| Cancer is among the most fatal diseases threatening humans worldwide. About12.7million cancer cases and7.6million deaths have occurred in2008according tothe latest global cancer statistics (CA Cancer J Clin.2011;61:69). Deregulation ofcell cycle and inactivation of apoptosis together lead to the malignant transformationand tumor development. In mammalian, the cell cycle is governed by the sequentialactivation of cyclin-dependent kinases (Cdks), thereby drives the orderly transitionof each phase. Abnormal upregulation of Cdk activity is the major biologicalcharacteristic in tumor cells, for example, in breast carcinoma, lung carcinoma,colorectal carcinoma, hepatocellular carcinoma and malignant lymphomas. Currently,Cdks have been proven to be important targets for anticancer therapy. Anotherhallmark of the tumor cells is the impaired apoptosis pathway, which is the mainreason for drug resistance, for instance, approximately80%of human hepatoma cellsoverexpress XIAP protein to inhibit the activation of caspases; over50%of humantumor cells enhance the expression of anti-apoptotic molecule Bcl-2; and in somecases, tumor cells resist TRAIL-induced apoptosis by expressing the decoy receptorsDcR1and DcR2. These mechanisms mentioned above directly or indirectlycontribute to the initiation and the development of cancer, and also increase thedifficulties for cancer treatment. Nowadays, poor therapeutic outcomes and seriousside effects, together with acquired resistance to multiple drugs, are commonproblems of cancer therapies. Therefore, there is an urgent need for novelcancer-targeted drugs with strong activity and high specificity, which has become thegreat challenge in the field of drug discovery and development.In this thesis, we choose Xylocydine, a broad-wide of Cdks inhibitor, as theleading compound to develop highly specific inhibitors of Cdk2, a critical regulatorof the G1/S phase transition and the S-phase progression in cell cycle. In this respect, we resolved the crystal structure of Cyclin A3-Cdk2-Xylocydine complex, anddetermined the modification site of Xylocydine basis on the crystal structureinformation. Then, we designed and synthesized a series of Xylocydine derivatives,and clarified their functional mechanisms against tumor cells.In this study, we have obtained three major innovative results:1) It is the first time that we have successfully resolved the crystal structure ofCyclin A3-Cdk2-Xylocydine complex (Resolution2.5);2) The information of Cyclin A3-Cdk2-Xylocydine structure suggested that theC6-Br position of Xylocydine is the potent modification stie. Then, wesynthesized twenty-four Xylocydine-derived compounds that substituted6-Brwith aryl and heteroaryl groups. The in vitro kinase assay showed that threecompounds,3h,3i and3j significantly inhibited Cyclin A-Cdk2activity, at leastincreasing20-folds selectivity compared to Cyclin B-Cdk1. The3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoniumbromide (MTT) assayshowed that3h-j exhibited inconspicuous cell killing effects, however, they allstrongly arrested the cell cycle at G1/S phase in HeLa cells. These data indicatethat3h-j may also inhibit the Cdk2activity in vivo, and thereby prevent theproliferation of tumor cells. To clarify the structure-activity relationship (SAR)between Cdk2and these inhibitors, we performed the molecular dockinganalysis, and the results showed that the region of the catalytic pocket in Cdk2formed by Lys33, Glu51, Phe80and Asp145amino acid residues confer a veryimportant opportunity for selectivity to Cdk2inhibitors. In addition, the spatialorientation of the purine ring, the hydrogen bonding between–NH2(XylocydineC4position) and Leu83(Cdk2) as well as-OH (Xylocydine C11position of thecrystal structure information) and Asp145(Cdk2) may play a crucial impact onthe activity of Cdk2inhibitors. These cases should be given full consideration inthe further work.3) By analyzing the cytotoxicity of Xylocydine derivatives using the MTT assay,we obtained an active compound, named JRS-15. JRS-15exhibited muchstronger cytotoxic and pro-apoptotic activity than its parent compound in various cancer cell lines, with IC50values ranging from12.42to28.25μM.Importantly, it is more potent for killing cancer than non-cancerous cells.Mechanistic studies showed that JRS-15treatment (5.0μM) dramaticallyarrested the cell cycle at G1/S phase, while under high concentration (25μM) itinduced apoptosis in HeLa cells. It triggered the translocation of both Bax andBak to the mitochondria, resulting in mitochondrial membrane potential (MMP)depolarization and the subsequent release of cytochrome c as well as the secondmitochondria-derived activator of caspase (Smac) into the cytosol. Thesequential activation of caspase-9and caspase-3eventually results in cellapoptosis. Caspase-8, an initiator caspase that is required to activate themembrane receptor-mediated extrinsic apoptosis pathway was not activated inJRS-15-treated cells. Further analysis showed that the levels of theanti-apoptotic proteins Bcl-xL and XIAP were significantly reduced uponJRS-15treatment. Furthermore, the caspase-9or pan-caspase inhibitor, andBcl-xL or XIAP overexpression all effectively prevented JRS-15-inducedapoptosis. Taken together, these results indicate that JRS-15induces cancer cellapoptosis by regulating multiple apoptotic proteins, and this compound maytherefore be a good candidate reagent for anticancer therapy. |
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