Application of small-molecule inhibitor buristatin for mitosis regulatory protein kinase BubR1
A mitosis and kinase technology, applied in the field of pharmaceutical compounds, can solve problems such as unknown relationships
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Embodiment 1
[0050] Example 1. Discovery of BubR1 kinase inhibitor bubristatin.
[0051] Whether BubR1 is a serine / threonine protein kinase has been an important topic of debate in the field of mitosis research for nearly 30 years. So far, no crystal structure information of BubR1 kinase has been reported. To resolve the controversy in the field, we employed a structural biology approach to answer the true biochemical function of BubR1. Given their conservation, we believe that the structures of their kinase domains share similarities. To this end, we simultaneously analyzed the structures of the kinase domains of human BubR1 (HsBubR1) and Drosophila melanogaster (Drosophila melanogaster) BubR1 (DmBubR1). The kinase region (720-1050aa) of HsBubR1 and the kinase region (1124-1460aa) of DmBubR1 were expressed and purified in an insect system, crystallized under classical protein kinase crystallization conditions, and X-ray diffraction analysis was performed to obtain crystal data. Finally...
Embodiment 2
[0054] Embodiment 2, the impact of BRT-1 on BubR1 kinase activity
[0055] 1. Experimental steps
[0056] 1) Prepare a basic phosphorylation reaction system (40 μL for each reaction), which contains the following components: kinase BubR1 (100 ng), kinase buffer (25 mM HEPES, 5 mM MgSO 4 , 50mM NaCl, 2mM EGTA, 1mM DTT, 0.01% Brij35, pH 7.2), 500μM ATP, substrate casein (2μg), 5μCi[γ- 32 P]-ATP, mix well. This step needs to be performed on ice, and different concentrations of the kinase inhibitor BRT-1 are added as needed.
[0057] 2) React in a 30°C water bath for 30 minutes.
[0058] 3) Terminate the reaction with 10 μL of 5×SDS-PAGE loading buffer (10% SDS; 0.5% bromophenol blue; 50% glycerol; 100 mM DTT), and cook the sample at 100° C. for 2 minutes.
[0059] 4) 25 μL of the sample was taken for SDS-PAGE electrophoresis and stained with Coomassie BrilliantBlue R250 (Coomassie BrilliantBlue R250).
[0060] 5) Put the dry glue made of SDS-PAGE in the Autoradiography Casse...
Embodiment 3
[0066] Example 3, BRT-1 inhibits BubR1 kinase activity and interferes with the process of cell mitosis
[0067] 1. Experimental steps
[0068] 1) Divide HeLa cells into live cell culture dishes;
[0069] 2) After 24 hours, transfect plasmid DNA: mCherry-tubulin and GFP-H2B (transfection plasmid cell density is 70%-80%);
[0070] 3) After 4 hours of transfection, replace with fresh medium and add Thymidine (final concentration: 2mM) to treat the cells for 14-16 hours;
[0071] 4) Thymidine was released, that is, the cells were washed 3 times with 37°C preheated PBS, and replaced with fresh medium to continue culturing;
[0072] 5) Turn on the Applied Precision Personal DV microscope and the constant temperature device to stabilize the temperature at 37°C;
[0073] 6) 8-9h after release, replace the cell culture medium with CO 2 -Independent Medium, add BRT-1 (3μM) or DMSO;
[0074] 7) Place the cells on the DV microscope stage (heated at 37° C.), and shoot in real time und...
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