RT Journal Article
SR Electronic
T1 Understanding inhibitor resistance in Mps1 kinase through novel biophysical assays and structures
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 112086
DO 10.1101/112086
A1 Yoshitaka Hiruma
A1 Andre Koch
A1 Nazila Hazraty
A1 Robbie P. Joosten
A1 René H. Medema
A1 Anastassis Perrakis
YR 2017
UL http://biorxiv.org/content/early/2017/02/27/112086.abstract
AB Monopolar spindle 1 (Mpsl/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 one such mutant, the Mps1 C604Y that raises 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) confirm that the C604Y mutant is not resistant to reversine, but also indicate that 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 bound to Cpd-5 at 2.2Å resolution, and compare the binding modes of Cpd-5, NMS-P715 and reversine bound to Mps1. The difference in steric hindrance between Tyr604 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 statement The 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.Mps1monopolar spindle 1Cpd-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)NMS-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-carboxamideKDdissociation constantIC50half maximal inhibitory concentrationFPfluorescence polarizationATPadenosine triphosphateKNL1kinetochore null protein 1Bub1/Bub3budding uninhibited by benzimidazoles 1 / budding uninhibited by benzimidazoles 3TMRtetramethylrhodamineKPi(inorganic) potassium phosphateGSTGlutathione S-transferaseWTwild-typeMSTmicroscale thermophoresis