Kinase experiment:

The kinase binding assay is used to assess compound binding to TTK by monitoring displacement of a fluorescently labeled, ATP site-directed kinase inhibitor (Kinase Tracer 236) from the kinase active site. Each 15 μL assay contains 5 nM TTK, variable amounts of test compound (Mps1-IN-1), 30 nM Kinase Tracer 236, 2 nM Eu-anti-GST Antibody, and 1% DMSO (residual from compound dilution) in Kinase Buffer A (50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35). Binding assays are initiated by addition of 5 μL of test compound (from 2-fold dilution series) to 5 μL of a kinase/antibody mixture, followed by addition of 5 μL of antibody. Assay plates are read using using standard Eu-based TR-FRET settings with excitation at 340 nm and emission monitored at 615 nm (donor) and 665 nm (acceptor). Emission intensities are measured over a 200 μs window following a 100 μs post-excitation delay[1].

Cell experiment:

U2OS cells expressing doxycycline-inducible PLK4 are plated in 96 well plates. A double thymidine block is performed using the following treatment regimen: thymidine for 18-20 hrs., release for 10 hrs. with doxycycline induction of PLK4 during this time, then a second thymidine block, followed by release. Six hours after the 2nd thymidine release, Mps1-IN-1 (or DMSO vehicle) is added and the proliferation of the cell populations is monitored with Cell Titer GLO assay[1].

Mps1-IN-1 is a selective Mps1 (monopolar spindle 1) kinase inhibitor with IC50 value of 367 nM.

MPS1 (monopolar spindle 1 kinase) is a crucial part of the spindle assembly checkpoint. It facilitates the formation of C-MAD2 (closed MAD2) conformer and the MCC (mitotic checkpoint complex) assembly, involved in the maintenance of chromosomal stability and tumor growth.

Mps1-IN-1 abolishes the SAC (spindle assembly checkpoint) function. In U2OS cells, dose-dependent treatment of Mps1-IN-1 decreases the time spent in mitosis with almost 100% cells starting anaphase in 20 minutes. In contrast, just 10% of DMSO-treated cells starting anaphase in the same period. Acceleration of mitosis kinetics in Mps1-IN-1 treated cells affects genomic stability and causes aneuploidy. In addition, Mps1-IN-1 treated cells shows decrease in kinetochore-bound Mad2 by 80%. Mps1-IN-1–treated cells spent roughly 40% less time in mitosis as compared to DMSO-treated cells. Moreover, Mps1-IN-1 disrupts the kinase activity of Aurora B. Mps1-IN-1 treatment lead to decrease in the phosphorylation status of Aurora B at Thr232 in a dose-dependent manner. Furthermore, Mps1-IN-1 increases multipolar cell divisions and decreases cell viability.

Reference:
[1].Kwiatkowski N, Jelluma N, Filippakopoulos P et al. Small-molecule kinase inhibitors provide insight into Mps1 cell cycle function. Nat Chem Biol. 2010 May;6(5):359-68.