The Study Of The Binding Modes For Cyclin-dependent Kinase4Selective Inhibitor

Due to the high sequence identity of protein kinase family and the common folding patternsof the ATP binding pocket, it is not easy to improve the selectivity of kinase inhibitors in drugdesign. Fascaplysin, an anti-cancer marine alkaloid from sponge has been shown markedselectivity inhibition for cyclin-dependent kinase4(CDK4) versus cyclin-dependent kinase2(CDK2). There are five different amino acids between the active binding site of CDK4and CDK2.And CDK4Thr102and CDK2Lys89, CDK4Glu144and CDK2Gln131may well be most responsible for theabove-mentioned selectivity of fascaplysin. The study of the binding mode of CDK4selectiveinhibitor will contributes to the structure-based drug design with higher selective inhibition and lesstoxic and side effects. Active natural human CDK4-cyclin D1and site mutation kinase complexsCDK4T102K-cyclin D1and CDKE144Q-cyclin D1and double site mutation kinase complexs can beprepared in vitro by using the molecular biology techniques such as gene cloning, prokaryoticexpression, purification and site-specific mutagenesis. The changes of the kinase inhibition amongthe fascaplysin binding to the nature and site-specific mutated kinases can be measured by using aluminescent ADP detection assay. Molecule docking results will give an intuitionistic exhibition ofthe binding modes of fascaplysin in the natural kinase and mutated kinase active binding pocketwhich is helpful in explainning the selective binding mechanism. Site-specific mutagenesis andkinase activity detection assays results: fascaplysin had the IC50of0.31μM for the natural CDK4-cyclin D1kinase complex. The Thr102Lys single site mutation led to a IC50value of16.36μMwhich was much higher than the Glu144Gln single site mutation with fascaplysin IC50value of3.365uM. Double site mutation led to the highest IC50value of47.56μM of fascaplysin. Themolecule docking results were consistent with these above-mentioned IC50variations whichindicated that Thr102and Glu144were a cause of great concern to CDK4selective inhibitor. Thisresearch method of exploring the binding mechanism of CDK4and inhibitors also can be used inother CDK4inhbitor binding modes. And the active human CDK4-cyclin D1kinase produced bythe prokaryotic expression can be used in the inhibitor high throughput screening in the laboratoryin the future.