Application of Dabrafenib in Inhibiting Necroptosis and Protecting the Liver
A programmed necrosis and inhibitor technology, applied in the field of medicine, can solve problems such as the unreported therapeutic use of dabrafenib
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Embodiment 1
[0041] Example 1 Dabrafenib inhibits RIP3 kinase activity and selectivity
[0042] The inhibitory activity of dabrafenib, vemurafenib, GDC-0879, SU11248 and necrostatin-1 on RIP3 protein was detected by isotope-labeled in vitro kinase activity detection system, and the inhibition of dabrafenib on other RIP family proteins RIP1, RIP2 and Inhibitory activity of RIP5, the study was commissioned by US ReactionBiology Corporation (Malvern, PA). The results are shown in Table 1 and figure 1 middle.
[0043] Table 1 shows the in vitro inhibitory activity of the listed 5 compounds on RIP3 and the results reported in the literature on B-RAF, B-RAF V600E and C-RAF inhibitory activity, among them, dabrafenib has a strong inhibitory effect on RIP3 and three kinds of Raf kinases, but although vemurafenib and GDC-0879 have strong inhibitory effects on RAF kinases, they have no effect on RIP3 Obvious inhibitory effect. In addition, the tyrosine kinase inhibitor SU11248 and the RIP1 inhib...
Embodiment 2
[0051] Example 2 Differential expression of RIP3 in different cells
[0052] In order to investigate whether dabrafenib can inhibit RIP3-dependent programmed necrosis, the expression of RIP3 mRNA and protein in three kinds of cells (HT29, U937 and A375) was detected. Such as figure 2 As shown, the expression of RIP3 was significantly different among the three types of cells, among which the expression of RIP3 mRNA and protein levels was the highest in HT29 cells, followed by U937 cells, and almost no RIP3 expression was detected in A375 cells.
Embodiment 3
[0053] Example 3 Dabrafenib selectively inhibits RIP3-dependent programmed cell necrosis
[0054] With 20ng / ml tumor necrosis factor α (Tumornecrosisfactorα, TNFα)+100nMSmac mimic (Smacmimetic)+20μM caspase inhibitor z-VAD (TSZ) and dabrafenib / SU11248 (negative control) / necrostatin-1 (positive control) respectively After 24 hours of treatment with RIP3-expressing HT29 cells and U937 cells, it was found that dabrafenib significantly inhibited the cell death induced by TSZ treatment, and had a concentration-dependent cytoprotective effect. For HT29 cells and U937 cells, in the dabrafenib+TSZ group, 10 μM dabrafenib made more than 80% of the cells survive, EC 50 About 0.4μM, comparable to the positive control compound RIP1 inhibitor Necrostatin-1, and significantly different from the negative control compound SU11248 ( image 3 ).
[0055] HT29 cells were used as the research object to observe under the light microscope. Compared with the untreated group (control), TSZ treatmen...
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