High Throughput Screening Of Inhibitors Of Tankyrase1/2in Wnt/β-catenin Pathway

The highly conserved Wnt/β-catenin signaling pathway plays essential roles in several biological events during the cell development and differentiation. In the fetal development, Wnt/β-catenin pathway is directly involved in directing the cell growth and differentiation and in regulation of the differentiation of organs such as cardiovascular system and central nervous system. In adult organisms, it nonetheless participate in the maintenance of normal tissues as well as in the proliferation and differentiation of stem cells. Deregulation of Wnt/β-catenin pathway has been observed in many different cancers cells, prompting the possibility of targeting Wnt/p-catenin pathway for cancer therapies. However, few identified members of the Wnt/β-catenin signaling pathway restrain the scope by which the proteins of Wnt/β-catenin signaling pathway could be drug-targeted. Several recent publications have demonstrated that in colon cancer the aberrant activation of Wnt/β-catenin signaling could be down-tuned by inhibiting Tankyrase1/2, which in turn results in the stabilization of Axin, degradation of β-catenin and consequently, down-regulation of the transcriptional activity of Wnt/β-catenin signaling pathway. Tankyrase1/2functions to maintain the telomere length and to prolong cell life span in conjunction with TRF1via regulate the telomerase activity. Excess expression of tankyrase1/2was detected in many different cancer cells and is postulated as the one of the contributing factors in the cell proliferation.Thus, inhibiting tankyrase activity could potentially down-regulate the Wnt/β-catenin signaling and the study on the regulation of Wnt/β-catenin signaling pathway will shed light on the cancer therapy.This study comprises three parts:the first part is on the virtual screening of inhibitors of Tankyrase1/2based on the published X-ray structure of Tankyrase1/2.118molecules out of13.3million molecules have scored significantly high in the scoring system such as Amber Score, NN Score and DG Score and employing of Dock, AutoDock Rigid Dock and AutoDock Flex Dock evaluations.The second part using isocaudarner deals with the construction of the cell-based reporter system for the high throughput screening, that is, optimal TCF/LEF repeats have been subcloned upstream of promoter miniP into pGL4.20vector by pET-28, which is proved highly responsive to the activation of Wnt/β-catenin signaling in this luciferase reporter system(160times higher than commercialization plasmid). In the third part,11hit compounds have been purchased and tested in vitro for their effects on the Wnt/β-catenin signaling pathway, resulting in identification of2agonists and6antagonists of Wnt/β-catenin signaling pathway(lower IC50, three compounds lower IC90). All the6antagonists shows no cytotoxic effect on the DLD-1cells and Hep-3B cells at24h, but exhibits pronounced antiproliferative effect on them at72h.The strategy and methods employed in this study exemplifies a novel approach in the Wnt inhibitor discovery. The results presented in this work include the construction of Cell Luciferase Reporter System and the identification of6inhibitors of tankyrase1/2, which are capable of inhibiting Wnt/β-catenin signaling pathway in the tested cancer cells. Furthermore, The Cell Luciferase Reporter System will serve as a prototype for further optimization in screening inhibitors against Wnt/p-catenin Signaling pathway.