In the NanoBRET assay MU1210 the following IC50 values were observed:
Figure 1: IC50 values from screening MU1210 and the negative control MU140 in the NanoBRET assay.
The Ki values as determined by NanoBRET assays in HEK293T cells were:
| Kinase | Ki MU1210 (µM) | |
| Ki MU140 (µM) |
| CLK1 | 0.084 | >10 |
| CLK2 | 0.091 | >10 |
| CLK4 | 0.023 | >10 |
| DYRK1A | 6.58 | >10 |
| DYRK1B | >10 | >10 |
| DYRK2 | 1.7 | >10 |
Table 1: Ki vales from screening MU1210 and the negative control MU140 in the NanoBRET assay.
After treatment of HeLa cells with MU1210 (VN339) for 3 hours the phosphorylation state of SR proteins was altered (Figure 2). MU1210 inhibited the phosphorylation of SRSF proteins in a dose-dependend manner, while the negative control MU140 had no effect on SRSF-phosphorylation.
Figure 2: Western blot analysis of phosphorylation state of SR proteins following treatment with MU1210 and its negative control MU140.
Treatment with MU1210 at 10 µM affected the alternative splicing of Mdm4 in MCF7 cells leading to an accumulation of the shorter Mdm4 form (Mdm4-S) (Figure 3). No changes were observed compared to the DMSO control, as well as the negative control MU140.
Figure 3: PCR showing alternative splicing following treatment with MU1210 (10 µM).
In vitro toxicity was assessed using a MTT assay. MU1210 was not toxic in cells at >1 µM after 24 hours. The negative control MU140 showed no significant toxicity after 24 hours, up to 35 µM (5). MU1210 toxicity was further assessed following 72 hours of treatment (Table 2).
| Cell line | Toxicity (µM) |
|
| MDA-MB-231 | 1.3 |
| MCF-7 | 1.2 |
| MCF-10a | 1.5 |
Table 2: Cellular toxicity in selected cell lines. Cells were treated with MU1210 for 72 hours. Cellular toxicity was assessed using a MTT assay.
MU1210 induced severe impairment of cell proliferation at >1µM over a longer time period (Figure 4). The high initial concentration of 5 µM in MEF cells caused precipitations of the probe in the wells due to the limited solubility of the compound.
Figure 4: Cell proliferation following treatment with MU1210 in selected cell lines.
Materials and Methods
Western blot analysis
For the dose dependence change of phosphor-SRSF proteins in Hela cells, 200,000 cells in 3 ml DMEM containing 10% FBS and Penicillin/Streptomycin were seeded in 6 well plates for 24h. The in DMSO diluted compounds were added and incubated for the stated time. Afterwards the cells were lysed mixed with SDS loading buffer, briefly heated and loaded onto a 12% SDS gel. After running the gel and blotting onto a nitrocellulose membrane, anti-phospho-SR antibody (Merck-Millipore, MABE50) was used to analyse the level of phosphorylated SRSF proteins. After washing steps, the membrane was incubated with anti-mouse antibodies coupled with horseradish peroxidase and analysed. Tubulin acted as a loading control.
Splicing analysis
The Mdm4 splicing in MCF7 cells is described in (5).
Cellular proliferation
For the proliferation assay cells were seeded between 100-200 cells/well in a 384 clear bottom plate (Nunc) in DMEM containing 10 % FBS and Penicillin/Streptomycin. After 24h compounds were added by an acoustic dispenser (ECHO) directly in each well. Proliferation was measured by determining the confluency over time in an Incucyte S3 automated microscope.
