44-1325G

Hayward D, Bancroft J, Mangat D, Alfonso-Pérez T, Dugdale S, McCarthy J, Barr FA, Gruneberg U
The Journal of cell biology 2019 Oct 7;218(10):3188-3199

Characterisation of CCT271850, a selective, oral and potent MPS1 inhibitor, used to directly measure in vivo MPS1 inhibition vs therapeutic efficacy.

Faisal A, Mak GWY, Gurden MD, Xavier CPR, Anderhub SJ, Innocenti P, Westwood IM, Naud S, Hayes A, Box G, Valenti MR, De Haven Brandon AK, O'Fee L, Schmitt J, Woodward HL, Burke R, vanMontfort RLM, Blagg J, Raynaud FI, Eccles SA, Hoelder S, Linardopoulos S
British journal of cancer 2017 Apr 25;116(9):1166-1176

Mitotic Checkpoint Kinase Mps1 Has a Role in Normal Physiology which Impacts Clinical Utility.

Martinez R, Blasina A, Hallin JF, Hu W, Rymer I, Fan J, Hoffman RL, Murphy S, Marx M, Yanochko G, Trajkovic D, Dinh D, Timofeevski S, Zhu Z, Sun P, Lappin PB, Murray BW
PloS one 2015;10(9):e0138616

Naturally Occurring Mutations in the MPS1 Gene Predispose Cells to Kinase Inhibitor Drug Resistance.

Gurden MD, Westwood IM, Faisal A, Naud S, Cheung KM, McAndrew C, Wood A, Schmitt J, Boxall K, Mak G, Workman P, Burke R, Hoelder S, Blagg J, Van Montfort RL, Linardopoulos S
Cancer research 2015 Aug 15;75(16):3340-54

Structure-based design of orally bioavailable 1H-pyrrolo[3,2-c]pyridine inhibitors of mitotic kinase monopolar spindle 1 (MPS1).

Naud S, Westwood IM, Faisal A, Sheldrake P, Bavetsias V, Atrash B, Cheung KM, Liu M, Hayes A, Schmitt J, Wood A, Choi V, Boxall K, Mak G, Gurden M, Valenti M, de Haven Brandon A, Henley A, Baker R, McAndrew C, Matijssen B, Burke R, Hoelder S, Eccles SA, Raynaud FI, Linardopoulos S, van Montfort RL, Blagg J
Journal of medicinal chemistry 2013 Dec 27;56(24):10045-65

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NIH3T3 fibroblast lysates, spiked with 1 ng active GST tagged-TTK protein (Invitrogen, Product # PV3792) (2-6) or Kinase dead mutant TTK protein (1) were resolved on a 10% polyacrylamide gel and transferred to PVDF. The membrane was blocked with a 3% BSA-TBST buffer for one hour at room temperature, and then incubated with the TTK (pTpS33/37) antibody for two hours at room temperature in 3% BSA-TBST buffer, following prior incubation with: no peptide (2), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphothreonine-containing peptide (4), a generic phosphoserine (5), or the phosphopeptide immunogen corresponding to TTK (pTpS33/37) (6). After washing, the membrane was incubated with goat F (ab')2 anti-rabbit IgG HRP conjugate (Product # ALI4404) and signals were detected using the Pierce SuperSignal™ reagent. The data show that the signal detected in the active TTK protein was selectively blocked by the phosphopeptide corresponding to TTK (pTpS33/37). The data also show the loss of phosphorylation signal on kinase dead protein indicating that the signal is phosphorylation site-specific.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 CCT271850 inhibits MPS1 autophosphorylation in cells. ( A ) Structure of CCT271850. ( B ) HCT116 cells transiently expressing Myc-tagged MPS1 were treated with increasing concentrations of CCT271850 for 2 h. Cell lysates were analysed for MPS1 autophosphorylation (at pT33pS37 and pT676 sites) and gel shift using immunoblotting. ( C ) HCT116 cells were synchronised with nocodazole for 24 h followed by treatment with indicated concentrations of CCT271850 for 2 h in the presence of MG132. Cell lysates were immunoprecipitated for endogenous MPS1 with phospho-specific anti-pT33pS37 antibodies and immunoblotted with anti-MPS1 antibodies. Total cell lysates were also immunoblotted with anti-MPS1 antibodies as a control for immunoprecipitation input (lower panel). ( D ) Immunofluorescence for MPS1 pT33pS37 localisation at kinetochores with and without treatment with CCT271850. Cells were treated with CCT271850 for 1 h, followed by treatment with nocodazole, MG132 and CCT271850 for an additional hour. Cells were fixed and stained as indicated.