IC values were obtained for an

IC50 values were obtained for an expanded version of the fragment library using the previously described mobility shift assay. Subsequently, Ki values were estimated from IC50 values to allow better comparison of the activity against targets which were measured at different substrate concentrations (Supplementary data Table 1). The methylpyrazole analogue 2 showed good activity and high ligand efficiency against both Aurora A and B, with a more modest Ki value against MPS1 (Table 1). Compound 3 demonstrated modest inhibition and good ligand efficiency against MPS1 and Aurora B but lower inhibitory activity against Aurora A (Table 1). In agreement with data provided from the single point assay (Fig. 3), GNE-317 australia 15 was only active against Aurora B displaying good ligand efficiency against this kinase (Table 1). The quinoline derivative 6 possessed reasonable activity against all three kinases but a reduction in LE was observed due to the increase in number of heavy atoms (Table 1). Introduction of 2-aminopyrimidine-based substituents (compounds 7, 8) resulted in an increase in potency compared to that observed for 3 (Table 1), as it would be expected by the observed trends in percentage inhibition at 100 µM (Fig. 3). Both the methylaminopyrimidine derivative 7 and cyclopropylaminopyrimidine 8 were potent in inhibiting all three kinases, maintaining high ligand efficiency (Table 1). Repositioning of the pyridine nitrogen by substituting the 4-pyridyl with a 3-pyridyl ring, compound 11, resulted in similar activity to that seen with 3, and removal of the nitrogen by replacement of the 4-pyridyl in 3 with a phenyl ring, compound 9, again gave comparable Ki values (Table 1). The pyrimidine analogue 10 showed a decrease in activity against MPS1 compared to compound 3, and a moderate increase in inhibitory activity against Aurora A and B (Table 1). Compound 12 showed selectivity for inhibition of Aurora B over Aurora A and MPS1 (Table 1). The binding mode of these biaryl fragments to MPS1 was elucidated using X-ray crystallography. The crystal structure of the N-methylpyrazole analogue 2 in complex with MPS1 was determined with a resolution of 3.00 Å (Fig. 4), and possessed the typical kinase tertiary structure. The positioning of the α C-helix and the DFG motif, and the presence of the catalytic C- and R-spine, suggest that MPS1 is in an active conformation.44, 49 However, the activation loop (M671 - V684) could not be modelled, indicating that this region is not ordered, and no salt bridge was observed between K553 and E571. Clear electron density for the ligand was observed, identifying key hydrogen bonds formed from the aminopyridinone NH to the carbonyl of E603 (2.8 Å) and from the NH of G605 to the carbonyl of the 3-aminopyridin-2-one (2.7 Å). Protein kinases frequently feature a hydrophobic channel that extends from the hinge region to the solvent exposed surface. Positioning of hydrophobic groups along this channel is a common strategy used to increase activity of kinase inhibitors. As substitution along this vector grants access to the solvent, it is also exploited to introduce solubilising groups. The amino acid sequence along this channel is less conserved between kinases than the hinge region and can also be exploited to gain selectivity. The crystal structure of compound 2 bound to MPS1 suggests that this channel is accessible through modification of the 3-amino position (Fig. 4). Owing to this observation and the attributes of the channel that extends from the hinge region, a compound library based on benzamido derivatives of compound 3 was synthesised including a sulfonamido-based derivative, compound 21 (Fig. 5). The amide bond formation reaction and SNAr substitution reactions shown in Fig. 5 have been previously reported for the preparation of compounds of this class. Compound 3 was selected as it displayed a less promiscuous kinase profile compared with 2, and it was hypothesised to have a kinase binding mode similar to that of fragment 2. A selection of members from this library was again screened against a panel of 26 protein kinases.