Construction of in vitro high-throughput screening system for activin receptor-like kinases 4, 5 and 7 inhibitors / 中国药理学与毒理学杂志

ABSTRACT

OBJECTIVE To establish an in vitro screening system for activin receptor-like kinase 4,5 and 7 (ALK4,ALK5 and ALK7) inhibitors.METHODS The insect expression systems for kinase domain of ALK4,5,7 and Smad2/3 proteins were established using the Bac to Bac baculovirus expression system.The desired proteins were expressed in Sf9 insect cells and purified by GST affinity.The screening system was composed of the kinase,Smad3 protein,ATP as well as the compound.The impact of the compound on the activities of ALK kinase domains was examined by measuring the amount of remnant ATP in the system as ALKs catalyzed the phosphorylation of Smad3 protein and consumed ATP during the process.The screening conditions were optimized,and validation of the screening system was conducted using known ALKs inhibitors.RESULTS All the reconstructed Bacmids were identified to be correct by PCR and restriction enzyme digestion.All the proteins were expressed in Sf9 insect cells after transfection,and purified proteins were achieved by GST affinity purification.For the screening system,the optimized kinase concentration and Smad3 concentration were 10 mg· L-1 and the optimized ATP concentration was 10 nmol·L-1.The Z'factor for ALK4,ALK5,and ALK7 kinase inhibitors screening system was 0.71,0.51 and 0.74,respectively.The well-known ALK inhibitor SB431542 inhibited the catalytic activities of ALK4,ALK5,and ALK7 with IC50 values of 22,188 and 91 nmol· L-1,respectively.CONCLUSION The in vitro screening system for ALK4,ALK5 and ALK7 inhibitors is successfully established.