ABSTRACT
Aim To discover specific telomerase inhibitors by high-throughput screening in the more than 2 000 strains of actinomycetes fermentation broth.Methods After verifying the three kinds of screening models for telomerase inhibitor by yeast PCR,the three yeast strains were inoculated overnight into yeast liquid culture which was lacking in histidine and contained 3-AT.Then concentration of yeast strains in culture liquid and dosage of screening sample were regulated for better adjustment to the high-throughput screening.At last the adjusted liquid culture containing yeast strain piped to 96-well plates,drug samples and control article were also added,then the yeast was incubated in the thermostat oscillator.At the same time OD_(595nm) of the yeast liquid was measured every 12 h by using Multifunctional Microplate Analyzer,and the samples whose inhibitory efficiency was over 0.45 were selected for secondary screening.Results The high-throughput screening method related to yeast three-hybrid system was effective in screening samples that had potential telomerase inhibitory ability.The initial concentration of yeast liquid OD_(595nm) was 0.04 and the best dosage of samples was 5 ml.14 active samples,whose inhibitory efficiency was over 0.45,were founded after primary and secondary screening.Conclusions High-throughput screening method related to yeast three-hybrid system is simple and stable in discovering telomerase inhibitors,and 14 new antitumor compounds are identified,which lays the foundation in the development of new anti-tumor agents.
ABSTRACT
Aim To isolate, purify, and chemically characterize a polysaccharide showing inhibition of the six-?-helix bundle formation of HIV-1 envelope glycoprotein gp41 from cultured broth of a streptomyces sp.strain. Methods Ethanol was used to precipitate polysaccharides and macromolecules from the broth.Proteins in the precipitate were removed by sevage method.Purification was carried out by DEAE-Cellulose and sephadex G-25 column chromatography. The chemical structure of the polysaccharide was determined with the combined application of HPLC,UV,IR and 1H-NMR spectroscopy, and the methods of periodate oxidation and Smith degradation,etc. Activity of anti-HIV-1 ~gp41 six-?-helix bundle formation was assayed withsandwich ELISA method.Results The purified polysaccharide,designated as SMP for Streptomyces polysaccharide,is neutral with a molecular weight of approximately 4855 Daltons. Sugar analysis showed SMP contains glucose and fructose residues in an approximate molar ratio of 22∶1 (10.96 to 0.48). The glycosidic linkages were estimated to be (1→4)-?-D-pyranoside as its main chain, and 1→6 linkage was attached to the main chain. Activity analysis revealed SMP markedly inhibited the six-?-helix bundle formation of HIV-1 glycoprotein gp41 and the IC_~50 was (145.48?7.25) mg?L~-1 .Conclusion Streptomyces polysaccharide SMP showing inhibition of the six-?-helix bundle formation of HIV-1 envelope glycoprotein gp41 was isolated and purified.