Monopolar spindle 1 (Mps1/TTK) is a protein kinase essential in mitotic checkpoint signaling, preventing anaphase until all chromosomes are properly attached to spindle microtubules. Mps1 has emerged as a potential target for cancer therapy, and a variety of compounds have been developed to inhibit its kinase activity. Mutations in the catalytic domain of Mps1 that give rise to inhibitor resistance, but retain catalytic activity and do not display cross-resistance to other Mps1 inhibitors, have been described. Here we characterize the interactions of two such mutants, Mps1 C604Y and C604W, which raise resistance to two closely related compounds, NMS-P715 and its derivative Cpd-5, but not to the well characterized Mps1 inhibitor, reversine. We show that estimates of the IC (employing a novel specific and efficient assay that utilizes a fluorescently labeled substrate) and the binding affinity ( ) indicate that, in both mutants, Cpd-5 should be better tolerated than the closely related NMS-P715. To gain further insight, we determined the crystal structure of the Mps1 kinase mutants bound to Cpd-5 and NMS-P715 and compared the binding modes of Cpd-5, NMS-P715, and reversine. The difference in steric hindrance between Tyr/Trp and the trifluoromethoxy moiety of NMS-P715, the methoxy moiety of Cpd-5, and complete absence of such a group in reversine, account for differences we observe Our analysis enforces the notion that inhibitors targeting Mps1 drug-resistant mutations can emerge as a feasible intervention strategy based on existing scaffolds, if the clinical need arises.

Monopolar spindle 1 (Mps1/TTK) is a protein kinase essential in mitotic checkpoint signalling, preventing anaphase until all chromosomes are properly attached to spindle microtubules. Mps1 has emerged as a potential target for cancer therapy, and a variety of compounds have been developed to inhibit its kinase activity. Mutations in the catalytic domain of Mps1 that give rise to inhibitor resistance, but retain catalytic activity and do not display cross-resistance to other Mps1 inhibitors, have been described. Here we characterize the interactions of two such mutants, Mps1 C604Y and C604W, which raise resistance to two closely related compounds, NMS-P715 and its derivative Cpd-5, but not to the well-characterised Mps1 inhibitor, reversine. We show that estimates of the IC50 (employing a novel specific and efficient assay that utilizes a fluorescently labelled substrate) and of the binding affinity (KD) indicate that in both mutants, Cpd-5 should be better tolerated than the closely related NMS-P715. To gain further insight, we determined the crystal structure of the Mps1 kinase mutants bound to Cpd-5 and NMS-P715, and compare the binding modes of Cpd-5, NMS-P715 and reversine. The difference in steric hindrance between Tyr/Trp604 and the trifluoromethoxy moiety of NMS-P715, the methoxy moiety of Cpd-5, and complete absence of such a group in reversine, account for differences we observe in vitro. Our analysis enforces the notion that inhibitors targeting Mps1 drug-resistant mutations can emerge as a feasible intervention strategy based on existing scaffolds, if the clinical need arises.Summary statementThe inhibition of specific Mps1 kinase inhibitors towards the wild-type protein and inhibitor-resistant mutants is explained by a novel specific activity assay, biophysical characterisation, and X-ray structures.AbbreviationsATPadenosine triphosphateBub1/Bub3budding uninhibited by benzimidazoles 1 / budding uninhibited by benzimidazoles 3Cpd-5Compound-5 (N-(2,6-diethylphenyl)-8-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide)FPfluorescence polarizationGSTGlutathione S-transferaseIC50half maximal inhibitory concentrationKDdissociation constantΔGcalccalculated Gibbs energy difference for ligand bindingKNL1kinetochore null protein 1KPi(inorganic) potassium phosphateMps1monopolar spindle 1MSTmicroscale thermophoresisNMS-P715N-(2,6-diethylphenyl)-1-methyl-8-({4-[(1-methylpiperidin-4-yl)carbamoyl]-2-(trifluoromethoxy)phenyl}amino)-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamideTMRtetramethylrhodamineWTwild-type