The new method to identify small molecules binding proteins by usingmutants and genome sequences

本研究旨在鉴定利用基因突变果蝇及CPT建立新药感受性评价方法的作用新的体内靶蛋白，采用T7噬菌体展示法结合晶体分离摆微天平（QCM）装置鉴定CPT-20-B连接肽（NSASRGGSQRGRGEH）。该肽测序显示了每个阵列以及人HshnRNP A1和黑腹果蝇DmHrb 87 F的同源性。CPT来自使用HshnRNP A1的HeLa细胞可熔化图片的珠下拉检查。CPT使HshnRNP A1-GST结合改变蛋白质，进行QCM相互作用分析，为HshnRNP A1，解离常数为82.7 nM。微生物敏感性试验结果表明，通过基因缺失的果蝇（Drosophila melanogaster），两株Hrb 87 FKG 02089和DmHrb 87 F基因突变株Hrb 87 FBG 02743对CPT的感受性高于野生株。拓扑异构酶I的作用是消除质粒DNA的超螺旋。在CPT和HshnRNP A1存在下观察到这种拓扑变化。CPT阻碍了所报告的topo I再生。此外，hnRNP A1也同样阻碍了拓扑I的再生。此外，CPT在二者共同存在下均增强了对HshnRNPA 1的topo I活性抑制，将上述结果与CPT结合，提出了HshnRNPA 1或DmHrb 87 F调节topo I激活的可能性。

The present study aimed at the identification of the action new and in vivo target protein of the establishment of the new medicine receptivity evaluation method that used gene mutation Drosophila melanogaster and CPT that used it.CPT-20-B connecting peptide (NSASRGGSQRGRGEH) is identified by using the T7 phage display method with crystal departure pendulum microbalance (QCM) device. This peptide sequencing showed each array and the homology of human HshnRNP A1 and Drosophila melanogaster DmHrb87F. CPT united from the bead pull-down examination that used HeLa cell meltable picture with HshnRNP A1. CPT made HshnRNP A1-GST united changing protein, did the QCM interaction analysis, and was HshnRNP A1 and was dissociation constant 82.7 nM.As a result of the microbial sensitivity test, by gene loss Drosophila melanogaster, two Hrb87FKG02089 and Hrb87FBG02743 that was the DmHrb87F gene mutant showed CPT high receptivity compared with the wild strain. Topo I has working that cancels a super-coil of plasmid DNA. This topology change was observed under CPT and the HshnRNP A1 existence. CPT obstructed the topo I revitalization as reported. Moreover, hnRNP A1 also similarly obstructed the topo I revitalization. In addition, CPT has improved the topo I active inhibition to HshnRNP A1 under both existence.HshnRNP A1 or DmHrb87F united from the above-mentioned result with CPT, and the possibility of adjusting the topo I revitalization was suggested.