Screening And Functional Identification Of Small Molecules Which Specifically Block The Interaction Between Hepatocellular Carcinoma Oncoprotein P28/Gankyrin And Rb

In 2000,a novel gene named gankyrin with repeated sequences coding "ankyrin' motif was cloned in human HCC by Japanese scientists Fujita and his colleagues using subtractive hybridization method.We found this gankyrin gene was identical to the p28 (Nas6p) gene reported by Hori in 1998 by searching the GenBank data,thus named it as p28^GANK in our work.P28^GANK(also known as PSMD10,p28,Nas6p,and Gankyrin),a non-ATPase subunit of the 19S/PA700 complex from the 26S proteasome,contains five ankyrin repeats mediating proteins interaction.20S proteasome is a protease degrading some incorrectly folding proteins or cell cycle regulators.Little is known about the mechanism how oncoprotein p28^GANK relates with proteins degradation by 26S proteasome.P28^GANK regulates the CDK4/CyclinD1/p16^INK4a/Rb1/E2F-1 pathway which connects with tumor development and progression,and p28^GANK-transfected cells are tumorigenic in nude mice. Recently,Nagao et al has identified MAGE-A4 binds to gankyrin and suppresses its oncogenic activity through yeast two-hybrid screen.In this article,we designed small molecules which block the interaction between P28^GANK and Rb in CDK4/CyclinD1/p16^INK4a/Rb1/E2F-1 signaling pathway.It has reported that an LxCxE motif in the C-terminal domain of P28^GANK interacts with N-terminal of pRb.Mutant in this motif established it can affect the interaction of P28^GANK and Rb.Polypeptide with LxCxE can block the interaction of P28^GANK and Rb,too. P28^GANK binding to pRb,presumably through the S6b ATPase of the 26S proteasome, increases the rate of pRb degradation.However,pRb might bind directly to the 20S core of the 26S proteasome and mediate its own degradation.Computer aid drug design(CADD) is a rapid developing frontier branches of science involving Chemistry,Biology,Computer Science,Informatics,Mathematics and Physics. Bases on the knowledge of drug and its macromolecule target,it predicts the interaction of receptor and ligand by analogizing and calculating.It directs and aids drug design to accelerate the process of drug exploitation.With the development of drug-design theory and methods,CADD plays a more and more important role in drug discovery and exploitation.It has been generally accepted that CADD is a potent drug-design method.There is no suitable pocket or active site on the surface of P28^GANK,but on Rb there is a pocket,although relatively shallow,for drug design.Therefore we choose Rb as target of drug design,hoping to find small molecules to block the interaction of P28^GANK and Rb specifically.We screened the library of SPECS and bought 71 small molecules with potential effect. Then we detect the biology function of them and found the number 6 small molecule effect. The EC50 is 3um and it can block the interaction of P28^GANK and Rb.