Exploration of the experimental conditions of drug affinity responsive target stability / 国际药学研究杂志

ABSTRACT

Objective To explore the optimal proteolysis condition for the new technology of target discovery, drug affinity responsive target stability (DARTS). Methods First, in order to determine the suitable pronase concentration range for DARTS, the extraction of human acute T lymphoblastic leukemia cells (Jurkat) were digested with a range of pronase concentrations (the mass ratio of pronase to protein was 1 100 to 1 10000) at room temperature and detected by SDS-PAGE and Coomassie brilliant blue staining. On this basis, in order to obtain the optimal proteolysis condition, we performed DARTS on α-ketoglutarate in combination with SDS-PAGE and gel staining, and observed the effect of different concentrations of pronase (1 500-1 5000) and different proteolysis time (5-30 min) on DARTS. The feasibility of the optimum condition was verified by using it on a target known small molecule, mycophenolic acid. Results When the protein extraction of Jurkat was hydrolyzed by a range of pronase concentrations, it was entirely hydrolysed by pronase 1 100 while showing no significant effect under the condition of pronase 1 10000. The effect of pronase 1 500 to 1 5000 on protein mixture was milder. And approximately 30%-60% of the protein was digested. The protein bands which were protected by α-ketoglutarate could be observed apparently under the conditon of pronase 1 1000 when the proteolysis time was about 15 min. Then we performed DARTS on mycophenolic acid utilizing this condition (pronase 1 1000, hydrolysed for 15 min) and obtained several visible protected protein bands between (4-7) ×104. Western blotting results showed that the target protein of mycophenolic acid, IMPDH1, was contained in the protected protein bands. Conclusion The optimal proteolysis condition for DARTS on protein mixture through α-ketoglutarate is obtained.