dihydroxy-2,2-dimethyl-3-oxo-2,3, 9,9a-tetrahydro-1H-imidazo[1,2-a]indol-9-yl)methyl)-1-isopropyl-[1,4]oxazino[3,4-b]quinazo line-3,6(1H,4H)-dione, tentatively. Six compounds including F04W2166A, F04W2166B, F05ZA896A, F05B151A, F05B151C and F05B151E showed good inhibitory activity towards proteasome in vitro with IC50 values of 3.1,1.5,0.1,2.5,20 and 10μg/mL, respectively. While F05B151C and F05B151D showed week inhibitory activity with IC50 values of 120 and 150μg/mL. F05B151F showed no significant proteasome inhibitory activity at concentration of 10 mg/mL.In addition, six compounds showed strong proteasome inhibitory activities were further tested on cell proliferation of colon cancer cell line HT-29 and human breast cancer cell line MDA-MB-231 and the results showed that F04W2166A, F04W2166B, F05ZA896A had strong anticancer activity, with dose-dependent inhibition, while F05B151A, C and E showed no significant activity.In this study, nine compounds were isolated and identified from 3 active fungal strains, among them F05B151C, F05B151E ware new natural product and F05B151E was a complex of two novel compounds shared same planar structure but different in stereo configuration. Six compounds were found to have strong proteasome inhibitory activities and this will lay a basis on the drug lead discovery of proeteasome inbibitors.