Therultikinase inhibition. Taken collectively, there Wnt Pathway is considerable evidence for c Src and c Abl dual kinase inhibitors to represent an important strategy in the combat of cancer. The design of novel c Abl/c Src inhibitors on the basis of different molecular scaffolds may improve therapeutic options in patients refractory to common protocols. In this regard, our research group carried out extensive studies on a new family of pyrazolo pyrimidines which we found to block c Abl and c Src phosporylation efficiently in the nanomolar range. This new class of inhibitors induce effectively apoptosis, reduce cell proliferation in different solid tumour cell lines such as epidermoid carcinoma A431 cells, the breast cancer 8701 BC cells, the osteosarcoma SaOS 2 cells and the prostate cancer PC3 cells.
In addition, this new class of inhibitors were well tolerated in engraftment experiments with the epidermoid carcinoma cell lines A431, and evidence has been obtained for these compounds to be potent inhibitors of angiogenesis due to reduced production of VEGF. Here we report the efficacy and the molecular pharmacology of 17 novel functionalized pyrazolopyrimidines that were studied on a panel of 11 different murine lung tumour progenitor cell lines that express stem cell markers and are derived from a cmyc/craf transgenic mouse model of lung cancer, as recently reported by us. The dual kinase inhibitors were also tested in the human lung adenocarcinoma cell line A549, the human hepatoma cell line HepG2 and the human colon cancer cell line CaCo2.
To improve an understanding of the mode of action of the most active inhibitors, whole genome expression analysis were carried out and subjected to advanced computational pathway analysis to enable generation of hypothesis and to validate further such data by protein expression studies. Overall, we report the effectiveness of 17 novel dual kinase inhibitors on a large panel of epithelial tumour cell lines and provide novel insight into the mode of action of these experimental drugs. Results Cancer stem cell markers The murine lung tumour cells A2C12, BetaD5, GammaA3 and GammaD12 were isolated from mice transgenic for c Myc and c Raf as described recently. For comparison the human lung cancer cell line A549, the human hepatoma HepG2 and the colon carcinoma CaCo2 cells were studied as well.
Initially, we investigated the expression of the cancer stem cell markers Cd34, Cd24a, Cd44, Cd133, Cd90, Podoplanin, Nestin and Discs, large homolog 7 . As shown in the supplementary table S1 expression of stem cell markers varied between the different cell lines, albeit expression was generally increased when compared to appropriate controls. These cells were used to investigate the effects of a series of dual kinase inhibitors on growth and resistance of tumours and to identify possible candidates for further preclinical development. c Abl/c Src dual kinase inhibitors As the 17 4 amino substituted pyrazolopyrimidine derivatives 4 5 and 9 23 were ATP competitive, the dual kinase inhibitors were tested for kinase inhibition and affinity to c Abl and c Src. The calculated Ki values for c Abl ranged in the nanomolar concentration. However, the affinity to c Src differed considerable according to the different substitutes. While Ki .