The Studies On Construction And Application Of Screening Model Of Proteasome Inhibitor

OBJECT: To construct a model of screening proteasome inhibitor from naturalproducts, and to screen 103 natural products using this model, then to study the effectof natural products which have distinct inhibiting action on proteasome. METHOD:HL-60 cells were cultured, total protein of HL-60 cells was extracted. The proteinconcentration was measured with Bradford method. Total protein, positive drug ornatural products, specific proteasome fluorescence substrate were mixed and reactedfor certain time, and then the fluorescence was detected. Proteasome activity isindicated by fluorescence value (A) produced by proteolysis by proteasomes. Thosenatural products showed low fluorescence value were selected. The inhibition actionof these positive natural products were further determined on the three proteasomeenzyme at different concentration, and IC50 was calculated. At the same time, WesternBlot technique was used for the further evaluation. RESULT: This method can beused for screening proteasome inhibitor in batches. The results of preliminary screeningof 103 natrual compounds showed that DM13, DM21, KZ4, KZ7 inhibited threeproteasome enzymes. The IC50 values on three proteasome activities(peptidyl-glutamyl-peptide hydrolyzing activity, chymotrypsin-like activity, trypsin-like activity) of DM13's were 18.7, 25.10 and 24.10 ug/ml respectively; of DM21'swere 32.45, 22.09 and 22.45 ug/ml respectively; of KZ4's were 55.39, 32.89 and42.40 nmol/ml respectively; and of KZ7's were 15.49, 13.24 and 21.34 nmol/mlrespectively. The results of Western Blot method proved KZ7's inhibiting actionon proteasome. CONCLUSION: The method is simple and convenient for evaluatingproteasome inhibiting activity of natural products. DM13, DM21, KZ4, KZ7 showedconcentration dependant inhibiting activity on the three proteasome enzyme,especially KZ7's inhibiting activity is very significant and worthy of further studying.